Impact of Nanosizing on Solubility and Dissolution Rate of Poorly Soluble Pharmaceuticals

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Impact of Nanosizing on Solubility and Dissolution Rate of Poorly Soluble Pharmaceuticals SHARAD B. MURDANDE,1 DHAVAL A. SHAH,2 RUTESH H. DAVE2 1 2

Drug Product Design, Pfizer Worldwide R&D, Groton, Connecticut 06340 Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, Brooklyn, New York 11201

Received 1 October 2014; revised 2 February 2015; accepted 26 February 2015 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24426 ABSTRACT: The quantitative determination of solubility and the initial dissolution rate enhancement of crystalline nanoparticles were critically investigated using a separation-based approach (ultracentrifugation and filtration). Four poorly soluble model compounds (griseofulvin, celecoxib, compound-X, and fenofibrate) were used in this investigation. The effect of the stabilizer concentration on the solubility of the unmilled compound was determined first to quantify its impact on the solubility and used for comparing solubility enhancement upon nanosizing. Methodologies were established for ultracentrifugation, ensuring satisfactory separation of crystalline nanoparticles. The data obtained using separation-based methodologies proved to be accurate, reproducible, and were in fair agreement with what would be predicted from the Ostwald–Freundlich equation. The dissolution studies under sink conditions were proved to be less efficient in quantifying the initial dissolution rate of crystalline nanoparticles. Nonsink dissolution experiments were able to reduce the high-dissolution velocity of nanoparticles and generated the best discriminative dissolution profile. The enhancement in initial dissolution rate was significantly less than that expected from the Noyes–Whitney equation based on surface area change. This discriminatory dissolution method can potentially C 2015 Wiley Periodicals, Inc. and the American be used further in the modeling of crystalline nanoparticles during drug development.  Pharmacists Association J Pharm Sci Keywords: nanoparticles; nanosuspension; solubility; nanotechnology; dissolution rate; thermodynamics

INTRODUCTION According to recent trends, the new chemical entities coming out of discovery laboratories in the pharmaceutical industry frequently have low aqueous solubility because of highthroughput screening.1,2 A number of formulation strategies have been developed to address the low aqueous solubility of pharmaceutical compounds.3,4 Among them, particle size reduction (i.e., via micronization) is a common strategy and has long been used as a means for improving the oral absorption of poorly soluble drugs. The method of particle size reduction to enhance the performance of food, drug, and spices is well known and is used routinely. However, the extent of dissolution rate enhancement required to improve the in vivo drug absorption for such poorly soluble drug candidates is greater than that which can be provided by micronization.5 Therefore, the further reduction of the particle size to the nanometer range should be considered as a means for improving the oral absorption of these drugs.6 Several published reports have shown that the nanoparticles have the potential to improve the oral absorption of poorly soluble drugs along with providing improvements in other biopharmaceutical properties (e.g., variable absorption in the fed vs. fasted state).7–9 Nanosizing refers to the process of reducing the active pharmaceutical ingredient (API) size to submicron range. Nanosuspensions are aqueous dispersions consisting of a mixture of API and stabilizers, such as surfactant and/or a polymer in water. Nanosuspensions can be prepared by wet meCorrespondence to: Sharad B. Murdande (Telephone: +860-715-5975; Fax: +860-715-4473; E-mail: [email protected]) This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. Journal of Pharmaceutical Sciences  C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association

dia milling,10 high-pressure homogenization,11 precipitation or a combination of the two approaches mentioned previously.12 Studies conducted in this report utilized nanosuspensions prepared by the wet media milling techniques. A solubility advantage for amorphous pharmaceuticals over their crystalline counterparts has been very well studied in the literature.13,14 However, solubility advantage for nanocrystals and their impact on thermal and kinetic properties has not been explored in detail. Recent reports on solubility and dissolution improvement upon nanosizing have reported variable results.15,16 The most common practice for the solubility determination reported was based on the separation by filtration to separate undissolved nanoparticles from the solution utilizing the membrane filters of various pore sizes (0.1, 0.22, and 0.45 :m pore size).17–19 A few reports have also shown the use of molecular weight cut-off filters,20 dialysis bag method,21 centrifugation, and ultracentrifugation22 for the separation of dissolved and undissolved nanocrystals during solubility measurement. However, confirmation of satisfactory separation of dissolved and undissolved fractions of crystalline nanoparticles has been ambiguous. Determining accurate solubility is a much needed tool in the preformulation study of any nanocrystalline particles of a poorly soluble compound. However, it becomes challenging to accurately measure solubility when the drug particle size is in the nanometer range. Complicating this is the tendency of nanoparticles that remain suspended in the solution even after centrifugation.4 Therefore, development of a method for precise solubility determination associated with nanocrystals was one of the objectives of this research. A discriminating in vitro drug dissolution test plays an important role in pharmaceutical product development as it may be used as a representative tool for the assessment of formulation performance in vivo. Results of the dissolution studies Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

