DEVELOPMENT OF FUROSEMIDE FLOATING MICROBALLOONS: IN-VITRO AND IN-VIVO EVALUATION
HTML Full TextDEVELOPMENT OF FUROSEMIDE FLOATING MICROBALLOONS: IN-VITRO AND IN-VIVO EVALUATION
Munija Pancheddula and Shayeda *
Department of Pharmaceutics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal - 506009, Telangana, India.
ABSTRACT: The present investigation deals with the development and evaluation of floating microballoons of furosemide to extend the gastric residence time (GRT) and prolong the drug release. In the present work, floating micro balloons of furosemide were formulated using eudragit RS 100, eudragit S 100, and HPMC K4M and ethylcellulose polymers by the solvent evaporation method. The prepared micro balloons were evaluated for their physicochemical properties, in-vitro drug release, and in-vitro buoyancy. The in-vitro release studies demonstrated that micro balloons of furosemide prepared using eudragit RS 100 along with eudragit S 100 in 1:1 ratio (FSDF10) shown the maximum amount of drug release; hence it is considered as the optimized formulation. The in-vitro release kinetics revealed that the optimized formulation releases the drug in zero-order manner based on the regression values of kinetic models. The optimized formulation was used for in-vivo evaluation. The in-vivo Radiographic study showed that the BaSo4 loaded optimized formulation remained buoyant up to 5.5 h in the stomach. The in-vivo pharmacokinetic study conducted in healthy albino rabbits revealed that the oral bioavailability of optimized formulation was increased significantly when compared to the marketed formulations. The increased bioavailability may be due to the floating mechanism of the dosage form in the stomach for longer duration.
Keywords: |
Furosemide, Solvent evaporation, Gastric residence time, In-vitro evaluation, In-vivo evaluation, Floating microballons
INTRODUCTION: Historically, oral drug administration has been the predominant route for drug delivery. During the past two decades, numerous oral delivery systems have been developed to act as drug reservoirs from which the active substance can be released over a defined period of time at a predetermined and controlled rate. However, this route has several physiological problems such as an unpredictable gastric emptying rate that varies from person to person a brief gastrointestinal transit time (8- 12 h).
The existence of narrow absorption window in the upper GIT for several drugs 1 these difficulties have prompted researchers to design a drug delivery system that can stay in the stomach for prolonged and predictable period. Attempts are being made to develop a drug delivery system that can provide therapeutically effective plasma drug concentration for a longer period, thereby reducing the dosing frequency and minimizing fluctuation in plasma drug concentration at a steady state by delivering the drug in a controlled and reproducible manner.
Gastroretentive systems can remain in the gastric region for several hours and hence significantly prolong the gastric residence time of drugs. Prolonged gastric retention improves bio-availability, reduces drug waste, and improves solubility for drugs that are less soluble in a high pH environment.
It has applications also for local drug delivery to the stomach and proximal small intestines. Microballoons are gastro retentive drug-delivery systems with a non-effervescent approach and considered as one of the most favorable buoyant systems with the unique advantages of multiple unit systems as well as better floating properties. Microballoons (Hollow microsphere) are in the strict sense, empty particles of spherical shape without core. These microspheres are characteristically free-flowing powders comprising of proteins or synthetic polymers, ideally having a size less than 200 micrometer 2. The slow release of drug at desired rate and better-floating properties of floating microspheres mainly depends on the type of polymer, plasticizer, the solvents employed for the preparation and the release of the drug can be modulated by optimizing polymer concentration and the polymer - plasticizer ratio.
The drug, furosemide is a benzoic-sulfonamide-furan. It is a diuretic with fast onset and short duration that is used for edema and chronic renal insufficiency. Furosemide is absorbed mostly in the stomach and upper small intestine, possibly due to its weak acidic properties (pKa 3.93) and is characterized by a short half-life (1-2 h). The narrow absorption window of furosemide leads to its low bioavailability (60-70%). The narrow absorption window of furosemide in the upper part of the gastrointestinal tract provides a rationale for developing a gastro retentive dosage form.
MATERIALS AND METHODS:
Materials: Furosemide was purchased from Yarrow chem. Products, Mumbai, India. Vitamin E TPGS, Eudragit RS 100, Eudragit S 100, HPMC K4M, Ethylcellulose, Ethanol, Dichloromethane chemicals of Laboratory-grade from SD Fine chemicals Pvt. Ltd., were used.
Methods:
Drug Excipient Compatibility Study:
Differential Scanning Calorimetry: The physicochemical compatibilities of the drug and the excipients were tested by differential scanning calorimetric (DSC) analysis. DSC thermograms of the drug alone and optimized formulation were derived from DSC (Perkin-Elmer, 4000). The instrument was calibrated with an indium standard. The samples (2-4 mg) were heated (20-300 °C) at a constant scanning speed (10 °C / min) in sealed aluminum pans, using nitrogen purged gas.
FTIR Spectroscopy: Drug-polymer compatibility studies were carried out using the FTIR spectrophotometer (Shimadzu) by KBr pellet technique. Pure drug and optimized formulation were subjected to FTIR study. Compatibility studies were carried out to know the possible interactions between furosemide and excipients used in the formulation. IR spectrum of pure drug and optimized formulation was seen in between 4000-400 cm-1.
Formulation Development: As the drug furosemide poorly water-soluble, before formulating it as floating microballoons, it was converted to freely soluble solid dispersion using vitamin E TPGS as a solubility enhancing carrier. The composition of solid dispersions prepared is given in below Table1. Solid dispersions were prepared by solvent evaporation method. Drug and carrier were dissolved in a suitable quantity of methanol, and solvent was slowly evaporated. The obtained solid residue was collected and evaluated.
TABLE 1: COMPOSITIONS OF SOLID DISPERSIONS OF FUROSEMIDE
S. no. | Materials | FSD1 | FSD2 |
1 | Furosemide | 10 mg | 10 mg |
2 | Vitamin E TPGS | 10 mg | 20 mg |
3 | Methanol | 10 mL | 10 mL |
Ratio of drug to polymer | 1:1 | 1:2 |
Evaluation of Solid Dispersions:
Saturation Solubility: Saturation solubility studies were conducted for prepared solid dispersions along with pure drug by adding an excess amount of drug in 2 mL of water and shaking it for 48-72 hours until equilibrium is attained. Then the solution is centrifuged and the supernatant is analyzed for amount of drug dissolved by spectrophotometrically at 276 nm.
