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Study on the dissolution improvement of albendazole using reconstitutable dry nanosuspension formulation
European Journal of Pharmaceutical Sciences 123 (2018) 70–78
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Study on the dissolution improvement of albendazole using reconstitutable dry nanosuspension formulation
T
Viktor Fülöpa, Géza Jakaba, Tamás Bozób, Bence Tótha, Dániel Endrésika, Emese Balogha, ⁎ Miklós Kellermayerb, István Antala, a b
Semmelweis University, Department of Pharmaceutics, Hőgyes Endre Street 7, Budapest H-1092, Hungary Semmelweis University, Department of Biophysics and Radiation Biology, Tűzoltó Street 37-47, Budapest H-1094, Hungary
A R T I C LE I N FO
A B S T R A C T
Keywords: Wet media milling Nanosuspension solidification Microcrystalline cellulose carrier Particle size distribution Solubility Dissolution rate Artificial rumen fluid
The aim of the study was to improve the solubility and dissolution rate of the poorly water soluble drug albendazole via surfactant assisted media milling process. Preparation of a nanosuspension and then post-processing with a solidification technique applied to improve the applicability of nanosuspension in a solid dosage forms carrier. The dry nanosuspension was obtained using microcrystalline cellulose as solid carrier after tray drying at 40 °C. Both reconstitution from the solid carrier and dissolution profile studies were investigated in biorelevant Artificial Rumen Fluid (ARF) at pH = 6.50 and dissolution media at pH = 1.20 and pH = 6.80. Reconstitution studies have demonstrated that the mean hydrodynamic diameter values of albendazole crystals released from the dry suspension were nanosized (intensity weighted hydrodynamic diameter values: 200.40 ± 2.318 nm in ARF at pH = 6.50, 197.17 ± 0.208 nm in dissolution medium at pH = 6.80). Thermodynamic solubility studies have indicated a 2.98 times increase in water solubility (144.41 ± 0.09 μg/ ml milled, 48.38 ± 0.01 μg/ml unmilled, 8.21 ± 0.02 μg/ml albendazole powder) in ARF at pH = 6.50, and 2.33 times in dissolution medium at pH = 6.8: (146.27 ± 0.28 μg/ml milled, 62.71 ± 0.04 μg/ml unmilled, 9.00 ± 0.01 μg/ml albendazole powder), and 13.65% increase at pH = 1.20 (1728.31 ± 3.31 μg/ml milled, 1559.41 ± 0.40 μg/ml unmilled, 1520.70 ± 1.39 μg/ml albendazole powder), dissolution rates have also increased. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) imaging investigations detected no albendazole nanocrystals on the surface of the carrier, which demonstrated the incorporation of albendazole into the microcrystalline cellulose solid carrier structure.
1. Introduction One of the major obstacles to the development of highly potent new drug candidates is the poor water solubility of these compounds. Approximately 30–40% of potential new chemical entities (NCE) identified by pharmaceutical companies (Pfizer, Merck) with high throughput screening (HTS) /combinatorial chemistry operations are poorly soluble in, which hinders their therapeutic application (Lipinski, 2003). The National Nanotechnology Initiative (NNI) defines Nanotechnology, “as research and development at the atomic, molecular, or macromolecular levels in the sub-100-nm range (w0.1–100 nm) to create structures, devices, and systems that have novel functional properties”(Morrow et al., 2007). According to European Medicines Agency (EMA), nanotechnology is defined as the production and application of structures, devices and systems by
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controlling the shape and size of materials at nanometre scale. The nanometre scale ranges from the atomic level at around 0.2 nm (2 Å) up to around 100 nm. A pharmaceutical nanosuspension is defined as very finely colloid, biphasic, dispersed, and solid drug particles in an aqueous vehicle, size below 1 μm, without any matrix material, stabilized by surfactants and polymers, and prepared by suitable methods for drug delivery applications, through various routes of administration like oral, topical, parenteral, ocular and pulmonary routes (Prabhakar and Bala Krishna, 2011). The particle-size distribution of the solid particles in nanosuspensions is usually less than 1 μm with an average particle size ranging between 200 and 600 nm (Chingunpituk, 2007). The potential benefits of the Nanosuspension Technology for poorly soluble drug delivery are increased drug dissolution rate, increased rate and extent of absorption, hence the bioavailability of drug (area under plasma versus time curve, onset time, peak drug level), reduced variability, and reduced fed/fasted effects, increased penetration
Corresponding author at: Department of Pharmaceutics, Semmelweis University, Hőgyes E. Street 7, Budapest H-1092, Hungary. E-mail address: [email protected] (I. Antal).
https://doi.org/10.1016/j.ejps.2018.07.027 Received 20 March 2018; Received in revised form 5 July 2018; Accepted 11 July 2018 0928-0987/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
capability of topical nanosuspension. Nanosuspension has low incidence of side effects by the excipients, increased resistance to hydrolysis and oxidation and increased physical stability to settling. Reduced administration volumes, essential for intramuscular, subcutaneous, and ophthalmic use. Finally they can provide the passive targeting (Ibrahim et al., 2012; Liu et al., 2011; Rabinow, 2004). Several production techniques are available to produce drug nanocrystals, such as precipitation, pearl milling (media milling), and highpressure homogenization (Gao et al., 2008). Solidification techniques transform nanosuspensions into solid dosage forms such as tablets, capsules, and pellets. Solid dosage forms increase the long term stability of nanosuspensions and improve patient compliance (Van Eerdenbrugh et al., 2008). Albendazole is an imidazole carbamate-ester, a broad-spectrum anthelmintic for the treatment of intestinal helminth infections. It also has anti-hydatid activity and is now recognized to have important application in treatment of human cystic and alveolar echinococcosis. The main targeted animals are cattle (dosage: 7.5–10 mg/kg) and sheep (dosage: 5 mg/kg) in animal healthcare (Campbell, 1990; Mckellar and Scott, 1990). This compound has a pH dependent, poor water solubility range from: 0.376 mg/ml in pH = 1.2 to 0.016 mg/ml in pH = 6.0 buffers, which is the lowest concentration achieved during the solubility studies of (Torrado et al., 1996). Albendazole has an octanol-water partition coefficient (Log P) value of 3.83 (Mottier et al., 2003), which is considered high, (Log P > 1.72 for reference compound metoprolol) according to BCS drug permeability classifications (Dahan et al., 2009). With low water solubility and high membrane permeability it is classified as a BCS Class II drug (Raimar Lödenberg, 2000). To overcome the drawbacks of the poor water solubility of ABZ, different formulation approaches have been adopted in the past such as liposomal entrapment (Wen et al., 1996), solid dispersions with polyvinylpyrrolidone (Kalaiselvan et al., 2006) and with Poloxamer 188 and PEG 6000 (Vidal et al., 2014). Inclusion complexes of ABZ with different CDs have been prepared, the significance of synthetized citrate derivate of β-CD should be highlighted, due to its high stability constant (García et al., 2014). Lipid based delivery systems of ABZ seem to be effective formulations, including self-microemulsifying drug delivery systems (SMEDDS) of ABZ to reach an marked water solubility improvement (Meena et al., 2012). Several particle size reduction methods have also been introduced but without solidification so far (Paredes et al., 2016). The low bioavailability of the API reduces the efficacy in hydatid. Pragmatic approaches for chemo-therapy of hydatid patients necessitate to focus on improved transport, targeting modulation of the physicochemical parameters and metabolic decomposition of benzimidazoles (Wen et al., 1996). Effective dose reduction, hence lower incidence of side effects, the ease of scale-up, low bath-to-batch variability can be mentioned as the main advantages of the utilizations of nanocrystals, while the most common disadvantages are high energy investment during manufacturing, immunotoxicity and non-specific uptake in reticuloendothelial system (RES) organs (Mitragotri et al., 2014). The aim of the work was to use surfactant assisted media milling to produce nanocrystals and to transform the milled albendazole nanosuspensions to solid form by wet granulation applying microcrystalline cellulose as carrier. Furthermore, the particle size distribution and Zetapotential of albendazole were studied after redispersion as well as the drug release and thermodynamic solubilities in various dissolution media. Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FT-IR) investigations have been performed to compare the final product to the active substance and to the carrier. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were utilized to compare the surfaces of the pure albendazole to the MCC carrier (Vivapur® 12), to their physical mixture and to the albendazole milled dispersion.
