- Email: [email protected]
Increasing the dissolution rate and oral bioavailability of the poorly water-soluble drug valsartan using novel hierarchical porous carbon monoliths
International Journal of Pharmaceutics 473 (2014) 375–383
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Increasing the dissolution rate and oral bioavailability of the poorly water-soluble drug valsartan using novel hierarchical porous carbon monoliths Yanzhuo Zhang a, * , Erxi Che b , Miao Zhang c , Baoxiang Sun c , Jian Gao a , Jin Han a , Yaling Song a a b c
School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China Pharmaceutical Division, Jiangsu Hengrui Pharma,Lianyungang 222047, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2014 Accepted 18 July 2014 Available online 19 July 2014
In the present study, a novel hierarchical porous carbon monolith (HPCM) with three-dimensionally (3D) ordered macropores (400 nm) and uniform accessible mesopores (5.2 nm) was synthesized via a facile dual-templating technique using colloidal silica nanospheres and Poloxamer 407 as templates. The feasibility of the prepared HPCM for oral drug delivery was studied. Valsartan (VAL) was chosen as a poorly water-soluble model drug and loaded into the HPCM matrix using the solvent evaporation method. Scanning electron microscopy (SEM) and specific surface area analysis were employed to characterize the drug-loaded HPCM-based formulation, confirming the successful inclusion of VAL into the nanopores of HPCM. Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) demonstrated that the incorporated drug in the HPCM matrix was in an amorphous state and the VAL formulation exhibited good physical stability for up to 6 months. In vitro tests showed that the dissolution rate of HPCM-based formulation was increased significantly compared with that of crystalline VAL or VAL-loaded 3D ordered macroporous carbon monoliths (OMCMs). Furthermore, a pharmacokinetic study in rats demonstrated about 2.4-fold increase in oral bioavailability of VAL in the case of HPCM-based formulation compared with the commercially available VAL preparation (Valzaar1). These results therefore suggest that HPCM is a promising carrier able to improve the dissolution rate and oral bioavailability of the poorly water-soluble drug VAL. ã 2014 Published by Elsevier B.V.
Keywords: Drug delivery Porous carbon monolith Dissolution rate Solubility Oral bioavailability
1. Introduction With the advent of combinatorial chemistry and high throughput in vitro pharmacology screening, the number of poorly watersoluble compounds has dramatically increased (Gardner et al., 2004; Lipinski, 2000). Active pharmaceutical ingredients (APIs) with poor aqueous solubility often demonstrate poor and erratic absorption when administrated orally due to the dissolution ratelimiting absorption in the gastrointestinal (GI) tract (Kawabata et al., 2011). It was reported that 70% of the potential drug candidates were discarded due to low bioavailability related with poor solubility in water before they ever reached the pharmaceutics department (Cooper, 2010). Hence, increasing the aqueous solubility and dissolution rate of poorly water-soluble APIs is a
* Corresponding author. Tel.: +86 516 83262116; fax: +86 516 83262116. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.ijpharm.2014.07.024 0378-5173/ ã 2014 Published by Elsevier B.V.
significant challenge to pharmaceutical scientists. Over recent decades, more and more strategies have been developed to overcome this obstacle for poorly water-soluble APIs, these strategies include reducing particle size to increase surface area (Gao et al., 2013; Merisko-Liversidge and Liversidge, 2011), enhancing the porosity (Hu et al., 2002), solubilization in surfactant systems (Chaubal, 2004), changing the drug crystalline state (Brough and Williams, 2013) and developing novel oral nanodrug delivery systems for immediate release (Nkansah et al., 2013; Zhang et al., 2012). During the past several years, porous materials (mesoporous silica, carbon nanotube, microporous hydroxyapatite, macroporous polymer, etc.) have been widely used in drug delivery field (Prakash et al., 2011; Tang et al., 2012). For the development of an inorganic carrier-based formulation to control drug release in the GI tract where no systemic absorption of the particles is desired, microparticles are preferred to nanoparticles, since cellular uptake is significantly lower making them toxicologically
376
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
less problematic (Sharma et al., 2012). Among the micrometersized porous materials, 3D ordered macroporous monoliths with well-defined surfaces, interconnected macroporous structures, and precisely controlled pore sizes in the sub-micrometer range have been proved to be useful as oral drug delivery platforms (Xie et al., 2012). Due to spatial confinement within the nanoscale pores, the encapsulated drug molecules are normally unable to form highly ordered crystals, instead remaining in microcrystalline or amorphous forms and leading to enhanced in vitro dissolution rate. However, 3D ordered macroporous monoliths generally lack mesopores in their matrices, which substantially limits their ability to highly disperse drugs down to the molecular level and the rate of mass transport (Wang et al., 2013). It is well known that mesoporous materials possess a larger inner surface area and pore volume to highly disperse the incorporated drug molecules, compared with ordered macroporous materials (Liang et al., 2008). Therefore, it is highly attractive to build up hierarchical porous architectures for 3D ordered macroporous monoliths by integrating small mesopore channels within interconnected macroporous matrices. Notably, HPCM is an important class of new-generation porous carbon materials, which exhibit a continuously interconnected macroporous structure and accessible uniform mesoporous porosity as walls, low mass density, high surface area and large pore volume (Huang et al., 2008; Wu et al., 2012). In addition to its mesopore channels being able to change the crystalline state of a drug to an amorphous one, the interconnected macropore and mesopore channels of HPCM may allow the dissolution media to easily penetrate into the particles and facilitate drug dissolution, compared with either a solely mesoporous equivalent or macroporous equivalent. All these properties demonstrate the potential and advantages of using HPCM in oral drug delivery. However, until now, no study has explored their potential in improving the dissolution and bioavailability of poorly water-soluble drugs following oral administration. In this study, a novel HPCM, with 3D ordered macropores (around 400 nm) and uniform accessible mesopores (around 5.2 nm), were successfully synthesized via a dual-templating approach and used as a potential carrier at first time. VAL (Fig. 1) is one of the angiotensin II receptor (AT1) antagonists recommended for treatment of hypertension, post-myocardial infarction or congestive heart failure (Chioléro and Burnier, 1998). It is a lipophilic compound (log P = 5.8) belongs to BCS class II drugs (low solubility and high permeability). The absolute bioavailability after oral administration of the solid dosage form of VAL is about 23% (Brookman et al., 1997). Therefore, improving the solubility and dissolution rate of such a drug is expected to enhance bioavailability and, hence, its therapeutic potency. The aim of this study was to develop a novel HPCM as an oral nano-drug carrier to improve the dissolution rate and increase the oral bioavailability of VAL. To achieve this goal, VAL-loaded HPCM was characterized in terms of morphology, macro-/mesoporous structures, specific surface area, physical state, solubility and in vitro dissolution. At the same time, the influence of the structural effect of the carbon
Fig. 1. Chemical structure of VAL.
matrix on the physicochemical properties of the VAL formulation was further studied by comparison with VAL-loaded OMCM. Finally, the in vivo pharmacokinetics of the VAL-loaded HPCM formulation was assessed in rats compared with the commercial VAL capsules. 2. Materials and methods 2.1. Materials Coarse VAL (purity more than 99%) and telmisartan were obtained from Huahai Pharma (Zhejiang, China) and used asreceived. Poloxamer 407 was kindly provided by BASF (Ludwigshafen, Germany). Tetramethyl orthosilicate, ammonium, furfuryl alcohol and oxalic acid were obtained from Kemiou Chemical Co. (Tianjin, China). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). PrestoBlue1 was purchased from Life Technologies (Carlsbad, CA, USA). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Solarbio (Beijing, China). Distilled water purified using a DW 200 purification system (Hitech Instruments, Shanghai, China) was used in all experiments. Commercially available VAL preparation (Valzaar1, Torrent Pharma, India) was chosen as the reference in the bioavailability study. 2.2. Cell line HT-29 human colon carcinoma cells were obtained from the American Type Culture Collection. The cells were cultured in DMEM medium, supplemented with 10% FBS, 100 U/ml penicillin, 1% (v/v) L-glutamine, 1% (v/v) non-essential amino acids and 0.1 mg/ml streptomycin at 37 C in a 5% CO2/95% air atmosphere. 2.3. Preparation of drug-loaded microparticles 2.3.1. Synthesis of spherical silica nanoparticles Nearly monodisperse spherical silica nanoparticles were prepared using a sol–gel method (Rao et al., 2005). Typically, 7.5 ml tetramethyl orthosilicate was mixed with 200 ml ethanol and the mixture was stirred at room temperature. After 10 min, 19 ml ammonium was added as a catalyst for the hydrolysis and condensation of tetramethyl orthosilicate. The resulting mixture was vigorously stirred for 20 h at 25 C. Finally, the obtained nanoparticles were collected by centrifugation, washed with ethanol, and dried in air to obtain the white silica powder. 2.3.2. Synthesis of HPCM and OMCM HPCM was synthesized using furfuryl alcohol as a carbon source, oxalic acid as a polymerization catalyst, silica nanospheres as a hard macroporous template and Poloxamer 407 as a soft mesoporous template. In a typical procedure, 0.9 g Poloxamer 407 was dissolved in 30 ml furfuryl alcohol containing 0.5% oxalic acid to obtain the carbon precursor. The precursor was stirred for 20 min prior to addition of 16 g silica nanospheres. The resulting suspension was stirred at 25 C for 30 min and then centrifuged at 4000 g for 20 min. After removing excess precursor solution, the resulting precipitate was heated to 90 C for 12 h to allow the polymerization of furfuryl alcohol. After polymerization, the polymer/silica composite was further heated to 400 C for 4 h under a nitrogen purge and subsequently heated to 700 C for 5 h to carbonize the polymer. Finally, the silica template in the carbon/ silica composite was dissolved in 5 M potassium hydroxide solution for 10 h to form HPCM. In addition, 3D OMCM was also synthesized following the above-mentioned procedure in the absence of Poloxamer 407 as the mesoporous template.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