In-vitro Dissolution Study: The drug release study was carried out using USP dissolution apparatus type XXIII basket type dissolution apparatus at 37 ± 0.5 °C and at 50 rpm using 900 ml of 0.1N HCl (pH 1.2) as a dissolution medium. 5 ml of sample solution was withdrawn at predetermined time intervals up to 12 h and the samples were filtered through Whatman filter paper, diluted suitably and analyzed spectrophotometrically with UV-Visible spectrophotometer at a maximum absorbance wavelength of 276 nm. Equal amount of fresh dissolution medium was replaced immediately after withdrawal of the test sample. The dissolution studies were performed and the average percentage drug release was calculated.
Formulation of Floating Microballoons: Solid dispersion prepared with 1:2 ratio of drug to vitamin E TPGS (FSD2) has shown improved solubility and dissolution and hence was chosen for preparing floating microballoons. The floating microballoons were formulated by solvent evaporation method. The polymer is dissolved in an organic solvent and the solid dispersion (10 mg) equivalent to 30 mg of drug is either dissolved or dispersed in the polymer solution.
The solution containing the drug is then emulsified into an aqueous phase containing suitable additive (surfactants /polymer) to form oil in water emulsion. After the formation of a stable emulsion, the organic solvent is evaporated either by increasing the temperature under pressure or by continuous stirring. Stirring was continued for 6 h under 3 blade propellers at 500 rpm, 40 °C until the smell disappears.
The solvent removal leads to polymer precipitation at the oil/water interface of droplets, forming cavity and thus making them hollow to impart the floating properties. Then microballoons are collected and washed with excess amount of distilled water to remove any remnants. Collected microballons were dried at room temperature and subjected for further evaluation.
TABLE 2: COMPOSITION OF FLOATING MICROBALLOONS OF FUROSEMIDE
S. no. | Materials | FSDF1 | FSDF2 | FSDF3 | FSDF4 | FSDF5 | FSDF6 | FSDF7 | FSDF8 | FSDF9 | FSDF10 | FSDF11 | FSDF12 | FSDF13 | FSDF14 | FSDF15 |
1 | Furosemide SD eqvt to 10mg | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 |
2 | Eudragit RS 100 | 10 | 10 | 10 | 20 | 20 | 20 | 10 | 10 | 20 | 20 | NA | NA | NA | NA | NA |
3 | Eudragit S 100 | 10 | 20 | 30 | 10 | 20 | 30 | 30 | 30 | 20 | 20 | NA | NA | NA | NA | NA |
4 | HPMC K4M | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | 10 | 10 | 10 | 20 | 20 |
5 | Ethylcellulose | NA | NA | NA | NA | NA | NA | NA | NA | NA | NA | 10 | 20 | 30 | 10 | 20 |
6 | Ethanol | 15 | 15 | 15 | 15 | 15 | 15 | 20 | 10 | 20 | 10 | 15 | 15 | 15 | 15 | 15 |
7 | Dichloromethane | 15 | 15 | 15 | 15 | 15 | 15 | 10 | 20 | 10 | 20 | 15 | 15 | 15 | 15 | 15 |
In-vitro Evaluation of Furosemide Micro-balloons:
Micromeritic Properties: Microballoons are evaluated by their micromeritic properties such as particle shape and size, bulk density, tapped density, Hausner’s ratio, and flow properties, which are determined by car’s index and angle of repose 3.
Particle Size Measurement: Particle size of prepared microballoons was measured using an optical microscope, and the mean particle size was calculated by measuring 100 particles with the help of a calibrated ocular micrometer 4.
Scanning Electron Microscope (SEM): The surface morphology and surface characteristics of best formulation were carried out by scanning electron microscope (SEM). Microballoons were scanned and examined under Electron Microscope connected with a fine coat, Ion sputter. The sample was loaded on a copper sample holder and sputter-coated with carbon followed by gold 5.
Tapped Density: Tapped density and com-pressibility index are calculated by measuring the change in volume using a bulk density apparatus; angle of repose is determined by the fixed funnel method. The compressibility/carr’s index was calculated using the following formula:
I = Vb – Vt / Vb × 100
Where Vb is the bulk volume, and Vt is the tapped volume. The value given below 15% indicates a powder which usually gives rise to good flow characteristics, whereas above 25% indicate poor flowability. Angle of repose of the micro balloons was determined by the fixed funnel method.
Percentage Yield: The prepared microballoons of all batches were accurately weighed. The weight of prepared microballoons was divided by the total amount of all the excipients and drugs used in the preparation of the microballoons, which give the total percentage yield of floating microballoons 6, 7, 8. It is calculated by using the following formula,
Percentage yield = Actual yield of product / Total weight of excipients and drug
Entrapment Efficiency: The amount of entrapped drug in the microballoons was calculated based on the total drug content and the unentrapped drug of the floating microballoons. The unentrapped drug was determined by taking one dose equivalent of floating microballoons and washed with 0.1N HCl to remove the free drug on the surface.
The drug content of microballoons was determined by dispersing 50 mg formulation (accurately weighed) in 10 ml 0.1 N HCl, followed by agitation with a magnetic stirrer for 12 h to dissolve the polymer and to extract the drug. Both the solutions of unentrapped drug and total drugs were filtered through a Whatman filter; the drug concentration was determined spectrophotometrically at 276 nm by making the desired dilution with 0.1N HCl9. Percentage entrapment efficiency was calculated as follows.
% Entrapment efficiency = Total drug content - unentrapped drug 100 / Total drug content
In-vitro Buoyancy: Microballoons were spread over the surface of a USP dissolution apparatus type II filled with 900 ml of 0.1 N HCl. The medium was agitated with a paddle rotating at 50 rpm for 12 h. The floating and the settled portions of microballoons were recovered separately. The microballoons were dried and weighed. Buoyancy percentage was calculated as the ratio of the mass of the microballoons that remained floating and the total mass of the microballoons 10.
Percentage buoyancy = Qf 100 / Qf + Qs
Where, Qf and Qs are the weight of the floating and the settled microballoons, respectively.
Drug Content: The drug content of each formulation equivalent to unit dose (30 mg) was determined by spectrophotometrically. Each formulation was taken and finely powdered in a glass mortar and dissolved in a solution of 0.1 N HCl for 6 h. The solution was then filtered, and absorbance was noted at 276 nm 11.