for nanocrystalline formulations have been documented in several reports where studies were carried out under sink conditions.23–25 At sink condition, nanocrystals dissolve instantaneously, which hinders the discrimination of dissolution behavior with respect to different particle size. Recently, a few reports have shifted the thinking in this direction and tried other approaches for carrying out the dissolution of nanosuspensions. Among these new approaches, turbidimetry is a reported method that can be used for real-time monitoring of small particle dissolution. Crisp et al.26 reported that the interfacial rate constant that is obtained from the turbidimetry method is independent of particle size; hence, it can quantitatively predict the dissolution rate for particle sizes down to the submicron range. In situ measurements using potentiometry27 and a solution calorimetry have also been investigated.28 In the case of the potentiometric methods, sensors are insensitive toward air bubbles and undissolved particles and can be a good tool for accurate dissolution studies. However, interference by the dynamics of dissolution and the requirement of preconditioned sensors with API are major drawbacks for this analytical method.27 Solution calorimetry also offers advantages over the classical dissolution testing approach by avoiding errors because of filtration and sample preparation. It is, however, a tedious method and requires a significant amount of additional time compared with the conventional methods.28 In the present study, the dissolution rate was examined in different nonsink conditions,29 for efficient discrimination of the dissolution rate enhancement with mean particle sizes that ranged from 80 nm to 11 :m. The present study primarily focused on investigating the thermodynamic properties (the change in the melting point, heat of fusion, and accurate solubility determination) and kinetic properties (the quantitative determination of initial dissolution rate enhancement with respect to particle size reduction upon nanosizing) for a nanosuspension by developing the suitable analytical methods.

MATERIALS AND METHODS Materials Four poorly soluble, nonionizable crystalline model compounds (griseofulvin, compound-X, celecoxib, and fenofibrate) were chosen for this study. Celecoxib and compound-X were received from Pfizer Inc. (Groton, Connecticut). Griseofulvin was obtained from Spectrum Chemicals (New Brunswick, New Jersey). Fenofibrate was purchased from Sigma–Aldrich (St. Louis, Missouri). All model compounds were used as received. The yttrium stabilized zirconium oxide beads were obtained from Glen Mills Inc. (Clifton, New Jersey). Hydroxypropylcellulose (HPC)–SL was obtained from Nisso America Inc. (New York, New York). Other laboratory chemicals such as docusate sodium, USP, acetonitrile, and trifluoroacetic acid (TFA) were purchased from Spectrum Chemicals and were of analytical grades. Nanosuspension Preparation Nanosuspensions of four model compounds were prepared by attrition media milling. The milling vehicle was a mixture of 1.25% (w/v) HPC–SL (stearic stabilizer) and 0.05% (w/v) docusate sodium (ionic stabilizer) in deionized water. Optimal concentration of stabilizers was determined from earlier experMurdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

iments. Yttrium-stabilized zirconium oxide beads were used as a grinding media (500 and 100 :m). The ratio of the grinding media to API in suspension was 3:1 (w/w). The milling speed was 1000 rpm. Temperature was maintained by the surrounded cold water outside of the milling chamber to compensate for the heat generated by high-speed milling. Targeted final concentration for nanosuspension was 50 mg/mL. The average milling time for all model compounds was 4 h. Milling was stopped after 4 h and the grinding media was allowed to settle down for 30 min. The suspension was separated by filtration through a coarse sintered glass filter (75 :m). The nanosuspension was harvested by removing the grinding media and stored under refrigerated conditions (4°C) to evaluate physical stability. Nanosuspensions were assayed for drug potency by HPLC. Characterization of Nanocrystal Particle Size and Zeta Potential Measurements A dynamic light scattering (DLS) instrument (Malvern Instruments Inc., Malvern, UK) was used to measure particle size and the size distribution of nanocrystalline suspensions. The samples were prepared by diluting 5–10 :L of nanosuspension with HPLC grade water. The sample was analyzed at 25°C, using a dispersant refractive index of 1.33. The attenuation and measurement settings were optimized automatically by resident software. The polydispersity index (PI) and count rate for the measurement were taken into consideration after the measurement to assess the quality of the nanosuspensions prepared. Particle size data obtained were expressed in terms of D10 , D50 , and D90 (10%, 50%, and 90% of the particles’ volume below certain sizes) along with the average particle size. In the current study, the zeta potential of the prepared nanosuspensions was measured in disposable-folded capillary cells (Model #DTS1061) along with the particle size measurement using the DLS instrument. Morphological Characterization Morphological characterization was performed using a polarized light microscope (PLM; Model BX60FS; Olympus BX60, Center Valley, Pennsylvania). The polarized microscopic images were taken using a camera from Diagnostic Instruments Inc. (Sterling Heights, Michigan) and the analysis was performed TM with the help of SPOT imaging advanced software. Samples were prepared by placing a 4-:L nanosuspension on a clean glass slide and covering it carefully with a clean glass cover slip. Nanocrystals were observed under 500x optical zoom in a dark field. Measurement of Thermal Properties Sample Preparation for Differential Scanning Calorimetry and X-ray Powder Diffraction The initial unmilled drug suspension was prepared by mixing micronized API with the stabilizer solution at 1000 rpm for 4 h at 25°C. The same stabilizer solution was used for both the unmilled and the nanosuspension preparation. Thus, the initial (unmilled) suspension was treated in the same manner as the nanosuspensions, with respect to the stabilizer concentration in the solution. Both the unmilled suspension and the nanosuspensions were transferred via an eppendorf vial and centrifuged using an Allegra 21 centrifuge, GS-15 (Beckman Coulter Inc., Schaumberg, Illinois). Centrifugation was carried DOI 10.1002/jps.24426

