Enhancing the solubility of Candesartan cilexetil-inclusion Complexation using

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1 International Journal of Drug Delivery 6 (2014) Original Research Article ISSN: Enhancing the solubility of Candesartan cilexetil-inclusion Complexation using β-cd B Sunitha Reddy 1, S Aruna Jyothi 1, A Navatha 1 *Corresponding author: B. Sunitha Reddy 1 Centre for Pharmaceutical Sciences, JNUTH, Kukatpally,Andhra Pradesh, India Abstract Candesartan is primarily used as Anti-Hypertensive which is poorly soluble drug. It is available as salt form of cilexetil i.e., Candesartan cilexetil included under class II of Biopharmaceutical classification system whose bioavailability is 15%.The aim of the study is to enhance the solubility of the Candesartan cilexetil using inclusion complexation technique with β-cd as complexing agent. The complexation is evaluated both in liquid and solid state. The phase solubility profiles in different media (Millipore water, O.1N HCl, PBS 7.4) showed A L- type. Binary Mixtures of Candesartan cilexetil with CDs were prepared in the different ratios of 1molar experimentally with three different techniques (Physical Mixture, Kneading method, co-evaporation method). The complexes were analyzed using Fourier transform infrared spectroscopy and X-Ray diffractometery. From the analysis the complexation was confirmed in the co-evaporation and kneading method. Invitro studies were performed for all the ratios prepared by different methods in order to define the most appropriate ratio and preparation method. The percentage drug release from different mixtures, Marketed product and API is given as follows: Co evaporated Mixture > Kneaded Mixture > > >Physical Mixture Marketed >API Keywords: Candesartan cilexetil, β-cd, phase solubility studies, XRD, FTIR. Introduction Candesartan cilexetil is a prodrug which is mainly used for treating Hypertension, Congestive heart failure [1]. It is practically insoluble in water and it has a bioavailability of 15%. To enhance the bioavailability of this hydrophobic drug solubility should be increased. There are many strategies to increase the solubility of poorly soluble drug among them complexation is one of the technique. The international union of pure and analytical chemistry (IUPAC) [2] defines an inclusion compound as a complex a complex in which one compound forms a cavity or in the case of a crystal, a crystal lattice containing spaces in the shape of long tunnels or channels in which molecular entities of a second chemical species are located. There is no covalent bonding between the guest and host; the attraction is because of vanderwaals forces. Inclusion complexation is formed by using a complexing agent like Cyclodextrin in order to improve the physico-chemical properties. Generally, with the consideration of CD molecule characteristics, drug molecules should fit the following requirements but not without exception to form an applicable complex with βcd [3]. - More than 5 atoms (C, P, S, and N) form the skeleton of the drug molecule; - Solubility in water of should be less than 10 mg/ml; - Melting point temperature is below 250 ÀC; - The molecule consists of less than 5 condensed rings; Based on these considerations one can assume the complexation of Candesartan cilexetil with the Cyclodextrins. Molecules with holes Cyclodextrins [4]: According to Stoddart- Cyclodextrins are all purpose molecular containers for organic, inorganic, organometallic, and metalloorganic compounds that may be neutral, cationic, anionic, or even radical. The ability of a cyclodextrin to form an inclusion complex with a guest molecule is a function of two key factors [5,6]. The first is stearic which depends on the relative size of the cyclodextrin to the size of the guest molecule or certain key functional groups within the guest. The Wrong size of the guest does not fit properly into the cavity on CD. Due to the limitation of size and apolar character of the CD cavity, the complexation is not suitable for all drugs. The second critical factor is the thermodynamic interactions between the different components of the system (cyclodextrin, guest, solvent). The main driving force for complex formation is thought to be the release of enthalpy rich water from the cavity due to the entrapping of guest molecules of CDs In our work inclusion complexes are prepared by three different methods (Physical mixture, Kneading Method, Co evaporation Method). Characterization of the binary mixtures was performed using phase solubility technique, FTIR, XRD. Solubility and dissolution measurements were also performed. The aim of this work was to evaluate the best molar ratio and also the effect of the This work is licensed under a Creative Commons Attribution 3.0 License.