2.3.3. Preparation of VAL-loaded HPCM The model drug VAL was loaded into the HPCM and OMCM samples using the solvent evaporation method. First, the synthesized HPCM powder (or OMCM) were micronized by grinding and passed through a 200 mesh sieve. Subsequently, a high concentration (240 mg/ml) of VAL solution was prepared by dissolving 7.2 g VAL in 30 ml ethanol solution (containing 0.3% Poloxamer 188). Then, 2.0 g HPCM (or OMCM) was immersed in 10 ml VAL solution and added to a round-bottom flask. After mechanical stirring for 4 h, the solvent was evaporated under reduced pressure in a rotary evaporator with a water bath set at a temperature at 40 C. Finally, the drug-loaded samples were further dried to remove the residual solvent in a vacuum oven at 25 C for 24 h. 2.4. Characterization of VAL-loaded HPCM 2.4.1. SEM study The particle morphology and macropore structure of the prepared porous carbon and drug-loaded samples were characterized by SEM. The micrographs were recorded on an S-4800 electron microscope from Hitachi (Tokyo, Japan). For SEM measurements, a small portion of the sample was adhered to an aluminum stud and sputter-coated with a thin layer of platinum prior to measurements being taken. 2.4.2. Transmission electron microscopy (TEM) study The mesoporous structure of the porous carbon samples was characterized by TEM. The micrographs of the particles were recorded on a Tecnai G2 electron microscope from FEI Instruments (Eindhoven, The Netherlands). For TEM measurements, a drop of a dilute ethanol dispersion of porous particles was placed on a carbon-coated copper grid and dried at room temperature. 2.4.3. Porosity characteristics Nitrogen adsorption measurements of the prepared samples were carried out using an ASAP 2010 accelerated surface area and porisimetry system from Micromeritics Instruments (Norcross, GA, USA). The calcined HPCM and OMCM samples were degassed at 200 C for 3 h prior to measurement, while the samples loaded with VAL and pure VAL powder were degassed at 40 C overnight in the degas port. During analysis, liquid nitrogen at 196 C maintained isothermal conditions. The Brunauer–Emmett–Teller (BET) specific surface area was calculated from adsorption isotherm data using ASAP 2010 software. 2.4.4. Differential scanning calorimetry (DSC) study DSC experiments were conducted to determine the melting point and the enthalpy of fusion of each sample using a Q2000 differential scanning calorimeter (TA Instruments, Brussels, Belgium). Two-point calibration using indium and tin was carried out to check the temperature axis and heat flow of the equipment. The accurately weighed samples (approximately 6 mg) were placed in standard aluminum pans and heated at a rate of 10 C/ min from 35 C to 160 C at a heating rate of 10 C/min. This was done in a dry nitrogen atmosphere at a flow rate of 40 ml/min, and an empty aluminum pan was used as a reference. All measurements were performed in triplicate. 2.4.5. Powder X-ray diffraction (PXRD) study PXRD was used to assess the degree of crystallinity of VAL after processing with HPCM. The analysis was carried out at ambient temperature using an X’Pert-Pro MPD diffractometer (PANlytical, The Netherlands) equipped with a Cu-Ka line as the source of radiation. An acceleration voltage and current of 40 kV and 30 mA were used. A sample of native crystalline VAL or VAL-loaded HPCM
377
(approximately 0.5 g) was loaded onto an aluminum sample holder and scanned in reflection mode between 4 and 45 (2u) with a scan speed of 0.06 2u/s and a step size of 0.02 . 2.5. Determination of drug loading in VAL formulation The level of drug loading in the VAL formulation was determined by dissolving an accurately weighed amount of VAL-loaded composites (about 10 mg) in 100 ml methanol. These suspensions were ultrasonicated for 15 min and subsequently put in a rotary mixer for 12 h. Then, the porous matrix was separated from the VAL solution by centrifugation (4000 g, 15 min). The supernatant was filtered with a 0.45 mm cellulose acetate filter, suitably diluted with mobile phase and then the drug concentration was determined by HPLC. The drug loading was calculated as: weight of drug in composites 100/weight of composites. Reverse-phase HPLC was performed on a Waters 2695 system consisting of a 1525 binary pump and a 2487 dual wavelength absorbance detector (Waters Corporation, USA). For the chromatographic separation of VAL, a Waters XBridgeTM analytical column (150 4.6 mm, 5 mm) was used, and the column oven was kept at 30 C. The wavelength for VAL detection was set at 225 nm. The determination was carried out by isocratical elution using an acetonitrile/water/acetic acid (50:50:0.1,v/v/v) mixture as the mobile phase at a flow rate of 1.0 ml/min. The calibration curve of VAL was linear (r2 = 0.99) over the concentration range of 0.2–25 mg/ml. 2.6. Solubility measurements The aqueous solubility of VAL was determined in purified water, 0.1 M HCl solution and phosphate buffer solution (PBS, pH 6.8) using the Higuchi and Connor method (Higuchi and Connors, 1965). Briefly, each VAL formulation (equivalent to 1 g VAL) was dispersed in 20 ml solvent and vortexed for 2 min to form a coarse suspension. Then, the resulting suspension was transferred to a screw-capped glass tube and incubated in a shaking water bath operated at 50 rpm at 37 C. After 48 h incubation, 2 ml aliquot of the incubated sample was filtered through a 0.45 mm cellulose acetate filter, and properly diluted in methanol prior to measuring the drug concentration by means of HPLC as described above. 2.7. Dissolution studies The dissolution of each VAL formulation was tested using a USP type II (paddle) dissolution apparatus (ZRS-8 G dissolution tester, Tianda Tianfa Technology Co., Ltd., China). Purified water, 0.1 M HCl (pH 1.2) and PBS (pH 6.8) were used as different dissolution media at a temperature of 37 0.1 C and a paddle speed of 100 rpm. Each prepared sample or the crude drug powder (equivalent to 20 mg VAL) was directly added to 900 ml dissolution medium, and 5 ml samples were withdrawn from the dissolution vessel at defined time intervals (5, 10, 15, 20, 30, 100 m, 45 and 60 min). The aliquot samples were filtered with a 0.45 mm cellulose acetate filter. The first 2 ml filtrate was discarded and the remainder was analyzed in an UV–visible spectrophotometer (UV2000, Unico, USA) at an absorption wavelength of 225 nm. Experiments were conducted in triplicate and the results were recorded as an average. 2.8. Accelerated stability test An accelerated stability study was carried out on the VAL formulation in accordance with ICH accelerated stress-stability conditions. Each sample was put into a screw-capped glass vial and then maintained at 40 C and 75% RH in a chamber kept at a
378
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
constant temperature and humidity. The samples were obtained after designated incubation times (1–3 and 6 months), and the crystallinity of the VAL-loaded HPCM sample was monitored by XRPD. In addition, the dissolution curves of VAL-loaded HPCM were also compared before and after storage. 2.9. In vitro cell viability assays HT-29 cells (4 103) were seeded onto 96-well microplates (Costars, Corning Inc., USA) containing DMEM with 10% FBS and then further incubated at 37 C in a 5% CO2/95% air atmosphere. After reaching 90% confluence, the cells were exposed to various concentrations of HPCM or OMCM (10, 25, 50, 75, 150 and 250 mg/ ml prepared in DMEM medium) for 48 h. Control cells were cultured with DMEM alone. After exposure, the culture medium was replaced with 10% PrestoBlue solution and the cells were incubated for an additional 30 min. Finally, the absorbance of each well was measured at 570 nm with a reference wavelength of 600 nm using a VictorTM Multilabel plate reader (PerkinElmer, USA). The cell viability was calculated as: absorbance of samples 100/absorbance of control.
2.10. Pharmacokinetic studies 2.10.1. Animal experiment and sample collection A pharmacokinetic study in Sprague-Dawley rats was designed to evaluate the VAL-loaded HPCM formulation by comparison with the commercial capsules (Valzaar1). The rats, weighing between 270 and 340 g, were housed in compliance with good laboratory practice (GLP) standard laboratory conditions and fed with commercial rodent chow and tap water. All in vivo experiments were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Institutional Animal Ethical Committee of Jiangsu Hengrui Pharma. The rats were fasted for at least 12 h prior to dosing but had free access to water and were randomly divided into 2 treatment groups (6 animals per group). The powder from commercial capsules and valsartanloaded HPCM were dispersed in an aqueous methylcellulose solution (0.1%, w/v) by simple vortexing for 30 s immediately prior to dosing. The dispersion was administered orally to rats at a single dose equivalent to 10 mg/kg of rat body weight via an oral gavage. At designated time intervals (0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h),
Fig. 2. SEM photographs of (a) OMCM and (b) HPCM; TEM photographs of (c) OMCM and (d) HPCM.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
the rats were anesthetized with ether and 0.5 ml blood samples were obtained by retro-orbital puncture, transferred to heparinized tubes, and then immediately centrifuged at 4000 g for 10 min to obtain plasma samples. The plasma samples were stored at 20 C for subsequent analysis. 2.10.2. Analysis of VAL in plasma samples To determine the VAL concentration, the plasma sample (100 ml) was mixed with 0.2 ml internal standard (telmisartan, 0.15 mg/ml in methanol) and vortex-mixed for 4 min. The sample was then centrifuged at 6000 g for 15 min. The supernatant was separated and evaporated at 40 C in a vacuum dryer. The residue was reconstituted with the mobile phase (100 ml), vortex-mixed for 1 min and centrifuged at 9000 g for 5 min. Then, a 20 ml aliquot of supernatant was injected into a 150 4.6 mm Shim-pack VP-ODS C18 column (Phenomenex, USA) and eluted isocratically, using an acetonitrile/phosphate solution (47:53, v/v) mixture as mobile phase, at a flow rate of 1.0 ml/min. The HPLC system (Shimadzu, Japan) equipped with a LC-10ATvp binary pump, an SCL-10Avp system controller and a RF 535 fluorescence detector
379
was used in this analysis. The effluent was monitored at excitation and emission wavelengths of 265 and 380 nm, respectively. The calibration curve for VAL was linear over the range 0.04–7.5 mg/ml, while the intra-day precision at three concentrations (0.04, 0.5, 7.5 mg/ml) was 4.1–12.7%, and the inter-day precision was less than 15%, showing acceptable precision and accuracy. The lower limit of quantification of VAL was 10 ng/ml. 2.10.3. Pharmacokinetic analysis Plasma levels of VAL in rats were plotted against time, and the pharmacokinetic parameters were calculated using a non-compartmental method via WinNonlin software (version 2.1; Pharsight Co., Mountain View, CA, USA). The area under the plasma concentration vs. time curve up to the last quantifiable time point, (AUC0 ! t), was obtained by the linear and log-linear trapezoidal method. The peak plasma concentration of drug (Cmax) and the time taken to reach the peak concentration (Tmax) were obtained directly from the plasma vs. time profile. The relative bioavailability (Fr) was calculated as: AUC0 ! t(test) 100/AUC0 ! t (reference).
Fig. 3. SEM photogrphs of (a) pure VAL, (b) VAL-loaded HPCM (low magnification), (c) VAL-loaded OMCM and (d) VAL-loaded HPCM.