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

out at 6882 RCF for 15 min (at 25°C). The supernatant was discarded from each vial and replaced with fresh DI water for washing the particles to remove excess stabilizer that was adsorbed on the surface of the crystalline particles. The washing procedure was repeated six times for each model compound. After discarding the supernatant liquid, wet particles from the bottom of the eppendorf were collected and subjected to X-ray powder diffraction (XRPD) analysis. The remaining portion was kept overnight in a vacuum dryer and subjected to differential scanning calorimetry (DSC) analysis afterwards. X-ray Powder Diffraction The crystalline properties of formulated nanosuspensions were assessed by XRPD using a Bruker D4 diffractometer (Bruker Bio Spin Corporation, Billerica, Massachusetts). The XRPD measurements were carried out in the standard measurement mode in the 22 range from 5° to 40°. The scan speed was 2°/min and the counting step was 0.02°. The X-ray source was CuK" ˚ the accelerating voltage was 40 kV, (wavelength = 1.5406 A); and the current was 40 mA. Samples were prepared by placing them in a silicon cavity wafer mount as described above. Data were collected and analyzed using the Bruker DIFFRAC Plus software suite (Bruker Bio Spin Corporation, Billerica, Massachusetts). Melting Point and Heat of Fusion Measurement Thermal properties (i.e., the melting point and the heat of fusion measurement) were investigated by a differential scanning calorimeter (DSC Q1000; TA Instruments, New Castle, Delaware). The instrument was calibrated using standard indium samples. Calibration of the heat capacity was performed using sapphire. The 5–10-mg samples of pure API, unmilled suspension, and nanosuspension were sealed in standard aluminum pans and subjected to analysis. The DSC parameters used were: a sample equilibration at 30°C, modulated for ±0.318°C every 60 s, isothermal for 5 min, and heated to 180°C/230°C with a ramp of 2°C/min. The DSC cell was purged with nitrogen at a flow rate of 50 mL/min. The data obtained were analyzed with the Universal Analysis Software (TA Instruments). All studies were performed in triplicate. Experimental Solubility Measurement HPLC Analysis Method The quantitative analysis of model compounds used in the solubility studies was performed by reverse-phase gradient HPLC (Agilent 1100 Series and Alliance 2695 Module). Analyses were performed using an Agilent 1100 Serial System (Agilent Technologies, Palo Alto, California) and Alliance 2695 Separation Module (Waters Corporation, Milford, Massachusetts) equipped with a 4.6 × 75 mm, 2.7 :m C18 column (Advanced Materials Technology, Wilmington, DE). The temperature of the column was kept at 45°C, and the wavelength of detection was set to 210 nm. The mobile phase consisted of a binary gradient of solvent A (acetonitrile) and solvent B (HPLC grade water with 0.05% TFA). The linear gradient started at 5% A and increased to 95% A in 8.5 min, followed by a return to the starting condition within 1.6 min and equilibrated at the starting condition for 2 min. The injection volume was either 5 or 10 :L. The flow rate was 1.0 mL/min, and the total run time was set for 13.10 min. This reverse-phase gradient method provided baseline resolution and excellent peak characteristics for DOI 10.1002/jps.24426

