int j pharm sci nanotech vol 9; issue 2 international … hydroxy propyl- -cyclodextrin (hpβcd)...

3170 Int J Pharm Sci Nanotech Vol 9; Issue 2 March April 2016

Research Paper

Development and Optimization of Atorvastatin Calcium-Cyclodextrin Inclusion Complexed Orally-Disintegrating Tablets with Enhanced Pharmacokinetic and Pharmaco-dynamic Profile Palem Chinna Reddy1&2, Narendar Reddy Dudhipala2, Satyanarayana Goda2 and Varsha B. Pokharkar1

1Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Erandwane, Pune-411 038, Maharashtra, India; and 2National Facilities in Engineering and Technology with Industrial Collaboration (NAFETIC) Centre, University College of Pharmaceutical Sciences, Kakatiya University, Warangal–506 009, Telangana, India.

Received December 12, 2015; accepted January 22, 2016

ABSTRACT

The content of the investigation was to study the influence of hydroxy propyl--cyclodextrin (HPβCD) complexed oral disintegrating tablets (ODTs) on enhancement of solubility, dissolution rate, pharmacodynamic activity and bioavailability of atorvastatin calcium (AT) by central composite design. Based on preliminary phase solubility studies, solubility was linearly increased and followed A

L-

type profile. Solid complexes were prepared by physical mixing, kneading, freeze and spray drying methods. Spray-dried product showed higher solubility and dissolution rate than other complexes. Amount of drug (X

1), amount of

HPβCD (X2) and amount of supradisintegrant (X

3) as

independent and solubility (Y1), disintegration time (Y

2) and

percent drug release in 15 min (Q15, Y3) as dependent responses. Drug- HPβCD complex formation was confirmed by FTIR, DSC and XRD. AT-HPβCD ODTs were developed and evaluated for physico-chemical properties,

stability and dissolution rate. Further, in vivo pharmacokinetic and pharmacodynamic studies were performed in rat model. The statistically optimized formulation showed 0.817 ± 0.06 mg/ml of solubility, 54 ± 2 sec of DT and 69 ± 2.4 % of Q15. The physical stability was studied for 6 months. No significant changes were detected in dissolution profile and drug content of tablets after 6 months during the stability studies. The in vivo studies of spray dried complexed tablets compared to AT in rats revealed that 3.3-folds improvement in oral bioavailability and there was significant reduction (p<0.01) in cholesterol and triglyceride levels and significant improvement (p<0.01) in HDL level. The results conclusively demonstrated that the AT-HPCD-ODT could be prepared with improved solubility and hypolipidemic activity by using central composite design.

KEYWORDS: Atorvastatin; Cyclodextrin complex; composite design; superdisintegrants.

Introduction

Atorvastatin calcium (AT) is a selective competitive HMG-CoA reductase inhibitor and potent lipid-lowering agent for the treatment of hyperlipidemia. AT is also prescribed in the treatment of benign prostatic hyperplasia and Alzheimer’s disease. After oral administration AT rapidly absorbed, but undergoes extensive first-pass metabolism in the gut wall and liver, that results in low oral bioavailability of about 14%. AT is very slightly soluble in water and pH 7.4 phosphate buffer, slightly soluble in ethanol, and freely soluble in methanol (Black et al., 1998; Kerc et al., 2004). Therefore, it is necessary to improve the solubility and dissolution rate of drug, which can lead to substantially enhancing its bioavailability.

In recent past, cyclodextrin complexation has been successfully used to improve solubility, dissolution rate, chemical stability and bioavailability of a number of poorly soluble drugs (Arima et al., 2001). Cyclodextrins are β-1, 4-linked cyclic oligosaccharides composed of seven D-glucopyranose units with relatively hydrophobic central cavity and having the capacity to entrap poorly soluble drug molecules to form reversible non-covalent inclusion complexes. This may improve physical and chemical properties of the incorporated guest molecule allowing, for example the improvement of solubility, stability (Rajewski and Stella, 1996) and bioavailability. However, it is known that the application of β-cyclodextrin in the pharmaceutical field is limited by its rather low aqueous solubility, which led to a search for more soluble derivatives of cyclodextrins (Szente and

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 9Issue 2March – April 2016

MS ID: IJPSN-12-12-15-POKHARKAR

3170

Reddy et al: Development and Optimization of Atorvastatin Calcium-Cyclodextrin OD Tablets 3171 Szejtli, 1999; Martin and Del, 2004; Masson and Loftsson, 1998).

Among industrially produced, standardized, and available β-cyclodextrin (βCD) derivatives, the most important ones are the heterogeneous, amorphous, highly water-soluble 2-hydroxypropylated β-cyclodextrins (HPβCD). They are widely used in pharmaceutical field owing to its ability to stabilize the drug molecules. Due to their heterogeneity, these products cannot be crystallized, which is an important advantage. It is soluble in cold water as well as in organic solvent. It is available in more than 95 % isomeric purity for injectable drug formulation (Loftsson and Brewster, 1996).

In the present scenario, statistical optimization is gaining importance in the formulation development. Response surface methodology (RSM) is an experimental design in which the factors involved either linear, quadratic or multiple and their relative importance can be assessed. It permits a thorough understanding of a process or formulation product and has important applications like optimization and in establishing the robustness and reproducibility of the product (Lewis et al., 1999). Central composite design (CCD) is an advanced from the factorial designs which have been widely used in RSM and optimization (Box and Wilson, 1951).

The objective of the present investigation was to improve the solubility, dissolution rate and pharmacodynamic activity of poorly-water soluble AT by complexation with HPβCD by spray-drying method based on preliminary studies. Finally, development of AT-HPβCD complex (spray dried) oral disintegrating tablets was developed and evaluated for physicochemical properties, dissolution rate by the application of central composite design. FTIR, DSC and X-ray diffraction studies confirmed the formation of the inclusion complexation and crystalline nature of the pure drug. Further, pharmacodynamic study in rats was carried out for hypolipidemic activity.

Materials and Methods

Materials

Atorvastatin Calcium was generously provided by Biocon Pharmaceutical Ltd., Bangalore, India. Hydroxypropyl -Cyclodextrin (HPCD) was a gift sample by M/s Roquette Ltd. (France). Dulbecco’s buffer (pH 6.8 and 7.4) was purchased from Himedia, Mumbai, India. All other chemicals and reagents used were of analytical grade.

