A Modified In Situ Method to Determine Release from a Complex Drug Carrier in Particle-Rich Suspensions
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Effective and compound-sparing methods to evaluate promising drug delivery systems are a prerequisite for successful selection of formulations in early development stages. The aim of the study was to develop a small-scale in situ method to determine drug release and supersaturation in highly concentrated suspensions of enabling formulations. Mesoporous magnesium carbonate (MMC), which delivers the drug in an amorphous form, was selected as a drug carrier. Five model compounds were loaded into the MMC at a 1:10 ratio using a solvent evaporation technique. The μDiss Profiler was used to study the drug release from MMC in fasted-state simulated intestinal fluid. To avoid extensive light scattering previously seen in particle-rich suspensions in the μDiss Profiler, an in-house-designed protective nylon filter was placed on the in situ UV probes. Three types of release experiments were conducted for each compound: micronized crystalline drug with MMC present, drug-loaded MMC, and drug-loaded MMC with 0.01% w/w hydroxypropyl methyl cellulose. The nylon filters effectively diminished interference with the UV absorption; however, the release profiles obtained were heavily compound dependent. For one of the compounds, changes in the UV spectra were detected during the release from the MMC, and these were consistent with degradation of the compound. To conclude, the addition of protective nylon filters to the probes of the μDiss Profiler is a useful contribution to the method, making evaluations of particle-rich suspensions feasible. The method is a valuable addition to the current ones, allowing for fast and effective evaluation of advanced drug delivery systems.
KEY WORDSrelease mesoporous supersaturation drug carrier μDiss Profiler
A major hurdle during drug development is the poor water solubility of discovery compounds; as many as 75% of the drugs under development suffer from low aqueous solubility (1). Methods to circumvent this, and hence increase the bioavailability of poorly water-soluble drugs (PWSDs), include the use of supersaturating drug delivery systems (SDDSs) (2). In these systems, the drug is typically delivered in its amorphous form, a high-energy form resulting in an increased apparent solubility (2,3). The addition of a crystallization inhibitor, e.g., a polymer, can stabilize the supersaturation. Common SDDSs are solubilizing formulations (e.g., self-emulsifying drug delivery systems promoting supersaturation during digestion), amorphous solid dispersions, and inorganic carriers loaded with the amorphous form of the drug (3). In 1992, mesoporous silica was introduced by the Mobil Oil company and since then several types of mesoporous material have been produced (4,5). Large pore volumes and high specific surface areas give these materials a variety of possible applications including improved drug dissolution of PWSDs (4,6). Once loaded into the carrier, the drug is in an amorphous form and recrystallization is inhibited through spatial constraints (7).
In 2013, a template-free mesoporous carrier, mesoporous magnesium carbonate (MMC), was introduced. The synthesis is performed under low temperatures, and no strong organic solvents nor surfactants are required during manufacturing (8). Magnesium carbonate is GRAS-listed by the FDA, and acute toxicity studies of MMC do not show any signs of toxicity in animals (9). MMC has also been shown to be a promising excipient in drug delivery systems by stabilizing the drugs in an amorphous state (10). MMC has proven to be compatible with a variety of drugs, indicating it as a new drug carrier with general applicability. Due to the diffusion-controlled release, the release rate can be tailored by altering the particle size of the MMC (11, 12, 13). Loading of MMC has so far been conducted by simple solvent evaporation, and studies of release from drug-loaded MMC have previously been performed using the traditional USP 2 dissolution bath (10,12). This does, however, demand significant amounts of material. As the sampling must also be done manually, it also limits the possibility of studying rapid release and time-sensitive events. The μDiss Profiler, a small-scale dissolution apparatus, enables in situ UV measurements of drug dissolution. This apparatus has been successfully used to determine dissolution rates and intrinsic dissolution rates (IDR) from powder, controlled suspensions, and compressed discs (14, 15, 16). It has also been used to study supersaturation obtained from SDDSs and release from drug-loaded MMC (11,17). However, particle-rich suspensions of MMC produce significant light scattering which interferes with the UV absorption measurements. To overcome this issue, we previously proposed a method to estimate the initial release rate from high concentrations of drug-loaded MMC in which the amount of MMC in the assay is gradually increased. The release rate is linearly dependent on the amount of drug-loaded MMC added, enabling extrapolation to higher concentrations (11). This provides an insight into the initial release rate in high concentrations of loaded carrier, but it does not provide information about the full release event, such as the degree of supersaturation and time to recrystallization. Therefore, we targeted the development of a methodology that would enable studies of both the immediate release and degree of supersaturation obtained from drug-loaded MMC. A small-scale in situ method was developed making use of the μDiss Profiler and protective filters. The method may also be applicable for studies of release from other complex formulations that produce particle-rich suspensions. It should be noted that the pore size of the nylon filter used herein (11 μm) suggests that the method might not be suitable for performance assessment of nanosuspensions.