Albendazole EP (micronized), (Sequent Scientific Ltd., India) was used as model drug. Particle size distribution measured by the supplier with sieve analysis demonstrated, that > 90% of the powder is under 30 μm. Laser diffraction measurements have been performed to confirm this result dispersing 3.00 w/w% albendazole into 0.50 w/w% Polysorbate 80 solution. Results have been interpreted by the mean of five individual measurements ± standard deviation values. Polysorbate 80 (Tween 80) (Molar Chemicals Kft., Hungary) as surface active agent for media milling, Dimethylpolysiloxane (Foamsol) (Kokoferm Kft., Hungary) as antifoaming agent, Coarse grade Microcrystalline cellulose with mean particle size of 180 μm, and bulk density of 0.33 g/ml (Vivapur® 12), (JRS Pharma GmbH & Co. KG, Germany) as carrier of the solid suspension. 2.1. Surfactant assisted media milling process The selection of the milling process parameters were based on preliminary studies involving relatively moderate agitation with high beads to powder loading ratio to achieve satisfactory particle size distributions. Retsch PM 100 planetary ball mill, with a stainless steel container of 50 ml in volume, were used for this process. Container loadings consisted of 0.5083 g, of pure albendazole powder (ρ albendazole = 1.3 g/ml, V albendazole = 0.391 ml) with 16.08 ml of 0.50 w/w % of polysorbate 80 surfactant solution, and 33.33 ml of d = 0.1 mm zirconia beads as the milling media. Beads to powder loading mass ratio was: 249.36. As for the milling parameters: milling speed was 400 rpm, and milling time was 120 min. Temperature control has been integrated as a part of the quality by design development of particle size reduction techniques (Nekkanti et al., 2015). Various milling programs have been compared in order to maintain the loading temperature as low as possible during the process. A d = 63 μm mesh size, stainless steel sieve, was utilized for separating the beads from the nanosuspension. All the equipment (planetary ball mill, container, d = 0.1 mm zirconia beads, and sieves) were provided by (Retsch Technology GmbH., Germany). 2.2. Solidification using wet-granulation technique The dried MCC carrier was weighted using a Kern ABT 320–4 M analytical balance, (ABT - KERN & SOHN GmbH, Germany) into a steel mortar and after every milling process the obtained nanosuspension was added to the carrier. Than mixed manually by kneading for 3 min, sieved through a 180 μm mesh size sieve and dried in a Labor-Innova drying chamber, (Labor-Innova Műszeripari Kft., Hungary) on 40 °C for 2 days. Solid suspension of unmilled albendazole was prepared the same way, but for the dispersing the albendazole an IKA RCT basic, heatable magnetic stirrer, (IKA® Works, Inc., USA) was employed. 2.3. Drug content and composition determination of milled and unmilled solid suspensions Since albendazole has pH dependent solubility, with a maximum of 0.376 mg/ml at pH = 1.2, it was evident to determine the amount of active content in highly diluted stock solutions at pH = 1.2. Three stock solutions have been prepared by weighing in 1 g of dispersions and diluted with 0.1 N hydrochloric acid to 2000 ml, stirred at 1000 rpm with IKA RCT basic, heatable magnetic stirrer, (IKA® Works, Inc., USA) for 24 h, sterile filtered using FilterBio® NY nylon membrane syringe filters, (LAB-EX Laborkereskedelmi Kft., Hungary) with pore sizes of 0.22 μm, and measured by UV-VIS spectrophotometry method. Mean Polysorbate 80 content of the dispersions were calculated from the added weight of suspensions after every milling and dispersing cycles, mean MCC (Vivapur® 12) concentration was calculated from the amount of dried carrier applied before wet granulation process. The 71
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2015). Particle size distributions of milled suspensions and released crystals from dry suspensions have been determined by Photon Correlation Spectroscopy (PCS) or Dynamic Light Scattering (DLS) method. Before dispersing the milled suspensions and dry suspensions in 100 ml of pH = 6.50 ARF, pH = 6.80 dissolution media and distilled water, the solutions were prefiltered using FilterBio® NY nylon membrane syringe filters, (LAB-EX Laborkereskedelmi Kft., Hungary) with pore sizes of 0.22 μm. Filtered solutions were mixed at 250 rpm speeds, and l = 3 cm long, d = 0.8 cm thick magnetic stirrer bars, conditioned at 37 °C with IKA RCT basic, heatable magnetic stirrer, (IKA® Works, Inc., USA). One dose of dispersion, containing 200 mg of albendazole and also one dose of milled suspension were then added to the filtered dissolution medium and mixed using the same 250 rpm speed. 5 ml of samples were taken with a syringe and filtered with Whatman® UNIFLO® PTFE membrane syringe filters with pore sizes of 0.45 μm, (Sigma-Aldrich Co., USA). The withdrawn samples were replaced with sterile filtered dissolution medium. 1 ml of the withdrawn samples were poured into a 6 G glass sizing cuvette with square aperture. Particle size distributions were determined with the instrument: Zetasizer nano ZS, (Malvern Instruments Ltd., UK). Measurement settings: Automatic mode, NIBS (Non-Invasive-Back-Scattering) laser angle 173°, 28–32 sub runs/measurement, run durations: 10 s. Sample chamber was heated to 37.0 °C, and equilibrating time was 180 s. Automatic laser positioning selected a 4.65 mm position from the bottom of the cuvette and for attenuation, attenuator 9 was selected automatically. Five individual measurements were performed, for every sample and the mean ± standard deviation values were reported for all the DLS parameters, including: intensity weighted mean hydrodynamic diameters (ZAVG d) and polydispersity indices (PDI) in this article. Particle size distribution comparison of granules and MCC Vivapur® 12 powder was also performed by sieve analysis method utilizing a Retsch sieve series (315 μm, 180 μm, 125 μm, 50 μm, 32 μm mesh size sieves) and a Retsch AS 200 control vibrational sieve shaker (Retsch Technology GmbH., Germany). Fractionation adjusted parameters, including amplitude 1.5 mm, and time 5 min were applied.
moisture contents of the end products were measured by a Scaltec Smo 01 evaporator, (Scaltec Instruments GmbH, Germany), with predetermined temperature of 95 °C and from 1.000 g of milled and unmilled solid suspension. It has already been reported, that water is retained in a porous material during wet granulation of MCC as a result of absorption and capillary effects. The material was characterized by irreducible saturation, which refers to liquid, that remains in porous bed, regardless of any further increase in pressure applied. For MCC and water, irreducible saturation was 90 w/w %, this indicates high extent of solid liquid interaction (Kleinebudde, 1997). According to the referred article, we have applied 85 w/w % of milled and unmilled suspension to the dried carrier, during wet granulation, not reaching the saturation value, but maximizing the drug content of the dispersion during a milling or dispersing cycle. Dose determinations were based on the drug content evaluations, by studying the therapeutic efficacy, toxicity pharmacokinetics of albendazole a 200 mg of dose was preferred (Dayan, 2003; Galia et al., 1999). 2.4. pH = 6.50 artificial rumen fluid (ARF) preparation Utilizing the same Kern ABT 320-4 M analytical balance, the following constituents were weighted: 9.80 g of Sodium Hydrogen Carbonate (Molar Chemicals Kft., Hungary), 9.30 g of Disodium Hydrogen Phosphate Dodecahydrate (Molar Chemicals Kft., Hungary), 0.47 g of Sodium Chloride (Molar Chemicals Kft. Hungary), 0.57 g of Potassium Chloride (Molar Chemicals Kft., Hungary), 0.06 g of Magnesium Chloride Anhydrate, (Sigma-Aldrich Co., USA) and were dissolved in 750 ml of demineralized water. Then 0.04 g of Calcium Chloride Anhydrate (Sigma-Aldrich Co., USA) was dissolved in 10 ml of demineralized water and this solution was then added to the first solution. 20 ml of 5 M acetic acid solution was diluted with demineralized water from Concentrated Acetic Acid (99.5%) (Molar Chemicals Kft., Hungary) and also added to the first solution. The volume was completed to 1000 ml with demineralized water. Finally, the pH should be in the range of 5.5 to 6.5. If it is not, 5 N Hydrochloric acid or 5 N Sodium Hydroxide solutions should be used (“Procedure to make 1 L of artificial rumen fluid,”). The pH value of the solution was measured with a Hanna pH 210 microprocessor pH meter, (Hanna Instruments Inc., Canada) and was exactly 6.50, no pH adjustments were required.
2.7. In vitro dissolution profile studies During the dissolution studies SR 8-Plus was used as dissolution bath, for sampling: Autoplus Maximizer and Autoplus MultiFill, (Teledyne Hanson Research Inc., USA). Predetermined dissolution parameters: mode: offline, collect only to rack type 16 × 100 test tubes, USP apparatus 2 paddle mode, rotation speed: 100 rpm, bath temperature: 37.5 °C, in vessels: 37.0 °C, volumes of the dissolution media were 900 ml. Dissolution profiles were investigated in pH = 1.2, pH = 6.50 ARF and pH = 6.80 dissolution media. Samples: 200 mg of albendazole powder, unmilled and milled Vivapur® 12 dispersions containing 200 mg of albendazole. A 5 ml rinse volume was set prior to every sampling, with a 60 s prestart time. Rinsed media were traced back to the bath. Also 5 ml of sample volumes were collected through P/N FIL10S-HR 10 μm pore size full flow filters (Quality Lab Accessories L.L.C., USA) placed on the probes, at sampling times: 5, 10, 15, 30, 45, 60, 90, 120, 180, 240 min after dropping the samples to the vessels. A 5 ml of media replacement was set after every sampling. Dissolution studies were performed in triplicates and the cumulative drug release (%) mean values ± SDs were calculated from the linear calibrations in every dissolution media, determined by spectrophotometry on the absorption maximum of albendazole, wavelength λmax = 291 nm, measured by a single beam 8453 UV–Vis spectrophotometer, (Agilent Technologies, USA).
2.5. pH = 6.80 dissolution medium preparation 250 ml of 0.2 M potassium dihydrogen phosphate solution was made from 6.80 g of Potassium Dihydrogen Phosphate (Sigma-Aldrich Co., USA) and was poured into a 1000 ml volumetric flask, 77. 0 ml of 0.2 M sodium hydroxide solution was made from 0.92 g Sodium Hydroxide (Molar Chemicals Kft., Hungary) and added to the previously mentioned solution then the volume was completed to 1000 ml with R water. The pH value of the solution was measured with a Hanna pH 210 microprocessor pH meter, and was exactly 6.80, no pH adjustments were required. 2.6. Particle size distribution and zeta-potential analysis The mean particle size, width of particle size distribution (span or polydispersity index PDI), the crystalline state, particle morphology, along with zeta potential are the main characteristics of nanosuspensions. The mean particle size and the width of particle size distributions could be measured by several techniques such as Laser Diffractometry (LD), Photon Correlation Spectroscopy (PCS) or Dynamic Light Scattering (DLS). Zeta potential gives certain information about the surface charge properties and further the long-term physical stability of the nanosuspensions. In order to obtain an electrostatically stabilized nanosuspension, a minimum zeta potential of ± 30 mV is required. In the case of a combination of electrostatic and steric stabilization, a minimum zeta potential of ± 20 mV is desirable (Yadollahi et al.,
2.8. Thermodynamic solubility studies Thermodynamic solubility studies were determined using a slightly modified version of the shake-flask method in pH = 1.20, pH = 6.50 72
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ARF and pH = 6.80 dissolution media. 10 mg of albendazole powder, milled and unmilled dispersions containing 10 mg of albendazole were accurately weighted into 5 ml volumetric vials with screw-caps, both 5 ml of dissolution media were poured on top of the samples. The contents of the vials were mixed at speed 800 rpm and heated to 37 °C using an Rt-5 power heatable magnetic stirrer, (IKA Work Inc., USA) and l = 0.8 cm long, d = 0.5 cm thick magnetic stirrer bars for 24 h, after the homogenization a sedimentation and equilibration cycle was performed for another 24 h on 37 °C as well. Samples were centrifuged with MicroGen 16, (Herolab GmbH, Germany) at speed 15,000 rpm for 5 min and filtered using FilterBio® NY nylon membrane syringe filters, with pore sizes of 0.22 μm. Every sample were measured three times, and the mean thermodynamic solubility values (μg/ml) ± SDs were calculated from the linear calibrations, determined by spectrophotometry at wavelength λmax = 291 nm.
shown in this paper.