Phase Solubility Studies

Phase solubility studies of AT with increasing concentration of hydroxypropyl-β-cyclodextrin (0.5 mM to 3 mM) were performed according to the method described by Higuchi and Connors (Higuchi and Connors, 1965). Briefly, excess amount of the AT was added to aqueous solutions containing HPβCD in a stoppered glass vials. The HPβCD solutions were prepared in distilled water, pH 1.2 and phosphate buffer (pH 7.4). The flasks were sealed and shaken for one week at 25 ± 1˚C to ensure

equilibrium. After equilibrium was attained, solutions was centrifuged at 4000 rpm for 10 min and the samples were filtered through a 0.45μm Millipore membrane filter and appropriately diluted and analyzed spectrophotometrically at 246 nm (Namdeo et al., 2014). The apparent stability constant (Ks) of the complexes was calculated from the slope of the phase solubility diagram (Ann and Nguyen, 2004).

K 1:1 = slope / So (1 – slope)

Preparation of Inclusion Complexes

All the binary mixtures were prepared in the 1:1 molar ratio between drug and HPβCD on the basis of the results obtained from the preliminary phase solubility studies.

Physical mixture

Physical mixture (1:1) was prepared by simple mixing AT and HPβCD using mortar and pestle for 10 min, the powders of both components previously sieved through a 250 μm sieve (Zingone and Rubessa, 2005).

Kneaded complex

HPβCD and distilled water were mixed together in a mortar so as to obtain homogeneous paste. AT was slowly added and the mixture was then ground for 15 min. During this process, an appropriate quantity of water was added to the mixture in order to maintain a suitable consistency. The resulting paste was dried in an oven at 45 °C for 48 h and the dried complex was pulverized into a fine powder and sieved through a 250 μm sieve (Stephane et al., 2005).

Spray-dried complex

Spray-dried product (1:1) was prepared by dissolving weighed quantity of atorvastatin calcium and HPβCD in 100 mL methanol. The resulting solution was stirred and subsequently spray-dried using laboratory scale spray dryer (Jay Instruments and system Pvt. Ltd, Mumbai, India), under the following conditions: flow rate of the solution 5mL/min; inlet temperature 78 oC; outlet temperature 50 oC; aspirator – 120 mmWC (Francisco et al., 2001).

Freeze-dried complex

Briefly, 1mM HPβCD was initially dissolved in distilled water and a 30% methanolic solution containing drug was then added stepwise to the aqueous solution of cyclodextrin derivative. It was stirred at 600 rpm for 6h until a stable suspension was formed on magnetic stirrer to obtain complexation equilibrium. All the clear solutions were frozen at – 20 °C and the frozen solutions were lyophilized in a freeze-dryer (Lyodel, Delvac Pumps Pvt. Ltd, India) to obtain a solid complex for 72 h and obtain a dry powder of AT-HP-β-CD-lyophilized.

Characterization of AT - HPβCD Complexes

Saturation solubility and Drug content

The saturation solubility of AT and different complexes were determined by equilibrating excess


powder in water and different buffer solutions (pH 1.2, 4.5 and 7.4) for 48 hrs on a mechanical shaker at 37°C. The samples were centrifuged at 4000 rpm for 10 min; supernatant was filtered through 0.45 μm membrane filter (Zingone and Rubessa, 2005). The filtrate was collected and assayed for AT spectrophotometrically at 246 nm. Drug content was determined by dissolving weighed amounts (20 mg) of inclusion complex in 10 mL of methanol. The solution was filtered, diluted and the drug content was determined spectrophotometrically at 246 nm.

Intrinsic Dissolution Rate (IDR)

In vitro IDR was measured using USP 30 dissolution apparatus type II. Pure drug and different batches of complexes were compressed in 13 mm IDR cell by using KBr press at 100 kg/cm2 for one min and was placed in 900 mL phosphate buffer pH 7.4. The dissolution medium was equilibrated at 37 ± 0.2 °C and stirred at a speed of 100 rpm. Aliquots were collected periodically and replenished with fresh dissolution media. Drug concentration was determined spectrophotometrically at 246 nm. Data analysis was carried out using PCP-Disso software (V3, Poona College of Pharmacy, Pune, India).

Central composite design - Design of experiments

In this method, a central composite design (CCD) was used to optimize the formulation variables of AT- HPβCD complexed oral disintegrating tablets, containing 3 factors and evaluated at 3 levels. The independent variables in our studies were amount of drug (X1), amount of HPβCD (X2) and amount of cross carmellose sodium as superdisintegrant (X3) for each factor an experimental range was selected based on the results of preliminary experiments (Table 1). The solubility of complex in mg/mL (Y1), disintegration time of tablet in sec (Y2) and percentage drug released at 15 min in pH 7.4 phosphate buffer (Y3:Q15) were included as responses. The experiments were designed by using DOE software (Version 9.0.0.1, Stat-Ease Inc., Minneapolis, MN, USA) at all 20 possible combinations and the layout of the design is shown in Table 2. The DOE software was used to give information not only on the critical values required to achieve the desired response but also the possible interactions of the selected independent variables on the dependent variables (Narendar and Kishan, 2015).

Preparation of tablets

Spray-drying method was selected based on the preliminary studies, and this method was used to prepare the AT-HPβCD complex as per table 2 (Francisco et al., 2001). Tablets were prepared by wet granulation technique and composition of a single tablet was shown in Table 3 and as per layout of the design. The tablets were evaluated for weight and thickness variation, friability, hardness, content uniformity and disintegration time.

TABLE 1

Variables in central composite design.

Variable Low level

Medium level

High level

X1:amount of drug (mg) 5 7.5 10 X2: amount of HPβCD (mg) 10 20 30 X3: amount of supradisintegrant (mg)

2 3 4

TABLE 2

Composition of AT HPβCD oral disintegrating tablets by Central composite design – variables and responses.

