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FormulationDevelopmentandTasteMaskingofRifaximinNanosuspensionARTICLEJANUARY2013

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Inventi Rapid: Pharm Tech Vol. 2013, Issue 4 [ISSN 0976-3783]

2013 ppt 877, CCC: $10 Inventi Journals (P) Ltd Published on Web 27/09/2013, www.inventi.in

RESEARCH ARTICLE

INTRODUCTION A surprisingly large proportion of solid active pharmaceutical ingredients (API) have poor aqueous solubility and therefore poor bioavailability. The bioavailability of these so-called brick dust API can now is improved by formulating them as nanosuspensions. [1] The dissolution rate of API is proportional to the surface areaavailable for dissolution as described by the NoyesWhitney equation and, in addition to the dissolution rate enhancement, an increase in the solubility of nanosized API is also expected as described by the OstwaldFreundlich equation. [2] The nanocrystals can be obtained either by particle size reduction of larger crystals (top-down approach) such as high-pressure homogenization [3] and media milling or by building up crystals by precipitation of dissolved molecules (bottom-up approach). [4] The top-down approach, however, is more frequently used, and this approach requires high energy and expensive equipment. Regarding the bottom-up method, in the last decade, supercritical fluid-based techniques have been widely investigated to obtain nanosized drug particles, such as supercritical anti-solvent precipitation (SAS)) or rapid expansion of a supercritical solution into a liquid solvent (RESOLV). [5] However, these methods are difficult to control and scale up and are expensive. Anti-solvent precipitation is an effective way to prepare micro or nano-size drug particles. In this method, briefly, the drug is first dissolved in the solvent, then; the drug solution is quickly introduced into the anti-solvent. Precipitation occurs immediately by a rapid desolvation of the drug. [6] Aqueous solutions containing some stabilizers, such as polymers and surfactants, are commonly used as the anti-solvent. Polymers, such as hydroxyl propyl methylcellulose (HPMC) and methylcellulose (MC), [6] and polyvidone (PVP), can form strong hydrogen bonds with the drugs, which can be adsorbed on the hydrophobic particle surface, inhibiting the crystal growth of the drugs. However, there are some basic problems associated with common anti-solvent precipitation techniques, i.e. it is difficult to maintain the

1M. C. E. Societys Allana College of Pharmacy, Pune-411001, Maharashtra, India. E-mail: [email protected] *Corresponding author

size of the particles produced after precipitation, usually with a rapid growth rate and, therefore, they are large and have a broad particle size distribution (PSD). Recently, the control of particle size and PSD has become relatively easy by using a static mixer [6] and a confined impinging jet reactor. These methods are all based on the classical theory of nucleation and crystal growth and they have been described in detail. [7] In the last decade, ultrasound has received much attention and has been used as an effective method of controlling the nucleation and crystallization process Ultrasound irradiation has been proved to be a feasible mixing method and it can intensify mass transfer and accelerate molecular diffusion. [8] Rifaximin is a Antibiotic, Antiinfective Agent, semi synthetic, rifamycin-based non-systemic antibiotic, meaning that the drug will not pass the gastrointestinal wall into the circulation as is common for other types of orally administered antibiotics. It is used to treat diarrhea caused by E. coli. Rifaximin is classified as a Class IV API (poorly soluble and highly permeable) by the Biopharmaceutics Classification System (BCS). Rifaximin have very bitter taste so having less patient compliance. The absolute oral bioavailability of this drug is reported to range from about 10% to 20%, depending in part on the dosage form. The dissolution is the rate-limiting factor for absorption. In this article, stable nanosuspensions were prepared by the precipitation ultrasonication method. The effects of the process parameters, such as the concentration of PVA in antisolvent and the time length of ultrasonication on the particle size of the nanosuspension were investigated systematically. The corresponding physical properties of the prepared rifaximin nanocrystals were characterized by scanning electronic microscopy (SEM), X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). The nanosuspension was also evaluated by particle size distribution and zeta potential distribution. [9]

MATERIALS AND METHODS Materials RFX (RFX) was received as gift sample from Lupin pharmaceutical (India), polyvinyl alcohol (PVA, Mw 3070 kDa, 88% alcoholysis) PEG 200 and acetone of analytical grade were purchased from Luba chemicals (MH).