2.9. Solid state characterization investigations
Temperature of the presuspension was 20.0 °C prior to milling. After 120 min of continuous milling opperation at a speed of 400 rpm, utilizing 66.67 v/v % of d = 0.1 mm zirconia beads as milling media, the temperature of the loading has massively increased from 20 °C to 36.5 °C. In order to minimize thermal stress, a 5 min long milling period was introduced, which yielded only 23.5 °C loading temperature. Our next task was to determine and integrate an appropriate cooldown cycle, which keeps the loading temperature as low as possible, while maintaining a reasonable process time. Loading temperature after 5 min of milling operation was registered at every minute to determine the cooldown rate and length of the ideal cooldown cycle. Loading temperature values were 23.0 °C, 22.5 °C, 21.5 °C, 21.0 °C and 20.5 °C respectively. Considering the results, a cyclic milling program has been developed, involving a 5 min long milling cycle, followed by a 5 min long cooldown and sedimentation cycle, also to accelerate the cooldown process the laboratory was air conditioned to 20 °C during the whole operation. Total process time was 240 min, with effective milling time of 120 min. Results of the reconstitution studies in every media have confirmed that the particle size distributions of albendazole milled suspensions and released crystals from the dry suspensions were in the nanosized range. Particle size distribution comparisons of milled suspension and dry suspensions have shown the overlapping curves pair-wise measured in different pH buffer solutions, but there was a slight difference between the particle size distributions of the milled suspensions and the solid suspensions (Fig. 1). All samples were measured five times, and the coefficient of variations were calculated (Table 1–2.). For monodisperse samples, where the coefficient of variation ((COV) < 20%) between DLS parameters and the mean particle size of the suspensions of unaggregated nanoparticles have diameters > 20 nm, the DLS produces highly reproducible and reliable measurements. Polydisperse nanoparticle solutions or stable solutions of aggregated nanoparticles (no visible particulates and no particle settling), typically the DLS measured diameters will be in the 100–300 nm range with a polydispersity index of 0.3 or below (‘Nanocomposix's guide to dynamic light scattering measurement and analysis’). According to the referred guide, our samples can be described as stable solutions of aggregated nanoparticles. With lower than 20% COV between the intensity weighted mean hydrodynamic diameter Z AVG d (nm) and the PDI values means, that the analytical method developed and applied to measure particle sizes in these solutions was reliable, reproducible, and have met built in Zetasizer software's quality criteria. Prior to wet granulation mean Zeta potential of the milled albendazole suspension was −35.8 ± 0.707 mV, after 100 times of dilution with sterile filtered distilled water. Post redispergation value was −24.0 ± 0.581 mV. Mean value increased by 11.8 mV due to wet granulation and especially tray drying. Cumulative undersize distributions of solid final products (containing milled, unmilled albendazole) compared to the MCC Vivapur® 12 carrier are slightly altered after the solidification (Fig. 2.).
2.10.2. SEM imaging SEM images were collected from the surfaces of individual particles operating JSM 6380LA Series Scanning Electron Micrpscope (JEOL Inc., USA). Accelerating voltages varied from 5 kV to 10 kV and spot sizes from 8 to 10 depended on the electrostatic charge-up of microstractured surfaces. 100–500 times of zoomed in images have been taken for particle size determinations and 1000–3000 times for particle surface comparisons. 3. Results and discussion 3.1. Particle size distribution, zeta-potential measurements and reconstitution studies
The assessment of the crystalline state and particle morphology together helps in understanding the polymorphic/amorphous or morphological changes that a drug might undergo when subjected to nanosizing. To track the crystalline amorphous transformation X-ray diffraction analysis (XRD) and differential scanning calorimetry (DSC) are the most commonly used methods. 2.9.1. DSC Comparison of the phase transitions and thermoanalytical behaviours of solid samples were investigated by Differential Scanning Calorimetry (DSC) method with the apparatus Seiko Exstar 6000/6200, (Seiko Instruments Inc. Japan). 3 mg of solid samples were accurately weighted into small aluminium pans and the empty pans were used as blanks to calculate the enthalpy (mJ/mg) values required for the phase transitions. Temperature of the sample chamber was fluctuated between 4 °C to 250 °C, heating speed was 10 °C/min, and investigations were carried out under air atmosphere. 2.9.2. FT-IR Physicochemical properties of MCC carrier Vivapur® 12, albendazole powder, granules of unmilled solid suspension and milled solid suspension were examined and compared with the apparatus Jasco FT/ IR-4200 spectrophotometer (Jasco Products Company, USA) equipped with Jasco ATR PRO470-H single reflection accessory. The measurements were performed in absorbance mode. The spectra were collected over a wavenumber range of 4000 to 800 cm−1. After 50 scans, the measurements were evaluated with the software (Spectra Manager-II, Jasco). 2.10. Microscopical investigations In order to get an actual understanding of particle morphology microscopic techniques are preferred. In search for milled albendazole nanocrystals on the surface of the milled dispersion, the surfaces of the albendazole powder, MCC (Vivapur® 12) carrier, their physical mixture and the albendazole milled dispersion have been scanned and compared. 2.10.1. AFM imaging Double sided tape was mounted on a metal AFM specimen disc (Ted Pella Inc., Redding, CA) and a small fraction of the microparticulate systems were poured on it. Unbound particles were removed with a stream of N2 gas. AFM images were collected from the surfaces of individual particles operating a Cypher S instrument (Asylum Research, Santa Barbara, CA) in non-contact mode at 1–2 Hz line-scanning rate in air, using a silicon cantilever (OMCL AC-160TS, Olympus, Japan, typical resonance frequency: 300–320 Hz). Temperature during the measurements was 29 ± 1 °C. AFM amplitude-contrast images are 73
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Fig. 1. Comparison of the PSDs by intensities (%) of milled albendazole suspensions and milled albendazole crystals released from Vivapur® 12 dispersions in various pH buffer solutions (n = 5). Table 1 Results of DLS parameters during reconstitution (n = 5). DLS parameters Sample
Milled susp. in pH = 6.50 Milled susp. in pH = 6.80 Released albendazole in pH = 6.50 Released albendazole in pH = 6.80 Released albendazole in distilled water
ZAVGd (nm)
PDI
Mean ± SD
COV (%)
Mean ± SD
COV (%)
124.20 ± 1.402
1.13
0.185 ± 0.007
3.78
134.15 ± 1.066
0.79
0.149 ± 0.009
6.03
200.40 ± 2.318
1.16
0.161 ± 0.017
10.56
197.17 ± 0.208
0.11
0.209 ± 0.007
3.37
140.10 ± 1.260
0.90
0.237 ± 0.010
4.22
Fig. 2. Particle size distributions of granules containing milled, unmilled albendazole and MCC-carrier (Vivapur® 12) powder. Table 2 Comparison of the DLS parameters during reconstitution (n = 5).
3.3. Drug content and composition determination studies
DLS parameters Sample
Released compared to suspension in pH = 6.50 Released compared to suspension in pH = 6.80
ZAVGd (nm)
Mean drug contents of milled and unmilled albendazole solid suspensions, have been calculated from the linear calibrations of albendazole in dissolution medium at pH = 1.2. Milled dispersion contained 22.98 ± 0.6% albendazole, while the unmilled one 22.08 ± 1.1%. The mean concentrations of polysorbate 80 was found to be 3.85% for both compostions, since equal amounts of suspensions were added to both compositions, while mean MCC (Vivapur® 12) amounts were 70.04% and 70.07% respectively, regarding to milled and unmilled dispersions. Moisture contents of the milled and unmilled end products were 3.14 w/w %, and 4.00 w/w % respectively. It has already been reported, that up to ∼5.6% of moisture the MCC characteristics allow the possibility of further processing (Sun, 2008).
PDI
Mean ± SD
COV (%)
Mean ± SD
COV (%)
162.30 ± 53.882
33.20
0.173 ± 0.0170
9.81
165.66 ± 44.560
26.90
0.179 ± 0.0420
23.48
3.2. In vitro dissolution studies The cumulative dissolution profiles studies investigated in 900 ml pH = 6.50 ARF have demonstrated that, the mean dissolution rates of the dry suspensions containing milled albendazole particles incorporated in MCC carrier were significantly increased compared to the dry dispersion containing unmilled albendazole and to the albendazole pure powder (Fig. 3.). The fitted parameters of Table 3 demonstrate marked increase in the dissolution profiles which follow first-order kinetics. The acceleration of the dissolution can be due to both the milling and presence of surfactant.
3.4. Analysis of thermodynamic solubilities of albendazole powder, milled and unmilled dispersions in various pH buffer solutions Albendazole powder alone showed the lowest mean solubility in pH = 6.50 ARF (8.21 ± 0.02 μg/ml). Wet granulation with 0.5 w/w % of polysorbate 80 increased the mean thermodynamic solubility of albendazole by 4.89 times in pH = 6.50 ARF and 5.97 times in pH = 6.8, where base solubility of ABZ was (9.00 ± 0.01 μg/ml). The investigated surfactant assisted media milling process further increased 74
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Fig. 3. Comparison of in-vitro dissolution profiles in ARF at pH = 6.50 dissolution media at pH = 6.80 and at pH = 1.2 (n = 3). Table 3 Fitted first-order dissolution rate constants (k) of milled, unmilled albendazole dispersions and pure albendazole powder (900 ml of ARF at pH = 6.50, at pH = 1.20 and pH = 6.80 dissolution media). Sample
kpH=1.20 (min−1)
kpH=6.50 (min−1)
kpH=6.80 (min−1)
Milled dispersion
0.174 (R = 0.9955) 0.166 (R = 0.9939) 0.009 (R = 0.9819)
0.282 (R = 0.9843) 0.196 (R = 0.9678) 0.024 (R = 0.9821)⁎
0.259 (R = 0.9903)
Unmilled dispersion Albendazole powder ⁎ ⁎⁎
0.181 (R = 0.9616) 0.015 (R = 0.9825)⁎⁎
Dissolution started at 51.15 min. Dissolution started at 15 min.
Fig. 5. Comparison of the thermoanalytical behaviours of solid samples.
albendazole and the MCC carrier. Also a major phase transition enthalpy change can be noticed between the solid samples, with the minimal value registered at the milled dispersion (2.00 mJ/mg). This indicated a partial crystalline-amorphous transition of albendazole due to the milling process. Mechanical stress and other sources of excess energy such as heat are inherent to milling and often lead to significant changes on the physical and chemical properties of pharmaceutical crystalline solids. Partial or complete transformation to the amorphous form, polymorphic transformations, and changes in chemical reactivity are among the frequently encountered changes produced by milling (Feng and Rodolfo Pinal, 2008).
Fig. 4. Mean thermodynamic solubility values of albendazole powder substance, milled and unmilled dispersions in various pH buffer solutions (n = 3).
3.6. FT-IR spectral evaluations
the mean solubility by 1.98 times in pH = 6.50 and 1.33 times in pH = 6.80 dissolution media (Fig. 4.), while only a small solubility change can be registered in pH = 1.20, + 2.55% due to wet granulation and + 11.11% to milling.
