Development of nanocrystal formulation of meloxicam

International Journal of Pharmaceutics 474 (2014) 151–156

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

Development of nanocrystal formulation of meloxicam with improved dissolution and pharmacokinetic behaviors Masanori Ochi a, * , Takaki Kawachi a , Eri Toita a , Issei Hashimoto a , Kayo Yuminoki a , Satomi Onoue b , Naofumi Hashimoto a a

Department of Pharmaceutical Physicochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka 422-8526, Japan b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 April 2014 Received in revised form 25 July 2014 Accepted 14 August 2014 Available online 17 August 2014

The present study aimed to develop nanocrystal formulations of meloxicam (MEL) in order to enhance its biopharmaceutical properties and provide a rapid onset of action. Nanocrystal formulations were prepared by wet-milling and lyophilization with hydrophilic polymers used as aggregation inhibitors. Aggregation inhibitors were selected based on high-throughput screening of crystal growth inhibition in supersaturated MEL solution. Supersaturation of MEL was observed in PVP K-30, HPC-SSL, and POVACOAT Type F solution. Although the particle size distributions of pulverized MEL with PVP K-30 (MEL/PVP), HPC-SSL (MEL/HPC), and POVACOAT Type F (MEL/POVA) were in the nanometer range following lyophilization, increases in micron-sized aggregates were observed after storage at 60 C for 21 days. The order of increased amount of aggregates was MEL/POVA MEL/HPC > MEL/PVP. These findings showed that hydrophilic polymers that inhibited crystal growth in supersaturated MEL solutions tended to prevent aggregation. The dissolution behavior of all nanocrystal formulations tested was markedly enhanced compared with that of unpulverized MEL. Oral administration of MEL/PVP showed a 2.0 h shortened Tmax and a 5.0-fold increase in bioavailability compared with unpulverized MEL. These findings showed that the MEL/PVP mixture was physicochemically stable and provided a rapid onset of action and enhanced bioavailability after oral administration. ã 2014 Elsevier B.V. All rights reserved.

Keywords: Nanocrystals Meloxicam Stabilization Supersaturation Dissolution Pharmacokinetic

1. Introduction Meloxicam (MEL) is a highly potent non-steroidal antiinflammatory drug (NSAID) used to treat rheumatoid arthritis (Furst, 1997), osteoarthritis (Hosie et al., 1996), and postoperative pain (Aoki et al., 2006). MEL selectively inhibits cyclooxygenase-2 isoenzyme (Pairet et al., 1998), consequently, MEL has effective anti-inflammatory and analgesic properties and low gastrointestinal toxicity. Despite these attractive pharmacological profiles, the onset of the pharmaceutical effects of MEL is slow due to slow oral absorption. A rapid onset of pharmaceutical effects is important to patients, particularly during acute exacerbation of rheumatism and osteoarthritis. Poor wettability and low solubility of MEL (ca. 0.6 mg/mL at both pH 1.2 and 4.0) (Ghorab et al., 2004) are believed to be as responsible for the slow oral absorption.

Abbreviations: HPC, hydroxypropyl cellulose; MEL, meloxicam; POVA, POVACOAT Type F; PVP, polyvinylpyrrolidone; XRPD, X-ray powder diffraction. * Corresponding author. Tel.: +81 72 866 3153; fax: +81 72 866 3153. E-mail addresses: [email protected], [email protected] (M. Ochi). http://dx.doi.org/10.1016/j.ijpharm.2014.08.022 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.

Consequently, the application of solubilization technologies could be a key for improving the pharmacological profile of MEL. A number of approaches have been made to enhance the aqueous solubility of poorly soluble drugs include salt formation (Serajuddin, 2007), nano-pulverization (Merisko-Liversidge et al., 2003), emulsification (He et al., 2010) and amorphous solid dispersion technique (Leuner and Dressman, 2000). Nanopulverization is one of the most simple and effective methods for improving the dissolution behavior and bioavailability of drugs. However, well-known problems associated with nanocrystal formulations include increased aggregation during preparation and storage (Abdelwahed et al., 2006; Quan et al., 2012). Increased aggregation leads to a reduction in surface area, causing a reduction in dissolution rate and systemic exposure. Therefore, the selection of an appropriate aggregation inhibitor is very important for the development of nanocrystal formulations. It has been reported that nanoparticles can be stabilized by covering the surface of the drug nanoparticles with suitable polymers (Peltonen and Hirvonen, 2010). Appropriate intermolecular interactions between the drug and polymer molecules on the surface of the nanocrystal are believed to be important for

