Ultra-high temperature treated whole cow’s milk standardized to less than 4% fat was acquired from Sainsbury’s, UK. First infant milk formula (cow’s milk-based formula) (Cow & Gate, UK) and smooth applesauce were acquired from Sainsbury’s, UK.
Ammonium acetate, acetonitrile (ACN), and methanol [high-performance liquid chromatography (HPLC) grade], 37% hydrochloric acid, sodium hydroxide, sodium chloride, sodium acetate trihydrate, glacial acetic acid were purchased from Fisher Scientific (UK). Water was ultra-pure (Milli-Q) laboratory grade.
Montelukast sodium pharmaceutical secondary standard certified reference material, sodium oleate, pepsin (from porcine), and maleic acid were obtained from Sigma-Aldrich Company Ltd., UK. Singulair® granules and chewable tablets (4 mg) (Merck Sharp & Dohme Ltd, UK) were obtained from UK and Belgium pharmacies. Sodium taurocholate (NaTC) (Prodotti Chimici Alimentari S.P.A., Italy), egg lecithin Lipoid EPCS (Lipoid GmbH, Germany), and glyceryl monooleate – Rylo Mg 19 (Danisco, Denmark) were obtained from the specified sources.
Regenerated cellulose [RC] membrane filters (13 mm, 0.45 µm) (Cronus®, UK), and glass microfiber syringe filters grade GF/D (13 mm, 2.7 µm) (Whatman™, UK), and porous full-flow polyethylene cannula filters (10 µm) (Quality Lab Accessories LCC, USA) were used. Polytetrafluoroethylene (PTFE) filter (13 mm, 0.45 μm) was purchased from Fisher Scientific (UK). Mackerey Nagel (MN) grade GF4 (MN GF-4) filters (15 mm, 1,4 μm) were purchased from VWR, UK. Glass microfiber filters grade GF/F (24 mm, 0.7 µm) and GF/D (24 mm, 2.7 µm) were from Whatman™, UK. Glass wool was obtained from Sigma-Aldrich Company Ltd., UK.
Media Used for Dissolution Studies
Adult and pediatric biorelevant media were freshly prepared for each experiment, as described by Maharaj et al. (12). The formula used for the pediatric fed state gastric media representative of newborns (Pnc-FeSSGF) was prepared as per the manufacturer’s instructions: 1 scoop of powder (approximately 4.5 g) was added to 30 mL of boiled cooled water. For the two-stage dissolution testing, double concentrated FaSSIF-V2 was prepared with an additional amount of sodium hydroxide to achieve the final composition of FaSSIF-V2 (pH 6.5) after its addition to the gastric phase.
In Vitro Dissolution Studies
µDISS Profiler™ Dissolution Studies
Dissolution studies were carried out using a μDISS Profiler™ (Pion Inc., MA). The 4 mg pediatric clinical dose of Singulair® granules and crushed chewable tablet were down-scaled according to a final experimental volume of 20 mL, considering in vitro age-appropriate volumes (i.e. infants 150 mL, children 200 mL, and adults 500 mL). For infants, children, and adults, the down-scaled doses were 0.53, 0.4, and 0.16 mg, respectively. Dissolution studies were conducted either with a single-stage approach or a two-stage approach as presented in Fig. 1.
For the single-stage dissolution set-ups, the formulation was added into 20 mL of dissolution medium. For the two-stage dissolution, the formulation was initially added to 10 mL of the fasted gastric medium (i.e. fasted simulated gastric fluid (FaSSGF) for adults and children scenarios, and infant fasted gastric fluid (Pi-FaSSGF) in the infant scenario). At 30 min, 10 mL of double-concentrated fasted intestinal simulated fluid (FaSSIF-V2) was added to the dissolution vessel.
Samples were stirred at 300 rpm for all experiments and the concentrations of drug dissolved were monitored for 4 h. The temperature for all experiments was maintained at 37 °C by circulating water through a heating block mounted to the μDISS profiler™. Drug quantification was performed in situ by UV fiber optic dip probes (path length 5 mm, with wavelength 300 to 400 nm) connected to the Pion® Rainbow Ultraviolet (UV) spectrometer system. Multiwavelength UV spectra were recorded at 30-s intervals. Calibration curves prepared in respective fresh media before the experiment (0.5 to 30 µg/mL), by appropriate dilutions of a 1 mg/mL stock solution of montelukast analytical standard in methanol. All experiments were performed in triplicate.