380
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
2.10.4. Statistical analysis All results were expressed as average standard deviation (SD) from at least three independent experiments. For statistical analysis, a Student’s t test or one-way analysis of variance (ANOVA) test were used. Differences were considered to be statistically significant when p < 0.05. 3. Results and discussion 3.1. Porous structure characterization In the present study, highly monodisperse silica colloidal spheres, serving as a template for macropores in the synthesis of both HPCM and OMCM materials, have a uniform particle size of about 380 nm (Fig. S1 in the Supporting Information). SEM analyses reveal the presence of macropores in both HPCM and OMCM samples, in a honeycomb structure, as can be seen in Fig. 2a and b. These pores are large 3D ordered arrays of spherical cavities, which are linked by interconnecting windows. The windows were formed as a result of the contact between the silica spheres prior to infiltration of the precursor solution. The macropore diameters and pore window sizes of both samples were about 400 nm and 120 nm, respectively, as measured from the SEM images. The internal wall structures of HPCM and OMCM were analyzed by TEM. Based on the TEM observation, the organization of the pore system is observed both on the surface and throughout the monoliths. Another important piece of information that can be obtained from TEM images is that the synthesized HPCM has a hierarchical porous structure. A representative TEM image acquired at high magnification revealed disordered, worm-like mesoporous channels inside the walls between the macropores of HPCM, as shown in Fig. 2d inset. The existence of such mesopore channels (derived from Poloxamer 407) can significantly increase the specific surface area, prevent congestion and agglomerate the encapsulated drug particles, and reducing the crystallinity of the encapsulated drug particles. In contrast, no mesopores could be observed for OMCM (Fig. 2c). It can be seen that the morphology of the coarse VAL particles were non-uniform with a broad particle size distribution from 5 to 75 mm (Fig. 3a). Interestingly, after incorporation into HPCM and OMCM by the solvent deposition method, large VAL crystals disappeared and the outer pores of both HPCM and OMCM were blocked, suggesting the successful incorporation of VAL into both carriers. The representative SEM micrographs of drug-loaded HPCM and OMCM are presented in Fig. 3c and d. Many VAL nanoparticles with a diameter of around 20 nm are homogeneously distributed in the macropores of OMCM. Apparently, the morphology of the drug-loaded HPCM particles was the same as the drug-loaded OMCM particles, while drug particles on the outer pores became few. This indicates that most of VAL was loaded in the mesopores of HPCM, in addition to encapsulate uniformly into the macropores of HPCM, as confirmed by the nitrogen sorption study. Low-temperature nitrogen adsorption/desorption measurements were performed to further examine the porosity and calculate the specific surface area of the prepared HPCM and OMCM. The nitrogen adsorption/desorption isotherms of the calcined HPCM and OMCM are shown in Fig. 4. HPCM exhibits a type IV isotherm with a H2 hysteresis loop according to the IUPAC classification (Fig. 4a), indicating the presence of mesoporosity within the macroporous structure of HPCM (Cychosz et al., 2012; Kruk et al., 2002). The corresponding pore size distribution data calculated from the adsorption branch of isotherms shows that the pores of HPCM are uniform and centered at about 5.2 nm. In contrast, OMCM exhibits a type II isotherm (Fig. 4b), which is typical for nonporous and macroporous materials (Ge et al., 2012). These results agreed well with those of the TEM observations. The
Fig. 4. Nitrogen adsorption/desorption isotherms of (a) HPCM and (b) OMCM.
data for the BET specific surface area, total pore volume, Barrett– Joyner–Halenda (BJH) pore size and degree of drug loading of the corresponding samples are listed in Table 1. As shown, HPCM possesses a higher surface area and larger pore volume compared with OMCM (due to the formation of a mesoporous structure, produced by the surfactant of Poloxamer 407), confirming its potential spatial dispersion ability as a drug carrier. The study of the change in the specific surface area, pore diameter and volume of the prepared porous matrices before and after loading provided important information about the space occupied by VAL molecules (Kinnari et al., 2011). After drug loading, a decrease in specific surface area, pore diameter and pore volume was observed and these changes were attributed to the successful incorporation of VAL into the cavities of each matrix (Ambrogi et al., 2007; Wani et al., 2012). Interestingly, the sharp drop in specific surface area and pore volume for VAL-loaded HPCM is excess to the specific surface area and pore volume of OMCM, suggesting a large number of VAL molecules had been adsorbed onto the internal surface of the mesopore channels. 3.2. Solid-state characterization The drug loading process maybe change the solid state of the drug and, therefore, PXRD measurements was performed on the coarse VAL, the physical mixture (mass ratio of VAL:carrier: Poloxamer 188 was 2.4:2:0.03) and VAL-loaded samples. It can be seen that coarse VAL exhibits characteristic peaks (which are similar to those reported in the literature, Yana et al., 2012) over the 2u range in PXRD spectra (Fig. 5a), whereas both HPCM and OMCM matrices show a broad amorphous band (Fig. 5b and c). As expected, VAL characteristic peaks are present in both physical mixtures (Fig. 5d and e). On the other hand, the characteristic peaks of VAL for the VAL-loaded OMCM sample (Fig. 5f) were still present but weaker than that of the physical mixture, which illustrates that VAL absorbed into the pore channels or on the external surface of OMCM was partially present as nanocrystals and partially present as amorphous form. However, the
Table 1 Carrier characterization before and after VAL loading (n = 3). Sample
SBET (m2/g)
Vt (cm3/g)
WBJH (nm)
Drug loading (%)
HPCM OMCM VAL-loaded HPCM VAL-loaded OMCM
1290 65 473 24 381 50 94 27
1.35 0.16 0.82 0.13 0.23 0.07 0.19 0.03
5.2 1.4 – 2.9 0.6 –
– – 53.7 1.8 52.9 2.3
SBET, BET specific surface area; WBJH, Barrett–Joiner–Halenda pore diameter; Vt, total pore volume.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
381
observed in the DSC thermogram of VAL-loaded HPCM (Fig. 6e), indicating that the drug entrapped in the mesopore channels of HPCM was in an amorphous state, which agreed well with the XRD results. The non-crystalline state of VAL-loaded HPCM will be beneficial to increase the solubility and dissolution rate of VAL (Brough et al., 2013; Vasconcelos et al., 2007; Zhang et al., 2011). 3.3. Drug dissolution profiles
Fig. 5. PXRD patterns of (a) pure VAL, (b) HPCM, (c) OMCM, (d) physical mixture1 (VAL\HPCM\Poloxamer 188), (e) physical mixture2 (VAL\OMCM \Poloxamer 188), (f) VAL-loaded OMCM, (g) VAL-loaded HPCM and (h) VAL-loaded HPCM after 6 months of storage.
In vitro dissolution tests were performed in order to demonstrate the ability of both HPCM and OMCM to increase the rate and extent of dissolution of VAL in simulated gastric/intestinal fluid. The in vitro dissolution curves for coarse VAL and different VAL formulations in different dissolution media are shown in Fig. 7. The observed dissolution of coarse VAL was very limited in both purified water and 0.1 M HCl solution (less than 10% within 60 min). The slow dissolution can be attributed to its larger crystal size and very low solubility. The saturated solubility of coarse VAL was 0.03 0.01 mg/ml in 0.1 M HCl solution and 0.14 0.02 mg/ml in purified water. At pH 6.8, however, the dissolution rate of coarse
characteristic crystalline peaks disappeared in the PXRD pattern of VAL-loaded HPCM (Fig. 5g) producing an amorphous halo pattern, revealing that the crystallinity of VAL-loaded HPCM was decreased dramatically. This is related to finite-size effects, preventing the drug molecules entrapped in the mesopore channels from rearranging themselves in a crystal lattice (Jackson and McKenna, 1996). It should be noted that VAL-loaded HPCM showed no sign of recrystallization during the storage period (Fig. 5h). As a result, the stability test showed that VAL-loaded HPCM exhibited good physical stability under accelerated storage conditions for at least 6 months. The DSC thermograms of coarse VAL, the physical mixture and VAL-loaded samples are depicted in Fig. 6. A single sharp endothermic peak in the case of coarse VAL was observed at 102.6 C (Fig. 6a, the endothermic value was 59.7 1.6 J/g), which corresponded to its melting point. As expected, a similar endothermic peak was also visible in the DSC thermogram of the physical mixtures (Fig. 6b and c), suggesting that both porous carbon matrices do not change the solid state of VAL in the physical mixtures. For the VAL-loaded OMCM sample, a small endothermic peak was observed at 102.4 C. Compared with coarse VAL, the VAL-loaded OMCM sample showed a significant reduction in the enthalpy of fusion (Fig. 6d, the endothermic value was 11.4 3.0 J/ g), indicating the decrease in crystallinity of the drug and also suggesting VAL was partly converted into an amorphous state. Unlike the VAL-loaded OMCM samples, no endothermic peak was
Fig. 6. DSC thermograms of (a) pure VAL, (b) physical mixture1, (c) physical mixture2, (d) VAL-loaded OMCM, (e) VAL-loaded HPCM and (g) HPCM.
Fig. 7. Dissolution of VAL from different formulations in (A) 0.1 M HCl, (B) purified water and (C) PBS; (a) pure VAL, (b) physical mixture1, (c) physical mixture2, (d) VAL-loaded OMCM, (e) VAL-loaded HPCM and (f) VAL-loaded HPCM after 6 months of storage. Each data point represents the mean SD (n = 3).
382
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
VAL was higher due to its weak acidity (the saturated solubility of coarse VAL in PBS was 15.7 0.21 mg/ml). As expected, in three different dissolution media, the dissolution profiles of both physical mixtures were similar to that of coarse VAL, which showed that the mechanical physical mixing of coarse VAL, porous carbon matrices (HPCM and OMCM) and Poloxamer 188 had little effect on the dissolution of VAL. In the cases of both VAL-loaded porous carbon-based formulations, however, a dramatic increase in both the dissolution rate and extent was seen compared with that of coarse VAL. For example, the accumulated amounts of dissolved coarse VAL, VAL-loaded OMCM and VAL-loaded HPCM in purified water were 4.6%, 63%, and 92%, respectively, after 20 min. This outstanding increase in drug dissolution may be attributed to the decrease in particle size and crystallinity of VAL present in the pores of OMCM or HPCM, together with the increase in specific surface area and improved dispersibility of VAL particles (Kinnari et al., 2011; Wang, 2009). For the VAL-loaded OMCM sample, the particle size of the drug incorporated in the macropores (around 400 nm) was significantly reduced compared with that of coarse VAL (5–75 mm). It is obvious that a dramatically decrease in the particle size down to the nano scale will accelerate the drug dissolution and increase the drug solubility (Nkansah et al., 2013) (the saturated solubility of VALloaded OMCM was 0.07 0.01 mg/ml in 0.1 M HCl solution and 0.26 0.03 mg/ml in purified water). Meanwhile, as a result of the particle size reduction, the effective surface area increases, thereby leading to a higher dissolution rate according to the Noyes– Whitney equation (Brough and Williams, 2013; Dokoumetzidis and Macheras, 2006). The same phenomenon was observed when nimodipine was loaded into ordered macroporous chitosan (Xie et al., 2012). Compared with VAL-loaded OMCM, VAL-loaded HPCM exhibited a faster dissolution rate, irrespective of pH conditions. Two factors probably accounted for this phenomenon: (1) the existence of a lot of mesopores inside the walls between the macropores allows dissolution media to flow quickly inside the porous matrix and to dissolve more the entrapped VAL. (2) VAL is completely in amorphous state in VAL-loaded HPCM but partly in crystalline state in VAL-loaded OMCM (confirmed by the PXRD and DSC studies). It is well known the formation of the amorphous form could significantly improve the dissolution and solubility of poorly water-soluble drugs compared with the relevant crystalline form because there is no lattice energy to be overcome (Vasconcelos et al., 2007; Babu and Nangia, 2011). The saturated solubility of VAL-loaded HPCM was 0.09 0.01 mg/ml in 0.1 M HCl solution and 0.31 0.02 mg/ml in purified water. It can be seen clearly from Fig. 7, the release profiles of VAL from VAL-loaded HPCM were similar to that of the freshly prepared ones within 6 months storage. This significant dissolution enhancing effect provided by HPCM will be particularly beneficial for the improved delivery of poorly water-soluble drugs belonging to the BCS class II
Fig. 9. Plasma concentration-time profiles of the VAL formulations tested; (&) commercial preparation and (&) VAL-loaded HPCM. Each data point represents the mean SD (n = 6).