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preventing aggregation. Chauhan et al. previously demonstrated that the crystal growth inhibition efficiency of a supersaturated drug solution correlated well with the stability of the amorphous solid dispersion (Chauhan et al., 2013). Screening polymers for their MEL crystal growth inhibition might help narrow down the number of aggregation inhibitor candidates for improving MEL nanocrystal formulations. However, to date, there has been no effort to screen crystal growth inhibitors for improving nanocrystal formulations. In this study, a 96-well filter-plate-based anti-precipitant screening approach was used because of its simplicity and applicability to automated high-throughput analysis (Yamashita et al., 2011). This study aimed to develop a physicochemically-stable MEL nanocrystal formulation with improved dissolution behavior in order to shorten the onset of pharmacological effects. The physicochemical properties were characterized by X-ray powder diffraction (XRPD), particle size distribution analysis before and after storage using a laser diffraction/scattering method, and dissolution testing in acidic and neutral media. The physical stability and dissolution profile results suggested the most suitable MEL nanocrystal formulation for in vivo testing. Pharmacokinetic profiling of MEL after oral administration of the nanocrystal formulation to rats was evaluated. 2. Materials and method

suspension (200 mL) was placed in each well of a 96-well filter plate (MultiScreen HTS solubility filter plate, 0.45 mm, Millipore). Then, 1 mL of 30 mg-MEL/mL solubilized in DMSO was added to each well using a pipette, then the plate was shaken in an IKA MS 3 digital MIXER (IKA, Staufen, Germany) for 1 h at 25 C. The samples were centrifuged at 500 g for 5 min, and the filtrates were collected in a 96-well plate. The filtrates were diluted with an equivalent volume of acetonitrile, then the concentration of MEL in each sample was measured using HPLC/UV. 2.4. Wet-milled formulation of MEL Nanocrystal formulations of MEL were prepared using a rotation/revolution mixer (NP-100; Thinky Company Ltd., Tokyo, Japan) as reported previously (Takatsuka et al., 2009). Briefly, 20 mg of MEL and 2.5 g of zirconia (zirconium oxide) balls (0.1 mm diameter; Nikkato Company, Ltd., Osaka, Japan) were weighed into the vessel of a rotation/revolution mixer, and 0.5 mL of hydrophilic polymer solution (40 mg/mL) was added. MEL suspension was nano-pulverized by two-step wet-milling as follows: the first step, 2000 rpm for 2 min with the polymer solution; the second step, 400 rpm for 1 min after the addition of 4.5 mL of distilled water. After nano-pulverization, each MEL suspension containing 20 mg of milled MEL and 20 mg of the polymer in a 10 mL vial was frozen in liquid nitrogen and freeze-dried using a FD-81 freeze dryer (Tokyo Rikakikai, Tokyo, Japan).

2.1. Materials 2.5. XRPD analysis Crystalline MEL was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Hydroxypropyl cellulose (HPC) was obtained from Nippon Soda Co., Ltd. (Tokyo, Japan). Polyvinylpyrrolidone (PVP) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Eudragit E PO ((poly(butyl methacrylate-co(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) 1:2:1), Eudragit L 100 (poly(methacylic acid-co-methyl methacrylate) 1:1) and Eudragit RL PO (poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.2) were provided by Evonik Industries (Darmstadt, Germany). POVACOAT Type F (polyvinyl alcohol/acrylic acid/methyl methacrylate copolymer) was kindly supplied by Daido Chemical Co., Ltd. (Kobe, Japan). Hydroxypropylmethylcellulose acetate succinate (HPMC-AS) was provided by Shin-Etsu Chemical (Tokyo, Japan). Acetonitrile (liquid chromatography grade) was purchased from Kanto Chemical (Tokyo, Japan). All other chemicals were purchased from commercial sources. 2.2. HPLC/UV analysis The concentration of MEL was determined by an absolute calibration method using a high-performance liquid chromatography (HPLC) system equipped with a diode array detector (Shimadzu, Kyoto, Japan). The HPLC system consisted of a LC20A solvent delivery unit with high-pressure flow-line selection valves, a SIL-20A auto sampler, a CTO-20A column oven, and a SPDM20A diode array detector connected to the LC solution software. Chromatography was conducted using a COSMOSIL 5C18-AR-II column (particle size: 5 mm, column dimensions: 4.6 mm 150 mm). Column temperature was maintained at 40 C, and the samples were separated using a mobile phase consisting of 20 mM phosphate buffer (pH 7.0) and acetonitrile (3/7, v/v) at a flow rate of 0.5 mL/min. The diode array detector was set at 360 nm. 2.3. Anti-precipitant screening by the solvent shift method Polymers were dissolved or homogeneous-dispersed in distilled water at 150 mg/mL. Hydrophilic polymer solution or