Run X1 X2 X3 Solubility DT (se) Q15 (%) 1 3.3 20 3 0.72 62 60.52 7.5 20 4.68 0.43 54 73.43 7.5 20 3 0.44 60 59.54 7.5 20 3 0.43 59 58.85 10 30 2 0.64 62 52.26 7.5 20 3 0.43 60 61.17 11.7 20 3 0.47 60 60.38 7.5 20 3 0.44 59 59.59 10 30 4 0.66 56 72.3

10 7.5 36.8 3 0.71 61 60.711 7.5 3.1 3 0.09 60 61.412 5 10 4 0.38 54 74.213 10 10 2 0.26 64 56.314 7.5 20 3 0.43 59 59.615 5 30 4 0.86 54 75.516 7.5 20 1.32 0.43 65 49.617 5 30 2 0.90 63 52.418 5 10 2 0.39 62 53.519 10 10 4 0.27 54 72.720 7.5 20 3 0.46 60 60.6

TABLE 3

Composition of AT HPβCD oral disintegrating single tablet.

S.no. Ingredients Amount (mg) 1. X1, X2 and X3 As per table 42. Lactose Monohydrate Diluent3. Microcrystalline Cellulose 20 4. Calcium carbonate 16 5 PVP K-30 2 6. Magnesium stearate 0.6

Total weight 110mg

In Vitro dissolution studies

The dissolution studies were performed using USP 30 type II dissolution test apparatus (TDT- 06P, Electrolab, India) in both phosphate buffer (pH 6.8) and 0.1N HCl. The tablets of different formulations were placed in the dissolution vessel containing 900mL phosphate buffer (pH 6.8) and 0.1N HCl maintained at 37 oC ± 0.5 oC and stirred at 50 rpm. Samples were collected periodically and replenished with a fresh dissolution medium. Concentration of drug was determined spectrophotometrically at 246 nm. Percent release of drug was calculated by using PCP-Disso software (V3, Poona College of Pharmacy, Pune, India). Similarly, this procedure was used for the dissolution studies of statistically optimized tablet formulation along with spray dried complex.

Reddy et al: Development and Optimization of Atorvastatin Calcium-Cyclodextrin OD Tablets 3173

Statistical analysis of the data and validation of the model

For the optimization of data, DOE software was used for the evaluation of the quality of fit of the model. Polynomial models including linear, 2FI and quadratic terms were initiated for all the dependent variables by employing regression analysis. Based on the comparison of several statistical parameters, such as the coefficient of R2, adjusted R2, predicted R2 and PRESS value the best fit model was selected. Further, analysis of variance was used to identify significant effects of factors on response regression coefficients. The Contour and 3-D surface response surface plots were used to illuminate the relationship between the dependent and independent responses (Narendar and Kishan, 2015). Finally, a numerical optimization technique i.e., desirability approach (based on desirability value) and a graphical optimization technique (overlay plots) were used to make new formulation with desired targeted responses. To validate the chosen optimal experimental design, the responses of experimental values were quantitatively compared with responses of predicted values by preparing new batch of formulation and relative error was calculated.

Fourier Transformed Infrared Spectroscopy (FTIR)

The formation of complex and interaction of excipients between the tablets formulations were analyzed by FTIR. The pure drug, pure HPβCD, physical mixture of optimized formulation, optimized spray dried and tablet formulation along with KBr were subjected to a pressure of 150 kg/cm2 in a KBr press (Spectra Lab. India.). The pellets were then analyzed by using FTIR (V 5300, JASCO, Japan) and the range from 4000 to 400 cm–1

was selected (Seoung et al., 2007).

Differential Scanning Calorimetry (DSC)

Thermal characteristics of drug and drug cyclodextrin complex were studied using differential scanning calorimeter equipped with an intracooler (METLLER Toledo DSC 821e module controlled by STARe software, Toledo GmbH, Switzerland). Indium/Zinc standards were used to calibrate the DSC temperature and enthalpy scale. The samples were hermetically sealed in aluminum pans and heated at a constant rate of 5 oC/min over a temperature range of 25 to 250 oC. Inert atmosphere was maintained by purging nitrogen gas at flow rate of 20 mL/min. An empty aluminum pan was used as reference (Longxiao and Suyan, 2006; Gladys et al., 2003).

Powder X-ray Diffractometry (PXRD)

The X-RD patterns were reported on X-ray Diffractometer (PW 1729, Philips, Netherl and) the samples were irradiated with monchromatized CuKα radiation (1.542 A°) and analyzed between 2-80° 2θ. The voltage and current used were 30 kV and 30 mA respectively. The range and the chart speed were 5 × 103

CPS and 10 mm/ °2θ, respectively (Nalluri et al., 2003).

Stability studies

The stability studies were carried out according to ICH guidelines. The statistically optimized tablet formulation was stored at 40° C/75 % RH for 180 days. Samples withdrawn after three months and six months were evaluated for disintegration time, drug content and dissolution rate.

In vivo Pharmacokinetic and Pharmacodynamic studies

The hypolipidemic activity and pharmacokinetic parameters of inclusion complex ODT formulation was determined in comparison with pure AT in healthy albino rats (Wistar strain) of either sex, weighing between 190 and 250g. The animals were procured from National Toxicology Center (Pune, India). General and environmental conditions were strictly monitored. The institutional animal ethics committee of Poona College of Pharmacy, Pune, India, approved the research protocol of the animal experimentation.

The animals were divided into four groups and each group was having six animals. The animals were starved for 18 hours and then injected 200 mg/kg Triton WR 1339 (iso octyl-polyoxyethylene phenol) intraperitonealy for three groups. Serum cholesterol levels increased sharply 2-3 times after 24h. Reference and test groups additionally received aqueous suspensions of pure drug and spray dried complex (equivalent to 10 mg/kg body weight) respectively, prepared by using 1% w/v gum acacia as a suspending agent.

Pharmacokinetic parameters of AT after administration of Statistically optimized spray dried ODT product and AT pure drug oral suspension were estimated for each rat by using a computer program, KINETICA 2000 (Version 3.0, Innaphase Corporation, Philadelphia, USA). Analysis was used to calculate the pharmacokinetic parameters, Cmax, tmax and area under the curve (AUC). Blood samples were collected under light ether anesthesia by retro orbital puncture at 0, 1, 2, 4, 6, 8, 12, 16, 20, 24 and 48 h. Collected samples were divided into two portions, one portion samples were analyzed for total cholesterol, triglycerides (TG) and high density lipoprotein (HDL) levels by the in vitro diagnostic kit (ACCUREX BIOMEDICAL PVT.LTD., Mumbai, India) and other portion is used to calculate the pharmacokinetic parameters, Cmax, tmax and area under the curve (AUC). The statistical analysis for the determination of differences in PK parameters and lipid profiles of treatment and control groups (hyperlipidemic and normal control) was done by one way ANOVA followed by Dunnet’s test and p<0.05 was taken as significant (Anshuman et al., 2005; Vogel and Vogel, 1997).

