Formulation and Evaluation of Nanosuspension of Ranolazine

Introduction

In the Biopharmaceutics Classification System (BCS), solubility is one of the significant parameters, and the most crucial element affecting a drug's bioavailability is its rate of dissolution. When taken orally, medications that are poorly water-soluble may require high dosages to attain therapeutic plasma concentrations. The main issue in developing formulations for novel chemical entities as well as for generic drugs is low water solubility. A chemical is considered weakly water-soluble if it dissolves in less than 1 part per 10,000 parts of water. Nanotechnology has been introduced to address issues associated with traditional approaches to improve solubility and bioavailability. For the drugs that are insoluble in both water and organic solvents, nanosuspension technology has been employed.¹

For drugs that are soluble in oil but insoluble in water, with a high log P value, high melting point, and high dosages, nanosuspension is more suitable. Additionally, drugs that are insoluble in both water and organic solvents can be dissolved using nano-suspension technology. Hydrophobic drugs such as Aceclofenac, Famotidine, Simvastatin, Atorvastatin, and Revaprazan, are manufactured as Nanosuspension. Surfactant-stabilized colloidal dispersions of nanosized drug particles are called Nanosuspension. They can alternatively be described as a biphasic system consisting of pure drug particles dispersed in an aqueous medium, each with a diameter smaller than 1 μm. Nanosuspensions can also be lyophilized or spray-dried and the nanoparticles from a nanosuspension can be incorporated into a solid matrix.²

The choice of excipients to be employed in the formulation depends on the type of technique used for the formulation of nanosuspension. The most common excipients used in the preparation of nanosuspension are i) stabilizers for preventing agglomeration of particles and for stabilization of nanosuspension, ii) surfactants for wetting particles, and iii) organic solvents for formulating the nanosuspension.³

Ranolazine, a piperazine derivative, is a new antianginal agent or anti-ischemic drug. Its IUPAC name is N‐(2,6‐ dimethylphenyl) ‐4(2‐hydroxy‐3‐[2‐meth‐oxyphenoxy] ‐propyl) ‐1‐piperazine acetamide. It is a selective inhibitor of cardiac late sodium channels. Blockage of these late sodium channels decreases the intracellular Na⁺ and Ca⁺², which leads to improved myocardial relaxation, diastolic function, and myocardial blood flow leading to its anti-anginal and anti-ischemic properties.

Ranolazine is designed to act without reducing heart rate or blood pressure, and is indicated for treatment of chronic angina in patients who failed to respond to prior angina therapies.⁴ It belongs to BCS class II drug (low solubility and high permeability). The bioavailability of Ranolazine ranges from 35 to 55%.

The low solubility of Ranolazine could be the reason for low absorption and thus lower bioavailability. Considering these factors, an attempt was made to enhance the solubility of Ranolazine by nanosuspension technique, thus achieving a possible enhancement in bioavailability.

Materials and Methods

Ranolazine was purchased from Yarrow chemicals. HPMC E15 and Eudragit L100 were purchased from Fisher Scientific, Mumbai. Soya lecithin, Tween 80, Ethanol, and HCl were purchased from Karnataka Fine Chem, Bengaluru.

Preformulation Studies

Determination of solubility of Ranolazine in different pH buffer medium

The solubility of Ranolazine was determined by adding excess amounts of Ranolazine in buffers with a pH range between 1.2 and 7.4 and distilled water at 37±0.5⁰C. After equilibrating the solutions for 48 hours, the solutions were filtered to obtain a clear solution. The absorbance was measured using UV spectrophotometer at 272 nm and concentration in µg/mL was determined. Solubility was determined in triplicates.⁵

Determination of λmax by ultraviolet spectrum

Accurately weighed 100 mg of Ranolazine was dissolved in 100 mL of 0.1N HCl buffer. From this stock solution-I, 10 mL solution was diluted to 100 mL with 0.1N HCl buffer to prepare stock solution-II. Further dilution was made to obtain the concentration of 10 µg/mL. The prepared solution was scanned on a UV/ Vis spectrophotometer between 200 and 400 nm. The maximum absorbance, λmax, obtained in the graph was considered as analytical wavelength for pure drug.6

Construction of calibration curve of Ranolazine (0.1N HCl buffer pH 1.2)

Accurately weighed 100 mg of Ranolazine was dissolved in 10 mL of 0.1N HCl and made up to 100 mL with 0.1N HCl buffer solution (1 mg/mL). Ten mL of the above solution was diluted up to 100 mL with 0.1N HCl buffer solution (0.1 mg/mL). From the above solution, further dilutions were made to prepare 2, 4, 6, 8 and 10 µg/mL solutions and analyzed by UV spectrophotometer at 272 nm. The graph of absorbance v/s concentration was plotted and r2 value of this graph was calculated.7