XRPD patterns were collected with a RINT diffractometer (Rigaku Co., Tokyo, Japan) with Cu Ka radiation generated at 40 mA and 40 kV. Data were obtained from 5 to 40 (2u ) at a step size of 0.2 and a scanning speed of 5 /min. 2.6. Particle size distribution The particle size distribution and specific surface areas of the MEL samples in water were determined by a laser diffraction/scattering method using a Mastersizer 2000 equipped with a Hydro 2000 mP (Malvern Instruments Ltd., Worcestershire, UK). Volume percent of micron size aggregate was calculated using the total sum of volume percent of particles with particle size bigger than 1 mm. MEL sample (16 mg) was suspended in 2.0 mL of distilled water and dispersed gently without sonication. The particle size distribution was expressed as the volume median diameter and SPAN factor defined as SPAN = (d90 d10)/d50, where d10,d50, and d90 are the particle diameters at 10%, 50%, and 90% of the cumulative volume, respectively. A high SPAN value indicates a wide size distribution. 2.7. Particle size stability study MEL samples were placed in sealed vials under 60 C for 21 days, then were subjected to particle size distribution analysis. 2.8. Dissolution test Dissolution tests were carried out in 900 mL of HCl solution (pH 1.2) using a Japanese Pharmacopeia dissolution apparatus I (NTR-VS6, Toyama Sangyo Co., Ltd., Osaka, Japan). The rotating basket method was used, with constant stirring at 100 rpm at 37 C. Each MEL sample was weighed to keep the total amount of MEL in the dissolution vessel constant at 15 mg. 1 mL of samples were collected at 7.5, 15, 30, 60, 90 and 120 min using a pipette. It was not replaced. The sampling site was 1 cm from the test vessel wall at ca. 25 mm below the surface of the medium. Samples were centrifuged at 15,000 g for 15 min to remove insoluble materials.


The concentration of MEL in the supernatant was determined as described in Section 2.2.

Table 1 Polymer screening. Polymer

2.9. Animals Male Sprague-Dawley (SD) rats (290.94–407.4 g in weight; Japan SLC, Shizuoka, Japan) were housed in the laboratory with free access to food and water. All procedures used in the present study were approved by the Ethical Review Committee of Setsunan University. 2.10. Plasma concentration of MEL after oral administration Male SD rats were fasted overnight before the experiment, but given free access to water. A dose equivalent to 1.0 mg-MEL/kg body weight was administered orally. The suspensions were prepared with distilled water; the dosing concentration was 0.5 mg-MEL/mL. Blood samples were collected from the jugular vein at 0, 0.5, 1, 2, 4, and 8 h after oral administration. Each blood sample was centrifuged at 3000 rpm for 10 min to prepare plasma samples. Plasma MEL concentrations were determined by HPLC/UV. Briefly, 75 mL of acetonitrile was added to 75 mL of plasma sample, and then centrifuged at 10,000 g for 5 min. The supernatant was analyzed on a Shimadzu HPLC system using a gradient mobile phase consisting of (A) 20 mM phosphate buffer (pH 7.0) and (B) acetonitrile (3/7, v/v). The gradient condition of the mobile phase was 0–0.5 min, 70% A; 0.5–5.5 min, 70–55% A; 5.51–6.0 min, 40% A. The flow rate was 0.5 mL/min. Cmax and the area under the curve of plasma MEL concentration were calculated using GastroPlus version 8.4 (Northern Science Consulting Inc.). 2.11. Statistical analysis Comparisons between two experimental groups were carried out using the F-test and unpaired Student’s t-test. A P value of less than 0.05 was considered statistically significant for all analyses. 3. Results and discussion 3.1. Selection of polymer In this study, 96-well filter-plate-based anti-precipitant screening was conducted to help identify the most promising polymer candidates for incorporation into non-aggregating MEL nanocrystal formulations. Anti-precipitant screening was conducted with the hydrophilic polymers Eudragit E PO, Eudragit L 100, Eudragit RL PO, HPMCAS-LF, HPMCAS-HF, POVACOAT Type F, HPC-SSL, HPC-L, PVP K-30, and PVP K-90. The dissolved concentration of MEL in the filtered solution with or without hydrophilic polymer is listed in Table 1. The concentration of MEL in the filtered solution was 7.49 1.86 mg/mL. After 1 h, the supersaturated solution decreased to a saturated solution in the absence of polymers due to precipitation and crystallization. In contrast, the dissolved concentrations of MEL in filtered solution in the presence of HPMCAS-LF, HPMCAS-HF, POVACOAT Type F, HPC-SSL, HPC-L, PVP K-30, and PVP K-90 solution remained above saturation level: the concentration of MEL after 1 h was 26.2 0.75, 25.0 3.17, 27.1 2.06, 39.2 7.54, 40.2 5.30, 103 17.3, and 79.0 25.9 mg/mL, respectively. The supersaturation ratio (dissolved concentration (C)/equilibrium solubility (Ceq)) of MEL in HPMCAS-LF, HPMCAS-HF, POVACOAT Type F, HPC-SSL, HPC-L, PVP K-30, and PVP K-90 solution was calculated to be 3.50, 3.34, 3.61, 5.23, 5.37,13.7, and 10.5 respectively. Generally, factors such as drug-polymer interactions, pH, and viscosity can significantly affect crystallization profiles (Chauhan et al., 2013). The viscosity and pH of the polymer solutions used are listed in Table 1. Because no correlation was observed between the