for which the oral bioavailability is limited by their poor dissolution rates. 3.4. In vitro cell viability HT-29 human colon carcinoma cells were treated with different porous carbon matrices (OMCM and HPCM) at different concentrations (10–250 mg/ml) for 48 h, and the cell viability was evaluated using Presto Blue reagent. As shown in Fig. 8, above 80% of cell viability was obtained, with no significant differences between results for both OMCM and HPCM. These suggest that both OMCM and HPCM are biocompatible with HT-29 cells and, therefore, well tolerated by the gastric/intestinal tract. 3.5. In vivo performance To further investigate the role of HPCM, the oral bioavailability of VAL-loaded HPCM-based formulation in Sprague-Dawley rats was compared with that of the commercial capsules (Valzaar1). The mean plasma VAL concentration after oral administration of each VAL formulation is shown in Fig. 9 and the corresponding pharmacokinetic parameters of VAL are listed in Table 2. As shown in Table 2, VAL-loaded HPCM demonstrated a higher Cmax of 4.71 1.56 mg/ml and a shorter Tmax of 1.42 0.38 h compared with oral commercial capsules (Cmax 2.32 1.28 mg/ml and Tmax 2.25 0.61 h). These results are in agreement with the dissolution studies showing that VAL was released from VAL-loaded HPCM much faster than that from crude VAL powder. The AUC0 ! 24 h value was also found to be significantly higher for the VAL-loaded HPCM formulation than for the commercial formulation. The high AUC0 ! 24 h value of VAL-loaded HPCM suggested that marked adsorption occurred after the quick release of VAL from HPCM in the GI tract. This underlines the important role of HPCM-based formulation in producing a high concentration gradient between the drug and the GI epithelium produced by a higher drug release. Interestingly, the relative bioavailability of VAL-loaded HPCM compared with the commercial formulation was 239.5 52.64%.
Table 2 Pharmacokinetic parameters of the VAL formulations tested (n = 6).
Fig. 8. Effect of HPCM and OMCM on HT-29 cell viability at various concentrations.
Parameters
VAL-loaded HPCM
Commercial capsules
Tmax (h) Cmax (mg/ml) AUC0 ! 24 h (mg h/ml) Fr (%)
1.42 0.38* 4.71 1.56* 33.26 9.20* 239.5 52.64
2.25 0.61 2.32 1.28 13.59 5.07 –
*
P < 0.05 compared with control formulation.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
4. Conclusions In summary, a novel HPCM with 3D ordered macropores (400 nm) and uniform accessible mesopores (5.2 nm) was designed, and the pharmaceutical performance of this material was studied. The VAL-loaded HPCM formulation showed a significantly higher drug release rate than pure VAL or VAL-loaded OMCM in simulated gastric/intestinal fluid. The pharmacokinetic data clearly showed that the VAL-loaded HPCM formulation produced a significant improvement in the bioavailability compared with the commercial capsules. HPCM may therefore be considered as a promising carrier to achieve enhanced dissolution rate and oral bioavailability of the poorly water-soluble drug VAL. Acknowledgement This work was partly funded by the National Basic Research Program of China(973 Program) (No. 2009CB930300). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.07.024. References Ambrogi, V., Perioli, L., Marmottini, F., Giovagnoli, S., Esposito, M., Rossi, C., 2007. Improvement of dissolution rate of piroxicam by inclusion into MCM-41 mesoporous silicate. Eur. J. Pharm. Sci 32, 216–222. Babu, N.J., Nangia, A., 2011. Solubility advantage of amorphous drugs and pharmaceutical cocrystals. Cryst. Growth Des. 1, 2662–2679. Brookman, L.J., Rolan, P.E., Benjamin, I.S., Palmer, K.R., Wyld, P.J., Lloyd, P., Flesch, G., Waldmeier, F., Sioufi, A., Mullins, F., 1997. Pharmacokinetics of valsartan in patients with liver disease. Clin. Pharmacol. Ther. 62, 272–278. Brough, C., Williams, R.O., 2013. Amorphous solid dispersions and nano-crystal technologies for poorly water-soluble drug delivery. Int. J. Pharm. 453, 157–166. Chaubal, M.V., 2004. Drug discovery today application of formulation technologies in lead candidate selection and optimization. Drug Discov. Today 9, 603–609. Chioléro, A., Burnier, M., 1998. Pharmacology of valsartan, an angiotensin II receptor antagonist. Expert Opin. Investig. Drugs 7, 1915–1925. Cooper, E.R., 2010. Nanoparticles: a personal experience for formulating poorly water soluble drugs. J. Control. Release 141, 300–302. Cychosz, K.A., Guo, X., Fan, W., Cimino, R., Gor, G., Tsapatsis, M., Neimark, A.V., Thommes, M., 2012. Characterization of the pore structure of threedimensionally ordered mesoporous carbons using high resolution gas sorption. Langmuir 28, 12647–12654. Dokoumetzidis, A., Macheras, P., 2006. A century of dissolution research: from Noyes and Whitney to the biopharmaceutics classification system. Int. J. Pharm. 321, 1–11. Gao, L., Liu, G., Ma, J., Wang, X., Zhou, L., Li, X., Wang, F., 2013. Application of drug nanocrystal technologies on oral drug delivery of poorly soluble drugs. Pharm. Res. 30, 307–324. Gardner, C.R., Walsh, C.T., Almarsson, Ö, 2004. Drugs as materials: valuing physical form in drug discovery. Nat. Rev. Drug Discov. 3, 926–934. Ge, J., Ding, H., Xue, X., 2012. A nanosheet-structured three-dimensional macroporous material with high ionic conductivity synthesized using glucose as a transforming template. Angew. Chem. Int. Ed. Engl. 51, 6205–6208.
383
Higuchi, T., Connors, K.A., 1965. Phase-solubility techniques. Adv. Anal. Chem. Instrum. 4, 117–212. Huang, Y., Cai, H., Feng, D., Gu, D., Deng, Y., Tu, B., Wang, H., Webley, P.A., Zhao, D., 2008. One-step hydrothermal synthesis of ordered mesostructured carbonaceous monoliths with hierarchical porosities. Chem. Commun. 23, 2641–2643. Hu, J., Rogers, T.L., Brown, J., Young, T., Johnston, K.P., Williams, R.O., 2002. Improvement of dissolution rates of poorly water soluble APIs using novel spray freezing into liquid technology. Pharm. Res. 19, 1278–1284. Kawabata, Y., Wada, K., Nakatani, M., Yamada, S., Onoue, S., 2011. Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications. Int. J. Pharm. 420, 1–10. Kinnari, P., Makila, E., Heikkila, T., Salonen, J., Hirvonen, J., Santos, H.A., 2011. Comparison of mesoporous silicon and non-ordered mesoporous silica materials as drug carriers for itraconazole. Int. J. Pharm. 414, 148–156. Kruk, M., Antochshuk, V., Matos, J.R., Mercuri, L.P., Jaroniec, M., 2002. Determination and tailoring the pore entrance size in ordered silicas with cage-like mesoporous structures. J. Am. Chem. Soc. 124, 768–769. Jackson, C.L., McKenna, G.B., 1996. Vitrification and crystallization of organic liquids confined to nanoscale pores. Chem. Mat. 8, 2128–2137. Liang, C., Li, Z., Dai, S., 2008. Mesoporous carbon materials: synthesis and modification. Angew. Chem. Int. Ed. Engl. 47, 3696–3717. Lipinski, C.A., 2000. Drug-like properties and the causes of poor solubility and poor permeability. J. Pharmacol. Toxicol. Methods 44, 235–249. Merisko-Liversidge, E., Liversidge, G.G., 2011. Nanosizing for oral and parenteral drug delivery: a perspective on formulating poorly-water soluble compounds using wet media milling technology. Adv. Drug Deliv. Rev. 63, 427–440. Nkansah, P., Antipas, A., Lu, Y., Varma, M., Rotter, C., Rago, B., El-Kattan, A., Taylor, G., Rubio, M., Litchfield, J., 2013. Development and evaluation of novel solid nanodispersion system for oral delivery of poorly water-soluble drugs. J. Control. Release 169, 150–161. Prakash, S., Malhotra, M., Shao, W., Tomaro-Duchesneau, C., Abbasi, S., 2011. Polymeric nanohybrids and functionalized carbon nanotubes as drug delivery carriers for cancer therapy. Adv. Drug Deliv. Rev. 63, 1340–1351. Rao, K.S., El-Hami, K., Kodaki, T., Matsushige, K., Makino, K., 2005. A novel method for synthesis of silica nanoparticles. J. Colloid. Interface Sci. 289, 125–131. Sharma, A., Madhunapantula, S.R.V., Robertson, G.P., 2012. Toxicological considerations when creating nanoparticle-based drugs and drug delivery systems? Expert Opin. Drug Metab. Toxicol. 8, 47–69. Tang, F., Li, L., Chen, D., 2012. Mesoporous silica nanoparticles: synthesis biocompatibility and drug delivery. Adv. Mater. 24, 1504–1534. Vasconcelos, T., Sarmento, B., Costa, P., 2007. Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discov. Today 12, 1068–1075. Wani, A., Muthuswamy, E., Savithra, G.H., Mao, G., Brock, S., Oupicky, D., 2012. Surface functionalization of mesoporous silica nanoparticles controls loading and release behavior of mitoxantrone. Pharm. Res. 29, 2407–2418. Wang, S., 2009. Ordered mesoporous materials for drug delivery. Micropor. Mesopor. Mater. 117, 1–9. Wu, Z.S., Sun, Y., Tan, Y.Z., Yang, S., Feng, X., Mullen, K., 2012. Three-dimensional graphene-based macro- and mesoporous frameworks for high-performance electrochemical capacitive energy storage. J. Am. Chem. Soc. 134, 19532–19535. Wang, Y., Zhao, Q., Hu, Y., Sun L, Bai L., Jiang, T., Wang, S., 2013. Ordered nanoporous silica as carriers for improved delivery of water insoluble drugs: a comparative study between three dimensional and two dimensional macroporous silica. Int. J. Nanomed. 8, 4015–4031. Xie, Y., Liu, Y., Wang, Y., Wang, S., Jiang, T., 2012. Chitosan matrix with three dimensionally ordered macroporous structure for nimodipine release. Carbohydr. Polym. 90, 1648–1655. Yana, Y.D., Sunga, J.H., Kima, K.K., Kimb, D.W., Kima, J.O., Leec, B.J., Yonga, C.S., Choi, H.G., 2012. Novel valsartan-loaded solid dispersion with enhanced bioavailability and no crystalline changes. Int. J. Pharm. 422, 202–210. Zhang, Y., Wang, J., Bai, X., Jiang, T., Zhang, Q., Wang, S., 2012. Mesoporous silica nanoparticles for increasing the oral bioavailability and permeation of poorly water soluble drugs. Mol. Pharm. 9, 505–513. Zhang, Y., Zhang, J., Jiang, T., Wang, S., 2011. Inclusion of the poorly water-soluble drug simvastatin in mesocellular foam nanoparticles drug loading and release properties. Int. J. Pharm. 410, 118–124.