In-vitro Drug Release Study: The drug release study was carried out using USP dissolution apparatus type XXIII basket type dissolution apparatus at 37 ± 0.5 ºC and at 50 rpm using 900 ml of 0.1 N HCl (pH 1.2) as a dissolution medium. 5 ml of sample solution was withdrawn at predetermined time intervals up to 12 h and the samples were filtered through whatman filter paper, diluted suitably and analyzed spectrphotometrically with UV-Visible spectrophotometer at a maximum absorbance wavelength of 276 nm. Equal amount of fresh dissolution medium was replaced immediately after withdrawal of the test sample 12, 13, 14, 15. The dissolution studies were performed and the average percentage drug release was calculated.
Drug Release Kinetic Studies: The mechanism of release was determined by fitting the release data to the various kinetic equations such as zero-order, first-order, higuchi and Korsmeyer-Peppas and finding the R2 values of the release profile corresponding to each model 16, 17 using PCP Disso v 3 software.
Stability Studies: Microballoons were herme-tically sealed in glass bottles and stored for 3 months at 4 ± 0.5 °C, room temperature and 40 ± 1 °C and 75% RH as per ICH guidelines 18,19 after every month, one bottle was used for evaluation. The microballoons were evaluated for physico chemical properties, drug content, percentage entrapment efficiency and percentage buoyancy and percentage of drug release.
In-vivo Evaluation Of Furosemide Micro-balloons: The experimental protocol to carry out in-vivo studies were reviewed and approved by the Institutional Animal Ethical Committee of University College of Pharmaceutical Sciences, Kakatiya University, Warangal, India (Registration No. IAEC/22/UCPSC/2018). The in-vivo per-formance of the optimized formulations was evaluated on healthy albino rabbits.
In-vivo Radiographic Studies: In-vivo floating behavior of optimized floating microballoons formulation was studied in healthy albino rabbits, weighing 1.5 kg to 2 kg. The 3 healthy male albino rabbits were used for the study. Animals were maintained for one week in the animal house to acclimatize them and were fed a fixed standard diet, under standard laboratory conditions (Temperature 25 ± 2 °C). To monitor the in-vivo transit behavior of the prepared floating microballoons. First X-rayy was taken for all the rabbits to ensure the absence of radio-opaque material in the stomach. Radiopaque microballoons were prepared by incorporating 500 mg of barium sulfate into the polymeric solution, and a similar procedure by which optimized microballoons were prepared was followed. Optimized microballoons prepared with BaSO4 equivalent to rabbit dose (3.5 mg/kg) were administered to rabbits with a sufficient amount of water. Gastric radiography is done at intervals of 0.5, 2.5, and 4. 5, 5.5 h in both fed and unfed state 20.
Fasting State: The BaSO4 loaded microballoons of furosemide were administered orally with a sufficient amount of water through a mouth gag introduced in between the two jaws of the rabbit. During the study, animals were not allowed to eat food, but the water was provided ad libitum, and in between radiographic imaging, animals are freed and allowed to move and carry out normal activities but not allowed to take any food.
Fed State: All the rabbits were fasted for 12 h before initiating the study and fed with a low-calorie diet. Half an hour later, BaSO4 loaded microballoons of furosemide were administered orally with a sufficient amount of water through a mouth gag introduced in between the two jaws of rabbit and at different time intervals, rabbits were exposed to X-ray imaging, and floating behavior was studied.
In-vivo Pharmacokinetic Evaluation of the Optimized Microballoons: Six healthy albino rabbits with body weight range of 1.5-2.5 Kg were selected through physical examination. An open-label, balanced, randomized, single-dose complete crossover study design in which six healthy albino rabbits received one treatment (product) each with a washout period of 7 days was designed and pharmacokinetic parameters are assessed. Healthy rabbits are divided into 2 groups (n=6 for each group). Group I animals are treated with optimized formulation (FSDF10) and group II animals are treated with marketed formulation (Lasix). At the predetermined time intervals of 0, 0.50, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 6.00, 8.00, 12.00, and 24.00 h, 0.5 ml of blood samples were withdrawn from marginal ear vein and analyzed using HPLC.
Pharmacokinetic parameters such as peak plasma concentration (Cmax), time at which Cmax occurred (Ttmax), area under the curve (AUC), biological half-life (t½), were calculated in each case using the data by kinetica TM 2000 software (Inna phase corporation, U. S. A) using the non-compartmental approach. Percent relative bioavailability of the optimized formulations with reference to the marketed preparation is studied 21.
RESULTS AND DISCUSSION:
Drug Excipient Compatibility Study:
Differential Scanning Calorimetry: DSC thermogram of the pure drug is shown in Fig. 1 endothermic peak was observed at 194.1°C indicates the drug melting point for the pure drug.
The shift in the endothermic peak of the drug was very less (190.5 °C), which indicates that the drug and polymers used were compatible with one another in the DSC of optimized formulation Fig. 2.
FIG. 1: DSC THERMOGRAM OF PURE DRUG
FIG. 2: DSC THERMOGRAM OF OPTIMIZED FORMULATION
FIG. 3: FTIR OF PURE DRUG
FIG. 4: FTIR OF OPTIMIZED FORMULATION
FTIR Spectroscopy: The drug-excipient compatibility study was done by Fourier transform infrared spectroscopy study. The prominent peaks of furosemide pure drug Fig. 3 were shown at 3286.09 cm-1 (due to –N-H), 1678.38 cm-1 (due to C=O), 2920.02 cm-1 (due to –CH2) and 1047 cm-1 (due to -C-O). These prominent peaks of the drug were also observed in the IR spectrum of optimized formulation of drug Fig. 4 with various recipients, which indicates that the drug was not interacted with the polymers used in the study, which confirms the stability of the drug.
Evaluation of Furosemide Solid Dispersion: Solubility and dissolution study of solid furosemide dispersion reveals that solid dispersion prepared with a 1:2 ratio of drug to vitamin E TPGS (FSD2) has shown more improved solubility and dissolution and hence chosen for preparing floating microballoons Table 3 and 4, Fig. 5.
