PREFORMULATION, FORMULATION DEVELOPMENT AND DRUG RELEASE STUDIES OF DIPYRIDAMOLE FLOATING MICROBALLOONS

PREFORMULATION, FORMULATION DEVELOPMENT AND DRUG RELEASE STUDIES OF DIPYRIDAMOLE FLOATING MICROBALLOONS

Seelam Ramya Krishna ^*, A. Ramu and S. Vidyadhara

Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur - 522510, Andhra Pradesh, India.

ABSTRACT: For the drugs possessing stomach and upper intestine as the absorption window, it has become high practical importance to improve their gastric residence time. The in vitro performance of the drug delivery being highly variable is ascertaining as gastric emptying is one of the complex mechanisms. Intense researches are being carried out for the development of multiparticulate systems, which are of greater importance than the single unit dosage forms in oral drug delivery. Floating microballoons offer more reproducible drug absorption, reduce the risk of local irritation, and improve the bioavailability of the drug. In the current research work, Dipyridamole, a BCS class – II drug was formulated as controlled release microballoons using ethyl cellulose as polymer and span 80 as the surfactant to improve the gastric retention of the drug. Preformulation studies viz. solubility, partition coefficient, micromeritics, and the drug excipient compatibility studies using Fourier transform infrared spectrophotometer and Differential Scanning Calorimeter were carried out. The emulsion solvent evaporation method was employed to develop controlled release dipyridamole floating microballoons. Different formulations of floating microballoons were formulated by considering five process and formulation factors viz. surfactant concentration, volume of solvent, volume of internal phase, polymer concentration, and rotational speed. All the formulations were subjected to in-vitro drug release studies, and the lowest and highest release rate was found to be 0.096 hr^-1 and 0.251 hr^-1 in the formulations F34 and F15, respectively. The Peppas n values of all the formulations were above 0.5, indicating the drug release mechanism was non-fickian diffusion.

Dipyridamole, Preformulation, Gastric transit time, Gastric emptying, Emulsion solvent evaporation

INTRODUCTION: Dipyridamole is an anti-platelet drug known chemically as methyl 2-({6-[bis(2- hydroxyethyl) amino]- 4, 8-bis(piperidin-1-yl)-[1, 3]diazino[5, 4-d]pyrimidin-2-yl}(2-hydroxy-ethyl) amino) ethan-1-ol. It has a chemical formula (C₂₄H₄₀N₈O₄) and a molecular weight of 504.6256 g/mol. It helps to keep blood flowing by stopping platelets from clumping together and by keeping heart blood vessels open.

Dipyridamole (DIP) is a platelet inhibitor. Clinically it is used as an antithrombotic agent. DIP has a short biological half-life of 2-3 h ^{1, 2}. Oral dosage forms of DIP exhibit variable absorption with limited bioavailability ranging from 11-44 %. DIP exhibits a pH-dependent solubility with good solubility at low pH (37°, 36.5 g/l at pH 1.0) and poor solubility at a higher pH (37°, 0.02 g/l at pH 7.0) ³.

The major absorption sites of DIP are stomach and duodenum ^1-3. Due to short biological half-life of 2 to 3 h, DIP should be frequently administered or as a controlled release dosage form. Dipyridamole appears to act in-vivo by synergistically modifying several biochemical pathways, including a) inhibition of platelet cAMP-phosphodiesterase; b) potentiation of adenosine inhibition of platelet function by blocking reuptake by vascular and blood cells, and subsequent degradation of adenosine; and possibly, c) potentiation of PGI₂ anti-aggregatory activity and enhancement of PGI₂ biosynthesis. These independent processes inhibit platelet function by increasing platelet cAMP through both a reduction in enzymatic cAMP-degradation and stimulation of cAMP formation via activation of adenyl cyclase by adenosine and possibly PGI₂. Only the inhibition of cAMP phosphodiesterase appears to be involved in the dipyridamole inhibition of isolated platelets in-vitro, since adenosine and PGI₂originate in-vivo from tissues other than platelets and any blood concentrations existing in-vivo will disappear before platelet-rich plasma has been prepared for in vitro platelet studies.