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Without polymer Eudragit E PO Eudragit L 100 Eudragit RL PO HPMCAS-LF HPMCAS-HF POVACOAT Type F HPC-SSL HPC-L PVP K-30 PVP K-90

MEL dissolved concentration (mg/mL)

7.49 1.86 6.69 1.12 5.67 0.05 2.79 0.27 26.2 0.75 25.0 3.17 27.1 2.06 39.2 7.54 40.2 5.30 103 17.3 79.0 25.9

Supersaturation number (C/Ceq)

1.00 0.89 0.76 0.37 3.5 3.34 3.61 5.23 5.37 13.7 10.5

Polymer solution pH

Viscosity (mPa s)

5.66 N.D. N.D. N.D. 6.04 5.37 6.52 6.13 5.6 5.62 7.05

0.94 N.D. N.D. N.D. 1.09 1.13 1.08 1.11 1.03 1.14 1.16

Data represent mean SD (n = 3). N.D.: not determined.

pH or the viscosity of the polymer solution and the precipitation profile of MEL, the polymer’s effect on MEL precipitation is largely due to drug–polymer interactions. The stabilization ability of polymers and the subsequent steady state particle size of drug nanocrystals depend on various interactions including ionic interactions, hydrogen bonding, dipole-induced forces, and van der Waals and London interactions. So far, the effect of functional groups on the stability of wet-milled nanocrystals has been considered only in few studies (Choi et al., 2005; Lee et al., 2005; Peltonen and Hirvonen, 2010). Thus, the reason why PVP K-30 provided the highest supersaturation ratio may be because PVP K-30 could inhibit MEL crystal growth by interacting with moieties associated with nucleation and crystal growth of MEL. The difference between HPC-SSL and HPC-L, PVP K-30 and PVP K-90 are degree of polymerization. Molecular wight of HPC-SSL, HPC-L, PVP K-30, and PVP K-90 were 15,000–30,000, 55,000–70,000, 40,000, and 630,000, respectively. Under the test conditions, the efficiency of crystal growth inhibition did not significantly change with the degree of polymerization. On the other hand, the difference between HPMCAS-LF and HPMCAS-HF is the ratio of acetyl and succinoyl substitution. The acetyl substitution of HPMCAS-LF and HPMCAS-HF were 5.0–9.0 and 10.0–14.0, respectively. The succinoyl substituent ratio of HPMCAS-LF and HPMCAS-HF were 14.0–18.0 and 4.0–8.0, respectively. Under the test conditions, the efficiency of crystal growth inhibition did not significantly change with the ratio of acetyl and succinoyl substitution. Polymers with a low degree of polymerization would be suitable for stabilizing nanocrystal formulations because polymers with high degree of polymerization could enhance flock aggregation (Golas et al., 2010). Based on these findings, nanocrystal formulations were prepared with PVP K-30 (MEL/PVP), HPC-SSL (MEL/HPC), and POVACOAT Type F (MEL/POVA). 3.2. Sample preparation and their characterization Nano-pulverization was conducted with a revolution/rotation mixer, NP-100 (Thinky Company Ltd., Tokyo, Japan). It is superior in quick pulverization with a few minutes and it can scale up from the mg order of compounds to the kg order (Takatsuka et al., 2009). The particle size distribution of unpulverized MEL and freeze dried nanocrystal formulations were evaluated by laser diffraction analysis (Fig. 1 and Table 2). These samples were dispersed gently without sonication in distilled water. The mass median diameter (D50) of unpulverized MEL was calculated to be 13.1 mm with SPAN factor of 2.19, in contrast, MEL/PVP, MEL/HPC, and MEL/POVA exhibited a D50 of as low as 119, 117, and 111 nm, respectively. The SPAN factors of MEL/PVP, MEL/HPC, and MEL/POVA were

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Fig. 1. Size distribution of MEL samples in water, as determined by laser diffraction. (a) Immediately after preparation and (b) after 60 C, 21 days. (I) MEL/PVP, (II) MEL/HPC, (III) MEL/POVA, and (IV) unpulverized MEL.