USP 2 Apparatus and Mini-Paddle Apparatus Dissolution Studies
Dissolution studies of 4 mg Singulair® granules or crushed chewable tablets were performed with a single-stage approach in either a USP 2 apparatus (Agilent 708-DS Dissolution apparatus; Agilent, USA) or a mini-paddle apparatus where TruAlign 200 mL vessels and electropolished stainless-steel mini-paddles were used (Agilent, USA). Experiments were conducted at 37 °C. Volumes were selected according to the respective age group tested (Table I). The agitation rate in USP 2 apparatus was set at 50 revolutions per minute (rpm). For the mini-paddle apparatus, the agitation rate was set based on a speed factor of 2.5 between the paddle and mini-paddle hydrodynamics to reflect the agitation rate used in the USP 2 apparatus (31). Sample collection (2 mL samples withdrawn with corresponding medium replacement) took place at 5, 15, 30, 45, 60, 75, 90, 120, 180, and 240 min, using a glass syringe (Fortuna Optima® fitted with a stainless tubing) through a cannula fitted with a 10 µm full-flow filter. After collection, aqueous-based samples were filtered through a 0.45-µm PTFE filter, treated (1000 µL of acetonitrile were added to 500 µL of the filtered sample), vortexed (HTZ, Cheshire, UK) for 1 min, and injected into the HPLC. Filter adsorption studies were performed in triplicate during preliminary studies. No adsorption issues onto the filters used in this study were observed. Milk- and formula-based samples were filtered through a 2.7-µm GF/D filter, treated with acetonitrile (1000 µL of acetonitrile was added to 500 µL of the filtered sample), vortexed for 1 min, and centrifuged (8000 rpm, 15 min, 4 °C) (Beckman Coulter J2-MC centrifuge, High Wycombe, UK). The supernatant was filtered through a 0.45-µm PTFE filter and injected into the HPLC. All experiments were performed in triplicate. To explore the dissolution performance of the drug products, and investigate if there were experimental issues (such as coning), dissolution tests were performed with Singulair® granules in FeSSGF media (500 mL) with the USP 2 apparatus at three agitation rate conditions; 50, 75, and 100 rpm (data not shown).
USP 4 Apparatus Dissolution Studies
Dissolution Studies Simulating Fasted and Fed State
Dissolution studies were conducted in an Erweka® flow-through dissolution tester (USP 4 apparatus; model DFZ720, Erweka GmbH, Germany) connected to an Erweka® Piston Pump (model HKP720). For the fasted state dissolution experiments, the USP 4 apparatus was equipped with Ø 22.6 mm cells (maintained at 37 ± 0.5 °C). A 5-mm size glass bead was positioned in the bottom of the cell, and glass beads (1 mm size) were added to the conical part of each cell. On the top of the cell, Whatman® glass microfiber filters were used (GF/F and GF/D with pore sizes of 0.7 µm and 2.7 µm, respectively). During the experiment, 4 mg Singulair® granules or 4 mg crushed chewable tablets were placed on top of the beads. Biorelevant media (adult and pediatric) were used as dissolution media. Duration of exposure to the various media and corresponding flow rates were appropriately selected to mimic the fasted and fed state transit times and gastrointestinal volumes of each subpopulation. Flow rates were selected to achieve a balance between the duration of the drug product exposure to the various media, and total fluid volumes, taking into account the lack of radial water flux in vitro (32). The in vitro conditions used are presented in Table II. Experiments were run in open-loop mode, with fresh media continuously passing through the cells (33). Samples were collected in volumetric cylinders and exchanged every 15 min, diluted, and assayed by HPLC. Experiments were run in triplicate.
For the fed state studies, USP 4 apparatus powder and granule cells were used (maintained at 37 ± 0.5 °C). A glass bead (5-mm diameter) was positioned in the apex of the flow-through cells. The lower conical part of the cell was filled with the formulation (4 mg Singulair® granules or 4 mg crushed chewable tablets). Glass wool (0.10 g) was placed on top of the formulation on the lower part of the cell. On the top of each cell, 0.30 g of glass wool was added, followed by an MN GF-4 1.4 μm filter. Experimental conditions are described in Table II.
Aqueous-based samples were filtered through a 0.45-µm PTFE filter, treated (1000 µL of acetonitrile was added to 500 µL of the filtered sample), and injected into the HPLC. Milk- and formula-based samples were treated (1000 µL of acetonitrile was added to 500 µL of the filtered sample), vortexed for 1 min, and centrifuged (8000 rpm, 15 min, 4 °C) and the supernatant was filtered through a 0.45-µm PTFE filter and injected into the HPLC.
Dissolution Studies Simulating Medicine Co-administration Practices in Infants
According to the British National Formulary for Children (BNF-C), Singulair® granules may be swallowed or mixed with cold, soft foods (not liquid), and taken immediately (25). In the present study, in addition to the fasted direct administration of formulation scenario (as described in the previous subsection), two additional scenarios were investigated: mixing formulation with formula milk or applesauce. These vehicles were selected due to their use of dosing vehicles for the administration of montelukast granules in the in vivo PK studies in infants (30,31,32). Because the prandial state of the patients was not reported in the in vivo study, both fasted and fed intestinal conditions were simulated in vitro. The USP 4 apparatus conditions for the simulation of medicine co-administration practices in infants are presented in Table III. In the scenarios where the drug was mixed with vehicles, each sample was prepared by the addition of the formulation to 5 mL of applesauce/formula measured with a plastic syringe, followed by mixing with a stainless-steel spatula. On the top of the USP 4 apparatus cell (Ø 22.6 mm cells, maintained at 37 ± 0.5 °C), Whatman® glass microfiber filters, GF/F (0.7 µm) and a GF/D (2.7 µm), separated by a layer of 0.10 g of glass wool were used for sample filtration. The remaining dissolution conditions (temperature, sampling points, sample collection, and treatment) were performed as described in the previous subsection.