Results and Discussion

Phase solubility studies and Stability constant

The phase solubility diagrams of AT: HPβCD was obtained by plotting the changes in guest solubility as a function of HPβCD concentration. Figure 1 illustrated


that the apparent solubility of AT increases linearly as a function of HPβCD over the entire concentration range and was characteristic of AL type of curve that suggested formation of a water-soluble complex. The slope values obtained were less than 1, which indicated that inclusion complex in the molar ratio of 1:1 between the guest and the host molecule was obtained irrespective of the pH.

Fig. 1 Phase solubility studies of AT with HPCD (Mean±SD, n=3).

The extent of complexation in aqueous media (i.e., the stability of the formed complex) was characterized by the stability constant Ks. The Ks determination was based on the solubility diagrams, which required calculations involving drug solubility. Hence, Ks values were calculated according to the equation of Higuchi and Connors from the initial straight-line portion of the solubility diagrams by assuming that a 1:1 complex was initially formed (the slope was less than 1). The apparent stability constants Ks of the 1:1 complexes at each pH value were calculated from the slopes of the solubility diagrams. The Ks values decreased with increase in the pH (Table 4). This phenomenon could be due to the ionization of the acidic atorvastatin at pH > pKa. Ionic form of the drug showed lower hydrophobicity and weaker interactions with the hydrophobic cavity of HPβCD than the unionized drug. The results reveal that the pH played an important role in determining the strength of complexation between drug and HPβCD.

TABLE 4

AT saturation solubility, slope, Ks and correlation coefficient (R2) from phase solubility diagrams (Mean±SD, n=3).

Buffer Solubility of ATC

Slope Ks (M-1) R2

(Mean ± SD) pH7.4 0.90 ± 0.09 0.192 102 0.999 Distilled Water 0.63 ± 0.09 0.169 122.4 0.998 pH 1.2 0.09 ± 0.01 0.042 265.2 0.996

Saturation solubility and Drug content of complex

The saturation solubility of pure drug, physical mixture, kneaded complex, freeze dried complex and spray-dried complex were obtained over a range of pH 1.2 to 7.4 as shown in Table 5. The saturation solubility of atorvastatin calcium increases from 0.09 ± 0.01 mg/mL to 0.90 ± 0.1 mg/mL (10- fold). This pH solubility profile is consistent with the ionization of the carboxyl group. This study supports interconversion kinetics, equilibrium, and solubility of the lactone and hydroxy forms of the atorvastatin sodium (Kearny et al., 1993). It can be seen that an increase of the solubility values was obtained from all the binary mixtures. This was probably due to the presence of hydrophilic cyclodextrin and a better wettability of the drug. Spray-dried product showed a 39-fold and 32- fold increase in water and phosphate buffer (pH 7.4), respectively than pure drug, attributable to the formation of inclusion complex. The drug content of physical mixture, kneaded, freeze-dried and spray-dried complexes were found to be 96.5%, 95.2%, 97.6% and 99.4% respectively and were in good agreement with the theoretical and actual drug content.

Intrinsic dissolution profile

The intrinsic dissolution studies of pure drug, physical mixture, kneaded product, freeze dried and spray dried product were performed in phosphate buffer pH 7.4. Pure AT showed a flux of 102 μg cm–2 h–1. The enhancement in intrinsic dissolution rate was dependent on the preparation method. The physical mixture and kneaded product showed a slight increase in the dissolution rate. This result was due to the solubilizing effect of the HPβCD and also by improved wettability of the drug. A significantly higher intrinsic dissolution rate was observed in spray dried product (258 μg cm–2 h–1) than other methods (freeze-dried, kneaded and physical mixture product showed 189, 170 and 161 μg cm–2 h–1, respectively) due to the formation of soluble inclusion complex, amorphisation of the drug and consequently higher solubility and better wettability. From the saturation solubility, drug content and intrinsic dissolution studies spray drying method exhibited better results than other methods; hence, spray dried inclusion complex was used for the preparation of ODT tablets.

Physical properties of tablets

The tablets prepared by wet granulation method using spray-dried complex were analyzed for the physical properties as per USP 30. Weight variation, thickness variation, hardness, friability and drug content were shown in Table 6. It showed good uniformity of weight and thickness of all the formulations. The tablets also exhibited good mechanical properties with regard to both friability and hardness.

Reddy et al: Development and Optimization of Atorvastatin Calcium-Cyclodextrin OD Tablets 3175 TABLE 5

Saturation solubility (mg/mL) of atorvastatin calcium complexes with HPβCD (Mean±SD, n=3).

Solvents SD Complex Kneaded product FD product Physical mixture Pure Drug

Phosphate buffer (pH 7.4) 28.76 ± 0.5 16.66 ± 0.59 19.43 ± 0.73 3.83 ± 0.09 0.90 ± 0.1 Distilled Water 24.78 ± 0.9 14.46 ± 0.67 16.19 ± 0.48 2.16 ± 0.05 0.63 ± 0.1 Acetate buffer (pH 4.5) 16.14 ± 0.2 10.12 ± 0.12 11.33 ± 0.35 1.98 ± 0.02 0.31 ± 0.2 0.1 N HCl (pH 1.2) 3.86 ± 0.16 2.83 ± 0.10 3.02 ± 0.13 0.40 ± 0.01 0.09 ± 0.01

SD-spray-dried; FD-Freeze-dried

TABLE 6

Physical properties of prepared tablets by design of approach.