Preformulation ^4-6 commences when a newly synthesized drug shows sufficient pharmacologic promise in animal models to warrants evaluation in man. These studies should focus on those physico-chemical properties of the new compound that could affect drug performance and the development of an efficacious dosage form. A thorough understanding of these properties may ultimately provide a rationale for formulation design, or support the need for molecular modification.

The aim of this study was to determine some of the physicochemical properties such as solubility, pKa, dissolution, melting point, assay development, stability in Solution, stability in solid-state, microscopy, bulk density, flow properties, and excipient compatibility.

Hence, formulating the Dipyridamole into microballoons helps in increasing gastric retention ^{7, 8} times, thereby maintaining constant plasma concentration. In the current research work, Dipyridamole was formulated as floating microballoons by emulsion solvent evaporation technique to improve its gastric residence time thereby enhancing its bioavailability.

In the current research work, one of the major quality characteristics of controlled release multiparticulate systems i.e. drug release profile and the release kinetics were studied.

MATERIALS: Dipyridamole drug was received as a gift sample from Mankind Pharma, Hyderabad. Ethylcellulose, span 80, methanol, diethyl ether, and liquid paraffin were purchased from SD Fine Chemicals Ltd, Mumbai.

Preformulation Studies:

Construction of Calibration Curve: 50 mg of Dipyridamole was dissolved in 50 mL of 0.1N HCl buffer placed in a 50 mL of volumetric flask (stock solution). From this stock solution, 10mL was taken and made upto 100ml using 0.1N HCL, to attain the working standard 100 µg/ml. From that, a series of dilutions were made to get 3, 6, 9, 12, 15µg/ml concentrations. The absorbance was measured using a double beam UV-visible spectro-photometer (Thermo Scientific) at wavelength maxima of 283nm. From the obtained absorbance values, a standard calibration curve was constructed.

Physical and Physicochemical Properties of Dipyridamole:

Solubility Determination: Shake flask method was utilized for determining the solubility of Dipyridamole. In this method, the drug was added to 10mL of 0.1M HCL and shaken at a predetermined time. Excess amount of Dipyridamole was added to a saturation level till the observation of the presence of undissolved drug. Orbital shaker was used to shake the flask for 24 h. After a specified time, the slurry was filtered, and the filtrate was collected for analysis. After suitable dilutions, the sample was analyzed using a UV-Visible spectrophotometer at the maximum wavelength of 283nm.

Partition Coefficient Determination: Shake flask method⁹ was used to determine the partition coefficient of Dipyridamole. 1gm of the drug was dissolved in a mixture of 10mL chloroform and 10mL water, both phases were then mixed together in a separating funnel and shaken for 1hr and was allowed to stand long enough for the phases to separate and the solute concentration was measured in each solvent after suitable dilutions by using UV-Visible spectrophotometer at the maximum wavelength of 283nm.

Melting Point Determination: The melting point of Dipyridamole was determined by using the Capillary method. A capillary tube that was sealed initially at one open end was filled with Dipyridamole drug sample. By repeated pounding of the capillary tube against, the powder was pushed to the bottom of the tube. About 2.0mm to 3.0mm of sample height was maintained for optimum results and reproducibility. Then the capillary tube was kept in the melting point apparatus, and the temperature at which the drug was melted was noted.

Micrometric Properties: The micromeritics¹⁰ properties such as particle size, angle of repose, compressibility index, and Hausner’s ratio were characterized for Dipyridamole.

Bulk Density: Into a 10mL measuring cylinder, 5gms of accurately weighed drug (Dipyridamole) was placed. Without disturbing the cylinder, the volume occupied by the drug was noted and the bulk density was calculated using the below equation (values expressed in gm/cm³).

