DESIGN AND DEVELOPMENT OF FLOATING PULSATILE DRUG DELIVERY OF LOSARTAN POTASSIUM

Objective: The objective of the present investigation was to the development of floating pulsatile drug delivery system of Losartan potassium (LP) tablets for obtaining no drug release during floating followed by pulsed, rapid drug release to achieve chronotherapeutic release. In hypertension, the risk of getting heart attacks early in the morning is high and therefore, there was need to develop drug delivery, which will release drugs at morning hours and provide efficacious therapy. LP is a short biological half-life (1.5-2.5h) and readily absorbed from the stomach and upper gastrointestinal tract. Methods: Tablet formulation was prepared by press coating of rapid release core tablets and core tablets were further top coated with a buoyant layer of HPMC K4M and sodium bicarbonate. Various grades of HPMC polymer (E5/E15/E50) were used for the pulsatile coating layer. The developed formulations were characterized for physical characteristics, floating lag time, floating time, release lag time, drug content, swelling index, in vitro dissolution studies, DSC and XRD. Results: The FTIR and DSC studies predicted that there was no chemical interaction between drug and excipients. The core tablet coated with HPMC E50 showed a high swelling index and release the drug 97.60±1.2% at 6h. Buoyant layer with 80 mg HPMC K4M and 25 mg sodium bicarbonate gave satisfactory floating lag time. Conclusion: The system showed an excellent lag phase followed by burst release in the distal small intestine, which gives site and time-specific delivery of LP acting as per chronotherapy for treatment of hypertension.


INTRODUCTION
Chronopharmaceutics, the drug delivery based on circadian rhythm, is recently gaining much attention worldwide. Various diseases like asthma, hypertension, and arthritis show circadian variation, which demands time scheduled drug release for effective drug action. To follow this principle, one must have to design the dosage form so that it can be given at a convenient time, e. g. bedtime for hypertension with the drug release in the morning would be a promising chronotherapeutic system. For this, a pulsatile release profile, where the drug is released completely after a defined lag time, is advantageous. A combination of floating and pulsatile principles of drug delivery system would have the advantage that a drug can be released in the distal small intestine after a defined time period of no drug release [1].
The gastroretentive floating pulsatile drug delivery system (GRFPDDS) is useful for those drugs, which have dependent pH solubility, poor bioavailability in the gastrointestinal tract (GIT) and narrow absorption window. Gastro retentive systems (GRS) can remained in the gastric region for several hours and hence significantly prolong the gastric residence time of drugs. Prolonged gastric retention improves bioavailability, reduces drug waste and improves the solubility of drugs that are less soluble in high pH environments [2,3]. Different approaches used for gastroprotection and to enhance the bioavailability of drugs such as floating drug delivery systems (FDDS), bioadhesive or mucoadhesive systems, swelling and expandable systems, high-density systems, raft systems, ion exchange resins, controlled osmotic systems etc. Floating pulsatile dosage forms, which are retained in the stomach for a long time (4-12 h) and not affected by variation in gastric pH, local environment or gastric emptying rate. These dosage forms may be either single unit (floating tablets) or multi particulates (beads, pellets, granules, microspheres) with the capability of gastro-retention. These systems are specifically beneficial for drugs, either absorbed from the stomach or requiring local delivery in the stomach [4].
It is well established that many of the body functions display circadian rhythms e. g. in peptic ulcer, the acid secretion is high in the afternoon and night, in case of asthma, precipitation of symptom during the night and early morning is high. Similarly, in cardiovascular disease, blood pressure is at its lowest during the sleep cycle and rises steeply during the early morning awaking period and in arthritis, the pain is more in the early morning. LP is an angiotensin II receptor antagonist used in the treatment of hypertension. The bioavailability of about 33% and plasma half-life ranging from 1.5 to 2.5 h. The other disorders that follow circadian rhythm are diabetic mellitus, angina pectoris, and rheumatic diseases [5]. Controlled releases that display a pulse release are mainly prepared by polymeric materials and systems that are made up of a reservoir and covered with a barrier. This barrier can be dissolved or eroded after which the drug is released. The release from such formulation can be modified to get a single pulse, double pulse, and multi-pulse release pattern [6].
The aim of the present work was to the development of a pulsatile drug release system of LP for the effective treatment of hypertension. LP is an angiotensin-II receptor antagonist drug which is mainly used to treat high blood pressure and it well absorbed after oral administration.

MATERIALS AND METHODS
Losartan Potassium was obtained as a gift sample from Macleod's Pharmaceuticals Mumbai, Microcrystalline cellulose, HPMC, sodium bicarbonate were purchased from Loba Chem. Pvt. Ltd. Mumbai, Lactose, Sodium starch glycolate, Cross povidone, Polyvinyl Pyrrolidone, magnesium stearate were purchased from Molychem Mumbai. All other reagents and chemicals used were analytical grade.