2 different preparation methods on the physicochemical properties of Candesartan cilexetil in various ph. Materials Candesartan cilexetil (MW: gms/mole) is a gift sample provided by Dr.ReddyÊs Lab, Hyd. β-cd (MW: 1135gms/mole) was supplied by Gangwal Chemicals Pvt. Ltd. Mumbai. All the chemicals and solvents used in this study are of Analytical grade. All the solutions are prepared using Millipore water. Methods Preparations of solid binary mixture Candesartan cilexetil β Cyclodextrin binary mixture is prepared in 1molar ratio (0.2:0.8, 0.4:0.6, 0.5:0.5, 0.6:0.4, and 0.8:0.2) as described below. Physical mixture Physical mixture of Candesartan cilexetil and Cyclodextrin (β-cd and HP-β-CD) were prepared by simply mixing powders with spatula for 15 min and then passed through sieve no. 60 and stored in air tight containers. Kneading method Kneaded product was obtained by adding small amount of water to β-cd/hp-β-cd placed in a mortar and mix it until a uniform paste is obtained to this Candesartan cilexetil was slowly added. Kneading is continued with the aid of the ethanol as cosolvent until a homogenous paste of the mixture is formed. The resulting paste was dried in hot air oven for 40 o c for 24 hrs and the dried complex was pulverized in to a fine powder and passed through sieve no 60 and stored in an air tight containers. Co-evaporation method Co-evaporated product was obtained by dissolving equimolar amount of β-cd/hp-β-cd and drug in 100ml of 50% ethanol. The solution was stirred up for complete dissolution of the powders or with the aid of ultrasonicator bath and the solvent was evaporated using a water bath at 45 o c. repeat the same procedure with 150ml of solvent. The obtained solid was ground, sieved through a sieve no. 60 and store in air tight containers. Phase solubility studies Phase Solubility studies were performed by preparing samples of 25ml solution of Millipore water with Candesartan cilexetil and β- CD, 25ml of solution of PBS 7.4 with Candesartan cilexetil and β- CD, 25ml of solution of 0.1N HCl with Candesartan cilexetil and β- CD. Approximately 5mg of drug were added to each 25 ml of solvent in test tube. Increasing amounts of Cyclodextrin (3, 6, 9, 12, 15 øm) is added to determine the change in the solubility of the Candesartan cilexetil. To form complexes in solutions these test tubes were placed in inorbital shaker for 72hrs to attain the equilibrium between the drug and the cyclodextrins. the samples were then filtered using whatman filter, The filtered samples were analyzed by UV spectrophotometer at λ max of 256nm, 270nm, 257nm in Millipore water, 0.1NHCl, Phosphate buffer solution ph 7.4 respectively. A graph is plotted with x-axis as concentration of β-cd and on y- axis concentration of Candesartan cilexetil. From the graph, slope and the intercept (s o ) are used to calculate the apparent stability constant (K). (Higuchi and Connors) 1 Melting Point The melting point of a pure substance is a characteristic of the substance being studied and any pure sample of the substance shall have same melting point. Therefore the melting point is a physical constant which can be used as a measure of purity and identity. Melting point range was studied by using a gallenkamp 220/240 volt melting point apparatus. Fourier Transform Spectroscopy Fourier transform Infrared Spectrophotometer (FTIR) has been employed as a useful tool to identify drug excipient interactions. FTIR from Alpha Bruker, consisting of Opus software is used. Using KBr pellet technique, a scan range of cm -1, the bands are identified. In these reports, the use of FTIR spectroscopy is to provide important information regarding the confirmation of inclusion complex formation of CDÊs with drug molecules[10,11]. X-Ray Diffractometery (Xrd) The powder X ray diffraction patterns of raw materials and binary mixtures were obtained at roo temperature using Bruker D8 Advance powder diffractometery, Japan, of Cu K radiation which is operated at voltage of 40Kv, current of 30 mamp, with a 2θ value of , Scan rate of 2 0 per minute, incrementing with [12]. Dissolution Studies The dissolution is performed for active ingredient, marketed drug and different binary mixtures prepared by different methods using Lab India dissolution apparatus. USP II apparatus is used for dissolution with 50rpm. Dissolution is performed in different dissolution media ( Millipore water, 01NHCl- ph 1.2, Phosphate buffer solution- ph 7.4 ) which is thermostated at 37À C. Powder Samples containing suitable amount of Candesartan cilexetil for sink condition were added to the surface of 900ml of the dissolution medium. Samples are withdrawn at 5, 10, 15, 30, 45, 60 minutes which are filtered using whatman filter paper and samples are analyzed using UV at λ max of 256nm, 270nm, 257nm in Millipore water, 0.1NHCl-pH 1.2, Phosphate buffer solution ph 7.4 respectively. The percentage drug release is calculated using after PAGE 180