TABLE 3: SOLUBILITY OF FUROSEMIDE SOLID DISPERSIONS
Formulation | Solubility (mg/mL) | |||
1 | 2 | 3 | Average | |
Pure drug | 0.01 | 0.02 | 0.015 | 0.015 |
FSD1 | 8.75 | 8.35 | 8.15 | 8.417 |
FSD2 | 12.57 | 15.54 | 13.45 | 13.853 |
TABLE 4: PERCENTAGE DRUG RELEASE OF FUROSEMIDE SOLID DISPERSIONS
Time (min) | Percentage of drug release | ||
Pure drug | FSD1 | FSD2 | |
0 | 0 | 0 | |
5 | 2.3 | 11.2 | 15.6 |
10 | 5.4 | 18.9 | 25.6 |
20 | 7.8 | 27.8 | 35.9 |
30 | 12.5 | 35.9 | 45.9 |
40 | 13.9 | 45.6 | 55.6 |
50 | 15.9 | 52.6 | 62.5 |
60 | 19.8 | 55.9 | 71.5 |
FIG. 5: DISSOLUTION PROFILES OF FUROSEMIDE SOLID DISPERSIONS
In-vitro Evaluation of Furosemide Micro-balloons: The particle size was measured using a calibrated optical microscope, and the average particle size of floating microballoons was found to be in the range of 120-180 μm Table 5 and 6. The measured tapped density bulk density, compressibility index, and angle of repose are within limits, which indicates good flow properties of microballoons Table 5 and 6. The floating microballoons were prepared and the percentage yield was calculated for all the formulations. The results of % yield are shown in Table 7. The percentage yield was in the range of 60-90 % for all the formulations. It was found to be less than 70% yield with ethyl cellulose and HPMC K4M, and for the optimized formulation, the yield was 84.5%. The entrapment efficiency of floating micro-balloons of furosemide was calculated, and the results are depicted in Table 7. The entrapment efficiency was in the range of 60-90% for all the formulations and was found to be 94.6% for optimized formulation.
The entrapment efficiency was low, with formulations prepared with ethyl cellulose and HPMC K4M. There was no effect of the solvent ratio observed in the % Entrapment Efficiency. The percentage buoyancy was calculated for all the formulations, and it was found that all the formulations were able to float on the dissolution medium (0.1 N HCl) over a period of 12 h. Even after 12 h of agitation of the dissolution medium, the microballoons continued to float without any apparent gelation. The high buoyancy of the microballoons is mainly due to the presence of pores and cavities which were formed during solvent evaporation. The percentage buoyancy was slightly less with formulations prepared with ethyl cellulose and HPMC K4M and decreased as the concentration of the polymers increased. This is because of the high viscosity of the polymer solution, which in turn is the reason for the less formation of pores and cavities in microballoons during solvent evaporation. The results of in-vitro buoyancy studies are shown in Table 7. The percentage buoyancy was in the range of 60-90 % for all the formulations and was found to be 91.2% for optimized formulation. The drug content of all the prepared formulations was found to be within the acceptable range of 90.0 -110.0%. Values obtained are given in Table 5 and 6.
TABLE 5: OBSERVATIONS OF IN-VITRO EVALUATION PARAMETERS OF FLOATING MICROBALLOONS OF FUROSEMIDE
Parameter | FSDF1 | FSDF2 | FSDF3 | FSDF4 | FSDF5 | FSDF6 | FSDF7 | FSDF8 |
Mean particle size (µm)٭٭ | 135.24±1.34 | 143.34±3.45 | 156.32±1.56 | 144.24±3.25 | 156.54±2.35 | 131.23±3.26 | 145.39±2.34 | 135.26±2.54 |
Bulk density* | 0.7±0.19 | 0.72±0.97 | 0.78±0.65 | 0.82±0.06 | 0.78±0.88 | 0.77±0.54 | 0.77±0.43 | 0.72±0.32 |
Tapped density* | 0.65±0.05 | 0.64±0.18 | 0.69±0.92 | 0.72±0.22 | 0.66±0.18 | 0.66±0.19 | 0.64±0.28 | 0.65±0.17 |
Compressibility index٭ | 7.14±0.18 | 11.11±0.26 | 11.54±0.19 | 12.20±0.92 | 15.38±0.94 | 14.29±0.22 | 16.88±0.91 | 9.72±0.84 |
Angle of repose٭ | 14.6±0.49 | 16.6±0.28 | 16.5±0.29 | 15.6±0.34 | 15.8±0.18 | 15.9±0.28 | 16.3±0.91 | 15.5±0.18 |
Drug content | 98.78±0.19 | 99.02±0.02 | 96.68±0.19 | 07.89±0.11 | 98.02±0.76 | 98.54±0.54 | 98.72±0.63 | 98.29±0.82 |
TABLE 6: OBSERVATIONS OF IN-VITRO EVALUATION PARAMETERS OF FLOATING MICROBALLOONS OF FUROSEMIDE
Parameter | FSDF9 | FSDF10 | FSDF11 | FSDF12 | FSDF13 | FSDF14 | FSDF15 |
Mean particle size (µm) ٭٭ | 125.35±2.38 | 127.35±3.24 | 121.35±3.36 | 128.35±3.36 | 145.32±3.69 | 161.23±3.38 | 125.64±3.39 |
Bulk density* | 0.77±0.76 | 0.78±0.82 | 0.81±0.95 | 0.76±0.33 | 0.78±0.28 | 0.75±0.18 | 0.82±0.19 |
Tapped density* | 0.66±0.21 | 0.69±0.28 | 0.71±0.84 | 0.68±0.93 | 0.68±0.84 | 0.65±0.83 | 0.71±0.89 |
Compressibility index٭ | 14.29±0.72 | 11.54±0.92 | 12.35±0.99 | 10.53±0.17 | 12.82±0.25 | 13.33±0.39 | 13.41±0.43 |
Angle of repose٭ | 16.2±0.38 | 14.2±0.32 | 15.5±0.48 | 16.3±0.11 | 15.8±0.17 | 16.1±0.81 | 16.5±0.11 |
Drug content | 99.04±0.07 | 99.38±0.06 | 98.06±0.17 | 99.08±0.04 | 97.67±0.11 | 98.03±0.18 | 94.58±0.17 |
*All values represent mean ± SD; n = 3 ٭٭ all values represent mean ± SD; n = 100
TABLE 7: PHYSICOCHEMICAL PROPERTIES OF PREPARED MICROBALLOONS OF FUROSEMIDE
Formulation Code | % Yield | % EE | % B |
FSDF1 | 85.4 | 85.4 | 81.5 |
FSDF2 | 84.3 | 94.5 | 86.5 |
FSDF3 | 86.2 | 96.5 | 86.9 |
FSDF4 | 77.8 | 96.5 | 91.2 |
FSDF5 | 82.5 | 92.5 | 82.2 |
FSDF6 | 94.3 | 95.8 | 88.9 |
FSDF7 | 82.5 | 91.5 | 87.5 |
FSDF8 | 93.6 | 91.2 | 85.6 |
FSDF9 | 82.7 | 91.5 | 82.5 |
FSDF10 | 84.5 | 94.6 | 91.2 |
FSDF11 | 72.6 | 71.2 | 67.5 |
FSDF12 | 67.5 | 66.5 | 67.8 |
FSDF13 | 68.9 | 64.5 | 66.9 |
FSDF14 | 66.5 | 66.3 | 65.2 |
FSDF15 | 62.5 | 63.5 | 65.3 |
FIG. 6: COMPARATIVE PHYSICOCHEMICAL PROPERTIES OF MICROBALLOONS OF FUROSEMIDE
Scanning Electron Microscope (SEM): The surface morphology of the floating microballoons was studied using a scanning electron microscope (SEM). The surface morphology of optimized formulation FSDF10 was shown in Fig. 7 from the SEM micrographs it is apparent that the furosemide loaded microballoons were predominantly spherical in appearance. The surface was observed to be smooth, dense, and less porous, whereas the internal core was highly porous and irregular with numerous depressions that are an expression of evaporation of water, ethanol, and dichloromethane. The less porous outer surface and highly porous internal surface supported the controlled release of drug from the microballoons and good buoyancy.