Bulk Density = Weight of sample / Volume of sample

Tapped Density: Into a 10mL measuring cylinder, 5gms of a drug (Dipyridamole) was placed. At an interval of 2 seconds, the cylinder was dropped onto a hard wooden surface from a height of one inch for 100 times. The final volume was recorded, and the tapped density was calculated by the following equation (values expressed in gm/cm³)

Tapped Density = Weight of sample / Tapped Volume of sample

Carr’s Index (%): The Carr’s index is an indication of the compressibility of a powder. Carr’s index is determined as the flow property of the blend depends on the carr’s index. A Carr’s index greater than 25% is considered to be an indication of poor flowability and below 15% of good flowability. It is calculated by the formula.

Carr's Index (%) = Tapped density-Bulk density / Tapped density × 100

Angle of Repose (θ): The angle of repose is indicative of the flowability of the substance. The funnel was adjusted in such a way that the stem of the funnel lies 2.5 cm above the horizontal surface. The sample powder was allowed to flow from the funnel, so the height of the pile just touches the tip of the funnel. The diameter of the pile was determined by drawing a boundary along the circumference of the pile and taking the average of three diameters. The angle of repose is calculated by using this formula:

tan θ = h/r

θ = tan^-1 h/r

Where θ is angle of repose, h is the height of the pile, r is the radius of the pile.

Hausner’s Ratio: The Hausner’s ratio is an indication of the compressibility of a powder and the flowability of the powder. A Hausner’s ratio greater than 1.25 is considered to be an indication of poor flowability. The observations for the flow properties determinations are recorded. It is calculated by the formula:

Hausner’s ratio = Tapped density / Bulk density × 100

Drug-Excipient Compatibility Studies:

FT-IR Spectroscopy: The physicochemical compatibility between Dipyridamole and ethylcellulose used in the research was carried out by subjecting to Fourier transform infrared spectrophotometer, Bruker. KBR pellet method was used to carry the physicochemical compatibility studies ¹⁰. The samples were prepared by mixing 100 mg of drug with 100mg of ethylcellulose used in the preparation of floating microballoons. These samples were scanned under diffuse reflectance mold, and spectra were recorded in the wave number region between 4000 cm^-1 to 400 cm^-1. The spectra obtained for the pure drug was compared with that of the physical mixtures of the drug with a polymer.

Differential Scanning Calorimetry Study: The physical incompatibilities of the drug and excipients can be evaluated quickly by Differential Scanning Calorimeter (DSC) as it changes in the appearance, the shift of melting endotherms and exotherms, and/or variations in the corresponding enthalpies of reaction. The DSC thermograms of pure drug and other excipients were recorded. The thermal analysis was performed in a nitrogen atmosphere at a heating rate of 10 °C/min over a temperature range of 50 °C to 300 °C.

Preparation of Floating Microballoons: ^{7, 11} Liquid paraffin was taken in a beaker, and 0.0% or 0.25% or 0.5% v/v of span 80 was added to it to get the oily phase. The oil phase was placed under the mechanical stirrer and set 400/550/700 rpm. In another beaker, the polymer is dissolved in a mixture of methanol and diethyl ether, to which drug was added and dissolved by placing on a vertex mixture for 2 min to get the organic phase. The organic phase was added drop by drop to the oil phase under stirring. Hollow microspheres were obtained by stirring continuously 4-5 h until the organic solvents were evaporated completely. To remove the traces of liquid paraffin, the obtained hollow microspheres were washed with petroleum ether and then dried. The compositions of various formulations were shown in Table 1.

TABLE 1: FORMULATION CODES OF DIPYRIDAMOLE BISULPHATE MICROBALLOONS

Characterization of Microballoons:

Drug Release Studies: ¹² USP II Dissolution test apparatus was used to perform the in-vitro dissolution studies for all the floating micro-balloons using 900mL of 0.1N HCl as the dissolution medium. The apparatus was set at 100 rpm, and the sample was withdrawn for every 30 min for the first 2 h and thereafter for every 1 h upto 12 h. After necessarily suitable dilutions, the samples were analyzed by using a UV-Visible spectrophotometer at the maximum wavelength of 283nm.