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Increasing the dissolution rate and oral bioavailability of the poorly water-soluble drug valsartan using novel hierarchical porous carbon monoliths Yanzhuo Zhang a, * , Erxi Che b , Miao Zhang c , Baoxiang Sun c , Jian Gao a , Jin Han a , Yaling Song a a b c
School of Pharmacy, Xuzhou Medical College, Xuzhou 221004, China School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China Pharmaceutical Division, Jiangsu Hengrui Pharma,Lianyungang 222047, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2014 Accepted 18 July 2014 Available online 19 July 2014
In the present study, a novel hierarchical porous carbon monolith (HPCM) with three-dimensionally (3D) ordered macropores (400 nm) and uniform accessible mesopores (5.2 nm) was synthesized via a facile dual-templating technique using colloidal silica nanospheres and Poloxamer 407 as templates. The feasibility of the prepared HPCM for oral drug delivery was studied. Valsartan (VAL) was chosen as a poorly water-soluble model drug and loaded into the HPCM matrix using the solvent evaporation method. Scanning electron microscopy (SEM) and specific surface area analysis were employed to characterize the drug-loaded HPCM-based formulation, confirming the successful inclusion of VAL into the nanopores of HPCM. Powder X-ray diffraction (PXRD) and differential scanning calorimetry (DSC) demonstrated that the incorporated drug in the HPCM matrix was in an amorphous state and the VAL formulation exhibited good physical stability for up to 6 months. In vitro tests showed that the dissolution rate of HPCM-based formulation was increased significantly compared with that of crystalline VAL or VAL-loaded 3D ordered macroporous carbon monoliths (OMCMs). Furthermore, a pharmacokinetic study in rats demonstrated about 2.4-fold increase in oral bioavailability of VAL in the case of HPCM-based formulation compared with the commercially available VAL preparation (Valzaar1). These results therefore suggest that HPCM is a promising carrier able to improve the dissolution rate and oral bioavailability of the poorly water-soluble drug VAL. ã 2014 Published by Elsevier B.V.
Keywords: Drug delivery Porous carbon monolith Dissolution rate Solubility Oral bioavailability
1. Introduction With the advent of combinatorial chemistry and high throughput in vitro pharmacology screening, the number of poorly watersoluble compounds has dramatically increased (Gardner et al., 2004; Lipinski, 2000). Active pharmaceutical ingredients (APIs) with poor aqueous solubility often demonstrate poor and erratic absorption when administrated orally due to the dissolution ratelimiting absorption in the gastrointestinal (GI) tract (Kawabata et al., 2011). It was reported that 70% of the potential drug candidates were discarded due to low bioavailability related with poor solubility in water before they ever reached the pharmaceutics department (Cooper, 2010). Hence, increasing the aqueous solubility and dissolution rate of poorly water-soluble APIs is a
* Corresponding author. Tel.: +86 516 83262116; fax: +86 516 83262116. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.ijpharm.2014.07.024 0378-5173/ ã 2014 Published by Elsevier B.V.
significant challenge to pharmaceutical scientists. Over recent decades, more and more strategies have been developed to overcome this obstacle for poorly water-soluble APIs, these strategies include reducing particle size to increase surface area (Gao et al., 2013; Merisko-Liversidge and Liversidge, 2011), enhancing the porosity (Hu et al., 2002), solubilization in surfactant systems (Chaubal, 2004), changing the drug crystalline state (Brough and Williams, 2013) and developing novel oral nanodrug delivery systems for immediate release (Nkansah et al., 2013; Zhang et al., 2012). During the past several years, porous materials (mesoporous silica, carbon nanotube, microporous hydroxyapatite, macroporous polymer, etc.) have been widely used in drug delivery field (Prakash et al., 2011; Tang et al., 2012). For the development of an inorganic carrier-based formulation to control drug release in the GI tract where no systemic absorption of the particles is desired, microparticles are preferred to nanoparticles, since cellular uptake is significantly lower making them toxicologically
376
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
less problematic (Sharma et al., 2012). Among the micrometersized porous materials, 3D ordered macroporous monoliths with well-defined surfaces, interconnected macroporous structures, and precisely controlled pore sizes in the sub-micrometer range have been proved to be useful as oral drug delivery platforms (Xie et al., 2012). Due to spatial confinement within the nanoscale pores, the encapsulated drug molecules are normally unable to form highly ordered crystals, instead remaining in microcrystalline or amorphous forms and leading to enhanced in vitro dissolution rate. However, 3D ordered macroporous monoliths generally lack mesopores in their matrices, which substantially limits their ability to highly disperse drugs down to the molecular level and the rate of mass transport (Wang et al., 2013). It is well known that mesoporous materials possess a larger inner surface area and pore volume to highly disperse the incorporated drug molecules, compared with ordered macroporous materials (Liang et al., 2008). Therefore, it is highly attractive to build up hierarchical porous architectures for 3D ordered macroporous monoliths by integrating small mesopore channels within interconnected macroporous matrices. Notably, HPCM is an important class of new-generation porous carbon materials, which exhibit a continuously interconnected macroporous structure and accessible uniform mesoporous porosity as walls, low mass density, high surface area and large pore volume (Huang et al., 2008; Wu et al., 2012). In addition to its mesopore channels being able to change the crystalline state of a drug to an amorphous one, the interconnected macropore and mesopore channels of HPCM may allow the dissolution media to easily penetrate into the particles and facilitate drug dissolution, compared with either a solely mesoporous equivalent or macroporous equivalent. All these properties demonstrate the potential and advantages of using HPCM in oral drug delivery. However, until now, no study has explored their potential in improving the dissolution and bioavailability of poorly water-soluble drugs following oral administration. In this study, a novel HPCM, with 3D ordered macropores (around 400 nm) and uniform accessible mesopores (around 5.2 nm), were successfully synthesized via a dual-templating approach and used as a potential carrier at first time. VAL (Fig. 1) is one of the angiotensin II receptor (AT1) antagonists recommended for treatment of hypertension, post-myocardial infarction or congestive heart failure (Chioléro and Burnier, 1998). It is a lipophilic compound (log P = 5.8) belongs to BCS class II drugs (low solubility and high permeability). The absolute bioavailability after oral administration of the solid dosage form of VAL is about 23% (Brookman et al., 1997). Therefore, improving the solubility and dissolution rate of such a drug is expected to enhance bioavailability and, hence, its therapeutic potency. The aim of this study was to develop a novel HPCM as an oral nano-drug carrier to improve the dissolution rate and increase the oral bioavailability of VAL. To achieve this goal, VAL-loaded HPCM was characterized in terms of morphology, macro-/mesoporous structures, specific surface area, physical state, solubility and in vitro dissolution. At the same time, the influence of the structural effect of the carbon
Fig. 1. Chemical structure of VAL.
matrix on the physicochemical properties of the VAL formulation was further studied by comparison with VAL-loaded OMCM. Finally, the in vivo pharmacokinetics of the VAL-loaded HPCM formulation was assessed in rats compared with the commercial VAL capsules. 2. Materials and methods 2.1. Materials Coarse VAL (purity more than 99%) and telmisartan were obtained from Huahai Pharma (Zhejiang, China) and used asreceived. Poloxamer 407 was kindly provided by BASF (Ludwigshafen, Germany). Tetramethyl orthosilicate, ammonium, furfuryl alcohol and oxalic acid were obtained from Kemiou Chemical Co. (Tianjin, China). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). PrestoBlue1 was purchased from Life Technologies (Carlsbad, CA, USA). Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from Solarbio (Beijing, China). Distilled water purified using a DW 200 purification system (Hitech Instruments, Shanghai, China) was used in all experiments. Commercially available VAL preparation (Valzaar1, Torrent Pharma, India) was chosen as the reference in the bioavailability study. 2.2. Cell line HT-29 human colon carcinoma cells were obtained from the American Type Culture Collection. The cells were cultured in DMEM medium, supplemented with 10% FBS, 100 U/ml penicillin, 1% (v/v) L-glutamine, 1% (v/v) non-essential amino acids and 0.1 mg/ml streptomycin at 37 C in a 5% CO2/95% air atmosphere. 2.3. Preparation of drug-loaded microparticles 2.3.1. Synthesis of spherical silica nanoparticles Nearly monodisperse spherical silica nanoparticles were prepared using a sol–gel method (Rao et al., 2005). Typically, 7.5 ml tetramethyl orthosilicate was mixed with 200 ml ethanol and the mixture was stirred at room temperature. After 10 min, 19 ml ammonium was added as a catalyst for the hydrolysis and condensation of tetramethyl orthosilicate. The resulting mixture was vigorously stirred for 20 h at 25 C. Finally, the obtained nanoparticles were collected by centrifugation, washed with ethanol, and dried in air to obtain the white silica powder. 2.3.2. Synthesis of HPCM and OMCM HPCM was synthesized using furfuryl alcohol as a carbon source, oxalic acid as a polymerization catalyst, silica nanospheres as a hard macroporous template and Poloxamer 407 as a soft mesoporous template. In a typical procedure, 0.9 g Poloxamer 407 was dissolved in 30 ml furfuryl alcohol containing 0.5% oxalic acid to obtain the carbon precursor. The precursor was stirred for 20 min prior to addition of 16 g silica nanospheres. The resulting suspension was stirred at 25 C for 30 min and then centrifuged at 4000 g for 20 min. After removing excess precursor solution, the resulting precipitate was heated to 90 C for 12 h to allow the polymerization of furfuryl alcohol. After polymerization, the polymer/silica composite was further heated to 400 C for 4 h under a nitrogen purge and subsequently heated to 700 C for 5 h to carbonize the polymer. Finally, the silica template in the carbon/ silica composite was dissolved in 5 M potassium hydroxide solution for 10 h to form HPCM. In addition, 3D OMCM was also synthesized following the above-mentioned procedure in the absence of Poloxamer 407 as the mesoporous template.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