FIG. 7: SEM IMAGE OF FUROSEMIDE LOADED OPTIMIZED MICROBALLOONS
In-vitro Drug Release Study: Dissolution studies of all the formulations were carried out using USP dissolution apparatus XXIII basket type dissolution apparatus. The dissolution profiles were compared among different formulations.
The cumulative percentage drug release was decreased with an increase in the polymer concentration. Based on the results of in-vitro drug release studies, it was found that FSDF10 has shown sustained drug release for 12 h with zero-order drug release. The results of the in vitro drug release profile were shown in Table 8, 9, and Fig. 8-10.
TABLE 8: PERCENTAGE DRUG RELEASE DATA OF FUROSEMIDE MICROBALLOONS
Time (H) | Percentage drug release | |||||||
FSDF1 | FSDF2 | FSDF3 | FSDF4 | FSDF5 | FSDF6 | FSDF7 | FSDF8 | |
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
0.5 | 25.6±0.02 | 20.6±0.02 | 15.6±0.12 | 20.4±0.93 | 6.5±0.32 | 7.2±0.98 | 17.8±0.32 | 16.5±0.54 |
1 | 44.2±0.23 | 34.5±0.32 | 25.9±0.24 | 37.2±0.26 | 11.2±0.42 | 10.2±0.45 | 26.5±0.18 | 25.6±0.18 |
2 | 64.5±0.42 | 51.3±0.47 | 44.2±0.43 | 57.6±0.47 | 21.2±0.09 | 13.5±0.57 | 43.5±0.38 | 44.5±0.17 |
3 | 87.5±0.21 | 63.8±0.98 | 58.6±0.66 | 65.5±0.32 | 26.5±0.58 | 21.3±0.17 | 58.7±0.97 | 61.2±0.23 |
4 | 98.5±0.22 | 75.4±0.26 | 66.9±0.85 | 75.6±0.82 | 35.4±0.41 | 31.2±0.83 | 66.9±0.11 | 71.2±0.24 |
6 | 100.1±0.31 | 82.5±0.32 | 74.5±0.41 | 87.9±0.17 | 53.2±0.95 | 46.5±0.37 | 73.5±0.81 | 73.5±0.38 |
8 | 100.2±0.04 | 86.4±0.33 | 99.7±0.02 | 65.4±0.73 | 58.4±0.88 | 85.6±0.92 | 87.6±0.52 | |
10 | 100.1±0.58 | 100.1±0.04 | 83.5±0.29 | 68.5±0.21 | 98.6±0.37 | 100.2±0.54 | ||
12 | 100.2±0.11 | 75.4±0.65 | 100.2±0.45 |
TABLE 9: PERCENTAGE DRUG RELEASE DATA OF FUROSEMIDE MICROBALLOONS
Time | Percentage drug release | ||||||
(H) | FSDF9 | FSDF10 | FSDF11 | FSDF12 | FSDF13 | FSDF14 | FSDF15 |
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
0.5 | 7.6±0.11 | 5.5±0.94 | 6.4±0.65 | 5.6±0.34 | 5.6±0.10 | 7.5±0.54 | 10.2±0.89 |
1 | 11.2±0.09 | 10.2±0.03 | 10.2±0.88 | 10.2±0.98 | 8.5±0.16 | 13.6±0.36 | 16.5±0.78 |
2 | 16.8±0.29 | 21.2±0.46 | 16.4±0.74 | 14.5±0.28 | 13.5±0.21 | 18.9±0.46 | 22.5±0.38 |
3 | 25.6±0.28 | 26.5±0.87 | 21.2±0.44 | 18.5±0.88 | 15.6±0.27 | 25.9±0.03 | 23.6±0.27 |
4 | 37.8±0.32 | 36.9±0.45 | 27.5±0.57 | 23.6±0.67 | 18.9±0.37 | 32.5±0.19 | 29.5±0.51 |
6 | 55.6±0.46 | 61.2±0.04 | 42.3±0.32 | 38.9±0.03 | 28.6±0.55 | 46.5±0.28 | 43.5±0.46 |
8 | 66.5±0.76 | 68.5±0.26 | 48.9±0.56 | 43.5±0.05 | 38.9±0.46 | 53.6±0.97 | 52.6±0.47 |
10 | 85.9±0.74 | 88.9±0.18 | 68.5±0.33 | 58.6±0.24 | 48.9±0.73 | 75.4±0.56 | 63.9±0.65 |
12 | 100.1±0.85 | 100.3±0.67 | 75.4±0.63 | 68.9±0.46 | 59.6±0.89 | 83.6±0.58 | 73.5±0.99 |
Release Kinetics of Floating Microballoons: Data of the in-vitro release of optimized formulation (FSDF10) was fit into kinetic models to explain the release kinetics of furosemide from microballoons. The kinetic models used were zero order, first order, Higuchi and Korsmeyer-peppas models. The in-vitro release kinetics revealed that the optimized formulation (FSDF10) releases the drug in a zero order manner based on the regression values Table 10.