Compatibility Studies (FT-IR)

Approximately 1 mg of Ranolazine was thoroughly mixed with about 100 mg of KBr in a ratio of 1:100 in a mortar. The mixture was manually pressed into a pellet in a die and the pellet was placed in a Fourier Transform Infrared (FTIR) Spectrophotometer (Shimadzu corporation 84005, Japan) and analyzed.8

Differential Scanning Calorimetry (DSC)

DSC analysis of the formulation was carried out by Perkin-Elmer-DSC 4000. The sample was heated at a heating rate of 10⁰C/min over the temperature range of 30-300⁰C.²

Preparation of Nanosuspension by Nanoedge method

Nanoedge is a combination of precipitation and high-pressure homogenization.⁹ The Nanoedge method includes two steps. In the first step, the drug was dissolved in ethanol at room temperature and this solution was added dropwise into aqueous solution containing different amounts of stabilizers and surfactant on a magnetic stirrer. The stirring was performed at room temperature and volatile solvents were allowed to evaporate. Then the suspension from step 1 was homogenized using RQT 127A/D high-pressure homogenizer (Remi Elektrotechnik Limited, Maharashtra) at the speed of 7000 rpm for 30 minutes until nanosuspension was formed.¹⁰ In total, eight formulations were prepared with varying concentrations of the two polymers, HPMC E15 and Eudragit L 100 as shown in Table 1.

Characterization of Prepared Nanosuspension

Fourier Transform Infrared Spectroscopy (FT-IR)

The FT-IR spectra of Ranolazine nanosuspensions, after separating it from the suspension were recorded to check drug polymer interaction stability.⁸

Differential Scanning Calorimetry

DSC analysis of the Ranolazine nanosuspension were carried out using a Diamond DSC (Perkin Elmer, USA) to evaluate any possible drug-polymer interaction (Figure 2). The analysis was performed at a rate of 10⁰C min-1 from 30 to 300⁰C temperature range under nitrogen flow of 25 mL min-1.

Drug Content

Drug content was determined by dissolving required amount of accurately weighed nanosuspension in methanol and diluted suitably in pH 0.1 HCl buffer. After 24 hrs, the solution was filtered and the drug content was determined by UV spectrophotometer at 272 nm.10 % Drug content = (Amount of drug obtained)/(theortical amount of drug) × 100

Drug Entrapment

Drug entrapment was determined by centrifugation method. The redispersed nanosuspension was centrifuged at 15,000 rpm for 40 min at 25⁰C to separate the free drug in the supernatant. The concentration of Ranolazine in the supernatant was determined by using a UV-Visible spectrophotometer at 272 nm after suitable dilution.¹¹

% Entrapment Efficiency = (Experimental drug content)/ (theortical drug content) × 100

Surface Morphology

Study Scanning electron microscopy (SEM) of the Ranolazine nanosuspension was performed to examine the surface morphology. The nanosuspension was mounted on metal stubs and the stub was then coated with conductive gold with a sputter coater attached to the instrument. The photographs were taken using a scanning electron microscopy (Hitachi X650, Tokyo, Japan) at different magnifications.¹²

Zeta Potential (Surface Charge)

The zeta potential of Ranolazine nanosuspensions was measured by Zeta sizer IV. To determine the zeta potential, nanosuspension samples were diluted with KCl (0.1 Mm) and placed in electrophoretic cell, where an electrical field of 15.2 Vcm^-1 was applied. Each sample was analyzed in triplicates.¹³

X-ray Diffraction (Crystalline state)

The ranolazine drug and nanosuspensions were investigated by XRD (Rigaku model smart lab 3KW, Japan). The generator was operated at a 40-KV tube voltage and 40-mA tube current and used Kα lines of copper as radiation source. The diffraction angle ranged from 0⁰ to 80⁰ (2ϴ) in the step-scan mode (Step width:1.0⁰ min-1).¹⁴

Saturation Solubility

The solubility was determined by adding excess amounts of nanosuspension in distilled water (1.5 mL of sample in 10 mL of distilled water) at 37±0.5⁰C. The solution was filtered to obtain clear solution. The absorbance was measured using UV spectrophotometer at 272 nm and concentration in µg/mL was determined. The sample was analyzed in triplicates.¹⁵

