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Supersaturation Potential of Ordered Mesoporous Silica Delivery Systems. Part 1: Dissolution Performance and Drug Membrane Transport Rates Tahnee Jade Dening, and Lynne S. Taylor Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00488 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018
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Molecular Pharmaceutics
Supersaturation Potential of Ordered Mesoporous Silica Delivery Systems. Part 1: Dissolution Performance and Drug Membrane Transport Rates
Tahnee J. Dening1, Lynne S. Taylor1,*
1. Department of Industrial and Physical Pharmacy, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States
*To whom correspondence should be addressed. Email:
[email protected]; Fax: +1 (765) 494-6545; Tel: +1 (765) 496-6614.
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Abstract
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Ordered mesoporous silica materials have shown great potential as oral drug delivery systems for
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poorly soluble drugs. However, the ability of these delivery systems to generate drug
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supersaturation has not been widely investigated, and the recently noted phenomenon of
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incomplete drug release is not well understood. Therefore, the aim of this study was to
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comprehensively evaluate the release of hydrophobic drug molecules into solution from ordered
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mesoporous silica, focusing on the extent and duration of drug supersaturation. The dissolution
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and supersaturation behavior of ritonavir, following loading into mesoporous SBA-15 silica
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particles, was investigated by undertaking simple in vitro dissolution studies in phosphate buffer
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pH 6.8 and fasted state simulated intestinal fluid, as well as membrane flux studies using a side-
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by-side diffusion cell apparatus. It was found that supersaturated ritonavir solutions were
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generated from ritonavir-loaded mesoporous SBA-15 particles, however, drug release was
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always incomplete, even under sink conditions. In addition, the percentage drug release was
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observed to decrease significantly as the theoretical supersaturation ratio and dose of ritonavir-
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loaded SBA-15 formulation increased. The data obtained suggest an equilibrium exists between
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drug adsorbed to the SBA-15 silica surface and free drug present in solution. The findings
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described herein are highly significant in aiding our understanding of ordered mesoporous silica
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as a supersaturating drug delivery system for bioavailability enhancement.
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Keywords. mesoporous; silica; SBA-15; hydrophobic drug; supersaturation; dissolution;
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incomplete release.
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Molecular Pharmaceutics
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Introduction
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Owing to the advent of combinatorial chemistry and high throughput screening, low aqueous
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solubility and high hydrophobicity are common characteristics of up to 90% of drug candidates
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currently under development.1-2 As a result, there is significant interest in the use of enabling
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formulation strategies to enhance drug solubility and boost drug absorption from the
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gastrointestinal tract. Such strategies can typically be classified into two broad categories.
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Solubilizing formulations enhance the crystalline solubility of the drug by using additives such
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as surfactants, cosolvents or complexing agents.3 In contrast, supersaturating formulations
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generate drug concentrations that are higher than the crystalline solubility.4 In this case, the
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chemical potential or thermodynamic activity of the solute exceeds that of the solute in a
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saturated solution, leading to the significant advantage of increased flux across the
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gastrointestinal membrane.5 Examples of supersaturating formulations include some lipid-based
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formulations,4, 6 salts7-8 and amorphous solid dispersions.9-10
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Amorphous solid dispersions, consisting of amorphous drug combined with a polymeric
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crystallization inhibitor, have attracted substantial attention owing to their ability to generate and
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maintain a supersaturated drug solution upon dissolution.11 Amorphous drug exists in a higher
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free energy state in comparison to the crystalline solid, owing to disruption of the crystal lattice.
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Spontaneous recrystallization to a more stable form is thermodynamically favored, and thus a
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polymer is typically used to impede crystallization from both the solid formulation and in
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solution following dissolution.12 Several amorphous solid dispersion products have been
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commercialized,13 and significant research efforts are ongoing to better understand these
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complex delivery systems.14-17
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As an alternative to polymer-based amorphous solid dispersions, mesoporous materials are being
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increasingly utilized to stabilize and deliver amorphous drugs via the oral route.18 The adsorption
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of drug molecules onto porous and nonporous silica has been described since the 1970s,19-21 and
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in 2001, ordered mesoporous silica (OMS) was first proposed as a vehicle for controlled drug
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delivery.22 OMS materials possess a regular arrangement of uniform pores with diameters of 2-
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30 nm, and have large specific surface areas (SSA), often in the range of 500-1000 m2/g.23 The
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large SSA of OMS materials leads to a high surface free energy, thus adsorption of drug
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molecules onto the silica surface allows the system to progress to a lower free energy state.12, 24
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As a result, adsorbed drug molecules exist in an amorphous and thermodynamically stable state,
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due to an overall decrease in the Gibbs free energy of the system.12,
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further enhanced due to a size-constraint effect on drug nucleation and crystal growth. Provided
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the mesopore diameter is small enough, spatial constraints prevent clusters of amorphous drug
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molecules reaching the critical nucleus size, and thus spontaneous drug crystallization within the
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mesopores is inhibited.12, 24
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Several studies have been undertaken to characterize the dissolution and release behavior of
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various hydrophobic drug molecules from OMS materials. Many of these studies have
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demonstrated the ability of OMS to enhance the rate and extent of drug dissolution in
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comparison to crystalline drug,27-31 however, few studies have examined the performance of
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OMS delivery systems under supersaturating (i.e. non-sink) conditions.32-34 Mellaerts et al.