Batch No. Weight (mg)a Thickness (mm)b Hardness (Kp)b Friability (%)a D.T. (Sec)b Drug Content (%)c

B1 109.5 ± 2.03 2.86 ± 0.84 4.50 ± 1.28 0.328 62 107.1 ± 2.4 B2 108.3 ± 1.88 2.92 ± 0.96 4.60 ± 1.18 0.301 54 101.5 ± 1.5 B3 110.1 ± 2.32 2.89 ± 1.01 4.70 ± 1.13 0.284 60 98.85 ± 1.8 B4 109.2 ± 1.64 2.91 ± 0.86 4.50 ± 0.84 0.265 59 99.4 ± 2.2 B5 109.5 ± 2.21 2.96 ± 1.02 5.10 ± 0.54 0.230 62 99.6 ± 1.4 B6 108.4 ± 1.84 2.9 ± 0.68 4.90 ± 0.68 0.195 60 102.5 ± 2.6 B7 109.8 ± 1.45 2.93 ± 0.94 4.60 ± 1.04 0.311 60 100.1 ± 0.98 B8 110.3 ± 2.12 2.8 ± 0.96 4.53 ± 1.11 0.310 59 100.1 ± 1.1 B9 109.9 ± 2.26 2.86 ± 1.01 4.640 ± 1.08 0.297 56 98.99 ± 1.2

B10 108.7 ± 1.40 2.96 ± 0.86 4.55 ± 0.83 0.261 61 99.8 ± 1.9 B11 109.2 ± 2.11 2.65 ± 1.02 4.73 ± 0.69 0.263 60 99.6 ± 1.2 B12 110.3 ± 2.41 2.68 ± 0.51 4.70 ± 0.58 0.188 54 102.4 ± 2.01 B13 109.6 ± 1.74 2.47 ± 0.93 4.61±1.11 0.312 64 102.1 ± 1.5 B14 109.5 ± 2.30 2.78 ± 0.84 4.57 ± 1.05 0.332 59 100.3 ± 1.2 B15 109.4 ± 2.21 2.86 ± 1.01 4.82 ± 1.00 0.281 54 99.85 ± 1.18 B16 110.0 ± 1.73 2.71 ± 0.63 4.64 ± 0.75 0.273 65 98.4 ± 1.04 B17 108.8 ± 2.10 2.86 ± 1.02 4.93 ± 0.88 0.244 63 99.16 ± 0.98 B18 109.5 ± 2.43 2.68 ± 0.71 4.92 ± 0.73 0.198 62 100.4 ± 1.4 B19 109.1 ± 1.69 2.86 ± 1.01 5.10 ± 0.54 0.310 54 99.23 ± 1.55 B20 110.2 ± 1.04 2.73 ± 0.73 4.88 ± 0.82 0.263 58 99.63 ± 1.26

All data expressed as Mean±SD, a-n=20, b-n=6, c-n=10

Statistical analysis of the data and validation of the model

From the response surface model, the regression equations (1-3) were obtained by Design Expert software over the range of independent variables from the sequence order as per in table 2 as follows:

Y1 = +0.44–0.082*X1+0.21* X2+2.93* X1 X2–0.01* X1 X3+0.07* X1

2+7.79*

X22+0.011* X3

2

Y2= +59.4–0.02*X1+0.20* X2+3.77* X3

Y3= +61.7–0.18*X1–0.40* X2+8.81* X3

In general, the sign and value of the quantitative effect represent tendency and magnitude of the term’s influence on the response, respectively (Sudhir et al., 2009). In regression equation positive value indicates an effect that favors the optimization due to synergistic effect, while a negative value indicates an inverse relationship or antagonistic effect between the factor and the response. Regression values represent the quantitative effect of process variables; X1, X2, X3 and their influence on the dependent responses; Y1, Y2 and Y3.

The responses of all the formulations were fitted to linear, interaction or quadratic models using DOE software. A quadratic model is suggested for solubility, linear model for DT and Q15. The calculated R2 (Table 7) values for all the responses ranged from 0.8717 to 0.9743 indicating a good model. The adjusted and predicted R2 values were 0.9494 and 0.8039 for Y1, 0.8694 and 0.8024 for Y2, 0.8958 and 0.8525 for Y3 respectively. In all the responses, the difference between the adjusted and predicted R2 values always below 0.2 and an agreement as per DOE requirement. When observed value of R2 was at least 0.80, it implied a good correlation and was found in all cases, indicating a good fit by model (Torrades et al., 2011). Analysis of variance and press values for the responses indicated was significant and valid for each of the responses Y1 (p<0.0011), Y2 (p<0.0229) and Y3 (p<0.0001) and hence was appropriate to represent the observed data, respectively.

The two-dimensional contour plots and three-dimensional response surface plots are shown in Figure 2. These plots are very useful to study the interaction effects of the factors on the responses, i.e., two factors on the response at one time. In all the presented figures, the third factor was kept at a constant level.


TABLE 7

Regression values represents the quantitative effect of process variables for responses.

Parameter Source R2 Adjusted R2 Predicted R2 Press p value

Y1 Linear 0.8717 0.8477 0.7815 0.17 0.0001 2FI 0.8807 0.8257 0.6035 0.30 < 0.0001 Quadratic 0.9734 0.9494 0.8039 0.15 0.0011

Y2 Linear 0.8900 0.8694 0.8024 43.23 0.0229 2FI 0.8963 0.8485 0.5306 102.70 0.0140 Quadratic 0.9080 0.8251 0.3228 148.18 0.0076

Y3 Linear 0.9122 0.8958 0.8525 171.80 < 0.0001 2FI 0.9243 0.8894 0.6555 401.34 0.5733 Quadratic 0.9531 0.9109 0.6629 392.72 0.1716

Fig. 2 Response surface plots (Contour and 3D plots) showing (i) the effect of independent variables on solubility (Y1), (A & B), (ii) disintegration time (Y2) (C&D) and (iii) Q15 % release (Y3) (E&F) of AT-HPCD ODTs.


When solubility (Y1) was indicated as the response, good correlation was shown between observed and predicted value with a p<0.0001. Magnitude of the positive coefficient (0.44) of the term is suggesting that the increased amount of drug and cyclodextrin in the complex formation could result in the enhancement of the solubility. The interactive term of X1X2 also plays major role in the enhanced solubility. But, alone the amount of supradisintegrant could not show any effect on the solubility.

In case of DT, the linear terms of variables exhibited synergetic effect based on integer value (59.4) and DT was ranging from 54 to 65 sec. This could be attributed to that better wettability due to presence of cyclodextrin.