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each model compound. Quantification of drug concentrations was performed by analyzing the peak area at retention using R R and Waters Empower Pro analytical Agilent Chemstation software. Thermodynamic Solubility Determination Solubility determination was carried out using the temperature cycling method with a rotating mixture equipped with ENVIRO GENE software (Scientific Industry Inc., Bohemia, NY). A three-step temperature cycling method was used for equilibrium solubility determination in which samples were rotated for 8 h at 40°C followed by 5 h at 15°C, which was then followed by 12 h at 25°C. To achieve an accurate measurement, the solubility determination was conducted in the presence of a stabilizer solution for both unmilled and nanocrystalline model compounds. The study provided more precise solubility data (compared with the solubility in the water), as both unmilled and nanocrystals were treated in the same manner.

r Aqueous solubility determination of model compounds Solubilities of the crystalline form of the model compounds were measured in DI water. An excess amount of API was dispersed in DI water and subjected to the temperature cycling method as described above. The samples were filtered using 0.1 :m anotop syringe filters and subjected to HPLC analysis. The aqueous solubility of all the model compounds was less than 10 :g/mL, except for the compound-X (47.2 :g/mL).

r Effect of nanosizing stabilizer on drug solubility The stabilizer can affect the drug solubility by altering the wetting property of the API. Crude suspensions were prepared with various stabilizer concentrations, that is, (1) no stabilizer, (2) 0.625% HPC-SL + 0.025% docusate sodium, (3) 1.25% HPC-SL + 0.05% docusate sodium, and (4) 2.5% HPC-SL + 0.1% docusate sodium. Samples were placed in a temperaturecontrolled rotary mixture equipped with ENVIRO GENE software for 25 h. At the end of the equilibration, it was very difficult to filter the nanosuspension samples through 0.1/0.02 :m filters because of higher concentration of particles and also the filter blockage was observed. Therefore, samples were subjected to ultracentrifugation at a speed of 313130 RCF at 25°C for 60 min in a 100% vacuum using a fixed angle rotor in a Beckman Coulter Ultracentrifuge (Beckman Coulter Inc.). Supernatants were collected immediately and subjected to HPLC analysis for equilibrium solubility determination. The same supernatants were filtered from 0.1 :m anotop syringe PTFE polypropylene filters (WhatmanTM ; GE Healthcare, Piscataway, NJ) and subjected to HPLC analysis to measure the equilibrium solubility.

r Nanocrystalline particle solubility in stabilizer solution Nanosuspensions of each compound were transferred to a Beckman Coulter Ultracentrifuge. The ultracentrifugation parameters were kept consistent throughout the study. After ultracentrifugation, the supernatants were collected immediately and transferred to an HPLC vial for solubility determination. The same supernatants of nanosuspensions were filtered from 0.1 :m anotop syringe filters for the particle size greater than 200 nm, and the supernatants for nanosuspensions with Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

particle size less than 125 nm were filtered from 0.02 :m anotop syringe filters (WhatmanTM ; GE Healthcare, Piscataway, NJ) for HPLC analysis. The first few milliliters of each sample were discarded to minimize losses because of adsorption. The discarded volume selected for each compound was determined by a separate filter validation experiment (data not shown). The solubility was also assessed from the dissolution profile by allowing the dissolution study to proceed until equilibrium was established. All solubility measurements were carried out in triplicate. Measurement of Dissolution Rate A dissolution apparatus (Distek 2100C; Distek Inc., North Brunswick, New Jersey) equipped with a USP type-II (paddle) was used to measure dissolution rates of nanocrystalline suspensions. Griseofulvin was chosen as a model drug for the dissolution study. The dissolution study was carried out by comparing unmilled suspension (micronized mean particle size of 11.3 :m) and nanosuspensions (mean particle sizes of 362 and 122 nm). Each dissolution study was carried out at 37±0.5°C under a 100-rpm paddle speed in a USP Type II apparatus for 2 h. The concentration of suspensions was made to 5 mg/mL by diluting 1 mL of each suspension with 10 mL of stabilizer solution [1.25% (w/v)/0.05% (w/v)-HPC-SL/docusate sodium]. Accordingly, 0.2 mL of suspension (equivalent to 1 mg of API) was used for the dissolution study under sink condition (a) and under reducing the sink condition factor (b).

r Dissolution under sink condition Each dissolution study was carried out in 900 mL DI water. The sink condition factor was maintained at 5.7× (calculated from experimentally determined solubility value for the griseofulvin). A 10-mL of a dissolution sample was withdrawn at intervals of 0, 2, 5, 7, 10, 20, 30, 45, 60, 90, and 120 min and replaced with same volume of dissolution media (preequilibrated at 37±0.5°). Samples were filtered through a 0.1-:m (unmilled 11.3 :m and 362 nm) and 0.02 :m (<125 nm) anotop syringe filter.