TABLE 10: RELEASE KINETICS OF FUROSEMIDE FLOATING MICROBALLOONS
Formulation | Release Kinetics Parameters | ||||
Zero-order | First-order | Higuchi model | Korse-meyer peppas | Hixon -crowell | |
RSF1 | 0.073 | 0.991 | 0.795 | 0.823 | 0.947 |
RSF2 | 0.551 | 0.989 | 0.962 | 0.960 | 0.979 |
RSF3 | 0.723 | 0.989 | 0.985 | 0.977 | 0.978 |
RSF4 | 0.476 | 0.992 | 0.947 | 0.952 | 0.979 |
RSF5 | 0.995 | 0.946 | 0.886 | 0.998 | 0.971 |
RSF6 | 0.981 | 0.984 | 0.901 | 0.990 | 0.993 |
RSF7 | 0.711 | 0.987 | 0.987 | 0.981 | 0.972 |
RSF8 | 0.690 | 0.986 | 0.976 | 0.965 | 0.973 |
RSF9 | 0.995 | 0.943 | 0.881 | 0.996 | 0.970 |
RSF10 | 0.999 | 0.947 | 0.887 | 0.983 | 0.973 |
RSF11 | 0.987 | 0.974 | 0.892 | 0.991 | 0.985 |
RSF12 | 0.987 | 0.978 | 0.890 | 0.991 | 0.986 |
RSF13 | 0.989 | 0.977 | 0.880 | 0.989 | 0.984 |
RSF14 | 0.979 | 0.969 | 0.908 | 0.989 | 0.981 |
RSF15 | 0.941 | 0.974 | 0.947 | 0.985 | 0.972 |
Stability Studies: The stability studies were conducted on the optimized formulation (FSDF10), the stability study was conducted for 3 months and the results were analyzed. No significant change was observed in microballons and was found to be stable at storage conditions for three months Table 11, 12.
TABLE 11: STABILITY DATA OF OPTIMIZED MICROBALLOONS OF FUROSEMIDE (FSDF10)
Optimized formulation FSDF10 | Bulk density | Tapped density | Compressibility index | Angle of repose | Mean particle
size (µm) |
Percentage buoyancy | Drug content |
1st Month | 0.77±0.81 | 0.68±0.27 | 11.52±0.91 | 13.9±0.31 | 126.31±3.02 | 81.2±0.87 | 99.35±0.01 |
2nd Month | 0.76±0.79 | 0.67±0.26 | 11.06±0.87 | 13.8±0.29 | 125.29±2.09 | 81.1±2.1 | 99.21±0.09 |
3rd Month | 0.75±0.76 | 0.66±0.24 | 11.05±0.85 | 13.5±0.24 | 124.25±1.89 | 80.09±1.9 | 99.05±0.11 |
TABLE 12: PERCENTAGE DRUG RELEASE OF OPTIMIZED MICROBALLOONS OF FUROSEMIDE (FSDF10) DURING STABILITY STUDIES
FSDF10 | 1st Month | 2nd Month | 3rd Month |
0 | 0 | 0 | 0 |
0.5 | 5.4±0.04 | 4.9±0.01 | 4.7±0.19 |
1 | 10.1±0.11 | 9.1±0.9 | 9.1±0.8 |
2 | 21.1±0.31 | 20.9±0.18 | 19.9±0.28 |
3 | 26.1±0.71 | 25.9±0.65 | 25.1±0.61 |
4 | 35.1±0.31 | 34.8±0.28 | 34.1±0.25 |
6 | 60.1±0.01 | 59.1±0.21 | 58.1±0.19 |
8 | 67.1±0.21 | 66.11±0.19 | 65.8±0.15 |
10 | 87.1±0.11 | 86.9±0.10 | 85.8±0.12 |
12 | 100.1±0.61 | 100±0.58 | 100±0.53 |
In-vivo Evaluation of Furosemide Micro-balloons:
In-vivo Floating Behaviour: The optimized floating microballoons formulation prepared was tested for in-vivo floating behavior in healthy albino rabbits. Radiographic images obtained at 0.5 h, 2.5 h, 4.5 h, and 5.5 h at unfed state and fed state are shown in Fig. 11 and 12, respectively. It was observed from the images that the formulation remained buoyant for up to 5.5 h in the stomach, indicating the uniform distribution of formulation in the stomach. But in the unfed state, the formulation remained buoyant in the stomach only up to 4.5 h; this is because in fasting condition, myoelectric migrating contraction forces the contents to duodenum from stomach. The forceful waves will remove all the contents of the stomach, including the dosage form. This will not take place in a fed state. Therefore from these studies, it was clearly observed that the floating microballoons should be given to patients after a standard diet.
FIG. 11: X-RAY IMAGES OF OPTIMIZED MICROBALLOONS OF FUROSEMIDE IN THE GASTRIC REGION OF RABBIT DURING UNFED STATE AT 0.5 H, 2.5 H, 4.5
FIG. 12: X-RAY IMAGES OF OPTIMIZED MICROBALLOONS OF FUROSEMIDE IN THE GASTRIC REGION OF RABBIT DURING FED STATE AT 0.5 h, 2.5 h, 4.5 h, 5.5 h
In-vivo Pharmacokinetic Study: The in-vivo pharmacokinetic study was conducted in healthy albino rabbits. In this study, the pharmacokinetic of optimized furosemide floating microballoons (FSDF10) were compared with IR tablets (Lithostat). The mean plasma concentration-time profile obtained from the study is shown in Fig. 13; various pharmacokinetic parameters were estimated such as Cmax, Tmax, AUC are given in Table 15. The mean tmax of reference formulation was 3 h. This indicates that the drug release from the reference formulation was rapid, while in the test formulation, the mean tmax was 5.5 h. This indicates that the test formulation was effective in delaying the peak plasma concentration, thus showing prolonged plasma concentration of furosemide from the floating microballoons.