Formulation Development and Taste Masking of Rifaximin Nanosuspension

M A M Danish1*, Azhar Shetsandi1, Kiran S Bhise1

Abstract: The aim of this study was to prepare and characterize rifaximin nanosuspensions to mask bitter taste and enhance the solubility of this drug. Nanosuspensions were prepared by the precipitation ultrasonication method. The effects of two important process parameters, i.e. the concentration of PVA in the anti-solvent and the time length of ultrasonication on the particle size of nanosuspensions were investigated systematically and the optimal values were 0.15% and 15 min, respectively. The particle size and zeta potential of nanocrystals were 129 nm and 23.9 mV, respectively. The morphology of nanocrystals was found to be flaky and spherical in shape by scanning electron microscopy (SEM) observation. The X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) analysis indicated that there was no substantial crystalline change in the nanocrystals compared with raw crystals. The taste of rifaximin was significantly masked and its solubility increased by reducing the particle size.

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RESEARCH ARTICLE

Methods 1. Preparation of RFX Nanosuspensions RFX nanosuspensions were prepared by the precipitationultrasonication method. Briefly, RFX was dissolved in a mixed solvent of PEG 200 and acetone (ratio of 1:1, v/v) to form a series of organic solutions containing 200 mg/ml of drug. PVA was dissolved in water to obtain a series of anti-solvents with the concentrations of 0.15%, 0.25%, 0.50%, 1.0% and 1.5% (w/v). Both solutions were passed through a 0.45 m millipore filter paper. The anti-solvent was cooled to below 3C in an ice-water bath. Then, 2 ml of organic solution was quickly introduced into 20 ml of the pre cooled anti-solvent at a stirring speed of 400 rpm. After thaximine anti-solvent precipitation, the samples were immediately transferred to a test tube (2 cm in diameter and 10 cm in length) and treated with an ultrasonic probe at ultrasonic power inputs 150 W for different time lengths ( 10, 15 and 30 min).

The probe with a tip diameter of 8mm was immersed 10 mm in the liquid, resulting in the wave traveling downwards and reflecting upwards. The period of ultrasound burst was set to 3 sec. with a pause of 3 sec. between two ultrasound bursts. During the process, the temperature was controlled using an ice-water bath. The obtained nanosuspensions were concentrated by centrifugation at 16,000 rpm for 40 min using an ultracentrifuge. After the centrifugation, the supernatant was replaced with 2ml of 0.2% PVA solution. The solid residue was redispersed using a bath sonicator and the final drug content was adjusted to 20 mg/ml (drug weight/volume of nanosuspension) using an appropriate volume of 0.2% PVA solution. Different process parameters were systematically investigated by 32 factor method to clarify their effects on the particle size of nanocrystals. The process parameters included the concentration of PVA in the anti-solvent and time length of ultrasonication. For long-term stability, RFX nanosuspensions were lyophilized. The nanosuspensions were rapidly frozen in liquid nitrogen and freeze-dried in a FD-1C-50 freeze-drier for 15 h. 2. Charactrization of Rfx Nanosuspension a. Entrapment Efficiency The % entrapment efficiency (% EE) of RFX in the nanosuspension was determined after sonication. For the removing free RFX the nanosuspension was subjected to centrifugation on a cooling ultracentrifuge at 1600 rpm for 45 min. The clear supernatant was siphoned off to separate the unentrapped drug. One ml of supernatant was taken and diluted with PEG-acetone system up to 10 ml and absorbance was recorded at 436 nm using UV spectrophotometer.

DrugEntrapment%

Massofdruginnanoparticle

Massofdrugusedinformulation

b. Cumulative % Drug Release The release of RFX from nanosuspension was determined using an USP dissolution apparatus II equipped with