The release of drugs from the tablets mainly affected by the amount of the X3, it showed synergistic effect on the drug release, as the amount varied the disintegration time changed. The other two variables not exhibited promised effect on the Y2 and Y3 responses (observations from regression equations 2 and 3).

Optimization of independent variables and validation of the AT-HPβCD complexed tablet

After analyzing the polynomial equations, depicting the dependent and independent variables, a further optimization and validation process by means of the design expert software was undertaken with desirable characteristics to probe the optimal formula solution of AT- HPβCD complexed tablet. This depended on the prescriptive criteria of a maximum solubility in mg/mL, minimizes the DT and targeted drug release in Q15%. Using a Design-Expert 9.0 software optimization process, recommended constraints of the independent variables were calculated by the desirability plot and the desirability was found to be 0.901. Based on the desirability, 5 mg of drug, 29.8 mg of HPβCD and 3.9 mg of cross carmellose sodium, which gives theoretical values of 0.82 mg/mL of solubility, 55.9 sec of DT and 70% of drug release in 15 min (Q15), respectively. Therefore, in order to confirm the predicted model, a new both of formulation was prepared according to the optimal factor levels. The observed optimized formulation had 0.817±0.06 mg/mL of solubility, 54.3±2.11 sec of DT and 69.2±2.42% of drug release in Q15%, which were in good agreement with the predicted values (Table 8). A comparison between these observed results and theoretical predictions indicated the reliability of CCD in predicting a desirable AT- HPβCD complexed tablet.

TABLE 8

Comparison of predicted and experimental values for validation of optimized formulation (Mean±SD, n=3).

Parameter Predicted value

Experimental value

Relative error (%)

Y1 (mg/mL) 0.82 0.817 ± 0.06 1.23

Y2 (Sec) 55.9 54.3 ± 2.11 2.94

Y3 (%) 70 69.2 ± 2.42 1.15

In vitro dissolution studies of tablets

In vitro dissolution study was carried out in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) for the prepared tablets and spray dried complex (statistically optimized) (Figure 3a-b). The percentage drug release from all the formulations of spray dried complex and tablets were found to be 40.4 to 68.3%, 64.2 to 82.4%; 83.5 to 94.2%, 90.1 to 100.8 % in 0.1 N HCl and phosphate buffer (pH 6.8) respectively (data not shown). The results reveal that the dissolution rate was faster in phosphate buffer (pH 6.8) as compare to acidic media; it might be due to acidic nature of drug (weak acid, pKa 4.46). In phosphate and acidic media, dissolution of tablet showed dramatic enhancement as compared to spray-dried complex product. This could be due to formation of inclusion complexation with HPβCD (Aly et al., 2003).

Fig. 3a Dissolution profiles of spray-dried complex and spray-dried complex tablet formulation in 0.1N HCl (pH 1.2) (Mean±SD, n=3).

Fig. 3b Dissolution profiles of spray-dried complex and spray-dried complex tablet formulation in phosphate buffer (pH 6.8) (Mean±SD, n=3).


FTIR Study

AT showed characteristic bands at 3063, 1658, 1593, 1226, 1159 and 841 cm–1. The IR spectra of HPβCD showed prominent absorption bands at 3414, 2933, 1164, 1083 cm–1. Physical mixture showed bands at 3387, 1658, 1585, 1313, 1157 and 1028 cm–1. There was slight shift in the absorption spectrum of the drug and it indicates that there was no strong interaction between the drug and the cyclodextrin molecule (Fig 4). The spectra of kneaded complex also showed the same bands. The IR spectra of spray-dried product revealed bands at 3364, 1651 and 1577 cm–1. These were attributed to interaction between the drug and OH groups of the cyclodextrin. Absence of bands at 3063, 1313 and 841cm–1 indicated that the vibrating and bending of the guest molecule (AT) was restricted due to the formation of an inclusion complex and the aromatic rings of atorvastatin being inserted into the cavity of the cyclodextrins (Narender and Tasneem, 2004; Sanjula et al. 2005).

Fig. 4 FTIR graphs of AT-HPβCD complexed ODTs: A) Pure Drug B) HPβCD C) Physical Mixture D) Spray-dried complex E) Spray-dried complex tablet.

DSC Studies

The DSC thermograms of pure drug, physical mixture, HPβCD, optimized spray-dried complex and tablet were shown in Figure 5. Pure drug characterized by a single, sharp melting endotherm at 158.57 oC (H –47.42 Jg-1). The DSC curve of HPβCD exhibited a very broad endothermal phenomenon between 60 °C and 120 °C due to loss of water. A broad endotherm was observed in the thermograms of physical mixture, spray dried complex due to loss of water during the heating cycle. However no endotherm characterizing the melting transition was noted in the thermogram of spray-dried complex and tablet suggesting that the complete

inclusion complex without free AT was formed. This is suggestive of the formation of an amorphous inclusion complex with the molecular encapsulation of the drug inside the HPβCD cavity. The previously published study on the DSC thermogram of nimesulide and cyclodextrin complex, have reported that the characteristic peak of nimesulide completely disappears and concluded that a true inclusion complex was formed (Narender and Tasneem, 2004).

Fig. 5 DSC thermograms of AT-HPβCD complexed ODTs: A) Pure Drug B) Physical mixture C) HPβCD D) Spray-dried complex E) Spray-dried complex tablet.

PXRD Studies

A supporting evidence for the formation of an amorphous inclusion complex between drug and HPβCD was obtained from powder X-ray diffraction pattern. The powder X-ray diffraction patterns of pure drug, physical mixture, kneaded product and spray-dried complex are shown in Fig. 6. The PXRD pattern of pure drug presented several diffraction peaks indicating the crystalline nature of the drug. The drug shows 2θ values at 9.15, 9.47, 10.26, 11.85, 12.19, 17.07, 19.48, 21.62, 22.96, 24.43 28.92 and 29.23. HPβCD presented an amorphous X-ray diffraction pattern. The PXRD patterned of physical mixture showed some of characteristic peaks, indicating the presence of AT in crystalline state. In contrast, spray-dried complex showed complete absence of sharp peak and some peaks with reduced intensity at 17.075, 19.48, 21.62, and 22.96, suggesting that the existence of amorphous state of drug. This reduction in crystallinity can be attributed to the spray-drying treatment. This phenomenon confirmed that atorvastatin calcium - HPβCD forms an efficient inclusion complex in the solid state (Sanjula et al., 2005).