Chromatographic Conditions for Drug Quantification
HPLC analysis was performed using a Hewlett Packard Series 1100 system equipped with an autosampler, temperature-regulated column compartment, quaternary pump, and diode array detector (DAD) (Agilent Technologies, UK). The chromatographic method used for the quantification of montelukast was a modification of the method by Raju NK et al. (34). A C18 column was used (Kromasil 100 Å C18 4.6 × 250 mm, 5 µm). The injection volume was 100 µL. The temperature of the column compartment and the sample tray was set at 20 °C. The mobile phase consisted of methanol: ammonium acetate buffer (50 mM; pH = 5.5) (90:10 v/v); the flow rate was 1 mL/min and the DAD detector was set at λ = 284 nm. The run time was set at 10 min. Quantification of montelukast in samples was performed with calibration curves of freshly prepared standard solutions (calibration curve range: 0.3–40 μg/mL). Standards were prepared in the medium of interest for each experiment by appropriate dilution of a 1 mg/mL stock solution of montelukast analytical standard in methanol. The limit of detection and the limit of quantification were 0.12 and 0.3 μg/mL, respectively.
Statistical Analysis of Dissolution Data
To describe and compare the obtained drug dissolution profiles, the linear trapezoidal method was used to calculate the area under the curve for each dissolution profile over 4 h (AUC0-4 h) using Microsoft Excel 2016, Office 365® (Microsoft, USA). This allowed the use of a single representative value of drug dissolution to compare the different scenarios tested.
Multivariate Statistical Analysis of In Vitro Dissolution Studies
Dissolution studies investigated in the µDISS profiler™, USP 2 apparatus, mini-paddle apparatus, and USP 4 apparatus (fasted and fed state with direct administration of the formulation) were analyzed with partial least squares regression (PLS-R) analysis. The PLS-R was used to correlate the AUC0-4 h (%diss⋅h) (dependent variable) with the explanatory variables that described the dissolution set-ups used in the experiments. PLS-R analysis was performed with XLSTAT Software (an add-in for Excel, Microsoft®). The explanatory variables were (i) gastrointestinal compartment (gastric, intestinal, gastric/intestinal); (ii) prandial state (fasted or fed); (iii) formulation (granules or chewable tablets); and (iv) hydrodynamics (µDISS profiler™, USP 2 apparatus, or mini-paddle and USP 4 apparatus). All explanatory variables were set as categorical variables. PLS-R analysis was grouped based on age: infants, children, and adults. For newborns, since the montelukast drug label does not recommend the use of this formulation in this age group, only exploratory dissolution studies were performed in the USP 4 apparatus; therefore, this age group was not included in the PLS-R analyses (26). The quality of the model was evaluated by the square of the coefficient of determination (R2) and goodness of prediction (Q2) (34,35,36). Values close to 1 are indicative of good fit and prediction power, respectively. Full cross-validation (leave-one-out procedure) was used to develop and evaluate the regression model. The optimum number of calibration factors for each model was selected based on the optimum predictability of the model and predicted residual error sum of squares (PRESS) (35,36,37). Standardized coefficients, that indicate the relative effect (positive or negative) of their corresponding variables on the response, were generated for each independent variable (35–37. The variable importance in projection (VIP) value was used to evaluate the importance of each factor on the model (35,36,37). Model variables with VIP values > 1 were evaluated as the most important in explaining the variation in the dependent variable, while values between 0.7 and 1 were considered to have a moderate impact. Values < 0.7 were deemed as not significant for the prediction of the dependent variable (35,36,37).
Impact of Medicine Co-administration Practices on Drug Dissolution Performance in Infants
Dissolution data from the studies simulating medicine co-administration practices in infants were analyzed. One-way analysis of variance (ANOVA) with a post hoc Tukey’s multiple comparisons test was performed (GraphPad Prism® v.6 Software) to investigate statistically significant differences (p < 0.05 noting significance level) in the in vitro AUC0-4 h obtained for the different dosing scenarios tested. Plasma concentration–time of montelukast granules (4 mg) co-administered with formula or applesauce to 3 infant cohorts (formula: 1 to 3 months; applesauce: 3 to 6 months and 6 to 24 months) were obtained from the literature (27,28,29,30). The observed PK profiles found in the literature were digitalized with the WebPlotDigitizer® v4.1 software. Non-compartmental PK data analysis of the mean plasma concentration–time profile for each age group was performed with PKSolver® (add-in program for Microsoft Excel®) (39). In vitro AUC0-4 h was compared to in vivo AUC0-24 h.