2.3.3. Preparation of VAL-loaded HPCM The model drug VAL was loaded into the HPCM and OMCM samples using the solvent evaporation method. First, the synthesized HPCM powder (or OMCM) were micronized by grinding and passed through a 200 mesh sieve. Subsequently, a high concentration (240 mg/ml) of VAL solution was prepared by dissolving 7.2 g VAL in 30 ml ethanol solution (containing 0.3% Poloxamer 188). Then, 2.0 g HPCM (or OMCM) was immersed in 10 ml VAL solution and added to a round-bottom flask. After mechanical stirring for 4 h, the solvent was evaporated under reduced pressure in a rotary evaporator with a water bath set at a temperature at 40 C. Finally, the drug-loaded samples were further dried to remove the residual solvent in a vacuum oven at 25 C for 24 h. 2.4. Characterization of VAL-loaded HPCM 2.4.1. SEM study The particle morphology and macropore structure of the prepared porous carbon and drug-loaded samples were characterized by SEM. The micrographs were recorded on an S-4800 electron microscope from Hitachi (Tokyo, Japan). For SEM measurements, a small portion of the sample was adhered to an aluminum stud and sputter-coated with a thin layer of platinum prior to measurements being taken. 2.4.2. Transmission electron microscopy (TEM) study The mesoporous structure of the porous carbon samples was characterized by TEM. The micrographs of the particles were recorded on a Tecnai G2 electron microscope from FEI Instruments (Eindhoven, The Netherlands). For TEM measurements, a drop of a dilute ethanol dispersion of porous particles was placed on a carbon-coated copper grid and dried at room temperature. 2.4.3. Porosity characteristics Nitrogen adsorption measurements of the prepared samples were carried out using an ASAP 2010 accelerated surface area and porisimetry system from Micromeritics Instruments (Norcross, GA, USA). The calcined HPCM and OMCM samples were degassed at 200 C for 3 h prior to measurement, while the samples loaded with VAL and pure VAL powder were degassed at 40 C overnight in the degas port. During analysis, liquid nitrogen at 196 C maintained isothermal conditions. The Brunauer–Emmett–Teller (BET) specific surface area was calculated from adsorption isotherm data using ASAP 2010 software. 2.4.4. Differential scanning calorimetry (DSC) study DSC experiments were conducted to determine the melting point and the enthalpy of fusion of each sample using a Q2000 differential scanning calorimeter (TA Instruments, Brussels, Belgium). Two-point calibration using indium and tin was carried out to check the temperature axis and heat flow of the equipment. The accurately weighed samples (approximately 6 mg) were placed in standard aluminum pans and heated at a rate of 10 C/ min from 35 C to 160 C at a heating rate of 10 C/min. This was done in a dry nitrogen atmosphere at a flow rate of 40 ml/min, and an empty aluminum pan was used as a reference. All measurements were performed in triplicate. 2.4.5. Powder X-ray diffraction (PXRD) study PXRD was used to assess the degree of crystallinity of VAL after processing with HPCM. The analysis was carried out at ambient temperature using an X’Pert-Pro MPD diffractometer (PANlytical, The Netherlands) equipped with a Cu-Ka line as the source of radiation. An acceleration voltage and current of 40 kV and 30 mA were used. A sample of native crystalline VAL or VAL-loaded HPCM
377
(approximately 0.5 g) was loaded onto an aluminum sample holder and scanned in reflection mode between 4 and 45 (2u) with a scan speed of 0.06 2u/s and a step size of 0.02 . 2.5. Determination of drug loading in VAL formulation The level of drug loading in the VAL formulation was determined by dissolving an accurately weighed amount of VAL-loaded composites (about 10 mg) in 100 ml methanol. These suspensions were ultrasonicated for 15 min and subsequently put in a rotary mixer for 12 h. Then, the porous matrix was separated from the VAL solution by centrifugation (4000 g, 15 min). The supernatant was filtered with a 0.45 mm cellulose acetate filter, suitably diluted with mobile phase and then the drug concentration was determined by HPLC. The drug loading was calculated as: weight of drug in composites 100/weight of composites. Reverse-phase HPLC was performed on a Waters 2695 system consisting of a 1525 binary pump and a 2487 dual wavelength absorbance detector (Waters Corporation, USA). For the chromatographic separation of VAL, a Waters XBridgeTM analytical column (150 4.6 mm, 5 mm) was used, and the column oven was kept at 30 C. The wavelength for VAL detection was set at 225 nm. The determination was carried out by isocratical elution using an acetonitrile/water/acetic acid (50:50:0.1,v/v/v) mixture as the mobile phase at a flow rate of 1.0 ml/min. The calibration curve of VAL was linear (r2 = 0.99) over the concentration range of 0.2–25 mg/ml. 2.6. Solubility measurements The aqueous solubility of VAL was determined in purified water, 0.1 M HCl solution and phosphate buffer solution (PBS, pH 6.8) using the Higuchi and Connor method (Higuchi and Connors, 1965). Briefly, each VAL formulation (equivalent to 1 g VAL) was dispersed in 20 ml solvent and vortexed for 2 min to form a coarse suspension. Then, the resulting suspension was transferred to a screw-capped glass tube and incubated in a shaking water bath operated at 50 rpm at 37 C. After 48 h incubation, 2 ml aliquot of the incubated sample was filtered through a 0.45 mm cellulose acetate filter, and properly diluted in methanol prior to measuring the drug concentration by means of HPLC as described above. 2.7. Dissolution studies The dissolution of each VAL formulation was tested using a USP type II (paddle) dissolution apparatus (ZRS-8 G dissolution tester, Tianda Tianfa Technology Co., Ltd., China). Purified water, 0.1 M HCl (pH 1.2) and PBS (pH 6.8) were used as different dissolution media at a temperature of 37 0.1 C and a paddle speed of 100 rpm. Each prepared sample or the crude drug powder (equivalent to 20 mg VAL) was directly added to 900 ml dissolution medium, and 5 ml samples were withdrawn from the dissolution vessel at defined time intervals (5, 10, 15, 20, 30, 100 m, 45 and 60 min). The aliquot samples were filtered with a 0.45 mm cellulose acetate filter. The first 2 ml filtrate was discarded and the remainder was analyzed in an UV–visible spectrophotometer (UV2000, Unico, USA) at an absorption wavelength of 225 nm. Experiments were conducted in triplicate and the results were recorded as an average. 2.8. Accelerated stability test An accelerated stability study was carried out on the VAL formulation in accordance with ICH accelerated stress-stability conditions. Each sample was put into a screw-capped glass vial and then maintained at 40 C and 75% RH in a chamber kept at a
378
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
constant temperature and humidity. The samples were obtained after designated incubation times (1–3 and 6 months), and the crystallinity of the VAL-loaded HPCM sample was monitored by XRPD. In addition, the dissolution curves of VAL-loaded HPCM were also compared before and after storage. 2.9. In vitro cell viability assays HT-29 cells (4 103) were seeded onto 96-well microplates (Costars, Corning Inc., USA) containing DMEM with 10% FBS and then further incubated at 37 C in a 5% CO2/95% air atmosphere. After reaching 90% confluence, the cells were exposed to various concentrations of HPCM or OMCM (10, 25, 50, 75, 150 and 250 mg/ ml prepared in DMEM medium) for 48 h. Control cells were cultured with DMEM alone. After exposure, the culture medium was replaced with 10% PrestoBlue solution and the cells were incubated for an additional 30 min. Finally, the absorbance of each well was measured at 570 nm with a reference wavelength of 600 nm using a VictorTM Multilabel plate reader (PerkinElmer, USA). The cell viability was calculated as: absorbance of samples 100/absorbance of control.
2.10. Pharmacokinetic studies 2.10.1. Animal experiment and sample collection A pharmacokinetic study in Sprague-Dawley rats was designed to evaluate the VAL-loaded HPCM formulation by comparison with the commercial capsules (Valzaar1). The rats, weighing between 270 and 340 g, were housed in compliance with good laboratory practice (GLP) standard laboratory conditions and fed with commercial rodent chow and tap water. All in vivo experiments were performed in accordance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and the experimental protocols were approved by the Institutional Animal Ethical Committee of Jiangsu Hengrui Pharma. The rats were fasted for at least 12 h prior to dosing but had free access to water and were randomly divided into 2 treatment groups (6 animals per group). The powder from commercial capsules and valsartanloaded HPCM were dispersed in an aqueous methylcellulose solution (0.1%, w/v) by simple vortexing for 30 s immediately prior to dosing. The dispersion was administered orally to rats at a single dose equivalent to 10 mg/kg of rat body weight via an oral gavage. At designated time intervals (0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12 and 24 h),
Fig. 2. SEM photographs of (a) OMCM and (b) HPCM; TEM photographs of (c) OMCM and (d) HPCM.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
the rats were anesthetized with ether and 0.5 ml blood samples were obtained by retro-orbital puncture, transferred to heparinized tubes, and then immediately centrifuged at 4000 g for 10 min to obtain plasma samples. The plasma samples were stored at 20 C for subsequent analysis. 2.10.2. Analysis of VAL in plasma samples To determine the VAL concentration, the plasma sample (100 ml) was mixed with 0.2 ml internal standard (telmisartan, 0.15 mg/ml in methanol) and vortex-mixed for 4 min. The sample was then centrifuged at 6000 g for 15 min. The supernatant was separated and evaporated at 40 C in a vacuum dryer. The residue was reconstituted with the mobile phase (100 ml), vortex-mixed for 1 min and centrifuged at 9000 g for 5 min. Then, a 20 ml aliquot of supernatant was injected into a 150 4.6 mm Shim-pack VP-ODS C18 column (Phenomenex, USA) and eluted isocratically, using an acetonitrile/phosphate solution (47:53, v/v) mixture as mobile phase, at a flow rate of 1.0 ml/min. The HPLC system (Shimadzu, Japan) equipped with a LC-10ATvp binary pump, an SCL-10Avp system controller and a RF 535 fluorescence detector
379
was used in this analysis. The effluent was monitored at excitation and emission wavelengths of 265 and 380 nm, respectively. The calibration curve for VAL was linear over the range 0.04–7.5 mg/ml, while the intra-day precision at three concentrations (0.04, 0.5, 7.5 mg/ml) was 4.1–12.7%, and the inter-day precision was less than 15%, showing acceptable precision and accuracy. The lower limit of quantification of VAL was 10 ng/ml. 2.10.3. Pharmacokinetic analysis Plasma levels of VAL in rats were plotted against time, and the pharmacokinetic parameters were calculated using a non-compartmental method via WinNonlin software (version 2.1; Pharsight Co., Mountain View, CA, USA). The area under the plasma concentration vs. time curve up to the last quantifiable time point, (AUC0 ! t), was obtained by the linear and log-linear trapezoidal method. The peak plasma concentration of drug (Cmax) and the time taken to reach the peak concentration (Tmax) were obtained directly from the plasma vs. time profile. The relative bioavailability (Fr) was calculated as: AUC0 ! t(test) 100/AUC0 ! t (reference).
Fig. 3. SEM photogrphs of (a) pure VAL, (b) VAL-loaded HPCM (low magnification), (c) VAL-loaded OMCM and (d) VAL-loaded HPCM.