MATERIAL AND METHODS
Five PWSDs were selected and MMC was synthesized in-house. MMC has been extensively characterized as a drug carrier in previous work and had a mean pore size of 5 nm, a specific surface area of 650 m2/g, and a total pore volume of 0.81 cm3/g (11,18). Hydroxypropyl methyl cellulose (HPMC) (Methocel E4M Premium CR) was generously donated by Colorcon (Dartford, UK). Fenofibrate, tolfenamic acid, tamoxifen, hydrocortisone, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, US). Tamoxifen and ketoconazole were purchased from Toronto Research Chemicals (Toronto, CAN). All drugs were used as received. The nylon net filter, 11-μm pore size, used in the protective filters were purchased from Merck Millipore (Billerica, MA, US). FaSSGF/FaSSIF/FeSSIF powder was purchased from biorelevant.com (Croydon, UK).
Micronization of the Crystalline Material
Each crystalline drug compound (461–500 mg) was micronized in a planetary ball mill (Model PM 100 Retsch, Germany), to reduce particle size and thereby increase the dissolution rate. The drug was added, together with ten small steel beads (Ø 5 mm) to the milling bowl, and milled for 20 min at 600 rpm.
Loading of the Carrier
Loading of the MMC was performed using a modified soaking method previously described by Zhang et al. (10). The drugs were loaded into the MMC at a 1:10 ratio by dissolving 1 mg/mL drug in ethanol, and then adding 10 mg/mL MMC to the solution. The loading degree used was chosen to ensure that the all compounds adsorbed into the MMC were in an amorphous state, and was furthermore intentionally kept low to enable challenging conditions (i.e., need for high amounts of MMC particles) during the release studies. The suspension was stirred on a magnetic stirrer at 500 rpm for 24 h. The ethanol was then evaporated at 70 °C during a time period of 24 h.
Characterization of Loaded Carrier
The thermal behavior of the drugs and drug-loaded MMC was determined with a Q2000 Differential Scanning Calorimeter (TA Instrument Co., USA). The DSC cell was calibrated with indium (melting temperature, Tm = 156.59 °C and heat of fusion, Hf = 28.57 J/g) and then purged with 50 mL/min of nitrogen. For determining the Tm of the crystalline drug, 2.2–4.3 mg was weighed into an aluminum pan that was then sealed with an aluminum lid containing pinholes. The samples were equilibrated at 0 °C and thereafter heated at 10 °C/min to 29–53 °C above the Tm of the drug. To increase the sensitivity, and confirm the absence of crystallinity, modulated DSC (mDSC) was performed on the drug-loaded MMC. In these measurements, 9.2–10.2 mg of drug-loaded MMC was weighed into the aluminum pans and each sample was equilibrated at 0 °C. It was then modulated at ± 0.5 °C every 60 s and heated at 1 °C/min to a temperature 20–53 °C above the Tm of the drug. Onset values of Tm are reported.
Drug Release Studies from Loaded MMC
Preparation of Dissolution Media
Fasted-state simulated intestinal fluid version 1 (FaSSIF) was prepared in accordance with the instructions from the manufacturer. The ready-to-use simulated intestinal fluid powder was dissolved in phosphate buffer (PhB), pH 6.5. To increase the complexity of the system and allow for studies of stabilization of supersaturation, a medium was prepared in which 0.01% w/w HPMC was pre-dissolved in PhB. Two modified versions of FaSSIF, at pH 7.5 and 8.5, were also made by adjusting the pH of the PhB (originally set to pH 6.5).
Evaluation of the Nylon Filters
Determination of Degradation Products by UPLC-MS
A shift in the UV spectrum occurred during the release of fenofibrate-loaded MMC, so UPLC-MS was used to determine the origin of the shift. Two samples were weighed into Eppendorf tubes, one with 8 mg fenofibrate-loaded MMC and the other with 0.7 mg crystalline fenofibrate and 7.2 mg MMC. Two milliliters of Milli-Q water was added to the tubes, which were then placed on a plate shaker at 37 °C for 24 h. The supernatants were recovered by centrifugation at 37 °C for 15 min at 23,000×g (Heraeus Megafuge 8R, ThermoScientific) and analyzed by obtaining a full MS spectrum (QTRAP 6500, AB Sciex). A stock solution of fenofibrate dissolved in DMSO was used as reference. Separation was performed on an Agilent 1290 LC-system, with a Waters BEH C18 2.1 × 50 mm (1.7 μm) column, and the run was made in positive mode. The mobile phase consisted of (A) 5% acetonitrile, 0.1% formic acid, and 94.9% Milli-Q water; and (B) 95% acetonitrile, 0.1% formic acid, and 4.9% Milli-Q water. A constant flow rate of 0.5 mL/min was used for the gradient elution as follows: A was constant at 99% for 1.0 min, then decreased linearly to 20% for 7.30 min. A was thereafter decreased to 10% for 0.2 min, kept constant for 0.5 min, and then increased to 99% for 0.5 min.