calculated to be 1.29, 1.01, and 1.35, respectively, suggesting that the milled particles were moderately homogeneous. Reducing the particle size increases the surface area. The specific surface area of unpulverized MEL, MEL/PVP, MEL/HPC, and MEL/POVA was calculated to be 0.674, 52.0, 52.3, and 63.0 m2/g, respectively, indicating that the specific surface areas of MEL/PVP, MEL/HPC, and MEL/POVA were increased 77.6-, 78.1-, and 94.0-fold, respectively, over unpulverized MEL. The physicochemical properties of the nanocrystal formulations were further characterized using XRPD. The XRPD pattern of unpulverized MEL indicated several characteristic peaks of most stable form (form I). Similar diffraction

patterns were also observed for MEL/PVP, MEL/HPC, and MEL/POVA (Fig. 2), indicating the high crystallinity of MEL in the formulation and maintenance of the most stable form. High physicochemical stability of a drug is indispensable for sufficient therapeutic efficacy and safe therapy. Nanocrystal formulations were stored at 60 C for 21 days; the physicochemical stability of the MEL samples before and after storage was studied by particle size distribution. Solubility and dissolution rate of nanoparticles are reduced considerably by increasing the specific surface area and micron-sized aggregates (Kipp, 2004). The specific surface area of MEL/HPC was decreased by 9.37% between initial and 60 C, for 21 days. In contrast, only 2.69% of decrease was observed about the specific surface area of MEL/PVP between initial and 60 C, for 21 days. Based on these findings, the physical stability of a nanocrystal formulation appears to be related to the crystal growth inhibition effect of polymers in supersaturated MEL solution. The micron-sized particles in the suspension of MEL/HPC and MEL/POVA were disappeared after strong sonication. Thus, micron-sized particles in suspension of MEL/HPC and MEL/POVA were thought to be aggregates (data not shown). A significant increase in micron-sized aggregates was observed in MEL/POVA (+7.80%) and MEL/HPC (+99.3%) after storage. In particular, the nanoparticle population completely disappeared in MEL/POVA, leaving a population with a D50 of 5.31 mm; the specific surface area decreased from 63.0 to 1.48 m2/g during this time. In contrast, the increase in micron-sized aggregates after storage was negligible in MEL/PVP (+0.72%). Similar results have often been obtained in studies on amorphous solid dispersion formulations (Chauhan et al., 2013; Ozaki et al., 2013). A correlation was observed in the crystal growth inhibition efficiency of a polymer in the solution state and its amorphous stabilization in the solid state. This correlation is likely due to the strength of drug-polymer interactions. The ratio of the number of molecules on the surface of a nanoparticle to the total number of molecules in a particle increases with a decrease in particle size. The number of intermolecular interaction of a molecule on the surface with neighboring molecules would be less than that of a molecule in the interior of a nanoparticle. Thus, the molecular mobility of a molecule on the surface of a nanoparticle would be higher than that of a molecule in the interior of a nanoparticle. The increase in molecular mobility of a surface molecule could be one reason for the decreased physicochemical stability and aggregation of nanocrystal formulations. Thus, stabilization of surface molecules of a drug nanoparticle with an appropriate polymer could stabilize the nanocrystal formulation. Consequently, the screening and identification of anti-precipitants could help identify aggregation

Table 2 Physicochemical properties of MEL nanocrystal formulations. Unpulverized MEL Nanocrystal formulation with

Polymer screening Supersaturation number (C/Ceq) Particle size distribution Soon after preparation D50 (mm) D90 (mm) Specific surface area (m2/g) After 60 C, 21 days D50 (mm) D90 (mm) Specific surface area (m2/g) XRPD Crystallinity

PVP

HPC

POVA

1.00

13.74

5.23

3.61

13.1 33 0.67

0.119 0.226 52

0.117 0.195 52.3

0.111 0.208 63

– – –

0.123 0.248 50.6

0.132 1.25 47.4

5.31 22.5 1.48

form I

form I

form I

form I

Fig. 2. X-ray powder diffraction patterns of MEL samples. (I) MEL/PVP, (II) MEL/HPC, (III) MEL/POVA, and (IV) unpulverized MEL.


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inhibitors for nanocrystal formulations. Although high-throughput anti-precipitant screening can help narrow down the candidates for stabilizing nanocrystal formulation, there are some limitations to this approach. For example, some combinations of polymers and drugs induce a polymorphic transformation of the drug (Lin et al., 2010; Pakula et al., 1977). Because the rate of crystal nucleation and the growth of metastable forms could be different from that of the most stable form, further clarification of crystallinity will be helpful for avoiding misinterpretation of the data. In addition, surfactants cannot be screened using the anti-precipitant method because their ability to prevent precipitation could lead to overestimation of the solubility of the drug. The most reliable factors stabilizing nanocrystal formulation are still under discussion; however, a combination identifying efficacious polymer-drug interactions and screening of anti-precipitants may be useful for developing accurate screening methods for nanocrystal formulation stabilizers.