3 60min. Dissolution studies are performed three times for each binary mixture in different media. Results and Discussions Phase Solubility Studies The phase solubility profiles obtained for Candesartan cilexetil: β- CD in different Medias Millipore water, 0.1NHCl-pH1.2, PBS 7.4 represented in Figure 1.The shape of all the solubility diagrams followed an A L type system (Higuchi and Connors 1965), in which a linear increase in the solubility of Candesartan cilexetil as the cyclodextrin concentration increases over the entire range. This linear host guest correlation with slope of less than 1 suggested the formation of first order soluble complexes with respect to Cyclodextrin concentration. The stability constants (K s ) are calculated using S o and slope calculated from the Phase solubility diagrams. Form the phase solubility studies indicates that stability constant depends on initial solubility which inturn depends on the ph of the medium. The ionized molecules with lower hydrophobicity should produce weaker interactions with the hydrophobic cavities of CDs than the unionized ones. Hence ionization plays an important role in complexation due to fact that he drug ionized form is less capable of displacing the water molecules of the CD cavity to yield more stable systems. XRD Table 1: Analytical parameters of Phase solubility study in Millipore water, 0.1NHCl, PBS 7.4 Cyclodextrin Medium S o *10-5 Equation K r 2 β CD Millipore Water 1.17μ0.22 y = x+1* ph 1.2 Buffer 0.402μ0.37 y = x + 4* ph 7.4 Buffer 1.48μ 0.12 y =0.016x+2* The XRD of powder samples complexed with β-cd IS shown in below figure. The characteristic and strong diffraction peaks of Candesartan cilexetil indicate the crystalline nature of drug. βcd has less diffraction peaks so less crystalline nature. The characteristic Candesartan cilexetil peaks are altered in the inclusion complexation. The characteristic peaks are completely decreased in the co evaporation method indicating that the Candesartan cilexetil- CDs inclusion complex constitutes a new solid state. Where as in the kneading method there are some evident peaks of the Candesartan cilexetil. FTIR Infrared spectroscopy was obtained using shimadzu FTIR spectrophotometer. The samples were analyzed using KBr pellet technique. Table 2: Melting point of Pure drug, β-cd and 1:1 binary mixtures Candesartan cilexetil: 169 o C βcd : 298 o C Inference 1:1 Kneaded Mixture 270 o C Confirms Complexation 1:1 Coevaporated Mixture 259 o C Confirms Complexation The IR spectrum of β-cd is characterized by intense bands at cm -1 ( cm -1 ), associated with the absorption of the hydrogen bonded OH groups of β-cd. CH-CH vibrations appear in the cm-1 region ( cm -1 ). In-vitro Studies Dissolution studies on various Candesartan cilexetil- Cyclodextrin mixtures were conducted to demonstrate the influence of the type of Cyclodextrin, and the complexation method on dissolution profile and the total amount of drug in solution. It is generally assumed that the complexes show higher dissolution as compared to that of pure drug. But the objective is to achieve higher solubility which is characteristic of inclusion complexes. Dissolution profiles of Candesartan cilexetil, and various binary systems of β CD in Millipore water, 0.1NHCl-pH1.2, PBS 7.4 are presented in the following tables and graphs. PAGE 181