380
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
2.10.4. Statistical analysis All results were expressed as average standard deviation (SD) from at least three independent experiments. For statistical analysis, a Student’s t test or one-way analysis of variance (ANOVA) test were used. Differences were considered to be statistically significant when p < 0.05. 3. Results and discussion 3.1. Porous structure characterization In the present study, highly monodisperse silica colloidal spheres, serving as a template for macropores in the synthesis of both HPCM and OMCM materials, have a uniform particle size of about 380 nm (Fig. S1 in the Supporting Information). SEM analyses reveal the presence of macropores in both HPCM and OMCM samples, in a honeycomb structure, as can be seen in Fig. 2a and b. These pores are large 3D ordered arrays of spherical cavities, which are linked by interconnecting windows. The windows were formed as a result of the contact between the silica spheres prior to infiltration of the precursor solution. The macropore diameters and pore window sizes of both samples were about 400 nm and 120 nm, respectively, as measured from the SEM images. The internal wall structures of HPCM and OMCM were analyzed by TEM. Based on the TEM observation, the organization of the pore system is observed both on the surface and throughout the monoliths. Another important piece of information that can be obtained from TEM images is that the synthesized HPCM has a hierarchical porous structure. A representative TEM image acquired at high magnification revealed disordered, worm-like mesoporous channels inside the walls between the macropores of HPCM, as shown in Fig. 2d inset. The existence of such mesopore channels (derived from Poloxamer 407) can significantly increase the specific surface area, prevent congestion and agglomerate the encapsulated drug particles, and reducing the crystallinity of the encapsulated drug particles. In contrast, no mesopores could be observed for OMCM (Fig. 2c). It can be seen that the morphology of the coarse VAL particles were non-uniform with a broad particle size distribution from 5 to 75 mm (Fig. 3a). Interestingly, after incorporation into HPCM and OMCM by the solvent deposition method, large VAL crystals disappeared and the outer pores of both HPCM and OMCM were blocked, suggesting the successful incorporation of VAL into both carriers. The representative SEM micrographs of drug-loaded HPCM and OMCM are presented in Fig. 3c and d. Many VAL nanoparticles with a diameter of around 20 nm are homogeneously distributed in the macropores of OMCM. Apparently, the morphology of the drug-loaded HPCM particles was the same as the drug-loaded OMCM particles, while drug particles on the outer pores became few. This indicates that most of VAL was loaded in the mesopores of HPCM, in addition to encapsulate uniformly into the macropores of HPCM, as confirmed by the nitrogen sorption study. Low-temperature nitrogen adsorption/desorption measurements were performed to further examine the porosity and calculate the specific surface area of the prepared HPCM and OMCM. The nitrogen adsorption/desorption isotherms of the calcined HPCM and OMCM are shown in Fig. 4. HPCM exhibits a type IV isotherm with a H2 hysteresis loop according to the IUPAC classification (Fig. 4a), indicating the presence of mesoporosity within the macroporous structure of HPCM (Cychosz et al., 2012; Kruk et al., 2002). The corresponding pore size distribution data calculated from the adsorption branch of isotherms shows that the pores of HPCM are uniform and centered at about 5.2 nm. In contrast, OMCM exhibits a type II isotherm (Fig. 4b), which is typical for nonporous and macroporous materials (Ge et al., 2012). These results agreed well with those of the TEM observations. The
Fig. 4. Nitrogen adsorption/desorption isotherms of (a) HPCM and (b) OMCM.
data for the BET specific surface area, total pore volume, Barrett– Joyner–Halenda (BJH) pore size and degree of drug loading of the corresponding samples are listed in Table 1. As shown, HPCM possesses a higher surface area and larger pore volume compared with OMCM (due to the formation of a mesoporous structure, produced by the surfactant of Poloxamer 407), confirming its potential spatial dispersion ability as a drug carrier. The study of the change in the specific surface area, pore diameter and volume of the prepared porous matrices before and after loading provided important information about the space occupied by VAL molecules (Kinnari et al., 2011). After drug loading, a decrease in specific surface area, pore diameter and pore volume was observed and these changes were attributed to the successful incorporation of VAL into the cavities of each matrix (Ambrogi et al., 2007; Wani et al., 2012). Interestingly, the sharp drop in specific surface area and pore volume for VAL-loaded HPCM is excess to the specific surface area and pore volume of OMCM, suggesting a large number of VAL molecules had been adsorbed onto the internal surface of the mesopore channels. 3.2. Solid-state characterization The drug loading process maybe change the solid state of the drug and, therefore, PXRD measurements was performed on the coarse VAL, the physical mixture (mass ratio of VAL:carrier: Poloxamer 188 was 2.4:2:0.03) and VAL-loaded samples. It can be seen that coarse VAL exhibits characteristic peaks (which are similar to those reported in the literature, Yana et al., 2012) over the 2u range in PXRD spectra (Fig. 5a), whereas both HPCM and OMCM matrices show a broad amorphous band (Fig. 5b and c). As expected, VAL characteristic peaks are present in both physical mixtures (Fig. 5d and e). On the other hand, the characteristic peaks of VAL for the VAL-loaded OMCM sample (Fig. 5f) were still present but weaker than that of the physical mixture, which illustrates that VAL absorbed into the pore channels or on the external surface of OMCM was partially present as nanocrystals and partially present as amorphous form. However, the
Table 1 Carrier characterization before and after VAL loading (n = 3). Sample
SBET (m2/g)
Vt (cm3/g)
WBJH (nm)
Drug loading (%)
HPCM OMCM VAL-loaded HPCM VAL-loaded OMCM
1290 65 473 24 381 50 94 27
1.35 0.16 0.82 0.13 0.23 0.07 0.19 0.03
5.2 1.4 – 2.9 0.6 –
– – 53.7 1.8 52.9 2.3
SBET, BET specific surface area; WBJH, Barrett–Joiner–Halenda pore diameter; Vt, total pore volume.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
381
observed in the DSC thermogram of VAL-loaded HPCM (Fig. 6e), indicating that the drug entrapped in the mesopore channels of HPCM was in an amorphous state, which agreed well with the XRD results. The non-crystalline state of VAL-loaded HPCM will be beneficial to increase the solubility and dissolution rate of VAL (Brough et al., 2013; Vasconcelos et al., 2007; Zhang et al., 2011). 3.3. Drug dissolution profiles
Fig. 5. PXRD patterns of (a) pure VAL, (b) HPCM, (c) OMCM, (d) physical mixture1 (VAL\HPCM\Poloxamer 188), (e) physical mixture2 (VAL\OMCM \Poloxamer 188), (f) VAL-loaded OMCM, (g) VAL-loaded HPCM and (h) VAL-loaded HPCM after 6 months of storage.
In vitro dissolution tests were performed in order to demonstrate the ability of both HPCM and OMCM to increase the rate and extent of dissolution of VAL in simulated gastric/intestinal fluid. The in vitro dissolution curves for coarse VAL and different VAL formulations in different dissolution media are shown in Fig. 7. The observed dissolution of coarse VAL was very limited in both purified water and 0.1 M HCl solution (less than 10% within 60 min). The slow dissolution can be attributed to its larger crystal size and very low solubility. The saturated solubility of coarse VAL was 0.03 0.01 mg/ml in 0.1 M HCl solution and 0.14 0.02 mg/ml in purified water. At pH 6.8, however, the dissolution rate of coarse
characteristic crystalline peaks disappeared in the PXRD pattern of VAL-loaded HPCM (Fig. 5g) producing an amorphous halo pattern, revealing that the crystallinity of VAL-loaded HPCM was decreased dramatically. This is related to finite-size effects, preventing the drug molecules entrapped in the mesopore channels from rearranging themselves in a crystal lattice (Jackson and McKenna, 1996). It should be noted that VAL-loaded HPCM showed no sign of recrystallization during the storage period (Fig. 5h). As a result, the stability test showed that VAL-loaded HPCM exhibited good physical stability under accelerated storage conditions for at least 6 months. The DSC thermograms of coarse VAL, the physical mixture and VAL-loaded samples are depicted in Fig. 6. A single sharp endothermic peak in the case of coarse VAL was observed at 102.6 C (Fig. 6a, the endothermic value was 59.7 1.6 J/g), which corresponded to its melting point. As expected, a similar endothermic peak was also visible in the DSC thermogram of the physical mixtures (Fig. 6b and c), suggesting that both porous carbon matrices do not change the solid state of VAL in the physical mixtures. For the VAL-loaded OMCM sample, a small endothermic peak was observed at 102.4 C. Compared with coarse VAL, the VAL-loaded OMCM sample showed a significant reduction in the enthalpy of fusion (Fig. 6d, the endothermic value was 11.4 3.0 J/ g), indicating the decrease in crystallinity of the drug and also suggesting VAL was partly converted into an amorphous state. Unlike the VAL-loaded OMCM samples, no endothermic peak was
Fig. 6. DSC thermograms of (a) pure VAL, (b) physical mixture1, (c) physical mixture2, (d) VAL-loaded OMCM, (e) VAL-loaded HPCM and (g) HPCM.
Fig. 7. Dissolution of VAL from different formulations in (A) 0.1 M HCl, (B) purified water and (C) PBS; (a) pure VAL, (b) physical mixture1, (c) physical mixture2, (d) VAL-loaded OMCM, (e) VAL-loaded HPCM and (f) VAL-loaded HPCM after 6 months of storage. Each data point represents the mean SD (n = 3).
382
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
VAL was higher due to its weak acidity (the saturated solubility of coarse VAL in PBS was 15.7 0.21 mg/ml). As expected, in three different dissolution media, the dissolution profiles of both physical mixtures were similar to that of coarse VAL, which showed that the mechanical physical mixing of coarse VAL, porous carbon matrices (HPCM and OMCM) and Poloxamer 188 had little effect on the dissolution of VAL. In the cases of both VAL-loaded porous carbon-based formulations, however, a dramatic increase in both the dissolution rate and extent was seen compared with that of coarse VAL. For example, the accumulated amounts of dissolved coarse VAL, VAL-loaded OMCM and VAL-loaded HPCM in purified water were 4.6%, 63%, and 92%, respectively, after 20 min. This outstanding increase in drug dissolution may be attributed to the decrease in particle size and crystallinity of VAL present in the pores of OMCM or HPCM, together with the increase in specific surface area and improved dispersibility of VAL particles (Kinnari et al., 2011; Wang, 2009). For the VAL-loaded OMCM sample, the particle size of the drug incorporated in the macropores (around 400 nm) was significantly reduced compared with that of coarse VAL (5–75 mm). It is obvious that a dramatically decrease in the particle size down to the nano scale will accelerate the drug dissolution and increase the drug solubility (Nkansah et al., 2013) (the saturated solubility of VALloaded OMCM was 0.07 0.01 mg/ml in 0.1 M HCl solution and 0.26 0.03 mg/ml in purified water). Meanwhile, as a result of the particle size reduction, the effective surface area increases, thereby leading to a higher dissolution rate according to the Noyes– Whitney equation (Brough and Williams, 2013; Dokoumetzidis and Macheras, 2006). The same phenomenon was observed when nimodipine was loaded into ordered macroporous chitosan (Xie et al., 2012). Compared with VAL-loaded OMCM, VAL-loaded HPCM exhibited a faster dissolution rate, irrespective of pH conditions. Two factors probably accounted for this phenomenon: (1) the existence of a lot of mesopores inside the walls between the macropores allows dissolution media to flow quickly inside the porous matrix and to dissolve more the entrapped VAL. (2) VAL is completely in amorphous state in VAL-loaded HPCM but partly in crystalline state in VAL-loaded OMCM (confirmed by the PXRD and DSC studies). It is well known the formation of the amorphous form could significantly improve the dissolution and solubility of poorly water-soluble drugs compared with the relevant crystalline form because there is no lattice energy to be overcome (Vasconcelos et al., 2007; Babu and Nangia, 2011). The saturated solubility of VAL-loaded HPCM was 0.09 0.01 mg/ml in 0.1 M HCl solution and 0.31 0.02 mg/ml in purified water. It can be seen clearly from Fig. 7, the release profiles of VAL from VAL-loaded HPCM were similar to that of the freshly prepared ones within 6 months storage. This significant dissolution enhancing effect provided by HPCM will be particularly beneficial for the improved delivery of poorly water-soluble drugs belonging to the BCS class II
Fig. 9. Plasma concentration-time profiles of the VAL formulations tested; (&) commercial preparation and (&) VAL-loaded HPCM. Each data point represents the mean SD (n = 6).