Fig. 4. Plasma MEL concentration–time profiles in rats after oral administration of MEL samples. , MEL/PVP; and , unpulverized MEL (1.0 mg-MEL/kg body weight). Each bar represents mean SD (n = 3–4).

3.3. Dissolution behavior of MEL nanocrystal

3.4. Pharmacokinetic profiling of a nanocrystal formulation

Dissolution is the most important factor for the rapid onset of pharmacological effects of a drug. However, unpulverized MEL dissolved extremely slowly in acidic and neutral media: after 120 min, 0.065 mg/mL of MEL dissolved from unpulverized MEL in acidic medium (Fig. 3) and 7.92 mg/mL dissolved in neutral medium. In contrast, all tested nanocrystal formulations showed improved dissolution behavior in acidic medium: the amount of dissolved MEL from MEL/PVP, MEL/HPC, and MEL/POVA at 7.5 min was 1.50, 1.71, and 2.72 mg/mL, respectively. Improved dissolution was also observed in neutral medium; for example, the amount of dissolved MEL after 10 min was 16.5 mg/mL (MEL/PVP), 15.5 mg/mL (MEL/HPC), and 10.2 mg/mL (MEL/POVA). These observations were consistent with previous reports, showing that the nanocrystal formulation of poorly soluble drugs could lead to a marked improvement in the dissolution properties (Onoue et al., 2009). Reducing the particle size of a compound increases its surface area and dissolution rate, as described by the Nernst–Brunner and Levich modifications of the Noyes–Whitney equation for dissolution (Dressman et al., 1998; Horter and Dressman, 2001). Solubility is also enhanced by reducing the particle size to the nanometer scale, according to the Ostwald– Freundlich equation (Kipp, 2004). Taking into account both its dissolution properties and physicochemical stability, MEL/PVP was the most suitable nanocrystal formulation for in vivo testing among the nanocrystal formulations tested.

According to the biopharmaceutics classification system (BCS) (Amidon et al., 1995), MEL is categorized into BCS class II and is identified as having low solubility and high membrane permeability (Nassab et al., 2006). Generally, the bioavailability of a BCS class II drug is rate-limited by its dissolution, and a small increase in dissolution rate sometimes results in a large increase in bioavailability (Lobenberg and Amidon, 2000). The marked increase in the dissolution rate and solubility of MEL/PVP was studied in rats to verify the pharmacokinetic behavior and enhanced bioavailability. Fig. 4 shows the plasma concentrationtime profile of MEL in rats after oral administration of MEL/PVP and unpulverized MEL (1.0 mg-MEL/kg); relevant pharmacokinetic parameters including Cmax, Tmax, AUC0–2, and AUC0–8 are listed in Table 3. Oral administration of unpulverized MEL to rats resulted in a gradual elevation in plasma MEL concentration, up to a Cmax of 0.43 mg/mL with a Tmax of 2.67 h. In contrast, the pharmacokinetic behavior of MEL/PVP was better than that of unpulverized MEL. Oral administration of MEL/PVP resulted in a rapid elevation in plasma MEL levels, up to a Cmax of 2.87 mg/mL with a Tmax of 0.63 h. Thus, the Cmax and AUC0–2 of MEL/PVP were 6.7- and 5.0-fold higher, respectively, and Tmax was shorter by 2.04 h, compared with unpulverized MEL-administered rats. The patients with rheumatoid arthritis and osteoarthritis often experience acute exacerbations of rheumatism and osteoarthritis. Since fast absorption and rapid onset of action were important for the relief of patient pain as early as possible, MEL/PVP could contribute to more efficacious treatment for the management of acute exacerbations. The mechanism of action of meloxicam, like that of other NSAIDs, is prostaglandin synthetase inhibition. Ibuprofen sodium dihydrate, fast dissolved salts of ibuprofen, could reduce median time to substantial pain relief 14 min earlier Table 3 Pharmacokinetic parameters of PVP/MEL following oral administration in rats. Unpulverized MEL

Fig. 3. Dissolution profiles of MEL samples in pH 1.2 solution. , MEL/PVP; &, MEL/HPC; 4, MEL/POVA; and , unpulverized MEL. Each bar represents mean SD (n = 3).

PVP/MEL

Cmax (mg/mL) Tmax (h)

0.43 0.10 2.67 1.15

2.87 0.62** 0.63 0.25*

AUC (mg h/mL) 0–2 h 0–8 h

0.35 0.21 2.21 0.50

4.15 0.85*** 11.21 2.97**

Cmax: maximum concentration; Tmax: time to maximum concentration; AUC: area under the curve of plasma MEL concentration vs. time from 0 to t (h) after oral administration. Data represent mean SD (n = 4). * P < 0.05 with respect to control (unpulverized MEL). ** P < 0.01. *** P < 0.001.