The characteristic bands of different structural moieties of albendazole can be easily identified comparing our results to previous investigations of reference materials. A broad band in the spectral range 3415–3190 cm−1, with peak at 3325, 3319–3317 cm−1, due to NeH stretching, from amine groups, overimposed on the vibrations of NeH bond from carbamate moiety. The sharp weak bands due to stretching of alkane-type CeH bonds from the propyl moiety appear around 2956 and 2865 cm−1. The C]O carbonyl bond bending appear as sharp band at 1712–1708 cm−1. The benzoimidazolyl part show two intense closed bands in the 1640–1590 cm−1 spectral range, with peaks at 1630 and 1595 cm−1. A characteristic band for the aromatic system is the band at 1523 cm−1, but as well the doublet bands at 1441 and 1422 cm−1. Other bands appear in the fingerprint region, below 950 cm-1, but are
3.5. Comparison of the phase transitions and thermoanalytical behaviours of the solid samples During the DSC investigations we've found that the melting point of the supplied pure albendazole powder was 197.7 °C and the enthalpy, that was required for this phase transition was 43.6 mJ/mg. The melting points of the albendazole in the unmilled and milled dry suspension formulations were 194.4 °C and 187.4 °C respectively (Fig. 5.). This sifting of the melting points indicates interactions between the 75
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Table 4 Comparison of the FT-IR absorbtion bands of various albendazole forms. Band assignmet for albendazole forms
Wavenumber (cm−1) ABZ I
eNH stretching Carbonyl group eCH3 absorption eCH stretching eC]N stretching Aromatic system eCH deformation Fingerprint region
ABZ II
range 3415–3188, peaks at 3325, 3319–3317 1712–1708 1443 2953, 2926 2957, 2912 1654, 1591 1629, 1578 1523, and duplet bands at 1441, 1422 1373 1377 881, 866, 846, 767, 755, 885, 863, 850, 805, 732, 597, 588, 520, 447 759, 728, 611, 597, 509
Albendazole EP
Unmilled
Milled
3331 1711 1443 2959, 2913 1616, 1586 1524,1441,1423 1374, 1377 887, 864, 847, 770, 755, 730, 598, 513, 449
3328 1711 1444 2958, 2909 1622, 1589 1524, 1443, 1426 1361 893, 866, 847, 770, 755, 730, 596, 525, 515, 466, 451
3332 1713 1445 2904 1623, 1588 1525, 1445, 1428 1361 894, 868, 847, 769, 752, 663, 591, 522, 510, 443
Fig. 6. FT-IR spectral evaluations of MCC (Vivapur® 12) carrier, albendazole powder, and granules containing unmilled and milled albendazole.
absorbed moisture of MCC at 1640 cm−1 is masked by the aromatic system of albendazole. The asymmetric eCH2 bending and wagging signal of MCC at 1440 and 1352 cm−1, finally the eCeOeCe stretching of the β-1,4-glycosidic linkage of MCC at 1120 cm−1 can be observed (Das et al., 2010) (Fig. 6.).
difficult to be asset to certain moieties. Previous investigations also revealed the spectral differences between the tautomeric forms of albendazole. When comparing form ABZ I to II, the bands corresponding to the eCH stretching vibration, to C]N stretching and to eCH deformation appeared slightly shifted. In addition, the bands in the fingerprint region (between 1500 and 600 cm−1) show marked differences between both solid forms, which can be used to identify and distinguish them. In particular, the spectrum of ABZ II shows shifted bands as well as new bands in comparison with that of ABZ I. ABZ I is characterized by bands at 885, 863, 850, 805, 759, 728, 611, 597 and 509 cm−1, whereas ABZ II exhibits characteristic bands at 881, 866, 846, 767, 755, 732, 597, 588, 520 and 447 cm−1 (Chattah et al., 2015; Trabdafirescu et al., 2016). As shown in Table 4, our sample consisted of the mixture of both tautomeric forms of albendazole. When subjected to nanosizing, a slight sifting of the band positions could be registered, nevertheless the recorded absorbance values have significantly decreased compared to albendazole powder, which also indicated a partial crystallineamorphous transition. Spectral analysis of the solid suspensions has also revealed the characteristic bands of MCC as well, masking each other with the vibrational signals of albendazole. From 3600 to 3000 cm−1 the stretching of the H-bonded eOH groups of MCC can be registered, masking the NeH stretching of albendazole, the eCeH stretching at 2900 cm−1 is a common band for both MCC and albendazole. The
3.7. AFM imaging Albendazole micronized powder contained small crystals, that form ~3 μm sized aggregates with one another. These small individual crystals were hard to detect on the surface of the microcrystalline carrier by AFM. Close examination of the surface of the milled dispersion (solid suspension) show no signs of nanoparticles. However a significant change can be detected comparing the surface roughness of the MCC to the milled dispersion. This microcrystalline surface change can be the effect of the wet granulation process (Fig. 7). 3.8. SEM imaging Same conclusions can be drawn, when taking a closer look at the SEM images. Albendazole micronized powder contains 3–8 μm sized aggregates that can be easily detected on the surface of the MCC carrier zooming in to the surface of the physical mixture with SEM. Also SEM 76
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Fig. 7. AFM amplitude-contrast images of microparticulate systems. (A) and (E) albendazole crystals; (B) and (F) Microcrystalline cellulose carrier (Vivapur® 12) powder; (C) and (G) milled albendazole dispersion (solid suspension containing albendazole nanocrystals); (D) and (H) albendazole: MCC 1:3 physical mixture.
Fig. 8. SEM images of microparticulate systems. (A) and (E) albendazole crystals; (B) and (F) Microcrystalline cellulose carrier (Vivapur® 12) powder; (C) and (G) milled albendazole dispersion (solid suspension containing albendazole nanocrystals); (D) and (H) albendazole: MCC 1:3 physical mixture.
solutions was reliable and reproducible. PSDs by intensities are overlapping each other pair wise considering suspensions and released albendazole crystals from the milled dispersions, which led us to the conclusion that there was no difference between the size distributions of albendazole crystals in pH = 6.50 and pH = 6.80. Unfortunately, there was a slight difference between the crystal size distributions of albendazole in the milled and dry suspension forms, COV values > 20% measured in both media (Table 2). We thought the reason behind this phenomenon is that Vivapur® 12 is a coarse grade MCC with a mean particle size of 180 μm therefore excellent flowability. These large carrier particles create smaller specific surface area and with smaller specific surface area, Vivapur® 12 can bind fewer nanocrystals on its surface than a fine powder with larger specific surface area would, which lead to some albendazole nanocrystal aggregation on the carrier's surface. This theory was quickly dismissed after the close examination
surface images indicated the change of surface morphology of MCC when adding the albendazole nanosuspension, but there is no sign of albendazole nanocrystals on the surface (Fig. 8.) 4. Discussion Albendazole nanocrystal reconstitution from microcrystalline cellulose carrier Vivapur® 12 was successful according to the released nanoparticles in dissolution media of pH = 6.50 ARF, pH = 6.80 and distilled water. Low COV values can be reported, between the intensity weighted mean hydrodynamic diameter (Z AVG d) values of dispersed milled suspension samples and albendazole nanocrystals released from dry suspension samples measured in both dissolution media and distilled water. These COV values are < 20%, which means that the analytical method developed and applied to measure particle sizes in these 77
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and comparison of the particle surfaces, because we couldn't detect any albendazole nanocrystals on the surfaces of the carrier neither with AFM nor with SEM. The drying process (tray drying at 40 °C) can be the main reason behind the small differences between the particle size distributions of albendazole in dry suspension and nanosuspension form. The pH values of the buffer solutions significantly influenced the solubility of albendazole, that was found to be 1520.70 ± 1.39 μg/ml in pH = 1.2, 8.21 ± 0.02 μg/ml in pH = 6.50 ARF, and 9.00 ± 0.01 μg/ml in pH = 6.80 medium. A major saturation solubility increase was achieved, when subjected to media milling and wetgranulation processes, 144.41 ± 0.09 μg/ml in pH = 6.50 ARF, and 146.27 ± 0.28 μg/ml in pH = 6.80. While only a small solubility change can be registered in pH = 1.20, + 2.55% due to wet granulation and + 11.11% to milling. The explanation of this phenomenon is that the base solubility of ABZ (1520.70 ± 1.39 μg/ml) is already so high in this acidic buffer solution, that the reduction of particle size shows little to no effect on further solubility gains. The pH dependence of the solubility of the albendazole is the main limiting condition of the cumulative drug release and thermodynamic solubility even from the milled solid suspension in alkaline buffer solutions.
soluble drugs and its application as a potential drug delivery system. J. Nanopart. Res. 10, 845–862. García, A., Leonardi, D., Salazar, M.O., Lamas, M.C., 2014. Modified β-cyclodextrin inclusion complex to improve the physicochemical properties of albendazole. Complete in vitro evaluation and characterization. PLoS One 9, 3–10. Nanocomposix's guide to dynamic light scattering measurement and analysis. [WWW Document], n.d. URL. https://webcache.googleusercontent.com/search?q= cache:4L8CzZFH6HYJ:https://www.researchgate.net/file.PostFileLoader.html%3Fid %3D56865cf86143254dd58b4569%26assetKey%3DAS%253A313041009217536% 25401451646200799+&cd=1&hl=hu&ct=clnk&gl=hu&client=firefox-b, Accessed date: 30 August 2017. Procedure to make 1 L of artificial rumen fluid. [WWW Document], n.d. URL. https:// www.ecow.co.uk/wp-content/uploads/2014/10/Procedure-for-making-1-L-ofartificial-rumen-fluid.pdf, Accessed date: 28 August 2017. Ibrahim, H.M., Ismail, H.R., Lila, A.E.A., 2012. Delivery system of amphotericin-B using box Behnken design. Int J Pharm Pharm Sci 4, 0–7. Kalaiselvan, R., et al., 2006. Optimiztion of drug-polymer mixing ratio in albendazolepolyvinylpyrrolidone solid dispersion by moisture absorption studies. Acta Pharm. Sci. 48, 141–151. Kleinebudde, P., 1997. The crystallite-gel_model for microcrystalline cellulose in wetgranulation, extrusion, and Spheronization. Pharm. Res. 14, 804–809. Lipinski, C., 2003. American pharmaceutical review poor aqueous solubility – an industry wide problem in drug discovery. Am. Pharm. Rev. 1–16. Liu, P., Rong, X., Laru, J., Van Veen, B., Kiesvaara, J., Hirvonen, J., Laaksonen, T., Peltonen, L., 2011. Nanosuspensions of poorly soluble drugs: preparation and development by wet milling. Int. J. Pharm. 411, 215–222. Mckellar, A., Scott, E.W., 1990. The benzimidazole anthelmintic agents - a review. J. Vet. Pharmacol. Ther. 13, 223–247. Meena, A.K., Sharma, K., Kandaswamy, M., Rajagopal, S., Mullangi, R., 2012. Formulation development of an albendazole self-emulsifying drug delivery system (SEDDS) with enhanced systemic exposure. Acta Pharma. 62, 563–580. Mitragotri, S., Burke, P.A., Langer, R., 2014. Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat. Rev. Drug Discov. 13, 655–672. Morrow, K.J., Bawa, R., Wei, C., 2007. Recent advances in basic and clinical nanomedicine. Med. Clin. North Am. 91, 805–843. Mottier, M.L., Alvarez, L.I., Pis, M.A., Lanusse, C.E., 2003. Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni : correlation with their octanol – water partition coefficients. Exp. Parasitol. 103, 1–7. Nekkanti, V., Marwah, A., Pillai, R., 2015. Media milling process optimization for manufacture of drug nanoparticles using design of experiments (DOE). Drug Dev. Ind. Pharm. 41, 124–130. Paredes, A.J., Llabot, J.M., Sánchez Bruni, S., Allemandi, D., Palma, S.D., 2016. Selfdispersible nanocrystals of albendazole produced by high pressure homogenization and spray-drying. Drug Dev. Ind. Pharm. 42, 1564–1570. Prabhakar, C., Bala Krishna, K., 2011. A review on nanosuspensions in drug delivery. Int. J. Pharma Bio Sci. 2, 549–558. Rabinow, B.E., 2004. Nanosuspensions in drug delivery. Nat. Rev. Drug Discov. 3, 785–796. Raimar Lödenberg, G.L.A., 2000. Modern bioavailability, bioequivalence and biopharmaceutics classification system. New scientific approaches to international regulatory standards. Eur. J. Pharm. Biopharm. 50, 3–12. Sun, C.C., 2008. Mechanism of moisture induced variations in true density and compaction properties of microcrystalline cellulose. Int. J. Pharm. 346, 93–101. Torrado, S., Torrado, S., Cadorniga, R., Torrado, J.J., 1996. Formulation parameters of albendazole solution. Int. J. Pharm. 140, 45–50. Trabdafirescu, C., Borcan, F., Dehelean, C., Szabadai, Z., Nicolov, M., Soica, C., 2016. Study of the interaction of albendazole with study of the interaction of albendazole with benzoic acid. Rev. Chim. 67, 2370–2374. Van Eerdenbrugh, B., Froyen, L., Van Humbeeck, J., Martens, J.A., Augustijns, P., Van Den Mooter, G., 2008. Alternative matrix formers for nanosuspension solidification: dissolution performance and X-ray microanalysis as an evaluation tool for powder dispersion. Eur. J. Pharm. Sci. 35, 344–353. Vidal, N.L.G., Castro, S.G., Sanchez Bruni, S.F., Allemandi, D.A., Palma, S.D., 2014. Albendazole solid dispersions: influence of dissolution medium composition on in vitro drug release. Dissolut. Technol. 21, 42–47. Wen, H., New, R.R.C., Muhmut, M., Wang, J.H., Wang, Y.H., Zhang, J.H., 1996. Pharmacology and efficacy of liposome-entrapped albendazole in experimental secondary alveolar echinococcosis and effect of co-administratration with cimetidine. Parasitology 113, 111–121. Yadollahi, R., Vasilev, K., Simovic, S., 2015. Nanosuspension technologies for delivery of poorly soluble drugs. J. Nanomater. 2015, 1–13.