4 Figure 1:X-Ray diffraction patterns of drug, βcd, 1:1 coevaporated mixture and 1:1 kneading method PAGE 182

5 Figure 2 :FTIR drug, βcd, 1:1 Coevaporated Mixture and 1:1 Kneaded Mixture C-OH bending appears as broad band at 1416cm -1. C-O stretching in alcohol appears at cm -1. C-H out of plane bending appears at cm -1 The prominent peak of the drug appears at 1728cm -1 due to the carbonyl group and methyl group at 2982cm -1 [14]. The C=O stretching of the drug disappears in the binary mixtures prepared by Kneading and co evaporation technique using βcd indicating that there is a complex formation between CD and candesartan cilexetil Table 3: The dissolution profile of Candesartan cilexetil in Millipore water, 0.1NHCl, PBS 7.4 Time Millipore water 0.1N HCl PBS (Min) Percentage release Percentage release Percentage release μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ μ0.3 PAGE 183

6 Figure 3: Time Vs Percentage of drug release of Candesartan cilexetil drug in Millipore water, 0.1NHCl, PBS-7.4 Candesartan cilexetil is a slightly acidic drug which is poorly soluble in Millipore water, acidic media, and Phosphate buffer solution. It is observed that an increase in the solubility of all media of different ratio of binary mixtures after complexation this is due to hydroxyl groups of cyclodextrins. The dissolution of active pharmaceutical ingredient and their binary mixtures in different Medias (Millipore water, 0.1NHCl ph-1.2, PBS 7.4) has been performed. API showed a very slow dissolution rate, the physical mixture showed small increase in dissolution than API other binary mixtures prepared by different methods has enhanced the solubility this can be attributed due to the formation of soluble complexes, improving the wettability or due to reduction in particle size. The dissolution rate is faster in the PBS 7.4 than Millipore water followed by 0.1NHCl. Percentage drug release was more from the Co-evaporated mixture > Kneaded mixture >>>> Physical mixture suggesting that solubility is also affected by the type of preparation method. The dissolution is also affected by the various ratios of drug and cyclodextrin, among all ratios 0.5:0.5 showed the maximum dissolution in PBS 7.4 After 60min percentage drug release from 0.5:0.5 (1:1) ratio in different media prepared by different methods Conclusions β Cyclodextrin has shown the complexation of the Candesartan cilexetil. The Inclusion complexation of the drug is influenced by the preparation methods of binary mixtures and also the ph. Among the different ratio of 1 molar 0.5:0.5 showed the maximum drug release. The drug release was maximum in the case of Co evaporation method followed by Kneading method. The solid state characterization was performed using Melting point (where the melting point of complexation is decreased when compared to β CD indicating the complexation is formed), FTIR, XRD. This methodology provides an important tool in predicting the bioavailability studies which in turn depend on the solubility of drug. Using this concept a BCS Class II drug can be changed to a BCS Class I drug. This concept had been further extended using HPβCD and comparing the results between the two Cyclodextrins. References [1]. KD Tripathi Essentials of medical pharmacology, fifth edition pg no: 453 [2]. Mc Naught and Wilkinson, Inclusion compound (inclusion complex). IUPAC Compendium of chemical technology 2 nd, October [3]. Frömming KH, Szejtli J. Cyclodextrins in pharmacy. Topics in inclusion science. Dordrecht: Kluwer Academic Publishers; 1994 [4]. Dr. Helena Dodziuk,institute of physical chemistry Cyclodextrin and their complexes published 2006 PAGE 184

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