r Dissolution under reducing sink condition factor The dissolution set up and sample preparation was maintained as described for the sink condition dissolution experiment. The only difference was with the use of the dissolution media. In order to reduce the sink condition factor, 40% and 55% of griseofulvin (based on its experimentally determined solubility value) were predissolved in DI water. After dissolving the appropriate amount of griseofulvin, the media was filtered through 0.22 :m Millipore Express Plus filter membrane. The dissolution studies were carried out in the dissolution media with a reduced sink condition factor of about 1.8× and 1.4×, respectively. A 10 mL of a dissolution sample was withdrawn at predefined intervals and replaced with same volume of dissolution media (pre-equilibrated at 37±0.5°). Samples were filtered through a 0.1 :m (unmilled 11.3 :m and 362 nm) and 0.02 :m (<125 nm) anotop syringe filter.

r Effect

of griseofulvin loading dose on dissolution under nonsink condition

Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

The dissolution set up was maintained as described for the sink condition dissolution experiment. The sample preparation and the dissolution media were different as compared with previous dissolution studies. The dissolution study was performed by generating two types of nonsink conditions.

r Dissolution media in which 25%, 40%, 55%, and 75% of r

griseofulvin was predissolved in DI water and the sample loading dose was of 10 mg of griseofulvin. Dissolution media in which 20%, 40%, and 75% of griseofulvin was predissolved in DI water and the sample loading dose was of 50 mg griseofulvin.

The dissolution media was filtered through a 0.22 :m Millipore Express PLUS filter membrane. The suspension samples were equivalent to either 10 or 50 mg of API (0.2 mL/1 mL with the original concentration of 50 mg/mL), and were used for the dissolution study to generate complete nonsink conditions. Samples were withdrawn at 0, 2, 5, 7, 10, 20, 30, 45, 60, 90, and 120 min and replaced with the same volume of dissolution media (preequilibrated at 37±0.5°) for the analysis of the drug concentration. Samples were filtered through a 0.1 :m (unmilled 11.3 :m and 362 nm) and 0.02 :m (<125 nm) anotop syringe filters. The average of the three replicate tests was reported for each of the dissolution studies.

RESULTS AND DISCUSSION Particle Size and Zeta Potential Measurement The mean particle sizes of wet-milled crystalline nanosuspensions of all four model compounds were found to be within 93– 362 nm range (Table 1) with an acceptable PI (<0.3). The average particle size data, along with their D10 , D50 , and D90 values, and PI and zeta potential values, were obtained from DLS. These data indicate that the wet-milled crystalline nanosuspensions were homogeneous with a narrow particle size distribution. Experimentally determined zeta potential values ranged from −22.7 to −30.3 mV, suggesting good physical stability of the formulated nanosuspensions.30 The stabilization of nanosuspensions can be attributed to two main factors. (1) The adsorption of HPC-SL on the drug particle provides stearic stabilization and (2) the docusate sodium provides electrostatic repulsion by forming the ionic charges at the drug particle surface. Morphological Characterization Morphological analysis by PLM micrographs demonstrated the presence of nanosized crystalline particles (data not shown). The images clearly show the presence of particles in the nanometer size range. X-ray Powder Diffraction X-ray powder diffraction was used to determine changes in the crystalline state of all model APIs in the nanosuspensions. X-ray diffractograms of crystalline nanosuspensions were compared with pure API and the stabilizer-treated unmilled API. The crystallinity in the nanosuspension is confirmed by the retention of characteristic peaks in X-ray diffractograms (data not shown). Moreover, the light microscopy data are also supportive to XRPD data for the crystallinity (data not shown). Dominant DOI 10.1002/jps.24426

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

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Table 1. Impact of Particle Size on Melting Point and Heat of Fusion Compounds

Size (nm)

PdI

Sample Name

MP (°C)

MP (°C)

Hf (J/g)

Hf (J/g)

Zeta Potential (−mV)

Griseofulvin

362

0.095

−24.1

0.210

10.9

−24.6

Celecoxib

341

0.272

26.1

−29.5

Fenofibrate

290

0.192

8.9

−30.3

Griseofulvin

122

0.131

15.2

−28.2

Comp.-X

93

0.172

111.8 96.6 101.7 90.7 100.5 74.5 95.4 86.5 111.8 96.6 101.7 67.6

15.2

243

219.1 217.8 207.5 205.6 162.1 160 80.4 79.2 219.1 215.5 207.5 202.2

1.3

Comp.-X

API NC API NC API NC API NC API NC API NC

34.1

−22.7

1.9 2.1 1.2 3.5 5.3

MP, melting point; Hf , heat of fusion; API, active pharmaceutical ingredient; NC, nanocrystals; PdI, polydispersivity index.