The mean biological half-life (t1/2 of furosemide from test and reference formulations was 18.72h and 7.45 h respectively. The difference observed here is due to prolonged absorption of test formulation; there is a prolonged continuous release of the drug into bloodstream. Therefore, the test formulation shows to have a longer half-life, i.e., the drug stays in the plasma for a longer time than the reference formulation. The lower half-life of reference preparation indicates the rapid removal of drugs from plasma, whereas the higher half-life of test formulation indicates prolonged release.
The mean area under plasma time curve AUC 0-t and AUC 0-α of reference formulation was 184.5458 ng/ml × h and 302.4 ng/ml × h and while AUC 0-t and AUC 0-α of the test formulation was 269.19583 ng/ml × h and 327.9 ng/ml × h, This indicates that the overall absorption of furosemide was more in the test formulation with respect to the reference product at the same dose. It was observed from the results that the oral bioavailability of optimized formulation (FSDF10) was increased significantly when compared to marketed formulation. Relative bioavailability with respective to marketed formulation was found to be 108.4, which is due to the prolonged gastric residence time of furosemide floating microballoons.
TABLE 13: PLASMA CONCENTRATION OF FUROSEMIDE CONVENTIONAL TABLETS (LASIX) IN RABBITS (N = 6) AT DIFFERENT TIME INTERVALS (REFERENCE FORMULATION)
Time (h) | Plasma concentration (ng/mL) | |||||||
Animal 1 | Animal 2 | Animal 3 | Animal 4 | Animal 5 | Animal 6 | Average | SD | |
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
0.5 | 4.5 | 4.6 | 4.8 | 5.2 | 4.9 | 4.7 | 4.78 | 0.25 |
1 | 5.5 | 5.7 | 6.2 | 5.8 | 5.9 | 6.2 | 5.88 | 0.28 |
1.5 | 7.2 | 7.4 | 7.5 | 7.1 | 6.9 | 7.5 | 7.27 | 0.24 |
2 | 10.5 | 10.6 | 11.2 | 10.9 | 11.6 | 10.8 | 10.93 | 0.41 |
2.5 | 12.5 | 13.2 | 13.9 | 14.2 | 12.8 | 13.2 | 13.30 | 0.64 |
3 | 15.2 | 15.5 | 16.2 | 15.5 | 16.3 | 15.8 | 15.75 | 0.43 |
4 | 12.5 | 13.2 | 13.8 | 14.5 | 15.2 | 15.6 | 14.13 | 1.19 |
6 | 10.5 | 11.2 | 10.8 | 10.9 | 11.3 | 12.5 | 11.20 | 0.70 |
8 | 8.5 | 8.8 | 8.9 | 9.2 | 9.5 | 8.8 | 8.95 | 0.35 |
12 | 6.5 | 6.6 | 6.8 | 7.1 | 6.2 | 6.4 | 6.60 | 0.32 |
24 | 4.2 | 4.5 | 4.4 | 4.8 | 5.2 | 5.3 | 4.73 | 0.45 |
TABLE 14: PLASMA CONCENTRATION OF FUROSEMIDE FLOATING MICROBALLOONS (FSDF10) IN RABBITS (N = 6) AT DIFFERENT TIME INTERVALS (TEST FORMULATION)
Time (h) | Plasma concentration (ng/mL) | |||||||
Animal 1 | Animal 2 | Animal 3 | Animal 4 | Animal 5 | Animal 6 | Average | SD | |
0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
0.5 | 3.2 | 3.5 | 3.8 | 3.9 | 3.3 | 3.3 | 3.50 | 0.29 |
1 | 4.5 | 4.9 | 5.1 | 4.2 | 4.8 | 5.1 | 4.77 | 0.36 |
1.5 | 6.8 | 7.2 | 6.9 | 6.5 | 6.6 | 7.5 | 6.92 | 0.38 |
2 | 8.9 | 9.1 | 8.8 | 9.3 | 9.4 | 9.6 | 9.18 | 0.31 |
2.5 | 11.5 | 12.5 | 13.5 | 10.2 | 11.5 | 11.9 | 11.85 | 1.11 |
3 | 14.8 | 15.2 | 16.5 | 14.5 | 13.5 | 13.8 | 14.72 | 1.08 |
4 | 16.9 | 17.5 | 18.2 | 16.2 | 14.9 | 15.5 | 16.53 | 1.24 |
6 | 18.5 | 21.2 | 23.2 | 21.2 | 19.2 | 21.2 | 20.75 | 1.68 |
8 | 14.5 | 15.5 | 14.3 | 12.9 | 13.5 | 16.8 | 13.92 | 1.04 |
12 | 12.5 | 13.2 | 13.5 | 10.5 | 12.6 | 15.5 | 13.80 | 1.05 |
24 | 3.5 | 3.5 | 3.5 | 3.8 | 3.8 | 3.5 | 3.60 | 0.15 |
FIG. 13: MEAN PLASMA CONCENTRATION TIME PROFILE OF FUROSEMIDE TEST (FSDF10) AND REFERENCE (LASIX) FORMULATIONS
TABLE 15: MEAN PHARMACOKINETIC PARAMETERS OF FUROSEMIDE AS REFERENCE AND TEST TABLETS IN RABBITS (N=6)
Pharmacokinetic parameter | Unit | Reference | Test |
Cmax | ng/mL | 15.75 | 20.75 |
Tmax | h | 3 | 6 |
AUC 0-t | ng/mL× h | 184.54 | 269.19 |
AUC 0-α | ng/mL × h | 302.4 | 327.9 |
T1/2 | h | 7.45 | 18.72 |
CONCLUSION: Gastro-retentive drug delivery system for furosemide was successfully prepared and evaluated by the solvent evaporation technique using eudragit RS 100, eudragit S 100, HPMC K4M, ethylcellulose polymers. From the drug-excipient compatibility studies, it was observed that, there was no interaction between drug and excipients used in the formulations. Prepared floating microballoons showed significant floating ability, good buoyancy, and sustained drug release. In vitro drug release of microballoons was influenced by polymers concentration. From the percentage loading efficiency and in-vitro drug release studies, it was observed that FSDF10 formulation exhibits greater drug loading efficiency and sustained release behavior. On fixing the in-vitro drug release data of optimized formulation to various kinetic models, it was found that it exhibits the zero-order kinetics. BaSO4 loaded optimized formulation FSDF10 selected for radiological study reveals that gastric retention time of floating microballoon in unfed state was 4.5 h, and in the fed state it was 5.5 h. In-vivo bioavailability study was conducted in rabbits optimized formulation (FSDF10) showed increased bioavailability when compared to the reference marketed tablets (Lasix) due to controlled floating technology.