5. Conclusions Surfactant assisted media milling and preparation of dry nanosuspension formulation as carrier was an effective method to improve dissolution rate and water solubility of poorly water soluble albendazole. The dissolution was improved due to both particle size reduction and presence of surfactant. Solid state characterization studies showed partial crystalline–amorphous transition of albendazole during the nanonization process. Microscopical surface characterization with AFM and SEM imaging demonstrated the incorporation of albendazole into the microcrystalline cellulose carrier which ensured also the reconstitution of nanocrystals. Declarations of interest None. References Campbell, W.C., 1990. Benzimidazoles: veterinary uses. Parasitol. Today 6, 130–133. Chattah, A.K., Zhang, R., Mroue, K.H., Pfund, L.Y., Longhi, M.R., Ramamoorthy, A., Garnero, C., 2015. Investigating albendazole desmotropes by solid-state NMR spectroscopy. Mol. Pharm. 12, 731–741. Chingunpituk, J., 2007. Nanosuspension technology for drug delivery. Walailak J. Sci. Technol. 4, 139–153. Dahan, A., Miller, J.M., Amidon, G.L., 2009. Prediction of solubility and permeability class membership: provisional BCS classification of the world's top oral drugs. AAPS J. 11, 740–746. Das, K., Ray, D., Bandyopadhyay, N.R., Sengupta, S., 2010. Study of the properties of microcrystalline cellulose particles from different renewable resources by XRD, FTIR, nanoindentation, TGA and SEM. J. Polym. Environ. 18, 355–363. Dayan, A.D., 2003. Albendazole, mebendazole and praziquantel. Review of non-clinical toxicity and pharmacokinetics. Acta Trop. 86, 141–159. Feng, Tao, Rodolfo Pinal, M.T.C., 2008. Process induced disorder in crystalline materials: differentiating defective crystals from the amorphous form of griseofulvin. J. Pharm. Sci. 97, 3207–3221. Galia, E., Horton, J., Dressman, J.B., 1999. Albendazole generics - a comparative in vitro study. Pharm. Res. 16, 1871–1875. Gao, L., Zhang, D., Chen, M., 2008. Drug nanocrystals for the formulation of poorly
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Study on the dissolution improvement of albendazole using reconstitutable dry nanosuspension formulation
T
Viktor Fülöpa, Géza Jakaba, Tamás Bozób, Bence Tótha, Dániel Endrésika, Emese Balogha, ⁎ Miklós Kellermayerb, István Antala, a b
Semmelweis University, Department of Pharmaceutics, Hőgyes Endre Street 7, Budapest H-1092, Hungary Semmelweis University, Department of Biophysics and Radiation Biology, Tűzoltó Street 37-47, Budapest H-1094, Hungary
A R T I C LE I N FO
A B S T R A C T
Keywords: Wet media milling Nanosuspension solidification Microcrystalline cellulose carrier Particle size distribution Solubility Dissolution rate Artificial rumen fluid
The aim of the study was to improve the solubility and dissolution rate of the poorly water soluble drug albendazole via surfactant assisted media milling process. Preparation of a nanosuspension and then post-processing with a solidification technique applied to improve the applicability of nanosuspension in a solid dosage forms carrier. The dry nanosuspension was obtained using microcrystalline cellulose as solid carrier after tray drying at 40 °C. Both reconstitution from the solid carrier and dissolution profile studies were investigated in biorelevant Artificial Rumen Fluid (ARF) at pH = 6.50 and dissolution media at pH = 1.20 and pH = 6.80. Reconstitution studies have demonstrated that the mean hydrodynamic diameter values of albendazole crystals released from the dry suspension were nanosized (intensity weighted hydrodynamic diameter values: 200.40 ± 2.318 nm in ARF at pH = 6.50, 197.17 ± 0.208 nm in dissolution medium at pH = 6.80). Thermodynamic solubility studies have indicated a 2.98 times increase in water solubility (144.41 ± 0.09 μg/ ml milled, 48.38 ± 0.01 μg/ml unmilled, 8.21 ± 0.02 μg/ml albendazole powder) in ARF at pH = 6.50, and 2.33 times in dissolution medium at pH = 6.8: (146.27 ± 0.28 μg/ml milled, 62.71 ± 0.04 μg/ml unmilled, 9.00 ± 0.01 μg/ml albendazole powder), and 13.65% increase at pH = 1.20 (1728.31 ± 3.31 μg/ml milled, 1559.41 ± 0.40 μg/ml unmilled, 1520.70 ± 1.39 μg/ml albendazole powder), dissolution rates have also increased. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) imaging investigations detected no albendazole nanocrystals on the surface of the carrier, which demonstrated the incorporation of albendazole into the microcrystalline cellulose solid carrier structure.
1. Introduction One of the major obstacles to the development of highly potent new drug candidates is the poor water solubility of these compounds. Approximately 30–40% of potential new chemical entities (NCE) identified by pharmaceutical companies (Pfizer, Merck) with high throughput screening (HTS) /combinatorial chemistry operations are poorly soluble in, which hinders their therapeutic application (Lipinski, 2003). The National Nanotechnology Initiative (NNI) defines Nanotechnology, “as research and development at the atomic, molecular, or macromolecular levels in the sub-100-nm range (w0.1–100 nm) to create structures, devices, and systems that have novel functional properties”(Morrow et al., 2007). According to European Medicines Agency (EMA), nanotechnology is defined as the production and application of structures, devices and systems by
⁎
controlling the shape and size of materials at nanometre scale. The nanometre scale ranges from the atomic level at around 0.2 nm (2 Å) up to around 100 nm. A pharmaceutical nanosuspension is defined as very finely colloid, biphasic, dispersed, and solid drug particles in an aqueous vehicle, size below 1 μm, without any matrix material, stabilized by surfactants and polymers, and prepared by suitable methods for drug delivery applications, through various routes of administration like oral, topical, parenteral, ocular and pulmonary routes (Prabhakar and Bala Krishna, 2011). The particle-size distribution of the solid particles in nanosuspensions is usually less than 1 μm with an average particle size ranging between 200 and 600 nm (Chingunpituk, 2007). The potential benefits of the Nanosuspension Technology for poorly soluble drug delivery are increased drug dissolution rate, increased rate and extent of absorption, hence the bioavailability of drug (area under plasma versus time curve, onset time, peak drug level), reduced variability, and reduced fed/fasted effects, increased penetration
Corresponding author at: Department of Pharmaceutics, Semmelweis University, Hőgyes E. Street 7, Budapest H-1092, Hungary. E-mail address: [email protected] (I. Antal).
https://doi.org/10.1016/j.ejps.2018.07.027 Received 20 March 2018; Received in revised form 5 July 2018; Accepted 11 July 2018 0928-0987/ © 2018 Elsevier B.V. All rights reserved.
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2. Materials and methods
capability of topical nanosuspension. Nanosuspension has low incidence of side effects by the excipients, increased resistance to hydrolysis and oxidation and increased physical stability to settling. Reduced administration volumes, essential for intramuscular, subcutaneous, and ophthalmic use. Finally they can provide the passive targeting (Ibrahim et al., 2012; Liu et al., 2011; Rabinow, 2004). Several production techniques are available to produce drug nanocrystals, such as precipitation, pearl milling (media milling), and highpressure homogenization (Gao et al., 2008). Solidification techniques transform nanosuspensions into solid dosage forms such as tablets, capsules, and pellets. Solid dosage forms increase the long term stability of nanosuspensions and improve patient compliance (Van Eerdenbrugh et al., 2008). Albendazole is an imidazole carbamate-ester, a broad-spectrum anthelmintic for the treatment of intestinal helminth infections. It also has anti-hydatid activity and is now recognized to have important application in treatment of human cystic and alveolar echinococcosis. The main targeted animals are cattle (dosage: 7.5–10 mg/kg) and sheep (dosage: 5 mg/kg) in animal healthcare (Campbell, 1990; Mckellar and Scott, 1990). This compound has a pH dependent, poor water solubility range from: 0.376 mg/ml in pH = 1.2 to 0.016 mg/ml in pH = 6.0 buffers, which is the lowest concentration achieved during the solubility studies of (Torrado et al., 1996). Albendazole has an octanol-water partition coefficient (Log P) value of 3.83 (Mottier et al., 2003), which is considered high, (Log P > 1.72 for reference compound metoprolol) according to BCS drug permeability classifications (Dahan et al., 2009). With low water solubility and high membrane permeability it is classified as a BCS Class II drug (Raimar Lödenberg, 2000). To overcome the drawbacks of the poor water solubility of ABZ, different formulation approaches have been adopted in the past such as liposomal entrapment (Wen et al., 1996), solid dispersions with polyvinylpyrrolidone (Kalaiselvan et al., 2006) and with Poloxamer 188 and PEG 6000 (Vidal et al., 2014). Inclusion complexes of ABZ with different CDs have been prepared, the significance of synthetized citrate derivate of β-CD should be highlighted, due to its high stability constant (García et al., 2014). Lipid based delivery systems of ABZ seem to be effective formulations, including self-microemulsifying drug delivery systems (SMEDDS) of ABZ to reach an marked water solubility improvement (Meena et al., 2012). Several particle size reduction methods have also been introduced but without solidification so far (Paredes et al., 2016). The low bioavailability of the API reduces the efficacy in hydatid. Pragmatic approaches for chemo-therapy of hydatid patients necessitate to focus on improved transport, targeting modulation of the physicochemical parameters and metabolic decomposition of benzimidazoles (Wen et al., 1996). Effective dose reduction, hence lower incidence of side effects, the ease of scale-up, low bath-to-batch variability can be mentioned as the main advantages of the utilizations of nanocrystals, while the most common disadvantages are high energy investment during manufacturing, immunotoxicity and non-specific uptake in reticuloendothelial system (RES) organs (Mitragotri et al., 2014). The aim of the work was to use surfactant assisted media milling to produce nanocrystals and to transform the milled albendazole nanosuspensions to solid form by wet granulation applying microcrystalline cellulose as carrier. Furthermore, the particle size distribution and Zetapotential of albendazole were studied after redispersion as well as the drug release and thermodynamic solubilities in various dissolution media. Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FT-IR) investigations have been performed to compare the final product to the active substance and to the carrier. Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) were utilized to compare the surfaces of the pure albendazole to the MCC carrier (Vivapur® 12), to their physical mixture and to the albendazole milled dispersion.