Stability Studies

The disintegration time, in vitro release and percentage drug content results (Table 9) reveal that after three months and six months there was no significant (p>0.05) difference in disintegration time, in vitro release and percentage drug content. Thus the complexation of AT with HPβCD was found to be stable.


Fig. 6 PXRD spectras of AT-HPβCD complexed ODTs: A) Pure Drug B) HPβCD C) physical mixture D) Spray-dried complex.

TABLE 9

Stability study of statistically optimized formulation during storage at different time intervals (Mean±SD, n=3).

Time Dissolution at the

end of 60 min. Disintegration

Time (sec) Drug

Content (%)

Day 0 99.0 ± 0.7 54.6 ± 2.3 99.6 ± 1.4 Day 90 99.7 ± 1.6 53.9 ± 2.6 99.5 ± 2.5

Day 180 98.2 ± 0.9 55.1 ± 3.1 99.2 ± 1.6

In vivo Pharmacokinetic and Pharmacodynamic Studies

A hypolipidemic drug like AT was known to reduce elevated total cholesterol and triglycerides (TG) levels in blood. At the same time they cause elevation of HDL levels, which promote the removal of cholesterol from peripheral cells and facilitate its delivery back to the liver (Vogel and Vogel, 1997). This pharmacodynamic effect is reported to be dose dependent hence, was used as a basis for the comparison of in vivo performance of

pure AT and spray-dried complexes with HPβCD. The serum lipid profiles of all the experimental groups at different time intervals are presented in Table 10. The results showed that there was significant decrease (p<0.01) in serum cholesterol and TG levels and increase (P<0.05) in HDL levels. Triton WR-1339 treated group (AP2) showed significant increase (p<0.01) in the cholesterol and TG levels as compared to reference group (AP3) and test group (AP4).

The mean plasma concentration–time profiles of AT following the application of the statistically optimized spray dried ODT formulation (AP4) and AT pure drug suspension (AP3) to rats are shown in Figure 7. The higher Cmax and tmax values were observed for AP4 formulation compared to the AP3 formulation. The mean Cmax and tmax for AT were calculated to be 67.9 ± 10.1 and 165.7 ± 16.2 ng mL−1; 1.86 ± 0.43 and 2.45 ± 1.03h, respectively, after administration of statistically optimized spray dried ODT formulation (AP4) and AT pure drug suspension (AP3). The mean AUC0-24 and AUCtotal for AT were found to be 127.1 ± 21.42 and 141.2 ± 23.56 ng-hmL−1; 419.5 ± 57.8 and 421.8 ± 59.7 ng-hmL−1 after administration of AP3 formulation and AP4 formulation formulations, respectively. The overall mean values of AUC0-n are 3.3 times higher for statistically optimized spray dried ODT formulation compared to the AT pure drug suspension (Table 11). The results suggest that statistically optimized spray dried ODT formulation showed greater bioavailability as the complex is having more solubility and dissolution rate. Thus the serum profiles of cholesterol and triglycerides were reduced significantly and the HDL was increased significantly. Therefore it can be concluded that the spray dried complex is having promising role in enhancing the solubility and thus subsequent improvement in the bioavailability of AT which could efficiently control the serum lipids.

TABLE 10

Serum Cholesterol, triglyceride and HDL profiles of various experimental groups at different time intervals (Mean ± SD, n= 6).

Group No.

Serum Cholesterol (h) Serum Triglyceride (h) Serum HDL (h)

0 6 24 48 0 6 24 48 0 6 24 48 AP2 44.6 ± 1.5 95.7 ± 3.3 254.2 ± 11.4 44.2 ± 2.4 65.3 ± 6.3 551.3 ± 16.3 727.4 ± 22.5 113.5 ± 8.2 28.3 ± 1.1 27.6 ± 1.1 29.4 ± 1.1 29.1 ± 2.1AP3 42.6 ± 2.6 61.3 ± 3.7 138.2 ± 7.8$ 47.5 ± 1.9 64.2 ± 7.5 382.4 ± 14.8$ 422.7 ± 19.1$ 88.9 ± 5.3$ 29.4 ± 2.1 32.3 ± 1.2 37.7 ± 1.9 32.6 ± 1.7AP4 49.2 ± 1.7 40.2 ± 2.3$ 48.4 ± 3.1# 46.4 ± 2.2 79.1 ± 7.2 172.5 ± 9.4#, * 193.6 ± 13.6#,* 87.1 ± 7.3$ 30.5 ± 1.4 44.6 ± 2.1 59.2 ± 1.9$ 40.1 ± 1.7AP 43.1 ± 0.9 42.8 ± 1.2 43.3 ± 2.3 45.4 ± 1.8 65.7 ± 4.3 62.9 ± 3.9 66.7 ± 6.1 66.4 ± 5.4 31.5 ± 1.2 30.3 ± 2.0 29.2 ± 1.2 29.8 ± 2.1

Fig. 7 Mean serum profiles of Atorvastatin calcium in healthy albino rats after administration of AT pure drug suspension (AP3) and Statistically optimized spray dried ODT (AP4) formulation (AP4), (Mean ± SD, n=6).


Conclusions

The inclusion complexes of AT with HPβCD could be prepared by the spray-drying method in a molar ratio of 1:1 and converted to ODT using cross carmellose sodium as superdisintegrant by applying central composite design The inclusion complexes were found to have improved solubility, in vitro drug release compared with the pure drug. The results clearly demonstrated a significant decrease in the hypolipidemic activity in rats. The physical stability was studied for 6 months and no significant changes were detected in drug content, disintegration time and dissolution profile of statistically optimized spray dried ODT tablets. The in vivo studies of spray dried complexed tablets compared to AT in rats revealed that there was significant reduction (p<0.01) in cholesterol and triglyceride levels and significant improvement (p<0.01) in HDL level. The Bioavailability of AT was 3.29 times higher for statistically optimized spray dried ODT formulation compared to the AT pure drug suspension. The results conclusively demonstrated that the AT-HPCD-ODT could be prepared with improved solubility, dissolution rate, hypolipidemic activity and bioavailability of AT by using central composite design.

Acknowledgements

One of the authors (Dr. Palem Chinna Reddy) acknowledges the financial support received from All India Council for Technical Education, New Delhi, India.