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investigated the release of itraconazole from mesoporous SBA-15 silica under supersaturating
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conditions in simulated gastric fluid, and observed that the concentration of itraconazole in
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solution was maintained well above the crystalline solubility for the duration of the experiment
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(60 min).32 However, despite a supersaturated solution being generated, the percentage release of
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Physical stability is
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itraconazole plateaued at approximately 55% when the drug load in SBA-15 was 20% w/w. The
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authors suggested a dynamic adsorption equilibrium had been reached, however, no further
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investigations were undertaken to explore this phenomenon. Significantly, such incomplete drug
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release from OMS is often a feature during dissolution under both sink27,
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supersaturating conditions.31-34, 38-41
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To better understand the potential of OMS materials as supersaturating oral drug delivery
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systems, this study aimed to comprehensively characterize the ability of mesoporous SBA-15
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silica to generate and maintain drug supersaturation in solution. Ritonavir was selected as the
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model compound, owing to its slow crystallization tendency from solution, thereby allowing
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supersaturation to be easily characterized in vitro. The hypothesis to be tested was that
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mesoporous SBA-15 silica can generate and maintain a supersaturated solution of ritonavir
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during dissolution under non-sink conditions, with the maximum achievable supersaturation
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being dictated by the amorphous solubility of ritonavir. Experimentally, the hypothesis was
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tested by monitoring the ritonavir concentration in solution during dissolution studies with
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ritonavir-loaded SBA-15 particles, and by conducting diffusion experiments, whereby drug flux
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across an artificial membrane was assessed and compared against the flux generated by neat
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amorphous ritonavir.
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Materials and Methods
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Materials
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Ritonavir and lopinavir were purchased from ChemShuttle (Hayward, CA). SBA-15 material
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with pore diameter 7.1 nm, pore volume 0.80 cm3/g and SSA 586 m2/g was supplied by Glantreo
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(Cork, Ireland). Hydroxypropyl methylcellulose (HPMC) (Pharmacoat® 606) was obtained from
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and
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Shin-Etsu Chemical Co. Ltd. (Tokyo, Japan). Fasted state simulated intestinal fluid (FaSSIF)
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powder was obtained from Biorelevant.com (London, United Kingdom). Polytetrafluoroethylene
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(PTFE) syringe filters with pore size 1.0 µm and diameter 4 mm were supplied by Tisch
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Scientific (North Bend, OH). Buffer salts and analytical grade solvents were obtained from
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Fisher Chemical (Fair Lawn, NJ). Regenerated cellulose membrane with a molecular weight cut-
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off (MWCO) of 6-8 kDa was acquired from Spectrum Laboratories Inc. (Rancho Dominguez,
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CA). The aqueous media used in all experiments was 50 mM sodium phosphate buffer pH 6.8
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with and without 5 µg/mL HPMC. HPMC was used to inhibit ritonavir crystallization during
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experiments where necessary.
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Methods
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Crystalline and Amorphous Solubility Measurements
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The crystalline solubility of ritonavir in 50 mM phosphate buffer pH 6.8 was determined by
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equilibrating an excess of crystalline ritonavir (polymorph form II) in 15 mL of medium using an
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agitating water bath (Dubnoff metallic shaking incubator, PGC Scientific, Palm Desert, CA) set
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at 37 °C for 48 h. The undissolved crystalline material was separated from solution by
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centrifugation at 21,100 × g for 30 min (37 °C) using a Sorvall™ Legend™ Micro 21R
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Microcentrifuge (ThermoFisher Scientific, Waltham, MA). The supernatant was diluted with
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acetonitrile and analyzed via high performance liquid chromatography (HPLC) using the method
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described below.
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The amorphous solubility of ritonavir was also determined using centrifugation. A methanolic
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solution of drug concentration 10 mg/mL was prepared, and 75 µL was added to 15 mL of 50
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mM phosphate buffer pH 6.8 at 37 °C under stirring at 300 rpm. To ensure nucleation and
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crystallization of ritonavir did not occur during the experiment, the amorphous solubility was
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also determined in the presence of 5 µg/mL HPMC. The turbid solutions were immediately
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centrifuged at 21,100 × g for 40 min (37 °C) to pellet the drug-rich phase. The supernatant was
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diluted with acetonitrile and analyzed via HPLC as described below.