rotating paddle. Accurately measured nanosuspension liquid equivalent to 200 mg of drug dose was put into a treated cellophane membrane which has been boiled and equilibrated in 0.1 M Sodium Lauryl Sulphate The bag was secured with two knots at each end and space was minimized as far as possible and was immediately placed into release apparatus containing 900 ml of the 6.8 phosphate buffer with addition of 1.5% SLS and kept at 370.5C with paddle stirring rate of 100 rpm. One ml sample was pipette out from the release medium at the time interval. 5, 15, 30, 45, 60, 90, 120, 180, 240, 360, 420 and 480min. and the volume of the samples were replaced by the fresh buffer to maintain sink condition. The samples were analyzed by UV-spectrophotometrically at 436 nm. c. Particle Size Analysis Mean particle size and size distribution of optimized batch of SLN was determined by dynamic light scattering using Zetasizer Ver. 6.34 (Malvern instrument Ltd., Malvern, UK) at room temperature. Before measurement, batches were diluted with filtered double distilled water until the appropriate concentration of particles was achieved to avoid multi-scattering. The diluted sample was filled in the Disposable transparent sizing cuvette. The size was measured at 25C. The dispersant Refractive index and Material refractive index was 1.330 and 1.59 respectively. The analysis was performed to obtain the Z-average size and the polydispersity index value. The width of the size distribution was indicated by the polydispersity index (PI). The particle size analysis data were evaluated using volume distribution to detect even a few large particles. d. Zeta Potential Charge on drug loaded droplet surface was determined using Zetasizer Ver. 6.34 (Malvern Instruments Ltd., Malvern, UK). Analysis time was kept for 60s and average ZP, charge and mobility of optimized batches of polymeric nanoparticles was determined. All measurements were done at 25C. e. SEM Studies of Nanosuspension for Surface Morphology SEM studies were performed for the nanosuspension. The lyophilized powder of nanosuspension was spread on a double-sided adhesive plate one side of which was stuck to a glass slide. Excess powder was removed and the slide was kept on the sample holder and the scanning electron micrographs were taken using an electron microscope JEOL- JSM- 6360A. (Welsh and Rhodes, 2001 have reported that these studies can be used to study the surface morphology of the dry nanosuspension powders). The SEM of nanosuspension at magnification 1000X has been depicted in Result and Discussion. f. Powder X-Ray Diffractometer RFX and formulation were subjected to PXRD using X-Ray Generator, D8 Advance, Germany. To study X-Ray Diffraction pattern, the sample was placed into aluminum holder and the instrument was operated between initial

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RESEARCH ARTICLE

and final 2 angle of 5-50 respectively in an increment of 0.1 2. g. Differential Scanning Calorimeter (DSC) The thermal behavior of RFX was examined by DSC, using a Shimadzu DSC-60 Differential Scanning Calorimeter. The system was calibrated with high purity sample of Indium. RFX and formulation were scanned at a heating rate of 10C/min over a temperature range of 50 to 300C

whereas others over the range of 10-100C. Peak transitions and enthalpy of fusion were determined for the samples using TA 60 integration software. h. FT-IR Spectroscopy Fourier Transform Infrared Spectroscopy (FT-IR) of the Nanosuspension was conducted using Jasco FTIR 4100 spectrophotometer (Jasco Tokyo Japan) and the spectrum was recorded in the wavelength region of 4000-400cm-1

Figure 1: Cumulative % drug release study

Figure 2: Powder X-Ray Diffraction of RFX, nanosuspension

Figure 3: Scanning electron microscopy Figure 4: Differential scanning calorimeter (DSC)

Figure 5: FT-IR spectroscopy

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RESEARCH ARTICLE

the FTIR spectrum was taken by mixing very small amount of the formulation with previously dried KBr at 160C for 30 min in the ratio of 1:3. i. In-vitro Evaluation of Bitter Taste of Nanosuspension Nanosuspension lyophilized powder (equivalent to 8 mg of RFX) were placed in volumetric flask with 25 ml of phosphate buffer pH 6.8 and stirred for 5 min. The mixture was filtered and filtrate was analyzed for RFX concentration at 430 nm by UV-visible spectrophotometer and that was compared with the threshold value. RESULT AND DISCUSSION Cumulative % Drug Release Study

The dissolution rate was carried out same as that of trial formula. The cumulative % drug release study indicated that after 7 hrs the 81.083 release is obtained by nanosuspension. % Entrapment Efficiency The % Entrapment efficiency is carried out same as of trial formula. An obtained result is 850.37. Powder X-Ray Diffraction of RFX Nanosuspension PXRD of RFX that exhibited numerous peaks at 2 value of 11.2, 11.8, 12.1 and 22.3 confirming the RFX to be Amorphous in nature. Whereas nanosuspension lyophilized powder exhibited characteristic peaks at 2

Figure 6: Size distribution by Intensity

Figure 7: Zeta potential distribution

Figure 8: 3D surface plot for drug release

Figure 9: 3D surface plot for entrapment efficiency

Table 1: % Entrapment Efficiency and Cumulative % Drug Release of Factorial Batches

S. No. Batches % Entrapment Efficiency* Cumulative % Drug Release* 1 FA1 330.22 53.6790.21 2 FA2 450.28 55.7600.46 3 FA3 570.21 65.1610.56 4 FB1 710.14 64.4890.54 5 FB2 830.17 72.8360.91 6 FB3 840.22 80.1240.40 7 FC1 400.91 51.3570.32 8 FC2 500.97 55.2360.21 9 FC3 360.55 59.5750.81