In vitro Dissolution Study

A dissolution study was carried out for pure drug and nanosuspension using USP II (paddle) apparatus in 0.1N HCl buffer, at a temperature of 37±0.5⁰C, 50 RPM. An accurately weighed amount of pure ranolazine and nanosuspension equivalent to 500 mg ranolazine were put into 900 mL 0.1N HCl buffer. A 5 mL of sample was taken out from the media at regular time intervals until 60 min and same volume of fresh medium was replaced to maintain the sink condition. The samples were filtered and drug release was analyzed in UV spectrophotometer at 272 nm.¹⁶

In vitro Drug Release Studies

Discussion

The drug solubility in different pH media is an important aspect as it directly influences the drug absorption throughout the GI tract. The solubility of drug was found to be 6.67 µg/mL in 0.1N HCl.

A calibration curve for Ranolazine was constructed in 0.1N HCl buffer. The absorption spectrum of Ranolazine was obtained between 200 and 400 nm with a concentration of 10 µg/mL in 0.1N HCl buffer solution using a UV spectrophotometer. The maximum peak, λmax, was observed at 272 nm, which was taken as the analytical wavelength. The FT-IR spectroscopy study was carried out to assess the compatibility between the drug, stabilizers and surfactant used in preparation of nanosuspension.

The FT-IR analyses were performed for the drug, stabilizers and surfactant and physical mixture of the drug, stabilizers and surfactant. Upon observing Figure 1, all the characteristic peaks of Ranolazine were present in the spectrum, indicating compatibility between the drug, stabilizer and surfactant and it can be concluded that the characteristic bands of Ranolazine were not affected after successful loading. It shows that there was no significant change in the chemical integrity of the drug. The DSC thermogram of pure Ranolazine showed sharp endothermic peak at 123.45o C, which corresponded to its melting point. The melting point reported for Ranolazine was 120-124o C indicating the crystalline nature of the drug.

The DSC thermogram of Ranolazine nanosuspension (Figure 2) showed endothermic peak at 120.10⁰C corresponding to the melting point. Thus, this study confirmed that there was no chemical interaction and presence of Ranolazine in the formulation.

The drug content was found to be in the range of 60.67±1.5% to 94.83±0.8% and drug entrapment was found to be in the range of 63.33±0.8% to 94.80±0.5% as shown in Table 2. Thus, this study confirmed that the drug content and drug entrapment increases with the increase in concentration of stabilizers.

Solubility of all the formulations was found to be in the range of 0.57 to 0.93 mg/mL. The XRD of the Ranolazine showed sharp peaks at 10.0⁰, 10.41⁰, 14.92⁰, 15.07⁰, 16.52⁰, 19.34⁰, 21.41⁰, 23.50⁰ and 24.64⁰. The XRD of Ranolazine nanosuspensions (Figure 3) showed different XRD compared to pure Ranolazine. The formulation showed peaks at 7.59⁰, 14.88⁰, 16.40⁰, 23.34⁰ and 24.55⁰. Thus, it is evident that the crystalline nature of the drug has been converted into amorphous form which can be demonstrated by the absence of the characteristic peaks and in the reduction of XRD peak intensity. Zeta potential of the formulation (F3) was observed and the value was found to be -3.15 mV, which indicates the prepared formulation to be stable.

Data of the dissolution studies is shown in the Table 3. The formulation prepared with HPMC E15 has shown good drug release at 60 min at concentration of 250 mg. Increase in the concentration above 250 mg retarded the release of drug. The formulations prepared with stabilizer at concentration of 1000 mg or below 1000 mg showed good drug release. Increase in the concentration of stabilizer above 1000 mg retarded the release of drug. As the concentration of stabilizers increase, the viscosity of the formulation also increases and stabilizers control the release of the drug. Thus the drug from the formulation gets released in a controlled manner.

Conclusion

The Ranolazine nanosuspension can be produced by Nanoedge method, incorporating HPMC E15 and Eudragit L100, Tween 80, lecithin, ethanol, and water. This method is economical and the manufacture of nanosuspension has been found to be simple with easy scalability. Based on the results of the present study, it can be concluded that nanosuspension improves solubility, and improves the site specificity of Ranolazine. Nanosuspension creates a new opportunity for poorly soluble drugs. Finally, based on the results, it can be concluded that the Ranolazine nanosuspensions exhibited immediate release and increased solubility compared to pure drug.

Conflict of interest

The authors have declared that no conflict of interest is linked with this work.

Acknowledgements

The authors wish to express their sincere gratitude to Department of Pharmaceutics, T. John College of Pharmacy, Bangalore, Karnataka, India for providing necessary facilities to carry out this research work.