Preformulation studies
The sample of LP was characterized for its physical state, colour and odour. The melting point was determined using a capillary tube method. Solubility study was carried out using different solvents. Fourier transforms infrared (FTIR) spectroscopy studies were used for the evaluation of physicochemical compatibility and interactions among drugs and excipients.

Preparation of rapid release core tablets (RRCT)
Core tablet was formulated by using various concentrations and different types of diluents and superdisintegrants. The drug, diluent, superdisintegrant and binder were weighed as per formulae given in [table 1]; these were then passed through sieve no.20 transferred to a polybag and blended for 5 min. To the homogeneous blend with magnesium stearate for 2 min. The resulting blend was compressed by using a rotary tablet compression machine to get a tablet of 110 mg weight with a diameter of 8 mm [7].

Preparation of the pulsatile release tablets (PRTs)
The optimized pulsatile release tablets were prepared by using different grades of HPMC (E5, E15 and E50) at different concentrations given in [table 2]. Dry coated tablet was prepared by placing 50% of pulsatile release layer in 10 mm die and rapid release core tablet (RRCT) layer was placed on it and then remaining quantity of pulsatile release layer was added in the cavity, so as to cover the RRCT and finally compressed using rotary tablet compression machine [8].

Preparation of floating pulsatile release tablets (FPRTs)
A 3 2 randomized full factorial design was used to optimize the composition of the buoyant layer. In the design two factors were evaluated, each at three levels and experimental trials were performed at all 9 possible combinations. The concentrations of HPMC K4M (X1) and sodium bicarbonate (X2) were selected as independent variables. All the nine possible combinations of buoyant powder were filled into the die individually, followed by the addition of optimized PRT and finally, compression was done. The optimized concentrations of HPMCK4M and sodium bicarbonate were incorporated in the floating tablet, which was used as the checkpoint of the regression analysis model. The 3 2

Optimization of formulation using factorial design
factorial designs for floating pulsatile tablet reported in [ A statistical model incorporating interactive and polynomial term was used to evaluate the responses.
Where Y is the dependent variable, β0 is the arithmetic mean response of the nine runs, and b1 is the estimated coefficient for the factor x1. The main effects (x1, x2) represent the average result of changing one factor at a time from its low to high value. The interaction terms (x1, x2) showed how the response changes when two factors are changed simultaneously. The polynomial terms (x1, x2) are included to investigate nonlinearity. From the data of floating lag time parameters for factorial composition F1 to F9 polynomial equation for the dependent variable has been derived. In this equation, the negative sign indicates a negative correlation and the positive sign indicates a positive correlation to the independent variables X1 and X2 [9,10].

Evaluation of RRCTs
Thickness and diameter are important parameters in the evaluation of tablet as it ensures uniformity of tablet size. The thickness of the tablet was determined using a Vernier caliper. Three tablets from formulation were used to measure thickness and diameter and average values were recorded in mm. For each formulation, the hardness of tablets was determined using the Monsanto hardness tester. Force of fracture recorded in kg/cm 2 [11].  Weight variation test 20 tablets were selected randomly, weighed individually and the average weight of tablet was determined. Individual tablet weight was then compared with the average value to find the deviation in weight. The weight variation for each tablet was determined using formula 1, Tablet passes the test if not more than two tablet falls outside permitted limit and in no case, any tablet shows variation equal to or greater than twice of permitted limit as per standard.

Drug content
The content uniformity test was carried out to ensure content of LP in each tablet i.e. to ensure dose uniformity. Ten tablets were weighed and calculate average weight and crushed in to powder in mortar-pestle. Powder triturate equivalent to 50 mg of LP was dissolved in 100 ml 0.1N HCL. Solution was filtered through whatmann filter paper no. 42 and 1 ml of the filtrate was diluted to the 10 ml with 0.1N HCL [12]. Absorbance of the resultant solution was measured using UV-Visible spectrophotometer at 205 nm wavelength. Content uniformity was calculated using formula 2, Where, C: Concentration, Au and As: Absorbance's obtained from standard preparation and assay preparation.

In vitro disintegration time
The disintegration test was carried out using USP in vitro disintegration test apparatus. Tablets were placed in each of the six tubes of the basket, contacting water maintained temperature at 37 °C±2 °C as per specified in the individual monograph. The time taken by the tablet for complete disintegration, leaving no tablet residue on wire screen, was recorded.

In vitro dissolution studies
The release rate of LP from rapid release core tablet was determined using USP dissolution testing apparatus II. The dissolution test was performed using 900 ml of 0.1N HCL at 37±0.5 °C and rotation 50 rpm, a sample volume of 5 ml was withdrawn at regular time intervals. The volume withdrawn was replaced by a fresh volume of dissolution medium to maintain sink condition. The filtered samples were analyzed spectrophotometrically at 205 nm wavelength. Drug content in the dissolution sample was determined [13].