for which the oral bioavailability is limited by their poor dissolution rates. 3.4. In vitro cell viability HT-29 human colon carcinoma cells were treated with different porous carbon matrices (OMCM and HPCM) at different concentrations (10–250 mg/ml) for 48 h, and the cell viability was evaluated using Presto Blue reagent. As shown in Fig. 8, above 80% of cell viability was obtained, with no significant differences between results for both OMCM and HPCM. These suggest that both OMCM and HPCM are biocompatible with HT-29 cells and, therefore, well tolerated by the gastric/intestinal tract. 3.5. In vivo performance To further investigate the role of HPCM, the oral bioavailability of VAL-loaded HPCM-based formulation in Sprague-Dawley rats was compared with that of the commercial capsules (Valzaar1). The mean plasma VAL concentration after oral administration of each VAL formulation is shown in Fig. 9 and the corresponding pharmacokinetic parameters of VAL are listed in Table 2. As shown in Table 2, VAL-loaded HPCM demonstrated a higher Cmax of 4.71 1.56 mg/ml and a shorter Tmax of 1.42 0.38 h compared with oral commercial capsules (Cmax 2.32 1.28 mg/ml and Tmax 2.25 0.61 h). These results are in agreement with the dissolution studies showing that VAL was released from VAL-loaded HPCM much faster than that from crude VAL powder. The AUC0 ! 24 h value was also found to be significantly higher for the VAL-loaded HPCM formulation than for the commercial formulation. The high AUC0 ! 24 h value of VAL-loaded HPCM suggested that marked adsorption occurred after the quick release of VAL from HPCM in the GI tract. This underlines the important role of HPCM-based formulation in producing a high concentration gradient between the drug and the GI epithelium produced by a higher drug release. Interestingly, the relative bioavailability of VAL-loaded HPCM compared with the commercial formulation was 239.5 52.64%.
Table 2 Pharmacokinetic parameters of the VAL formulations tested (n = 6).
Fig. 8. Effect of HPCM and OMCM on HT-29 cell viability at various concentrations.
Parameters
VAL-loaded HPCM
Commercial capsules
Tmax (h) Cmax (mg/ml) AUC0 ! 24 h (mg h/ml) Fr (%)
1.42 0.38* 4.71 1.56* 33.26 9.20* 239.5 52.64
2.25 0.61 2.32 1.28 13.59 5.07 –
*
P < 0.05 compared with control formulation.
Y. Zhang et al. / International Journal of Pharmaceutics 473 (2014) 375–383
4. Conclusions In summary, a novel HPCM with 3D ordered macropores (400 nm) and uniform accessible mesopores (5.2 nm) was designed, and the pharmaceutical performance of this material was studied. The VAL-loaded HPCM formulation showed a significantly higher drug release rate than pure VAL or VAL-loaded OMCM in simulated gastric/intestinal fluid. The pharmacokinetic data clearly showed that the VAL-loaded HPCM formulation produced a significant improvement in the bioavailability compared with the commercial capsules. HPCM may therefore be considered as a promising carrier to achieve enhanced dissolution rate and oral bioavailability of the poorly water-soluble drug VAL. Acknowledgement This work was partly funded by the National Basic Research Program of China(973 Program) (No. 2009CB930300). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.07.024. References Ambrogi, V., Perioli, L., Marmottini, F., Giovagnoli, S., Esposito, M., Rossi, C., 2007. Improvement of dissolution rate of piroxicam by inclusion into MCM-41 mesoporous silicate. Eur. J. Pharm. Sci 32, 216–222. Babu, N.J., Nangia, A., 2011. Solubility advantage of amorphous drugs and pharmaceutical cocrystals. Cryst. Growth Des. 1, 2662–2679. Brookman, L.J., Rolan, P.E., Benjamin, I.S., Palmer, K.R., Wyld, P.J., Lloyd, P., Flesch, G., Waldmeier, F., Sioufi, A., Mullins, F., 1997. Pharmacokinetics of valsartan in patients with liver disease. Clin. Pharmacol. Ther. 62, 272–278. Brough, C., Williams, R.O., 2013. Amorphous solid dispersions and nano-crystal technologies for poorly water-soluble drug delivery. Int. J. Pharm. 453, 157–166. Chaubal, M.V., 2004. Drug discovery today application of formulation technologies in lead candidate selection and optimization. Drug Discov. Today 9, 603–609. Chioléro, A., Burnier, M., 1998. Pharmacology of valsartan, an angiotensin II receptor antagonist. Expert Opin. Investig. Drugs 7, 1915–1925. Cooper, E.R., 2010. Nanoparticles: a personal experience for formulating poorly water soluble drugs. J. Control. Release 141, 300–302. Cychosz, K.A., Guo, X., Fan, W., Cimino, R., Gor, G., Tsapatsis, M., Neimark, A.V., Thommes, M., 2012. Characterization of the pore structure of threedimensionally ordered mesoporous carbons using high resolution gas sorption. Langmuir 28, 12647–12654. Dokoumetzidis, A., Macheras, P., 2006. A century of dissolution research: from Noyes and Whitney to the biopharmaceutics classification system. Int. J. Pharm. 321, 1–11. Gao, L., Liu, G., Ma, J., Wang, X., Zhou, L., Li, X., Wang, F., 2013. Application of drug nanocrystal technologies on oral drug delivery of poorly soluble drugs. Pharm. Res. 30, 307–324. Gardner, C.R., Walsh, C.T., Almarsson, Ö, 2004. Drugs as materials: valuing physical form in drug discovery. Nat. Rev. Drug Discov. 3, 926–934. Ge, J., Ding, H., Xue, X., 2012. A nanosheet-structured three-dimensional macroporous material with high ionic conductivity synthesized using glucose as a transforming template. Angew. Chem. Int. Ed. Engl. 51, 6205–6208.
383
Higuchi, T., Connors, K.A., 1965. Phase-solubility techniques. Adv. Anal. Chem. Instrum. 4, 117–212. Huang, Y., Cai, H., Feng, D., Gu, D., Deng, Y., Tu, B., Wang, H., Webley, P.A., Zhao, D., 2008. One-step hydrothermal synthesis of ordered mesostructured carbonaceous monoliths with hierarchical porosities. Chem. Commun. 23, 2641–2643. Hu, J., Rogers, T.L., Brown, J., Young, T., Johnston, K.P., Williams, R.O., 2002. Improvement of dissolution rates of poorly water soluble APIs using novel spray freezing into liquid technology. Pharm. Res. 19, 1278–1284. Kawabata, Y., Wada, K., Nakatani, M., Yamada, S., Onoue, S., 2011. Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications. Int. J. Pharm. 420, 1–10. Kinnari, P., Makila, E., Heikkila, T., Salonen, J., Hirvonen, J., Santos, H.A., 2011. Comparison of mesoporous silicon and non-ordered mesoporous silica materials as drug carriers for itraconazole. Int. J. Pharm. 414, 148–156. Kruk, M., Antochshuk, V., Matos, J.R., Mercuri, L.P., Jaroniec, M., 2002. Determination and tailoring the pore entrance size in ordered silicas with cage-like mesoporous structures. J. Am. Chem. Soc. 124, 768–769. Jackson, C.L., McKenna, G.B., 1996. Vitrification and crystallization of organic liquids confined to nanoscale pores. Chem. Mat. 8, 2128–2137. Liang, C., Li, Z., Dai, S., 2008. Mesoporous carbon materials: synthesis and modification. Angew. Chem. Int. Ed. Engl. 47, 3696–3717. Lipinski, C.A., 2000. Drug-like properties and the causes of poor solubility and poor permeability. J. Pharmacol. Toxicol. Methods 44, 235–249. Merisko-Liversidge, E., Liversidge, G.G., 2011. Nanosizing for oral and parenteral drug delivery: a perspective on formulating poorly-water soluble compounds using wet media milling technology. Adv. Drug Deliv. Rev. 63, 427–440. Nkansah, P., Antipas, A., Lu, Y., Varma, M., Rotter, C., Rago, B., El-Kattan, A., Taylor, G., Rubio, M., Litchfield, J., 2013. Development and evaluation of novel solid nanodispersion system for oral delivery of poorly water-soluble drugs. J. Control. Release 169, 150–161. Prakash, S., Malhotra, M., Shao, W., Tomaro-Duchesneau, C., Abbasi, S., 2011. Polymeric nanohybrids and functionalized carbon nanotubes as drug delivery carriers for cancer therapy. Adv. Drug Deliv. Rev. 63, 1340–1351. Rao, K.S., El-Hami, K., Kodaki, T., Matsushige, K., Makino, K., 2005. A novel method for synthesis of silica nanoparticles. J. Colloid. Interface Sci. 289, 125–131. Sharma, A., Madhunapantula, S.R.V., Robertson, G.P., 2012. Toxicological considerations when creating nanoparticle-based drugs and drug delivery systems? Expert Opin. Drug Metab. Toxicol. 8, 47–69. Tang, F., Li, L., Chen, D., 2012. Mesoporous silica nanoparticles: synthesis biocompatibility and drug delivery. Adv. Mater. 24, 1504–1534. Vasconcelos, T., Sarmento, B., Costa, P., 2007. Solid dispersions as strategy to improve oral bioavailability of poor water soluble drugs. Drug Discov. Today 12, 1068–1075. Wani, A., Muthuswamy, E., Savithra, G.H., Mao, G., Brock, S., Oupicky, D., 2012. Surface functionalization of mesoporous silica nanoparticles controls loading and release behavior of mitoxantrone. Pharm. Res. 29, 2407–2418. Wang, S., 2009. Ordered mesoporous materials for drug delivery. Micropor. Mesopor. Mater. 117, 1–9. Wu, Z.S., Sun, Y., Tan, Y.Z., Yang, S., Feng, X., Mullen, K., 2012. Three-dimensional graphene-based macro- and mesoporous frameworks for high-performance electrochemical capacitive energy storage. J. Am. Chem. Soc. 134, 19532–19535. Wang, Y., Zhao, Q., Hu, Y., Sun L, Bai L., Jiang, T., Wang, S., 2013. Ordered nanoporous silica as carriers for improved delivery of water insoluble drugs: a comparative study between three dimensional and two dimensional macroporous silica. Int. J. Nanomed. 8, 4015–4031. Xie, Y., Liu, Y., Wang, Y., Wang, S., Jiang, T., 2012. Chitosan matrix with three dimensionally ordered macroporous structure for nimodipine release. Carbohydr. Polym. 90, 1648–1655. Yana, Y.D., Sunga, J.H., Kima, K.K., Kimb, D.W., Kima, J.O., Leec, B.J., Yonga, C.S., Choi, H.G., 2012. Novel valsartan-loaded solid dispersion with enhanced bioavailability and no crystalline changes. Int. J. Pharm. 422, 202–210. Zhang, Y., Wang, J., Bai, X., Jiang, T., Zhang, Q., Wang, S., 2012. Mesoporous silica nanoparticles for increasing the oral bioavailability and permeation of poorly water soluble drugs. Mol. Pharm. 9, 505–513. Zhang, Y., Zhang, J., Jiang, T., Wang, S., 2011. Inclusion of the poorly water-soluble drug simvastatin in mesocellular foam nanoparticles drug loading and release properties. Int. J. Pharm. 410, 118–124.