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RESEARCH ARTICLE

values of 15.42, 16.98, 18.9 and 20.94. X-ray diffraction hasbeen used to analyze potential changes in the inner structure of RFX crystals. It was confirmed that no crystalline change was found in the nanocrystals, because their powder X-ray diffraction patterns were consistent with the pattern of the raw crystals and spherical crystal. However, the differences in the relative intensities of their peaks might be attributed to the differences in the crystallinity of the samples. Scanning Electron Microscopy The morphology of nanocrystals was found to be spherical and flaky in shape by scanning electron microscopy (SEM) observation. The surface topography of spherical crystals showed agglomerates of small crystal nuclei by SEM observation. Differential Scanning Calorimeter (DSC) In order to further confirm the physical state, DSC was also performed to analyze the different samples. In each case, a DSC scan of each sample showed a single sharp endothermic peak described to the melting of the drug which also indicated that there was no substantial crystalline change. However, the melting point of the nanocrystals and spherical crystals was lower than that of the raw crystals and this might be due to the size reduction in the crystals. FT-IR Spectroscopy The FT-IR of RFX nanosuspension showed all the peaks of drug and Excipients. In-vitro Evaluation of Bitter Taste of Nanosuspension For drug concentration analysis sample subjected to UV and drug concentration in filtrate after 5 min of stirring was found to be 5.8 g. Size Distribution by Intensity Mean particle size of optimized batch was found to be 124.2 nm. Zeta Potential Distribution Zeta potential of optimized batch was found to be -23.9 (mV).

CONCLUSION From the present study it can be concluded that, the nanosuspension of RFX can be formulated which can be converted to lyophilized powder for long term stability.

Formulation of RFX into nanosuspension mask its bitter taste, enhances the solubility and dissolution as well as permeability. Concentration of nano sized particles has marked effects on the dissolution of RFX from nanosuspension. REFERENCES AND NOTES 1. Barrett E Rabinow. Nanosuspensions in drug delivery. Nature

Reviews Drug Discovery, 3:785-796, 2004. 2. Raval Patel. Preparation and Characterization of Nanoparticles

for Solubility and Dissolution Rate Enhancement of Meloxicam. Intl R J of Pharmaceuticals, 1(2):42-49, 2011.

3. RA Nash. Suspensions. Encyclopedia of pharmaceutical technology, 2:2045-3032 ,2002,

4. Huabing Chen, Chalermchai Khemtong. Nanonization strategies for poorly water-soluble drugs. Drug Discovery Today, 2010.

5. Atul Pathak, Suresh P Vyas. Nano-vectors for efficient liver specific gene transfer. Int J Nanomedicine, 3(1):3149, 2008.

6. Jianxin Zhanga, Matthew Bunkera. Nanoscale thermal analysis of pharmaceutical solid dispersions. International Journal of Pharmaceutics, 380:170173, 2009.

7. Y Sugimoto, P Pou, M Abe, P Jelinek, R Perez, S Morita and O Custance. Nanosuspensions. Nature, 446:64-68, 2007.

8. Dengning Xiaa, Peng Quan. Preparation of stable nitrendipine nanosuspensions using the precipitationultrasonication method for enhancement of dissolution and oral bioavailability. European Journal of Pharmaceutical Sciences, 40:325334, 2010.

9. Robert Steffen M D, David A Sack. Therapy of Travelers Diarrhea With Rifaximin on Various Continents. The American Journal of Gastroenterology, 98:5-9, 2003

Acknowledgments Authors are thankful to Lupin limited, Pune, for the gift sample of RFX.

Table 2: Size Distribution by Intensity

Size (r.nm) % Intensity Width (r.nm) Z-Average (r.nm): 201.3 Peak 1: 124.2 60.3 39.66

PdI: 0.586 Peak 2: 533.9 35.9 191.6

Table 3: Zeta Potential Distribution

Mean (mV) Area (%) Width (mV) Zeta Potential (mV): -23.9 Peak 1: -23.9 100.0 6.34 Zeta Deviation (mV): 6.34 Peak 2: 0.00 0.0 0.00

Conductivity (mS/cm): 0.201 Peak 3: 0.00 0.0 0.00

Cite this article as: M A M Danish, Azhar Shetsandi, Kiran S Bhise. Formulation Development and Taste Masking of Rifaximin Nanosuspension. Inventi Rapid: Pharm Tech, 2013(4):1-5, 2013.

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