peaks at 22 angle were observed in the nanosuspension formulations suggesting the conservation of the crystalline property of all model compounds, though reductions in both heat of fusion and in melting point were observed (Table 1). Determination of Thermal Properties Differential Scanning Calorimetry A DSC analysis was performed for the investigation of thermal properties and further confirmation of the crystalline state after particle size reduction. The thermograms of the nanocrystalline samples were compared with the unmilled API to confirm the crystallinity of the API. All the nanocrystalline formulations showed an endothermic melting event. Here, the nanocrystals of griseofulvin (mean particle size 362 nm), produced a relatively sharp but slightly shifted endothermic melting peak at 217.8°C (from 219.1°C), along with a shift in Hf (from 111.8 J/g to 96.6 J/g). The observed peak shift has been reported to be in agreement with the particle size reduction.31,32 For all other APIs, the observed changes in the melting point and heat of fusion with respect to particle size reduction are presented in Table 1. For the particle sizes between 290 and 362 nm, the melting point reduction was approximately 1.2°C– 2.1°C and the heat of fusion reduction was around 8.9–26.1 J/g. To further evaluate the thermal changes, crystalline nanosuspensions were formulated down to 100 nm particle size. A further reduction in both the melting point (3.5°C and 5.3°C) and the heat of fusion (15.2 and 35 J/g) was observed when the particle size was reduced close to 122 and 93 nm, respectively. The phenomena of change in the melting point and the heat of fusion because of the reduction in particle sizes can also be explained by the Gibbs–Thomson equation.33,34 Equilibrium Solubility Crystalline nanoparticles of very poorly soluble compounds (solubility <10 :g/mL) for which even a small error in solubility measurement can have a significant impact on the results. In addition, it is also important to study the filter binding capacity of a drug molecule to avoid underestimating the actual solubility data. Selection of the analytical method (i.e., UV/HPLC) for quantitative analysis also plays an important role. In the current research, we established a systematic approach to accurately measure the solubility of crystalline nanoparticles in order to estimate the improvement in solubility that can be DOI 10.1002/jps.24426

achieved upon nanosizing. Saturation solubilities for the pure API were experimentally determined in DI water at room temperature. The RSD values for the solubilities were within 2% of the reported values for the crystalline drugs. Solubility studies were also performed for unmilled drugs with different concentrations of the stabilizer solution. The reported data suggest that the solubility number rises proportionally with the concentration of the stabilizer. The concentration [1.25% (w/v) HPC SL + 0.05% (w/v) docusate sodium] at which the nanosuspension formulation had been made have very less impact on the solubility (data not shown). So, it is fair to compare the solubility results in the stabilizer solution for both the milled and unmilled particles. The solubility enhancement observed can be considered solely because of the particle size. The average solubility (n = 3) of unmilled API and nanocrystals in the stabilizer solution [1.25% (w/v) HPC SL + 0.05% (w/v) docusate sodium] is shown in Table 2. The solubility values determined from the supernatant of the ultracentrifugation and ultracentrifugation plus filters in the case of griseofulvin were 8.12 versus 7.63 :g/mL for unmilled API. Whereas in the case of celecoxib, solubility values were 1.45 versus 1.00 :g/mL, and for fenofibrate, 1.53 versus 0.74 :g/mL. The reported data were the average number of triplicate measurements along with the standard deviations. There was no significant difference in the solubility between nanocrystals and the unmilled API in both cases (i.e., ultracentrifugation and ultracentrifugation +0.1 :m filter). The solubility enhancement ratio was found to be minimal when the particle size was reduced from 292 to 362 nm. The experimental solubility enhancement ratio based on particle size was also compared with the theoretical solubility enhancement ratio described by the Ostwald–Freundlich equation. Note that for the theoretical calculation, the interfacial tension is equal to the surface tension of water of 72 dyne/cm2 has been made in the Ostwald–Freundlich equation.35 The results for solubility enhancement were similar with what would be expected based on the Ostwald–Freundlich equation (data not shown). In studying the impact of particle size on solubility, when the size is reduced to close to 100 nm, the nanosuspensions were prepared for griseofulvin and compound-X with mean a particle size of 122 and 93 nm, respectively. The solubility measurements were performed using a 0.02 :m syringe filter instead of 0.1 :m because of a further reduction in the particle size (Table 2). The solubility for griseofulvin was 10.30 versus 8.12 :g/mL (ultracentrifugation) and 9.99 versus 7.63 :g/mL Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

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Table 2. Solubility of Crystalline Nanoparticles

Compounds

Sample

Solubility Unmilled Drug (:g/mL)