The mean and standard deviation were determined for all measurements. The area under the curve (AUC) was calculated for the release profiles using GraphPad Prism 7.03, and one-way ANOVA analysis was performed on the AUC values for each compound (GraphPad Software, Inc., San Diego, US). Tukey’s multiple comparison test with a single pooled variance was performed to determine differences between the three setups. Limit for significance was set to p < 0.05.
RESULTS AND DISCUSSION
Characterization of the Loaded Carrier
The PWSDs were selected on the basis of their physicochemical properties. Tm values for all compounds are shown in Table I. No Tm was detected for compounds loaded into the MMC (Supplementary data, Fig.S2). This lack of drug crystallinity when incorporated to the MMC is consistent with previously published results (10,11). For all compounds, a solvent (water) endotherm was observed in the thermograms (Supplementary data, Fig.S2). This indicates that the drug-loaded MMC had absorbed some water during storage. However, the absorbed water did not seem to affect the stability of the system, since no recrystallization was detected.
Drug Release Studies from Loaded MMC
Evaluation of Nylon Filters
An important aspect when selecting filter material is the potential adsorption of the drug and/or carrier. This work focused on supersaturated systems, and the degree of adsorption of the drug to the filters was not evaluated. For one of the compounds studied (hydrocortisone), non-saturated conditions were applied. The micronized hydrocortisone with MMC present reaches a plateau equivalent to the amount of drug added, 360 μg/mL (i.e., 5.5 mg drug in 15 mL FaSSIF) (Fig. 2b). This indicates limited adsorption of this lipophilic drug onto the filter. However, compound-specific adsorption to the filter material needs to be evaluated when, e.g., sink conditions are used.
The release profiles from the drug-loaded MMCs were compound dependent. Supersaturation was observed during the release of ketoconazole and hydrocortisone (Fig. 2a, b). The degree of supersaturation was higher for ketoconazole than hydrocortisone and can be explained by the amount that had to be added in relation to the Sapp of the drug. For ketoconazole, amounts equivalent to 12 × Sapp were used, whereas only 1.6 × Sapp was used for hydrocortisone, because of the large difference in solubility between the two drugs. The 15.1-fold higher solubility of hydrocortisone requires large amounts of drug to be added into the vial, which means that the number of MMC particles must also be significantly higher to study supersaturation. The higher supersaturation level of ketoconazole compared to hydrocortisone also explains why ketoconazole crystallizes more rapidly.
The release of one of the drugs, hydrocortisone, was incomplete during the time course of the experiment (Fig. 2b), even though the amount added was lower than the solubility of the compound. About 360 μg/mL of hydrocortisone (9% of 4 mg/mL drug-loaded MMC) was added, but only about 320 μg/mL was released (Supplementary data, Tab. S1). Incomplete release from MMC has also been reported by Zhang et al., who speculate that it can be due to the diffusion time through different particle sizes (12). The MMC in our study was polydisperse in terms of size, and this may have affected the amount released from the MMC.
Physicochemical Properties and Apparent Solubility of Compounds Investigated
M w a
Sapp (μg/mL) FaSSIFb
80.5 ± 0.02
16.4 ± 0.3
221.4 ± 0.35
454.3 ± 32.1
147.5 ± 0.22
30.0 ± 0.8
212.2 ± 0.20
66.7 ± 0.8
97.1 ± 0.21
152.9 ± 10.8
Determination of Degradation Products by UPLC-MS
Small-scale methods to evaluate promising drug delivery systems are a prerequisite for successful selection of formulations in early development stages. In this work, we present a method with the μDiss profiler and protective nylon filters to study release from particle-rich suspensions of a complex carrier-mediated system. The setup allows for determination of immediate release and studies of supersaturation in suspensions of 4–8 mg/mL of drug-loaded carrier. The effect of a stabilizer, HPMC, could also be determined. During the drug release of one compound, fenofibrate, a change in the UV spectra was detected, which also showcases the versatility of the method. In conclusion, the method is applicable to studies of a variety of compounds and formulations, filling an existing gap in the current evaluation of promising drug delivery systems.
The authors thank Dr. André Mateus for his assistance with the UPLC-MS measurements and Erik Petersson for his help with the initial experimental work. Colorcon (Dartford, UK) is acknowledged for their generous donation of the Methocel E4M Premium CR used in the study.
This study was financially supported by the Swedish Research Council (grant no. 2014-3929).
Compliance with Ethical Standards
Maria Strømme declares she is the inventor of MMC and holds shares in the company Disruptive Materials.
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