Declaration of Interest

The authors report no conflicts of interest.

References Aly AM, Qato MK and Ahmad MO (2003). Enhancement of the

dissolution rate and bioavailability of glipizide through cyclodextrin inclusion complex. Pharm Tech 54-62.

Ann MS and Nguyen BN (2004). Effect of hydroxypropyl-β-Cyclodextrin-complexation and pH on solubility of Camptothecin. Int J Pharm 284: 61-68.

Anshuman AA, Mahadik KR and Anant P (2005). Spray-dried amorphous solid dispersions of simvastatin, a low tg drug: in vitro and in vivo evaluations. Pham Res 22(6): 990-998.

Arima H, Yunomae K, Miyake K, Irie T, Hirayama F and Uekama K (2001). Comparative studies of the enhancing effects of cyclodextrins on the solubility and oral bioavailability of tacrolimus in rats. J Pharm Sci 90: 690-701.

Black AE, Michael WS and Roger NH (1998). Metabolism and excretion studies in mouse after single and multiple oral doses of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor Atorvastatin. Drug Met Dis 26: 755-763.

Box GPE and Wilson KB (1951). On the experimental attainment of optimum conditions. J Royal Stat Soc Ser B 13: 1-45.

Francisco V, Catarina F and Philippe M (2001). Influence of the preparation method on the physicochemical properties of tolbutamide/cyclodextrin binary systems. Drug Develop Ind Pharm 27(6): 523-532.

Gladys G, Claudia G and Marcela L (2003). The effect of pH and triethanolamine on sulfisoxazole complexation with hydroxypropyl-β-cyclodextrin. Eur J Pharm Sci 20: 285–293.

Higuchi T and Connors K (1965). Phase-solubility techniques. Adv Anal Chem Instrum 4: 117-212.

Kearny AS and Crawford LF (1993). The Interconversion kinetics, equilibrium and solubilities of the lactone and hydroxy acid forms of the HMG-CoA reductase inhibitors. Pharm Res 10(10): 1461-1465.

Kerc K, Salobir M and Bavee S (2004). Atorvastatin calcium in a pharmaceutical form composition thereof and pharmaceutical formulation comprising atorvastatin calcium. US pat 2004/0138290A1.

Lewis GA, Mathieu D and Phan Tan Lui R (1999). Editors. Pharmaceutical experimental design. New York: Marcel Dekker Inc.

Loftsson T and Brewster M (1996). Pharmaceutical applications of Cyclodextrins: A. drug solubilization and stabilization. J Pharm Sci 85: 1017-1025.

Longxiao L and Suyan Z (2006). Preparation and characterization of inclusion complexes of prazosin hydrochloride with β-cyclodextrin and hydroxypropyl- β-cyclodextrin. J Pharm Biomed Anal 40:122-127.

Martin EM and Del V (2004). Cyclodextrins and their uses: A review. Process Biochem 39: 1033-1046.

Masson M and Loftsson T (1998). Stabilization of ionic drugs through Complexation with ionic and non-ionic cyclodextrins. Int J Pharm 164: 45-55.

Nalluri BN and Chowdhary KPR (2003). Physicochemical characterization and dissolution properties of nimesulide cyclodextrin binary systems. AAPS Pharm Sci Tech 4(1): 6-17.

Namdeo RJ, Ramesh SK and Sameer JN (2014). Dual Wave length Spectrophotometric method for simultaneous estimation of atorvastatin calcium and felodipine from tablet dosage form. Adv Chem 1-6.

Narendar Dudhipala and Kishan Veerabrahma (2015). Pharmacokinetic and pharmacodynamic studies of nisoldipine-loaded solid lipid nanoparticles developed by central composite design. Drug Dev Ind Pharm 41(12): 1968-77.

Narender Reddy M and Tasneem R (2004). β-Cyclodextrin complexes of celecoxib: molecular-modeling, characterization, and dissolution studies. AAPS Pharm SciTech 6(1): 1-10.

Rajewski RA and Stella VJ (1996). Pharmaceutical applications of cyclodextrins-2. In vivo drug delivery. J Pharm Sci 85: 1142-1169.

Sanjula B, Mona D and Kanchan K (2005). Physicochemical characterization, in vitro dissolution behavior and pharmacodynamic studies of rofecoxib-cyclodextrin inclusion compounds. Preparation and properties of rofecoxib hydroxypropyl β-cyclodextrin inclusion complex: A technical note. AAPS Pharm Sci Tech 6(1): 83-90.

Seoung Wook J, Min-Soo K, Jeong-Soo K, Hee Jun P, Sibeum L, Jong-Soo W and Sung-Joo H (20070. Preparation and characterization of simvastatin/ hydroxypropyl--cyclodextrin inclusion complex using supercritical antisolvent (SAS) process. Eur J Pharm Biopharm 66: 413-421.

Stephane G, Siham BZ, Pierre M, Isabelle F and Alain A (2005). Melarsoprol-Cyclodextrins inclusion complexes. Int J Pharm 306:107-121.

Sudhir V, Yan L, Rajeev G and Diane JB (2009). Quality by design approach to understand the process of nanosuspension preparation. Int J Pharm 377: 185-198.

Szente L and Szejtli J (1999). Highly soluble cyclodextrin derivatives: chemistry, properties, and trends in development. Adv Drug Deliv Rev 36: 17-38.

Torrades F, Saiz Z and García-Hortal JA (2011). Using central composite experimental design to optimize the degradation of black liquor by fenton reagent. Desalination 268: 97-102.

Vogel HG and Vogel WH (1997). Drug Discovery and Evaluation: Pharmacological Assays. Springer-Verlag. Berlin Heidelberg, 604-611.

Zingone G and Rubessa F (2005). Pre-formulation study of the inclusion complex warfarin-β-Cyclodextrin. Int J Pharm 291: 3-10.

Address correspondence to: Prof. Varsha B. Pokharkar, Department of Pharmaceutics, Poona College of Pharmacy, Bharati Vidyapeeth University, Pune-411038, India. Phone: + 91-20-2543 7237 E-mail: [email protected], [email protected];

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int j pharm sci nanotech vol 9; issue 2 international … hydroxy propyl- -cyclodextrin (hpβcd)...

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