Solubility of Nanocrystal Drug (:g/mL)

Solubility Ratio (Nanocrystal/ Unmilled Drug)

Size (nm) 362

UC

8.12 ± 0.14

8.17 ± 0.28

1.01

UC + 0.1 :m filter

7.63 ± 0.89

8.40 ± 0.25

1.1

UC

65.17 ± 1.58

63.41 ± 1.26

0.97

UC + 0.1 :m filter

74.44 ± 3.07

68.86 ± 10.73

0.93

UC

1.45 ± 0.51

1.50 ± 0.17

1.03

UC + 0.1 :m filter

1.00 ± 0.03

1.11 ± 0.03

1.11

Griseofulvin Comp.-X Celecoxib

UC

1.53 ± 0.21

1.66 ± 0.66

1.08

UC + 0.1 :m filter

0.74 ± 0.27

0.82 ± 0.26

1.11

UC

8.12 ± 0.14

10.30 ± 0.18

1.26

Fenofibrate Griseofulvin

UC + 0.02 :m filter

7.63 ± 0.89

9.99 ± 0.15

1.3

UC

65.17 ± 1.58

89.06 ± 6.36

1.36

Comp.-X

243 341 290 122 93

UC, ultracentrifugation.

(ultracentrifugation + filter). In the case of compound-X, the solubility from the ultracentrifuge was 89.06 versus 65.17 :g/mL. The maximum solubility enhancement observed upon nanosizing was about 30% compared with unmilled API. A similar enhancement of 15% in the solubility upon nanosizing was also reported when the particle size was reduced from 158 to 700 nm.22 On the basis of the theoretical calculations of the Ostwald–Freundlich equation, in order to get 30% enhancement in thermodynamic solubility, the required particle size should be approximately 160 nm. Therefore, the observed solubility enhancement achieved upon nanosizing is in fair agreement with what can be expected, based on the Ostwald– Freundlich equation. The reported solubility enhancement ratios are shown in Table 2. Note that the solubility determination studies were conducted by maintaining precision in the generated data (i.e., separate filter validation experiments; each measurement was carried out running a fresh calibration curve for all the model compounds, along with the samples during each quantitative analysis). Therefore, the reported data were accurate enough to understand the solubility behavior of nanocrystals when the particle size was reduced to approximately 100 nm.

Dissolution Studies Particle size reduction also has a major impact on the dissolution rate of crystalline drugs. As per the Noyes–Whitney equation, a positive effect (i.e., increase in the dissolution rate with increase in the surface area) can be observed in the dissolution rate when the particle size is reduced. Therefore, an accurate measurement of enhancement in the dissolution rate is important in order to understand the observed in vivo pharmacokinetic results obtained using nanosuspension relative to unmilled crystalline suspension. Initially the dissolution behaviors of nanosuspensions were determined by maintaining perfect sink condition following the guidelines in European Pharmacopeia (Fig. 1a). A comparison of dissolution profiles was made among the 122, 362 nm, and 11.3 :m size griseofulvin suspensions. From the dissolution profiles, it is clear that nanocrystals dissolved instantaneously (>80% API dissolved in <2 min for both nanocrystalline suspensions). However, the micronized unmilled suspension showed a slow release compared with the nanosuspensions. It took more than 20 min for the micronized suspension to release 80% of the API. Hence, we can get a comparable

Figure 1. In vitro dissolution studies (griseofulvin: 10 mg loading dose): comparing unmilled API, 362 and 122 nm size of particles: (a) sink condition dissolution and (b) reducing sink condition factor (1.8×, 40% saturation and 1.4×, 55% saturation). Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

DOI 10.1002/jps.24426

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Figure 2. Nonsink dissolution (griseofulvin:10 mg loading dose): comparing unmilled API, 362 and 122 nm size of particles: (a) 25% and 40% saturation and (b) 55% and 75% saturation.

idea of the dissolution profile using the traditional sink condition dissolution method. An improvement in the dissolution velocity upon nanosizing is well explained in multiple reports.36,37 However, initial linear regions in sink conditions lacked discrimination for the initial dissolution rate enhancement. Also, nanocrystals with particle sizes of 362 and 122 nm had superimposable dissolution profiles. This concludes that the sink condition dissolution profile is insufficient in providing actual discrimination for the initial dissolution rate enhancement and its relation to the observed improvement in various PK parameters. For further discrimination and quantification of the initial dissolution rate, studies were carried out by reducing the sink condition factor as described in the method section. The dissolution profiles for the reduced sink condition factor are presented in Figure 1b. A clear discrimination was observed between micronized and nanocrystals in the initial region of the dissolution profile. However, the dissolution profiles for both different sized nanocrystals were superimposable on each other. The initial dissolution rate ratios were calculated from dissolution at the first couple of time points (<5 min into the dissolution process).38 In order to further discriminate the dissolution profiles of nanosized griseofulvin, studies were performed by generating the two types of nonsink conditions as described in the Materials and Methods section. The dissolution profiles are shown in Figures 2 and 3. The calculated initial dissolution rate ratios are reported in Table 3. All of the dissolution profiles with