Evaluation of pulsatile release tablets
Pulsatile release tablets were evaluated for parameters like diameter, thickness, hardness, uniformity of weight, in vitro drug release as per the procedure mentioned for evaluation of core tablet.

Swelling index
The swelling properties of tablets were determined by placing the tablets in the dissolution test apparatus, in 900 ml of 0.1 N HCL at 37±0.5 °C. The tablets were removed periodically from the dissolution medium, blotted to remove an excess of water and weighed [14]. Swelling characteristics were expressed in terms of percentage of water uptake (WU %), calculated by formula 3.
%Water uptake = Weight of swollen tablet-Initial weight of tablet Weight of tablet × 100 … . 3

In vitro dissolution studies
The release rate of LP from pulsatile release tablets was determined using USP dissolution testing apparatus II (TDT 08L Electrolab Mumbai, Maharashtra, India). The dissolution study was performed using the same procedure for core tablet [15,16].

Evaluation of floating-pulsatile release tablet (FPRT)
FPRT was evaluated for parameters like diameter, thickness, hardness, uniformity of weight, in vitro drug release as per the procedure mentioned for evaluation of core tablets.

In vitro buoyancy studies
The in vitro buoyancy test was conducted by placing the tablet in 900 ml 0.1N HCL maintained pH 1.2, 37±0.5 °C temperature and speed of rotation at 50 rpm. The time is taken by tablet to float on surface after its introduction in the medium (floating lag time) and entire duration, during tablet remained float on the surface of the medium (total floating time) were recorded by visual observation [17,18].

Fourier transform infrared spectroscopy study (FTIR)
FTIR absorption spectrum of TA was determined by fourier transform infrared spectrophotometer FTIR (Jasco 4100, Shimadzu, Japan). Briefly, about 2 mg of sample was ground thoroughly with previously dried KBr at 120 °C for 30 min; uniformly mixed with drug sample and kept in the sample holder, spectra were recorded over the wavenumber 400-4000 cm -1

Powder X-ray diffractometry (PXRD)
. Infrared spectrums of pure drug and optimized batches were recorded.
PXRD studies were carried out in order to study any change in the crystalline nature of the pure drug and optimized formulation [19,20]. DSC studies provide qualitative and quantitative information about physical and chemical changes of a compound that involve endothermic and exothermic processes, or changes in heat capacity. Thermograms of LP and optimized formulation were obtained using DSC instrument (TA Instruments SDT-2960, USA) equipped with an intra-cooler. Indium standard was used to calibrate the DSC temperature and enthalpy scale. The powder samples of the formulation was kept in the aluminum pan and heated at a constant rate 50 °C/min, over a temperature range of 100 °C to 300 °C. Inert atmosphere was maintained by purging nitrogen at the flow rate of 100 ml/min [21,22].

Stability studies
The stability studies were carried out according to (ICH) Q1A (R2) guidelines (FDA, 2003). The optimized formulation was subjected to accelerated stability studies at 40±2 °C/75±5% RH for a period of 2 mo in stability chamber. Tablets were packed in amber-colored wide mouth glass bottle hermetically sealed. The samples were taken out at 30 and 60 d and evaluated for the physical parameters, % drug content, in vitro % drug release study, floating lag time and floating time [23].

Preformulation studies
The sample of LP was found to be white to off white crystalline powder having a bitter taste. Melting point of LP was found to be in the range of 263-265 °C. LP was found to be soluble in water, methanol, ethanol and acetonitrile. The FTIR spectrum of LP exhibits a characteristic peak at 760 cm -1 ,1000 cm -1 , 1462 cm -1 , 1575 cm -1 and 2995 cm -1

Evaluation of powder blends
due to chloride moiety, secondary hydroxyl group, aromatic ring, nitrogen moiety and an aliphatic chain respectively.
The powder blend of all the formulations batches (1-6) were subjected for various pre-compressional evaluation parameters such as angle of repose, bulk and tapped density, compressibility index and Hausner's ratio. Results of all the pre-compression parameters performed of the blends for all batches shown in [table 5]. The prepared powder blends were within the limit of all specified values and showed good powder characteristics. Angle of repose was found to be in the range of 23.48° to 29.78°. The powder blends of all formulations showed good flow property. The bulk density and tapped density of blends were found to be in the range of 0.4647 to 0.4852 g/cm 3 and 0.5076 to 0.55 g/cm 3, respectively. This indicates a good packing capacity of blends. Carr's index was calculated and the results were shown in [table 5]. It was found to be in the range of 5.79 to 14.54 indicates that, the powder blend has required flow property. Values of Carr's index below 15% usually show good flow characteristics. Hausner's ratio of all formulations was found to be 1.06 to 1.17 indicates the good flow properties of blends.