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The crystalline and amorphous solubility of ritonavir was also measured in FaSSIF, using the
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methods described above.
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Estimation of Ritonavir Molecular Dimensions
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The molecular dimensions of the ritonavir molecule were measured using Mercury CSD 3.10.1
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software (Cambridge Crystallographic Data Centre, Cambridge, United Kingdom) and the single
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crystal structure with a reference code of YIGPIO. The “measure distance” tool was used to
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determine the distance between the atoms furthest apart in approximately orthogonal directions.
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The surface area for adsorption was then estimated from the product of the two longest
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dimensions.
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Preparation of Ritonavir-Loaded SBA-15 Formulations
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Ritonavir was loaded into SBA-15 material using a solvent incipient wetness impregnation
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method.32 A concentrated methanolic solution of the drug (25 mg in 400 µL) was added
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dropwise to 75 mg SBA-15 to achieve the desired drug loading, and intensively mixed with a
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spatula. The solvent was subsequently removed from the sample by drying in an oven at 40 °C
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for 24 h, followed by 48 h under reduced pressure at ambient temperature. The drug content was
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confirmed using a solvent extraction technique, whereby a known quantity of sample was
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dispersed in methanol with the aid of ultrasonication (Cole Parmer Ultrasonic Cleaner Model
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8892, Vernon Hills, IL) for 30 min. Samples were then centrifuged at 21,100 × g for 10 min and
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the supernatant was diluted with acetonitrile prior to HPLC analysis.
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X-Ray Powder Diffraction (XRPD) Analysis
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Drug crystallinity in the ritonavir-loaded SBA-15 formulations was analyzed by measuring the
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X-ray powder diffraction pattern using a Rigaku Smartlab™ diffractometer (Tokyo, Japan).
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Samples were scanned over the range 5 to 40° 2θ at a scanning rate of 4°/min and step size of
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0.02°. The voltage and current were set at 40 kV and 44 mA, respectively.
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Dissolution Experiments
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To evaluate the supersaturation potential of SBA-15, dissolution experiments were performed in
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50 mM phosphate buffer pH 6.8 media. The buffered solution (75 mL) was equilibrated in a
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jacketed-vessel maintained at 37 °C under stirring at 300 rpm. The ritonavir-loaded SBA-15
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formulation was dosed at the amorphous solubility of ritonavir, to generate a maximum
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supersaturation ratio (S) of 7.8. This required 9.4 mg sample to be added to the medium. The
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formulation was also dosed at half the amorphous solubility of ritonavir (i.e. S of 3.9), which
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required 4.7 mg sample per dissolution experiment. Dissolution was monitored over 120 min,
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and 0.7 mL sample aliquots were withdrawn at fixed time points. Samples were centrifuged at
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21,100 × g for 10 min (37 °C) and the supernatant was diluted with acetonitrile and analyzed via
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HPLC.
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For the sequential dilution dissolution study, 50 mL buffered solution was equilibrated as
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described above. Approximately 6.3 mg sample was added to the dissolution medium to generate
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a supersaturated solution of ritonavir (S of 7.8), and 0.5 mL sample aliquots were withdrawn
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every 15 min over a 180 min period. Every 30 min, fresh phosphate buffer (pre-equilibrated to
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37 °C) was added to dilute the medium, such that the theoretical maximum concentration of
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ritonavir in solution was decreased in increments and was approximately equal to the equilibrium
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crystalline solubility of ritonavir during the final 30 min. The maximum supersaturation ratio
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was decreased by dilution as follows: S=7.8 at 0 min; S=3.9 at 30 min; S=2.6 at 60 min; S=1.9 at
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90 min; S=1.6 at 120 min; S=1.3 at 150 min; S=1 at 180 min. Samples were prepared for HPLC
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analysis as described above.
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For dissolution experiments conducted at total drug concentrations above the amorphous
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solubility of ritonavir, samples were prepared for analysis using a filtration method. This
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technique allows for the quantification of both free drug in solution and drug-rich nanodroplets
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formed when the amorphous solubility is exceeded; the drug-rich nanodroplets formed by
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ritonavir were estimated using dynamic light scattering (Zetasizer Nano-ZS, Malvern, United
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Kingdom) to have a Z-average diameter of 740 ± 45 nm (PDI 0.23 ± 0.12) when the
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concentration of ritonavir in solution was 56 µg/mL. Previous studies have also confirmed the
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formation of ritonavir nanodroplets of size