nonsink conditions showed significant discrimination between micronized and nanocrystals. Nonsink conditions (loading dose 10 mg) with saturation levels of 25% and 40% were not sufficient enough for discrimination of the initial dissolution rate enhancement (Fig. 2a). All the dissolution profiles started to be discriminating with each other as the saturation level increased to 55%. When the saturation level reached 75%, maximum discrimination was observed (1.8 vs. 3.6) for the initial dissolution rate ratios between both the nanocrystals (Fig. 2b). The dissolution profiles with nonsink conditions and higher loading doses (i.e. 50 mg) showed discrimination in all three saturation levels (Fig. 3). The findings of the current study justified the use and the need for a nonsink dissolution approach to accurately evaluate the dissolution rate enhancement upon nanosizing. Current dissolution practices (i.e., sink condition) as reported in the literature justifies their results by citing the Noyes–Whitney equation,39,40 which is most popular for studying the dissolution behavior of particles greater than a few microns. Crisp et al.26 have also reported that when particle size is reduced to nanometer range, the interfacial phenomena plays a more important role compared with the surface area, so the dissolution rate predicted from the Noyes–Whitney equation will not correlate well with the observed data. However, our findings for the dissolution rate enhancement are not in close agreement with respect to surface area change. The calculated surface area increases upon nanosizing were much larger than the observed dissolution rate enhancement upon nanosizing for griseofulvin (Fig. 4).

CONCLUSIONS

Figure 3. Nonsink dissolution (griseofulvin: 50 mg loading dose): comparing initial dissolution rate enhancement for unmilled API, 362 and 88 nm size of particles at 20%, 40%, and 75% saturation level. DOI 10.1002/jps.24426

We have established a reliable method for measuring the solubility of crystalline nanoparticles obtained from the wet-milling technique. The significant improvement in the separation of undissolved nanoparticles was achieved by ultracentrifugation. The observed increase in the solubility upon nanosizing was only marginal and in close agreement with the theory. Therefore, any improvement in in vivo pharmacokinetics that can be achieved with nanoparticles can be expected to be mainly because of the dissolution rate enhancement. The enhancement in dissolution achieved upon nanosizing is best studied under nonsink conditions. However, factors that contribute to the Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 3. Initial Dissolution Rate Enhancement at Various Saturation Levels Ratio Dissolved in 2 min (%) Sink Condition Factora 5.7× 1.8× 1.4×

362 nm

122 nm

122 nm

Unmilled

362 nm

122 nm

Unmilled

Unmilled

362 nm

38 38 24

79 76 86

79 86 98

2 2 3.6

2.1 2.2 4

1 1.1 1.11

362 nm

122 nm

122 nm

Nonsink Conditionb

Saturation Level 20% 40% 55% 75%

Unmilled

362 nm

122 nm

Unmilled

Unmilled

362 nm

14 11.7 13 18

44 35 36 33

49 49 49 65

3.1 3 2.76 1.8

3.5 4.2 3.76 3.6

1.1 1.3 1.3 2

362 nm

88 nm

88 nm

Nonsink Conditionc

Saturation Level 20% 40% 75%

Unmilled

362 nm

88 nm

Unmilled

Unmilled

362 nm

15 12 7

19 16 10

29 30 22

1.2 1.3 1.4

2 2.4 3.15

1.5 1.75 2.4

Solubility of griseofulvin in dissolution media; 6.27 :g/mL. Nonsink dissolution; 10 mg griseofulvin in 900 mL of dissolution media. Nonsink dissolution; 50 mg griseofulvin in 900 mL of dissolution media.

a b c

ACKNOWLEDGMENTS The authors would like to acknowledge Ravi M. Shanker, Ph.D and Jigna D. Patel, Ph.D for their helpful discussion.

REFERENCES

Figure 4. Comparing predictive initial dissolution rate enhancement ratio upon surface area change with experimentally determined initial dissolution rate enhancement ratio.

dissolution rate enhancement are not directly related to the surface area increase for crystalline nanoparticles. Further studies are needed to understand the impact of surface area change and the surface-free energy change that occurs upon nanosizing on the dissolution rate enhancement. Murdande, Shah, and Dave, JOURNAL OF PHARMACEUTICAL SCIENCES

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