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in subjects with moderate-to-severe pain after extraction of third molars (Schleier et al., 2007), and these findings would provide further support for the effectiveness of fast-absorbing formulation in improving the therapeutic potential of MEL/PVP in pain patients. Previously, Alladi and Shastri attempted to improve therapeutic efficacy of MEL using fast-dissolving formulation containing oil surfactant, Gelucire 44/14 (Alladi and Shastri, 2014). Similar to our outcomes, the enhanced dissolution behavior was observed. Furthermore, rapid release of MEL with improved dissolution resulted in rapid in vivo anti-inflammatory activity. However, the toxic potential of Gelucire 44/14 was not acceptable for chronic use because treatment of Caco-2 cells with Gelucire 44/14 resulted in decreased cell viability and reduced expression and activity of P-glycoprotein (Sachs-Barrable et al., 2007). In contrast, PVP has been used as ingredients in the marketed drugs and the safety of PVP K-30 was confirmed. Safety information, taken together with dissolution and pharmacokinetic behaviors, suggested that newly developed MEL/PVP may provide safe medication with rapid onset of action. Moreover, nanocrystal formulation has the advantage of reducing food effects of drugs (Jinno et al., 2006); therefore, MEL/PVP might be useful for treating patients with anorexia caused by severe pain. 4. Conclusion In the present study, three nanocrystal formulations of MEL were prepared using wet-milling technology and their physicochemical properties were characterized. There were significant improvements in the dissolution behavior of all the MEL nanocrystal formulations prepared. Although a significant increase in aggregation was observed in MEL/POVA and MEL/HPC after storage at 60 C for 21 days, the increase in aggregation in MEL/PVP was negligible. This hydrophilic polymer helps maintain supersaturation and tends to prevent nanoparticle growth. According to the pharmacokinetic profiling of MEL/PVP, the rapid and highly systemic exposure of MEL after oral administration of MEL/PVP was significantly enhanced compared to that of unpulverized MEL. These observations suggest that the nano-pulverization approach could be a promising formulation strategy for enhancing the bioavailability of MEL and providing a rapid onset of pharmacological effects for the treatment of osteoarthritis. References Abdelwahed, W., Degobert, G., Stainmesse, S., Fessi, H., 2006. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv. Drug Deliv. Rev. 58, 1688–1713. Alladi, S., Shastri, N.R., 2014. Semi solid matrix formulations of meloxicam and tenoxicam: an in vitro and in vivo evaluation. Arch. Pharm. Res. . Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12, 413–420. Aoki, T., Yamaguchi, H., Naito, H., Shiiki, K., Izawa, K., Ota, Y., Sakamoto, H., Kaneko, A., 2006. Premedication with cyclooxygenase-2 inhibitor meloxicam reduced postoperative pain in patients after oral surgery. Int. J. Oral Maxillofac. Surg. 35, 613–617. Chauhan, H., Hui-Gu, C., Atef, E., 2013. Correlating the behavior of polymers in solution as precipitation inhibitor to its amorphous stabilization ability in solid dispersions. J. Pharm. Sci. 102, 1924–1935. Choi, J.-Y., Yoo, J.Y., Kwak, H.-S., Uk Nam, B., Lee, J., 2005. Role of polymeric stabilizers for drug nanocrystal dispersions. Curr. Appl. Phys. 5, 472–474.