Microballoons prepared in this study provide a promising gastro retentive drug delivery system to deliver furosemide with sustained-release in order to improve oral drug bioavailability. Thus, the prepared floating microspheres may prove to be potential candidates for multiple-unit delivery devices adaptable to any intragastric condition.
ACKNOWLEDGEMENT: Authors are thankful to the Principal, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, for providing necessary facilities.
CONFLICTS OF INTEREST: There are no conflicts of interest.
REFERENCES:
- Gupta G and singh A: Short review on stomach specific drug delivery system. International Journal of Pharm Tech Research 2012; 4(4): 1527-45.
- Vyas SP and Khar RK: Targeted and controlled drug delivery novel carrier system. New Delhi CBS Publishers and Distributors 2002; 417-54.
- Gattani YS, Kawtikwar PS and Sakarkar DM: Formulation and evaluation of gastroretentive multiparticulate drug delivery system of aceclofenac. Int J Chem Tech Res 2009; 1: 1-10.
- Sarkar BS, Tanwar SS, Soni P and Jain P: Formulation, characterization and in-vitro evaluation of floating microspheres of esomeprazole. Int J of Bioassay 2012.
- Pusp RN, Myung KC and Hoo KC: Preparation of floating microspheres for fish farming. International Journal of Pharmaceutics 2007; 341: 85-90.
- Jain SK, Awasthi AM, Jain NK and Agrawal GP: Journal of Controlled Release 2007; 107(2): 300-09.
- Awasthi R and Kulkarni GT: Development and characterization of amoxicillin loaded floating micro-balloons for the treatment of Helicobacter pylori induced gastric ulcer. Asian J of Pharma Sci 2013; 8: 174-80.
- Shah M, Jadhav N and Agrawal YK: Fullerenes Nanotubes and Carbon Nanostructures 2009; 17(5): 528-47.
- Whitehead L, Collett JH and Fell JT: Amoxicillin release from a floating dosage form based on alginates. Int. J. Pharm 2000; 210: 45-49.
- Mali AD and Bathe RS: An updated review on microballoons for better approach in gastroretnton. Asian J Res Pharm Sci 2015; 5(3): 188-92.
- Patel A, Ray A and Thakur RS: In-vitro evaluation and optimization of controlled release floating drug delivery system of metformin hydrochloride. DARU 2000; 2: 57-64.
- Rani U and Nagaraju R: A review on approaches of floating microspheres 2015: 4(12); 646- 55.
- Singh BN and Kim KH: Floating drug delivery systems: an approach to oral controlled drug delivery via gastric retention. J Control Release 2000; 63: 235-59.
- Dinarvand R, Mirfattahi S and Atyabi F: Preparation characterization and in-vitro drug release of isosorbide dinitrate microballoons. J Microencapsul 2002; 19: 73-81.
- Abrol S, Trehan A and Katare OP: Formulation, characterization and in-vitro evaluation of silymarin-loaded lipid microballoons. Drug Deliv 2004; 11: 185-91.
- Wu PC, Tsai MJ, Huang YB, Cheng JS and Tsai YH; In-vitro and in-vivo evaluation of potassium chloride sustained release formulation prepared with saturated poly-glycolyed glycerides matrices. Int J Ph 2002; 243: 119-24.
- Polli JE, Rehki GS, Augsburger LL and Shah VP; Methods to compare dissolution profiles and a rationale for wide dissolution specification for metoprolol tartrate tablets. J Pharm Sci 1997; 86: 690-700
- Mathews BR: Regulatory aspects of stability testing in Europe. Drug Dev Ind Pharm 1999; 25(7): 831-56.
- Singhal P, Kumar K and Shubhini A: Saraf formulation and evaluation of sustained release microballoons of furosemide. International Journal of Pharmaceutical Sciences Review and Research 2011; 6(1): 75-82.
- Gangadharappa H, Srirupa B, Anil G, Gupta V and Kumar P: Development in-vitro and in-vivo evaluation of novel floating hollow microspheres of rosiglitazone maleate. Der Pharmacia Lettre 2011; 3(4): 299-16.
- Mandal UK and Chatterjee B: Faria gias senjoti gastro-retentive drug delivery systems and their in-vivo success: a recent update. Asian Journal of Pharmaceutical Sciences 2016; 11: 575-84.
- Bagre A, Awasthi S and Kori ML: Clarithromycin loaded floating eudragit microsphere for anti h. Pylori Therapy In-vitro and In-vivo Assessment Journal of Chemical and Pharmaceutical Research 2017; 9(4): 270-76.
- Fentie M, Belete A and Gebre-Mariam T: Formulation of sustained release floating microspheres of furosemide from ethylcellulose and hydroxypropyl methylcellulose polymer blends. J Nanomed Nanotechnol 2015; 6: 1.
- Vinodbhai PKA, Gohel MCB, Parikh RKB, Bariyac MS, Rajeshvari N and Suthar D: Sustained release floating microspheres of acyclovir: formulation, optimization, characterization and in-vitro Int J Drug Dev and Res Jan-March 2011; 3(1): 242-51.
- Husseiny RA, Lila AAS, Abdall HMFSDF and Hanaa A: Elghamry formulation and evaluation of sustained release floating microspheres. International Journal of Trend in Research and Development 4(3): 2394-33.
How to cite this article:
Pancheddula M and Shayeda: Development of furosemide floating microballoons: in-vitro and in-vivo evaluation. Int J Pharm Sci & Res 2020; 11(5): 2248-61. doi: 10.13040/IJPSR.0975-8232.11(5).2248-61.
All © 2013 are reserved by the International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
Article Information
33
2248-2261
787
926
English
IJPSR
M. Pancheddula and Shayeda *
Department of Pharmaceutics, University College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana, India.
shayeda_ucpsc@yahoo.com
26 June 2019
05 February 2020
02 April 2020
10.13040/IJPSR.0975-8232.11(5).2248-61
01 May 2020