Albendazole EP (micronized), (Sequent Scientific Ltd., India) was used as model drug. Particle size distribution measured by the supplier with sieve analysis demonstrated, that > 90% of the powder is under 30 μm. Laser diffraction measurements have been performed to confirm this result dispersing 3.00 w/w% albendazole into 0.50 w/w% Polysorbate 80 solution. Results have been interpreted by the mean of five individual measurements ± standard deviation values. Polysorbate 80 (Tween 80) (Molar Chemicals Kft., Hungary) as surface active agent for media milling, Dimethylpolysiloxane (Foamsol) (Kokoferm Kft., Hungary) as antifoaming agent, Coarse grade Microcrystalline cellulose with mean particle size of 180 μm, and bulk density of 0.33 g/ml (Vivapur® 12), (JRS Pharma GmbH & Co. KG, Germany) as carrier of the solid suspension. 2.1. Surfactant assisted media milling process The selection of the milling process parameters were based on preliminary studies involving relatively moderate agitation with high beads to powder loading ratio to achieve satisfactory particle size distributions. Retsch PM 100 planetary ball mill, with a stainless steel container of 50 ml in volume, were used for this process. Container loadings consisted of 0.5083 g, of pure albendazole powder (ρ albendazole = 1.3 g/ml, V albendazole = 0.391 ml) with 16.08 ml of 0.50 w/w % of polysorbate 80 surfactant solution, and 33.33 ml of d = 0.1 mm zirconia beads as the milling media. Beads to powder loading mass ratio was: 249.36. As for the milling parameters: milling speed was 400 rpm, and milling time was 120 min. Temperature control has been integrated as a part of the quality by design development of particle size reduction techniques (Nekkanti et al., 2015). Various milling programs have been compared in order to maintain the loading temperature as low as possible during the process. A d = 63 μm mesh size, stainless steel sieve, was utilized for separating the beads from the nanosuspension. All the equipment (planetary ball mill, container, d = 0.1 mm zirconia beads, and sieves) were provided by (Retsch Technology GmbH., Germany). 2.2. Solidification using wet-granulation technique The dried MCC carrier was weighted using a Kern ABT 320–4 M analytical balance, (ABT - KERN & SOHN GmbH, Germany) into a steel mortar and after every milling process the obtained nanosuspension was added to the carrier. Than mixed manually by kneading for 3 min, sieved through a 180 μm mesh size sieve and dried in a Labor-Innova drying chamber, (Labor-Innova Műszeripari Kft., Hungary) on 40 °C for 2 days. Solid suspension of unmilled albendazole was prepared the same way, but for the dispersing the albendazole an IKA RCT basic, heatable magnetic stirrer, (IKA® Works, Inc., USA) was employed. 2.3. Drug content and composition determination of milled and unmilled solid suspensions Since albendazole has pH dependent solubility, with a maximum of 0.376 mg/ml at pH = 1.2, it was evident to determine the amount of active content in highly diluted stock solutions at pH = 1.2. Three stock solutions have been prepared by weighing in 1 g of dispersions and diluted with 0.1 N hydrochloric acid to 2000 ml, stirred at 1000 rpm with IKA RCT basic, heatable magnetic stirrer, (IKA® Works, Inc., USA) for 24 h, sterile filtered using FilterBio® NY nylon membrane syringe filters, (LAB-EX Laborkereskedelmi Kft., Hungary) with pore sizes of 0.22 μm, and measured by UV-VIS spectrophotometry method. Mean Polysorbate 80 content of the dispersions were calculated from the added weight of suspensions after every milling and dispersing cycles, mean MCC (Vivapur® 12) concentration was calculated from the amount of dried carrier applied before wet granulation process. The 71
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2015). Particle size distributions of milled suspensions and released crystals from dry suspensions have been determined by Photon Correlation Spectroscopy (PCS) or Dynamic Light Scattering (DLS) method. Before dispersing the milled suspensions and dry suspensions in 100 ml of pH = 6.50 ARF, pH = 6.80 dissolution media and distilled water, the solutions were prefiltered using FilterBio® NY nylon membrane syringe filters, (LAB-EX Laborkereskedelmi Kft., Hungary) with pore sizes of 0.22 μm. Filtered solutions were mixed at 250 rpm speeds, and l = 3 cm long, d = 0.8 cm thick magnetic stirrer bars, conditioned at 37 °C with IKA RCT basic, heatable magnetic stirrer, (IKA® Works, Inc., USA). One dose of dispersion, containing 200 mg of albendazole and also one dose of milled suspension were then added to the filtered dissolution medium and mixed using the same 250 rpm speed. 5 ml of samples were taken with a syringe and filtered with Whatman® UNIFLO® PTFE membrane syringe filters with pore sizes of 0.45 μm, (Sigma-Aldrich Co., USA). The withdrawn samples were replaced with sterile filtered dissolution medium. 1 ml of the withdrawn samples were poured into a 6 G glass sizing cuvette with square aperture. Particle size distributions were determined with the instrument: Zetasizer nano ZS, (Malvern Instruments Ltd., UK). Measurement settings: Automatic mode, NIBS (Non-Invasive-Back-Scattering) laser angle 173°, 28–32 sub runs/measurement, run durations: 10 s. Sample chamber was heated to 37.0 °C, and equilibrating time was 180 s. Automatic laser positioning selected a 4.65 mm position from the bottom of the cuvette and for attenuation, attenuator 9 was selected automatically. Five individual measurements were performed, for every sample and the mean ± standard deviation values were reported for all the DLS parameters, including: intensity weighted mean hydrodynamic diameters (ZAVG d) and polydispersity indices (PDI) in this article. Particle size distribution comparison of granules and MCC Vivapur® 12 powder was also performed by sieve analysis method utilizing a Retsch sieve series (315 μm, 180 μm, 125 μm, 50 μm, 32 μm mesh size sieves) and a Retsch AS 200 control vibrational sieve shaker (Retsch Technology GmbH., Germany). Fractionation adjusted parameters, including amplitude 1.5 mm, and time 5 min were applied.
moisture contents of the end products were measured by a Scaltec Smo 01 evaporator, (Scaltec Instruments GmbH, Germany), with predetermined temperature of 95 °C and from 1.000 g of milled and unmilled solid suspension. It has already been reported, that water is retained in a porous material during wet granulation of MCC as a result of absorption and capillary effects. The material was characterized by irreducible saturation, which refers to liquid, that remains in porous bed, regardless of any further increase in pressure applied. For MCC and water, irreducible saturation was 90 w/w %, this indicates high extent of solid liquid interaction (Kleinebudde, 1997). According to the referred article, we have applied 85 w/w % of milled and unmilled suspension to the dried carrier, during wet granulation, not reaching the saturation value, but maximizing the drug content of the dispersion during a milling or dispersing cycle. Dose determinations were based on the drug content evaluations, by studying the therapeutic efficacy, toxicity pharmacokinetics of albendazole a 200 mg of dose was preferred (Dayan, 2003; Galia et al., 1999). 2.4. pH = 6.50 artificial rumen fluid (ARF) preparation Utilizing the same Kern ABT 320-4 M analytical balance, the following constituents were weighted: 9.80 g of Sodium Hydrogen Carbonate (Molar Chemicals Kft., Hungary), 9.30 g of Disodium Hydrogen Phosphate Dodecahydrate (Molar Chemicals Kft., Hungary), 0.47 g of Sodium Chloride (Molar Chemicals Kft. Hungary), 0.57 g of Potassium Chloride (Molar Chemicals Kft., Hungary), 0.06 g of Magnesium Chloride Anhydrate, (Sigma-Aldrich Co., USA) and were dissolved in 750 ml of demineralized water. Then 0.04 g of Calcium Chloride Anhydrate (Sigma-Aldrich Co., USA) was dissolved in 10 ml of demineralized water and this solution was then added to the first solution. 20 ml of 5 M acetic acid solution was diluted with demineralized water from Concentrated Acetic Acid (99.5%) (Molar Chemicals Kft., Hungary) and also added to the first solution. The volume was completed to 1000 ml with demineralized water. Finally, the pH should be in the range of 5.5 to 6.5. If it is not, 5 N Hydrochloric acid or 5 N Sodium Hydroxide solutions should be used (“Procedure to make 1 L of artificial rumen fluid,”). The pH value of the solution was measured with a Hanna pH 210 microprocessor pH meter, (Hanna Instruments Inc., Canada) and was exactly 6.50, no pH adjustments were required.
2.7. In vitro dissolution profile studies During the dissolution studies SR 8-Plus was used as dissolution bath, for sampling: Autoplus Maximizer and Autoplus MultiFill, (Teledyne Hanson Research Inc., USA). Predetermined dissolution parameters: mode: offline, collect only to rack type 16 × 100 test tubes, USP apparatus 2 paddle mode, rotation speed: 100 rpm, bath temperature: 37.5 °C, in vessels: 37.0 °C, volumes of the dissolution media were 900 ml. Dissolution profiles were investigated in pH = 1.2, pH = 6.50 ARF and pH = 6.80 dissolution media. Samples: 200 mg of albendazole powder, unmilled and milled Vivapur® 12 dispersions containing 200 mg of albendazole. A 5 ml rinse volume was set prior to every sampling, with a 60 s prestart time. Rinsed media were traced back to the bath. Also 5 ml of sample volumes were collected through P/N FIL10S-HR 10 μm pore size full flow filters (Quality Lab Accessories L.L.C., USA) placed on the probes, at sampling times: 5, 10, 15, 30, 45, 60, 90, 120, 180, 240 min after dropping the samples to the vessels. A 5 ml of media replacement was set after every sampling. Dissolution studies were performed in triplicates and the cumulative drug release (%) mean values ± SDs were calculated from the linear calibrations in every dissolution media, determined by spectrophotometry on the absorption maximum of albendazole, wavelength λmax = 291 nm, measured by a single beam 8453 UV–Vis spectrophotometer, (Agilent Technologies, USA).