Dressman, J.B., Amidon, G.L., Reppas, C., Shah, V.P., 1998. Dissolution testing as a prognostic tool for oral drug absorption: immediate release dosage forms. Pharm. Res. 15, 11–22. Furst, D.E., 1997. Meloxicam: selective COX-2 inhibition in clinical practice. Semin. Arthritis Rheum. 26, 21–27. Ghorab, M.M., Abdel-Salam, H.M., El-Sayad, M.A., Mekhel, M.M., 2004. Tablet formulation containing meloxicam and beta-cyclodextrin: mechanical characterization and bioavailability evaluation. AAPS PharmSciTech 5, e59. Golas, P.L., Louie, S., Lowry, G.V., Matyjaszewski, K., Tilton, R.D., 2010. Comparative study of polymeric stabilizers for magnetite nanoparticles using ATRP. Langmuir 26, 16890–16900. He, C.X., He, Z.G., Gao, J.Q., 2010. Microemulsions as drug delivery systems to improve the solubility and the bioavailability of poorly water-soluble drugs. Expert Opin. Drug Deliv. 7, 445–460. Horter, D., Dressman, J.B., 2001. Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Adv. Drug Deliv. Rev. 46, 75–87. Hosie, J., Distel, M., Bluhmki, E., 1996. Meloxicam in osteoarthritis: a 6-month, double-blind comparison with diclofenac sodium. Br. J. Rheumatol. 35, 39–43. Jinno, J., Kamada, N., Miyake, M., Yamada, K., Mukai, T., Odomi, M., Toguchi, H., Liversidge, G.G., Higaki, K., Kimura, T., 2006. Effect of particle size reduction on dissolution and oral absorption of a poorly water-soluble drug, cilostazol, in beagle dogs. J. Control. Release 111, 56–64. Kipp, J.E., 2004. The role of solid nanoparticle technology in the parenteral delivery of poorly water-soluble drugs. Int. J. Pharm. 284, 109–122. Lee, J., Lee, S.J., Choi, J.Y., Yoo, J.Y., Ahn, C.H., 2005. Amphiphilic amino acid copolymers as stabilizers for the preparation of nanocrystal dispersion. Eur. J. Pharm. Sci. 24, 441–449. Leuner, C., Dressman, J., 2000. Improving drug solubility for oral delivery using solid dispersions. Eur. J. Pharm. Biopharm. 50, 47–60. Lin, S.Y., Hsu, C.H., Ke, W.T., 2010. Solid-state transformation of different gabapentin polymorphs upon milling and co-milling. Int. J. Pharm. 396, 83–90. Lobenberg, R., Amidon, G.L., 2000. Modern bioavailability, bioequivalence and biopharmaceutics classification system. New scientific approaches to international regulatory standards. Eur. J. Pharm. Biopharm. 50, 3–12. Merisko-Liversidge, E., Liversidge, G.G., Cooper, E.R., 2003. Nanosizing: a formulation approach for poorly-water-soluble compounds. Eur. J. Pharm. Sci. 18, 113–120. Nassab, P., Rajkó, R., Szabó-Révész, P., 2006. Physicochemical characterization of meloxicam–mannitol binary systems. J. Pharm. Biomed. Anal. 41, 1191–1197. Onoue, S., Takahashi, H., Kawabata, Y., Seto, Y., Hatanaka, J., Timmermann, B., Yamada, S., 2009. Formulation design and photochemical studies on nanocrystal solid dispersion of curcumin with improved oral bioavailability. J. Pharm. Sci. 99, 1871–1881. Ozaki, S., Kushida, I., Yamashita, T., Hasebe, T., Shirai, O., Kano, K., 2013. Inhibition of crystal nucleation and growth by water-soluble polymers and its impact on the supersaturation profiles of amorphous drugs. J. Pharm. Sci. 102, 2273–2281. Pairet, M., van Ryn, J., Schierok, H., Mauz, A., Trummlitz, G., Engelhardt, G., 1998. Differential inhibition of cyclooxygenases-1 and -2 by meloxicam and its 40 -isomer. Inflamm. Res. 47, 270–276. Pakula, R., Pichnej, L., Spychala, S., Butkiewicz, K., 1977. Polymorphism of indomethacin. Part I. Preparation of polymorphic forms of indomethacin. Pol. J. Pharmacol. Pharm. 29, 151–156. Peltonen, L., Hirvonen, J., 2010. Pharmaceutical nanocrystals by nanomilling: critical process parameters, particle fracturing and stabilization methods. J. Pharm. Pharmacol. 62, 1569–1579. Quan, P., Shi, K., Piao, H., Liang, N., Xia, D., Cui, F., 2012. A novel surface modified nitrendipine nanocrystals with enhancement of bioavailability and stability. Int. J. Pharm. 430, 366–371. Sachs-Barrable, K., Thamboo, A., Lee, S.D., Wasan, K.M., 2007. Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoprotein mediated efflux of rhodamine 123 partially due to modifying P-glycoprotein protein expression within Caco-2 cells. J. Pharm. Pharmaceut. Sci. 10, 319–331. Schleier, P., Prochnau, A., Schmidt-Westhausen, A.M., Peters, H., Becker, J., Latz, T., Jackowski, J., Peters, E.U., Romanos, G.E., Zahn, B., Ludemann, J., Maares, J., Petersen, B., 2007. Ibuprofen sodium dihydrate, an ibuprofen formulation with improved absorption characteristics, provides faster and greater pain relief than ibuprofen acid. Int. J. Clin. Pharmacol. Ther. 45, 89–97. Serajuddin, A.T.M., 2007. Salt formation to improve drug solubility. Adv. Drug Deliv. Rev. 59, 603–616. Takatsuka, T., Endo, T., Jianguo, Y., Yuminoki, K., Hashimoto, N., 2009. Nanosizing of poorly water soluble compounds using rotation/revolution mixer. Chem. Pharm. Bull. 57, 1061–1067. Yamashita, T., Ozaki, S., Kushida, I., 2011. Solvent shift method for anti-precipitant screening of poorly soluble drugs using biorelevant medium and dimethyl sulfoxide. Int. J. Pharm. 419, 170–174.