In vitro dissolution studies
In vitro drug release studies reveals that, formulations containing cross povidone (CT4-CT6) showed fast dissolution than the sodium starch glycolate. Dissolution of LP RRCTs increases with an increase in the concentration of sodium starch glycolate and cross povidone due to rapid swelling and disintegration. The tablets prepared with cross povidone have maximum drug release as these tablets have less disintegration time owing to the formation of larger pores with continuous network in dissolution media and swelling provide enough pressure for faster disintegration, additionally, it also have the capability to swell at least twice of its original volume, when contact with dissolution fluid.
Among six formulations of LP inner core tablets, it was observed that, when cross povidone used in a concentration of 10 % provides rapid disintegration and maximum dissolution rate. Latha K et al. reported that, Hydroxypropyl methylcellulose (HPMC) forms a firm gel but does not hydrate quickly while MCC is a good disintegrant and acts by a wicking mechanism. Hence, the drug was immediately released from the tablet core after the rupture of the surrounding outer layer containing MCC alone and admixture of HPMC100KM and MCC [7]. In vitro drug release profiles for all the batches of RRCTs of LP are given in [ fig. 1].

Evaluation of pulsatile release tablets
All the tablets formulations were subjected for evaluation of parameters like shape, thickness, diameter, hardness, . The hardness of all the formulations was almost uniform and the tablets possess good mechanical strength. From the results, it was revealed that lag time goes on increasing with increasing viscosity grades of HPMC polymer and concentration of polymer. Bajpai et al. reported that coating with carboxymethyl cellulose sodium showed the least lag time because of its high permeation and high water-absorbing character, which leads to quick hydration of core tablet that evolves gas generating internal pressure. Also, carboxymethyl cellulose sodium has a lower extent of cross-linking and gelling character than other three polymers that provided a lesser extent of mechanical strength to its coat [24].

Swelling index
The swelling behavior of optimized PRT containing HPMC E50 was compared with other PRTs containing HPMC E5 and HPMC E15 at the same concentration i.e. 280 mg. The results indicated that the swelling front erodes faster for PRTs coated with HPMC E5 as compare to PRTs coated with HPMC E15 and E50 due to their marked viscosity properties shown in [ fig. 5]. The thickness of the swelled layer of PRT coated with HPMC E50 was higher as compared with PRTs with HPMC E5 and HPMC E15. This was due to the complete penetration of the solvent and high viscosity of the HPMC E50. Raghavendra et al. reported that the ethylcellulose showed highest lag time, which is based upon the hydration of outer barrier layer or water penetration through the outer barrier layer. The formulated bilayered tablets achieved a burst release after 3-4 h lag time [26]. Dhakar VK et al. showed that the direct correlation between swelling and lag time was observed and found that the formulations having maximum swelling indices showed the higher lag time. The FTIR spectrum of formulation F9 shows a broad peak at 3753.23 cm -1, indicating the presence of the primary alcoholic OH group. A peak is shown at 1542 cm -1 and 775.19 cm -1, depicting nitrogen and chloride moiety. One sharp peak of aryl chloride was observed at 1117 cm -1 . In comparison with the pure drug, the absorption peak of the spectra for LP in FPDDS form optimized formulation batch F9 showed no significant shift of characteristic peaks suggesting that there is no interaction between drug and excipients. IR spectra of LP, physical mixture and optimized formulation batch F9 as shown in [ fig. 7-9].

Stability studies
Short term stability study was performed for formulation CT-6 at 40±2.0 °C and RH 75%±5% for two months. The samples were analyzed for diameter, thickness, weight variation, hardness, disintegration time, percent drug content and in vitro drug release studies. The results of all parameters indicated that no significant difference in parameters observed before and after stability studies shows in [table 9]. Short term stability studies of FPRTs were performed of the optimized formulation F9, indicated that there was no significant difference in physical parameters, floating lag time and floating time shown in [table 10]. Optimized formulation (F9) showed good stability at accelerated stability conditions. From the experimental findings it can be concluded that floating pulsatile tablets of Losartan potassium can give efficient therapy by reducing dose and dosing frequency and provide chronotherapy for an effective management of morning surge of hypertension. Finally, it may be concluded that floating pulsatile drug delivery system offers a valuable dosage form treatment of hypertension.

ACKNOWLEDGMENT
Anilkumar J. Shinde and co-authors are thankful to Macleod's Pharmaceuticals Mumbai India for the gift sample of Losartan hydrochloride.

AUTHORS CONTRIBUTIONS
The author designed and performed the experiment analyzed data and prepared the manuscript. All authors played an equal role in completing this research work.