2.5. pH = 6.80 dissolution medium preparation 250 ml of 0.2 M potassium dihydrogen phosphate solution was made from 6.80 g of Potassium Dihydrogen Phosphate (Sigma-Aldrich Co., USA) and was poured into a 1000 ml volumetric flask, 77. 0 ml of 0.2 M sodium hydroxide solution was made from 0.92 g Sodium Hydroxide (Molar Chemicals Kft., Hungary) and added to the previously mentioned solution then the volume was completed to 1000 ml with R water. The pH value of the solution was measured with a Hanna pH 210 microprocessor pH meter, and was exactly 6.80, no pH adjustments were required. 2.6. Particle size distribution and zeta-potential analysis The mean particle size, width of particle size distribution (span or polydispersity index PDI), the crystalline state, particle morphology, along with zeta potential are the main characteristics of nanosuspensions. The mean particle size and the width of particle size distributions could be measured by several techniques such as Laser Diffractometry (LD), Photon Correlation Spectroscopy (PCS) or Dynamic Light Scattering (DLS). Zeta potential gives certain information about the surface charge properties and further the long-term physical stability of the nanosuspensions. In order to obtain an electrostatically stabilized nanosuspension, a minimum zeta potential of ± 30 mV is required. In the case of a combination of electrostatic and steric stabilization, a minimum zeta potential of ± 20 mV is desirable (Yadollahi et al.,
2.8. Thermodynamic solubility studies Thermodynamic solubility studies were determined using a slightly modified version of the shake-flask method in pH = 1.20, pH = 6.50 72
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ARF and pH = 6.80 dissolution media. 10 mg of albendazole powder, milled and unmilled dispersions containing 10 mg of albendazole were accurately weighted into 5 ml volumetric vials with screw-caps, both 5 ml of dissolution media were poured on top of the samples. The contents of the vials were mixed at speed 800 rpm and heated to 37 °C using an Rt-5 power heatable magnetic stirrer, (IKA Work Inc., USA) and l = 0.8 cm long, d = 0.5 cm thick magnetic stirrer bars for 24 h, after the homogenization a sedimentation and equilibration cycle was performed for another 24 h on 37 °C as well. Samples were centrifuged with MicroGen 16, (Herolab GmbH, Germany) at speed 15,000 rpm for 5 min and filtered using FilterBio® NY nylon membrane syringe filters, with pore sizes of 0.22 μm. Every sample were measured three times, and the mean thermodynamic solubility values (μg/ml) ± SDs were calculated from the linear calibrations, determined by spectrophotometry at wavelength λmax = 291 nm.
shown in this paper.
2.9. Solid state characterization investigations
Temperature of the presuspension was 20.0 °C prior to milling. After 120 min of continuous milling opperation at a speed of 400 rpm, utilizing 66.67 v/v % of d = 0.1 mm zirconia beads as milling media, the temperature of the loading has massively increased from 20 °C to 36.5 °C. In order to minimize thermal stress, a 5 min long milling period was introduced, which yielded only 23.5 °C loading temperature. Our next task was to determine and integrate an appropriate cooldown cycle, which keeps the loading temperature as low as possible, while maintaining a reasonable process time. Loading temperature after 5 min of milling operation was registered at every minute to determine the cooldown rate and length of the ideal cooldown cycle. Loading temperature values were 23.0 °C, 22.5 °C, 21.5 °C, 21.0 °C and 20.5 °C respectively. Considering the results, a cyclic milling program has been developed, involving a 5 min long milling cycle, followed by a 5 min long cooldown and sedimentation cycle, also to accelerate the cooldown process the laboratory was air conditioned to 20 °C during the whole operation. Total process time was 240 min, with effective milling time of 120 min. Results of the reconstitution studies in every media have confirmed that the particle size distributions of albendazole milled suspensions and released crystals from the dry suspensions were in the nanosized range. Particle size distribution comparisons of milled suspension and dry suspensions have shown the overlapping curves pair-wise measured in different pH buffer solutions, but there was a slight difference between the particle size distributions of the milled suspensions and the solid suspensions (Fig. 1). All samples were measured five times, and the coefficient of variations were calculated (Table 1–2.). For monodisperse samples, where the coefficient of variation ((COV) < 20%) between DLS parameters and the mean particle size of the suspensions of unaggregated nanoparticles have diameters > 20 nm, the DLS produces highly reproducible and reliable measurements. Polydisperse nanoparticle solutions or stable solutions of aggregated nanoparticles (no visible particulates and no particle settling), typically the DLS measured diameters will be in the 100–300 nm range with a polydispersity index of 0.3 or below (‘Nanocomposix's guide to dynamic light scattering measurement and analysis’). According to the referred guide, our samples can be described as stable solutions of aggregated nanoparticles. With lower than 20% COV between the intensity weighted mean hydrodynamic diameter Z AVG d (nm) and the PDI values means, that the analytical method developed and applied to measure particle sizes in these solutions was reliable, reproducible, and have met built in Zetasizer software's quality criteria. Prior to wet granulation mean Zeta potential of the milled albendazole suspension was −35.8 ± 0.707 mV, after 100 times of dilution with sterile filtered distilled water. Post redispergation value was −24.0 ± 0.581 mV. Mean value increased by 11.8 mV due to wet granulation and especially tray drying. Cumulative undersize distributions of solid final products (containing milled, unmilled albendazole) compared to the MCC Vivapur® 12 carrier are slightly altered after the solidification (Fig. 2.).
2.10.2. SEM imaging SEM images were collected from the surfaces of individual particles operating JSM 6380LA Series Scanning Electron Micrpscope (JEOL Inc., USA). Accelerating voltages varied from 5 kV to 10 kV and spot sizes from 8 to 10 depended on the electrostatic charge-up of microstractured surfaces. 100–500 times of zoomed in images have been taken for particle size determinations and 1000–3000 times for particle surface comparisons. 3. Results and discussion 3.1. Particle size distribution, zeta-potential measurements and reconstitution studies
The assessment of the crystalline state and particle morphology together helps in understanding the polymorphic/amorphous or morphological changes that a drug might undergo when subjected to nanosizing. To track the crystalline amorphous transformation X-ray diffraction analysis (XRD) and differential scanning calorimetry (DSC) are the most commonly used methods. 2.9.1. DSC Comparison of the phase transitions and thermoanalytical behaviours of solid samples were investigated by Differential Scanning Calorimetry (DSC) method with the apparatus Seiko Exstar 6000/6200, (Seiko Instruments Inc. Japan). 3 mg of solid samples were accurately weighted into small aluminium pans and the empty pans were used as blanks to calculate the enthalpy (mJ/mg) values required for the phase transitions. Temperature of the sample chamber was fluctuated between 4 °C to 250 °C, heating speed was 10 °C/min, and investigations were carried out under air atmosphere. 2.9.2. FT-IR Physicochemical properties of MCC carrier Vivapur® 12, albendazole powder, granules of unmilled solid suspension and milled solid suspension were examined and compared with the apparatus Jasco FT/ IR-4200 spectrophotometer (Jasco Products Company, USA) equipped with Jasco ATR PRO470-H single reflection accessory. The measurements were performed in absorbance mode. The spectra were collected over a wavenumber range of 4000 to 800 cm−1. After 50 scans, the measurements were evaluated with the software (Spectra Manager-II, Jasco). 2.10. Microscopical investigations In order to get an actual understanding of particle morphology microscopic techniques are preferred. In search for milled albendazole nanocrystals on the surface of the milled dispersion, the surfaces of the albendazole powder, MCC (Vivapur® 12) carrier, their physical mixture and the albendazole milled dispersion have been scanned and compared. 2.10.1. AFM imaging Double sided tape was mounted on a metal AFM specimen disc (Ted Pella Inc., Redding, CA) and a small fraction of the microparticulate systems were poured on it. Unbound particles were removed with a stream of N2 gas. AFM images were collected from the surfaces of individual particles operating a Cypher S instrument (Asylum Research, Santa Barbara, CA) in non-contact mode at 1–2 Hz line-scanning rate in air, using a silicon cantilever (OMCL AC-160TS, Olympus, Japan, typical resonance frequency: 300–320 Hz). Temperature during the measurements was 29 ± 1 °C. AFM amplitude-contrast images are 73
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Fig. 1. Comparison of the PSDs by intensities (%) of milled albendazole suspensions and milled albendazole crystals released from Vivapur® 12 dispersions in various pH buffer solutions (n = 5). Table 1 Results of DLS parameters during reconstitution (n = 5). DLS parameters Sample
Milled susp. in pH = 6.50 Milled susp. in pH = 6.80 Released albendazole in pH = 6.50 Released albendazole in pH = 6.80 Released albendazole in distilled water
ZAVGd (nm)
PDI
Mean ± SD
COV (%)
Mean ± SD
COV (%)
124.20 ± 1.402
1.13
0.185 ± 0.007
3.78
134.15 ± 1.066
0.79
0.149 ± 0.009
6.03
200.40 ± 2.318
1.16
0.161 ± 0.017
10.56
197.17 ± 0.208
0.11
0.209 ± 0.007
3.37
140.10 ± 1.260
0.90
0.237 ± 0.010
4.22
Fig. 2. Particle size distributions of granules containing milled, unmilled albendazole and MCC-carrier (Vivapur® 12) powder. Table 2 Comparison of the DLS parameters during reconstitution (n = 5).
3.3. Drug content and composition determination studies
DLS parameters Sample
Released compared to suspension in pH = 6.50 Released compared to suspension in pH = 6.80
ZAVGd (nm)
Mean drug contents of milled and unmilled albendazole solid suspensions, have been calculated from the linear calibrations of albendazole in dissolution medium at pH = 1.2. Milled dispersion contained 22.98 ± 0.6% albendazole, while the unmilled one 22.08 ± 1.1%. The mean concentrations of polysorbate 80 was found to be 3.85% for both compostions, since equal amounts of suspensions were added to both compositions, while mean MCC (Vivapur® 12) amounts were 70.04% and 70.07% respectively, regarding to milled and unmilled dispersions. Moisture contents of the milled and unmilled end products were 3.14 w/w %, and 4.00 w/w % respectively. It has already been reported, that up to ∼5.6% of moisture the MCC characteristics allow the possibility of further processing (Sun, 2008).
PDI
Mean ± SD
COV (%)
Mean ± SD
COV (%)
162.30 ± 53.882
33.20
0.173 ± 0.0170
9.81
165.66 ± 44.560
26.90
0.179 ± 0.0420
23.48
3.2. In vitro dissolution studies The cumulative dissolution profiles studies investigated in 900 ml pH = 6.50 ARF have demonstrated that, the mean dissolution rates of the dry suspensions containing milled albendazole particles incorporated in MCC carrier were significantly increased compared to the dry dispersion containing unmilled albendazole and to the albendazole pure powder (Fig. 3.). The fitted parameters of Table 3 demonstrate marked increase in the dissolution profiles which follow first-order kinetics. The acceleration of the dissolution can be due to both the milling and presence of surfactant.