Drug Release Kinetic Studies: ¹³ The data obtained from in-vitro release studies were fitted to various kinetic equations to find out the mechanism of drug release. The kinetic models used are:

Qt = K₀ t (zero-order equation)

lnQ_t = ln Q₀ - K₁ t (first-order equation)

Q_t = K_h t_1/2 (Higuchi equation)

Where Q_t is the amount of drug release in time t, Q₀ is the initial amount of drug in the microballoons and K₀, K₁, and Kh are rate constants of zero order, first order, and Higuchi equations respectively. Further to confirm the mechanism of drug release, the first 60% of drug release was fitted in the Korsemeyer-Peppas model.

M_t / M_∞= k tⁿ

where M_t is the amount of drug release at time t and M_∞ is the amount release at time t=∞; thus M_t / M_∞ is the fraction of drug released at time t, k is the kinetic constant, and n is the diffusion exponent which can be used to characterize both mechanisms for both solvent penetration and drug release.

RESULTS AND DISCUSSION:

Preformulation Studies:

Construction of Calibration Curve: The calibration of Dipyridamole was carried out by using a double beam UV spectrophotometer (Thermo Scientific) in 0.1N HCL. The absorbances were measured at λ max of 288nm shown in Table 2. The linear co-efficient was found to be closer to 1 (i.e. 0.999) at a concentration range between 3-15µg/mL. The regression equation generated was y=0.055x as shown in Fig. 1.

FIG. 1: CALIBRATION CURVE FOR DIPYRIDAMOLE

TABLE 2: CALIBRATION CURVE DATA FOR DIPYRIDAMOLE

Physical and physicochemical properties of Dipyridamole: In the preformulation studies, the physical and physicochemical properties of Dipyridamole were determined for solubility, melting point, partition coefficient, and micromeritics and the results were shown in Table 3 which were all correlated with literature values.

TABLE 3: PHYSICAL AND PHYSICOCHEMICAL PROPERTIES OF DIPYRIDAMOLE

Drug-Excipient Compatibility Studies:

FT-IR Spectroscopy: Compatibility studies of drug and polymer were conducted by employing I.R spectral studies. IR spectra of pure Dipyridamole and the physical mixtures of drugs and polymers were shown in Fig. 2, 3, and 4, respectively. As shown in Table 4, the characteristic peaks of the Dipyridamole were observed with the spectra of Dipyridamole and the physical mixtures. It was inferred from the FT-IR results that no interaction existed between the drug and polymers as the identical principle peaks were observed in all the cases.

TABLE 4: FT-IR DRUG EXCIPIENT COMPATIBILITY STUDIES

FIG. 2: FT-IR SPECTRA OF DIPYRIDAMOLE

FIG 3: FT-IR SPECTRA OF DIPYRIDAMOLE AND ETHYL CELLULOSE PHYSICAL MIXTURE

FIG. 4: FT-IR SPECTRA OF DIPYRIDAMOLE AND SPAN 80 PHYSICAL MIXTURE

DSC Studies: In the current work, DSC has been used to study the physical and chemical interaction between the drug and the excipients used. Drug excipient interactions play a vital role with respect to the release of drug from the formulation amongst others. It was observed in the DSC graphs that there was no chemical interaction ¹³ between dipyridamole and the polymer used as there wasn’t any difference in the melting point of dipyridamole which was found to be 163.1 °C for dipyridamole alone and 161.2 °C for dipyridamole when mixed with ethyl cellulose as shown in Fig. 5 and Fig. 6, respectively.

Characterization of Microballoons: Drug Release Studies:

FIG. 7: DRUG RELEASE PROFILES OF (A) F1 TO F4; (B) F5 TO F8; (C) F9 TO F12 AND (D) F13 TO F16             

FIG. 8: DRUG RELEASE PROFILES OF (E) F17 TO F20; (F) F21 TO F24; (G) F25 TO F28 AND (H) F29 TO F32

FIG. 9: DRUG RELEASE PROFILES OF (I) F33 TO F36 AND (J) F37 TO F41

All the formulated dipyridamole microballoons were subjected to in-vitro drug release studies, and the drug release was found to be controlled to a maximum in case of F34 with a release rate constant of 0.096 hr^-1 and F15 showed rapid drug release at a rate of 0.251 hr^-1. The drug release profiles of F1 to F16, F17 to F32, and F33 to F41 were represented in Fig. 7, 8, and 9, respectively.

FIG. 10: EFFECT OF POLYMER CONCENTRATION AND METHANOL CONCENTRATION ON RELEASE RATE CONSTANT

FIG. 11: EFFECT OF INTERNAL PHASE VOLUME AND SPEED OF ROTATION ON RELEASE RATE CONSTANT

TABLE 5: ANOVA FOR DRUG RELEASE RATE OF THE DIPYRIDAMOLE MICROBALLOONS

From Fig. 10 it was inferred that upon an increase in the polymer concentration, the drug release rate was found to be decreased. This might be attributed to the increased path length at high polymer concentrations for the diffusion of an entrapped drug, which leads to a decrease in the drug release rate ⁷. Upon an increase in the concentration of methanol in the internal phase, the drug release rate was found to be decreased. At a high concentration of methanol, the slow evaporation rate results in the controlled formation of microballoons with a tight surface and thus might have lead to a decrease in drug release rate.

Fig. 11 illustrates that upon an increase in the volume of the internal phase, there was an increase in the drug release rate. Due to the less viscosity of the dispersed phase, the high internal phase volumes resulted in microballoons with a smaller size. As more surface area comes in contact with dissolution medium in the smaller particles, there was an increase in the drug release rate ⁷. The drug release rate was found to be decreased with the increase in the speed of rotation. The high speed of rotation resulted in microspheres with smaller size. Small size particles have more surface area, so the contact with the dissolution medium was more, which lead to an increase in drug release rate.

It was inferred from Table 5 that there was no effect of surfactant on the drug release rate as the p-value found to be 0.3399 which is more than the probable p-value (>0.100) indicating that the effect of Factor D on the drug release rate was not significant

Drug Release Kinetic Studies: The data of the drug release kinetics of all Dipyridamole micro-balloon formulations were represented in Table 6, and it was inferred that all the formulations were found to have followed first-order kinetics of drug release. The exponent (n value) form Peppas plots of all the formulations were found to be above 0.5, thus indicating all the formulations followed non-Fickian diffusion ¹⁴ as their drug release mechanism.

TABLE 6: DRUG RELEASE KINETICS OF DIPYRIDAMOLE MICROBALLOONS

CONCLUSION: In an aim to develop floating microballoons, dipyridamole was studied extensively for its preformulation parameters. Five different formulation and process variables viz. polymer concentration, surfactant concentration, the volume of internal phase, the concentration of methanol in the internal phase, and speed of rotation was considered for the formulation of dipyridamole floating microballoons. In the current research work, the emulsion solvent evaporation technique was employed to formulate floating microballoons as 41 formulations. All the formulations were subjected to drug release and release kinetic studies effectively, and the results were suitably analyzed.

ACKNOWLEDGEMENT: I wish to express my sincere thanks to my supervisor Dr. A. Ramu, for providing invaluable guidance, comments, and suggestions throughout the course of the project. I place on record my sincere gratitude to Dr. S. Vidyadhara, for providing me with all the necessary facilities.

CONFLICTS OF INTEREST: The authors declare that there are no conflicts of interest.

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    How to cite this article:

    Krishna SR, Ramu A and Vidyadhara S: Preformulation, formulation development and drug release studies of dipyridamole floating microballoons. Int J Pharm Sci & Res 2020; 11(9): 4637-47. doi: 10.13040/IJPSR.0975-8232.11(9).4637-47.

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