3.4. Analysis of thermodynamic solubilities of albendazole powder, milled and unmilled dispersions in various pH buffer solutions Albendazole powder alone showed the lowest mean solubility in pH = 6.50 ARF (8.21 ± 0.02 μg/ml). Wet granulation with 0.5 w/w % of polysorbate 80 increased the mean thermodynamic solubility of albendazole by 4.89 times in pH = 6.50 ARF and 5.97 times in pH = 6.8, where base solubility of ABZ was (9.00 ± 0.01 μg/ml). The investigated surfactant assisted media milling process further increased 74
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Fig. 3. Comparison of in-vitro dissolution profiles in ARF at pH = 6.50 dissolution media at pH = 6.80 and at pH = 1.2 (n = 3). Table 3 Fitted first-order dissolution rate constants (k) of milled, unmilled albendazole dispersions and pure albendazole powder (900 ml of ARF at pH = 6.50, at pH = 1.20 and pH = 6.80 dissolution media). Sample
kpH=1.20 (min−1)
kpH=6.50 (min−1)
kpH=6.80 (min−1)
Milled dispersion
0.174 (R = 0.9955) 0.166 (R = 0.9939) 0.009 (R = 0.9819)
0.282 (R = 0.9843) 0.196 (R = 0.9678) 0.024 (R = 0.9821)⁎
0.259 (R = 0.9903)
Unmilled dispersion Albendazole powder ⁎ ⁎⁎
0.181 (R = 0.9616) 0.015 (R = 0.9825)⁎⁎
Dissolution started at 51.15 min. Dissolution started at 15 min.
Fig. 5. Comparison of the thermoanalytical behaviours of solid samples.
albendazole and the MCC carrier. Also a major phase transition enthalpy change can be noticed between the solid samples, with the minimal value registered at the milled dispersion (2.00 mJ/mg). This indicated a partial crystalline-amorphous transition of albendazole due to the milling process. Mechanical stress and other sources of excess energy such as heat are inherent to milling and often lead to significant changes on the physical and chemical properties of pharmaceutical crystalline solids. Partial or complete transformation to the amorphous form, polymorphic transformations, and changes in chemical reactivity are among the frequently encountered changes produced by milling (Feng and Rodolfo Pinal, 2008).
Fig. 4. Mean thermodynamic solubility values of albendazole powder substance, milled and unmilled dispersions in various pH buffer solutions (n = 3).
3.6. FT-IR spectral evaluations
the mean solubility by 1.98 times in pH = 6.50 and 1.33 times in pH = 6.80 dissolution media (Fig. 4.), while only a small solubility change can be registered in pH = 1.20, + 2.55% due to wet granulation and + 11.11% to milling.
The characteristic bands of different structural moieties of albendazole can be easily identified comparing our results to previous investigations of reference materials. A broad band in the spectral range 3415–3190 cm−1, with peak at 3325, 3319–3317 cm−1, due to NeH stretching, from amine groups, overimposed on the vibrations of NeH bond from carbamate moiety. The sharp weak bands due to stretching of alkane-type CeH bonds from the propyl moiety appear around 2956 and 2865 cm−1. The C]O carbonyl bond bending appear as sharp band at 1712–1708 cm−1. The benzoimidazolyl part show two intense closed bands in the 1640–1590 cm−1 spectral range, with peaks at 1630 and 1595 cm−1. A characteristic band for the aromatic system is the band at 1523 cm−1, but as well the doublet bands at 1441 and 1422 cm−1. Other bands appear in the fingerprint region, below 950 cm-1, but are
3.5. Comparison of the phase transitions and thermoanalytical behaviours of the solid samples During the DSC investigations we've found that the melting point of the supplied pure albendazole powder was 197.7 °C and the enthalpy, that was required for this phase transition was 43.6 mJ/mg. The melting points of the albendazole in the unmilled and milled dry suspension formulations were 194.4 °C and 187.4 °C respectively (Fig. 5.). This sifting of the melting points indicates interactions between the 75
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Table 4 Comparison of the FT-IR absorbtion bands of various albendazole forms. Band assignmet for albendazole forms
Wavenumber (cm−1) ABZ I
eNH stretching Carbonyl group eCH3 absorption eCH stretching eC]N stretching Aromatic system eCH deformation Fingerprint region
ABZ II
range 3415–3188, peaks at 3325, 3319–3317 1712–1708 1443 2953, 2926 2957, 2912 1654, 1591 1629, 1578 1523, and duplet bands at 1441, 1422 1373 1377 881, 866, 846, 767, 755, 885, 863, 850, 805, 732, 597, 588, 520, 447 759, 728, 611, 597, 509
Albendazole EP
Unmilled
Milled
3331 1711 1443 2959, 2913 1616, 1586 1524,1441,1423 1374, 1377 887, 864, 847, 770, 755, 730, 598, 513, 449
3328 1711 1444 2958, 2909 1622, 1589 1524, 1443, 1426 1361 893, 866, 847, 770, 755, 730, 596, 525, 515, 466, 451
3332 1713 1445 2904 1623, 1588 1525, 1445, 1428 1361 894, 868, 847, 769, 752, 663, 591, 522, 510, 443
Fig. 6. FT-IR spectral evaluations of MCC (Vivapur® 12) carrier, albendazole powder, and granules containing unmilled and milled albendazole.
absorbed moisture of MCC at 1640 cm−1 is masked by the aromatic system of albendazole. The asymmetric eCH2 bending and wagging signal of MCC at 1440 and 1352 cm−1, finally the eCeOeCe stretching of the β-1,4-glycosidic linkage of MCC at 1120 cm−1 can be observed (Das et al., 2010) (Fig. 6.).
difficult to be asset to certain moieties. Previous investigations also revealed the spectral differences between the tautomeric forms of albendazole. When comparing form ABZ I to II, the bands corresponding to the eCH stretching vibration, to C]N stretching and to eCH deformation appeared slightly shifted. In addition, the bands in the fingerprint region (between 1500 and 600 cm−1) show marked differences between both solid forms, which can be used to identify and distinguish them. In particular, the spectrum of ABZ II shows shifted bands as well as new bands in comparison with that of ABZ I. ABZ I is characterized by bands at 885, 863, 850, 805, 759, 728, 611, 597 and 509 cm−1, whereas ABZ II exhibits characteristic bands at 881, 866, 846, 767, 755, 732, 597, 588, 520 and 447 cm−1 (Chattah et al., 2015; Trabdafirescu et al., 2016). As shown in Table 4, our sample consisted of the mixture of both tautomeric forms of albendazole. When subjected to nanosizing, a slight sifting of the band positions could be registered, nevertheless the recorded absorbance values have significantly decreased compared to albendazole powder, which also indicated a partial crystallineamorphous transition. Spectral analysis of the solid suspensions has also revealed the characteristic bands of MCC as well, masking each other with the vibrational signals of albendazole. From 3600 to 3000 cm−1 the stretching of the H-bonded eOH groups of MCC can be registered, masking the NeH stretching of albendazole, the eCeH stretching at 2900 cm−1 is a common band for both MCC and albendazole. The
3.7. AFM imaging Albendazole micronized powder contained small crystals, that form ~3 μm sized aggregates with one another. These small individual crystals were hard to detect on the surface of the microcrystalline carrier by AFM. Close examination of the surface of the milled dispersion (solid suspension) show no signs of nanoparticles. However a significant change can be detected comparing the surface roughness of the MCC to the milled dispersion. This microcrystalline surface change can be the effect of the wet granulation process (Fig. 7). 3.8. SEM imaging Same conclusions can be drawn, when taking a closer look at the SEM images. Albendazole micronized powder contains 3–8 μm sized aggregates that can be easily detected on the surface of the MCC carrier zooming in to the surface of the physical mixture with SEM. Also SEM 76
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Fig. 7. AFM amplitude-contrast images of microparticulate systems. (A) and (E) albendazole crystals; (B) and (F) Microcrystalline cellulose carrier (Vivapur® 12) powder; (C) and (G) milled albendazole dispersion (solid suspension containing albendazole nanocrystals); (D) and (H) albendazole: MCC 1:3 physical mixture.
Fig. 8. SEM images of microparticulate systems. (A) and (E) albendazole crystals; (B) and (F) Microcrystalline cellulose carrier (Vivapur® 12) powder; (C) and (G) milled albendazole dispersion (solid suspension containing albendazole nanocrystals); (D) and (H) albendazole: MCC 1:3 physical mixture.
solutions was reliable and reproducible. PSDs by intensities are overlapping each other pair wise considering suspensions and released albendazole crystals from the milled dispersions, which led us to the conclusion that there was no difference between the size distributions of albendazole crystals in pH = 6.50 and pH = 6.80. Unfortunately, there was a slight difference between the crystal size distributions of albendazole in the milled and dry suspension forms, COV values > 20% measured in both media (Table 2). We thought the reason behind this phenomenon is that Vivapur® 12 is a coarse grade MCC with a mean particle size of 180 μm therefore excellent flowability. These large carrier particles create smaller specific surface area and with smaller specific surface area, Vivapur® 12 can bind fewer nanocrystals on its surface than a fine powder with larger specific surface area would, which lead to some albendazole nanocrystal aggregation on the carrier's surface. This theory was quickly dismissed after the close examination
surface images indicated the change of surface morphology of MCC when adding the albendazole nanosuspension, but there is no sign of albendazole nanocrystals on the surface (Fig. 8.) 4. Discussion Albendazole nanocrystal reconstitution from microcrystalline cellulose carrier Vivapur® 12 was successful according to the released nanoparticles in dissolution media of pH = 6.50 ARF, pH = 6.80 and distilled water. Low COV values can be reported, between the intensity weighted mean hydrodynamic diameter (Z AVG d) values of dispersed milled suspension samples and albendazole nanocrystals released from dry suspension samples measured in both dissolution media and distilled water. These COV values are < 20%, which means that the analytical method developed and applied to measure particle sizes in these 77
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and comparison of the particle surfaces, because we couldn't detect any albendazole nanocrystals on the surfaces of the carrier neither with AFM nor with SEM. The drying process (tray drying at 40 °C) can be the main reason behind the small differences between the particle size distributions of albendazole in dry suspension and nanosuspension form. The pH values of the buffer solutions significantly influenced the solubility of albendazole, that was found to be 1520.70 ± 1.39 μg/ml in pH = 1.2, 8.21 ± 0.02 μg/ml in pH = 6.50 ARF, and 9.00 ± 0.01 μg/ml in pH = 6.80 medium. A major saturation solubility increase was achieved, when subjected to media milling and wetgranulation processes, 144.41 ± 0.09 μg/ml in pH = 6.50 ARF, and 146.27 ± 0.28 μg/ml in pH = 6.80. While only a small solubility change can be registered in pH = 1.20, + 2.55% due to wet granulation and + 11.11% to milling. The explanation of this phenomenon is that the base solubility of ABZ (1520.70 ± 1.39 μg/ml) is already so high in this acidic buffer solution, that the reduction of particle size shows little to no effect on further solubility gains. The pH dependence of the solubility of the albendazole is the main limiting condition of the cumulative drug release and thermodynamic solubility even from the milled solid suspension in alkaline buffer solutions.
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