PMC:7561592 / 9184-21218 JSONTXT

{"project":"2_test","denotations":[{"id":"33063245-32592045-28135","span":{"begin":1124,"end":1126},"obj":"32592045"},{"id":"33063245-29380369-28136","span":{"begin":5192,"end":5194},"obj":"29380369"},{"id":"33063245-15497676-28137","span":{"begin":5737,"end":5739},"obj":"15497676"},{"id":"33063245-20726533-28138","span":{"begin":8016,"end":8018},"obj":"20726533"},{"id":"33063245-32592045-28139","span":{"begin":10752,"end":10754},"obj":"32592045"}],"text":"MATERIALS AND METHODS\n\nMaterials\nAmmonium acetate (high-performance liquid chromatography (HPLC) grade), 37% hydrochloric acid, sodium hydroxide, sodium chloride, glacial acetic acid, potassium dihydrogen phosphate, acetonitrile (HPLC grade) and methanol (HPLC grade) were purchased from Fisher Scientific (UK). Trifluoroacetic acid (TFA) (HPLC grade), montelukast sodium (pharmaceutical secondary standard) and mesalazine (≥ 99%) were obtained from Sigma-Aldrich Company Ltd. (UK). Water was ultra-pure (Milli-Q) laboratory grade. Regenerated cellulose (RC) membrane filters (0.45 μm) (Cronus®, UK), and filter papers (0.45 μm), polytetrafluoroethylene (PTFE) filters (0.45 μm) and glass microfiber (GF/D) filters (2.7 μm) (Whatman®, UK) were used. Porous full flow polyethylene cannula filters (10 μm) were obtained from Quality Lab Accessories LCC (USA). Nine different soft foods and drinks were used as co-administration vehicles. These were chosen based on differences in their composition, physicochemical properties and drug solubility in each vehicle; these factors are extensively described in our previous study (11). Orange squash, milk U.H.T. full fat and orange juice were purchased from The Co-Operative (UK). Blackcurrant squash was from Lucozade Ribena Suntory Ltd. (UK). First Infant Milk (cow’s milk-based formula) was from Cow \u0026 Gate (UK). Applesauces were Bramley applesauce Colman’s of Norwich (referred to as ‘applesauce UK’) from Unilever (UK) and Apfelmark applesauce (referred to as ‘applesauce DE’) from Bauck Hof (Germany). Plain yoghurt from Yeo Valley (UK) and Greek yoghurt from Fage (Greece) were also used. The four formulations studied were kindly donated by AstraZeneca (UK). Product information is summarised in Table I.\nTable I Information of the Formulations Used in This Study\nActive principal ingredient (API) Brand Manufacturer Formulation type/release mechanism Excipients Dose tested (mg) Administration recommendations (26)\nMesalazine Salofalk® Dr. Falk Pharma (UK) Granules/delayed release pH-dependent Aspartame (E 951), carmellose sodium, cellulose, citric acid, silica, hypromellose, magnesium stearate, Eudragit L 100, Eudragit NE 40 D containing 2% nonoxynol 100, povidone K 25, simeticone, sorbic acid, talc, titanium dioxide (E 171), triethyl citrate, vanilla custard flavour 135 Granules should be placed on the tongue and washed down with water without chewing.\nPentasa® Ferring (UK) Granules/extended release pH-independent coating Ethylcellulose, povidone 135 Granules should be placed on the tongue and washed down with water or orange juice, without chewing. Contents of one sachet should be weighed and divided immediately before use; any remaining granules should be discarded.\nMontelukast Singulair® Merck Sharp \u0026 Dohme Ltd. (UK) Granules/immediate release Mannitol, hydroxy-propyl cellulose, magnesium stearate 4 Granules may be swallowed or mixed with cold, soft foods (not liquid), and taken immediately.\nActavis® Actavis (UK) Chewable tablets/immediate release Lactose monohydrate, aspartame 5 Tablet should not be taken with food; should be taken at least 1 h before or 2 h after food.\n\nMethods\n\nDissolution Media Preparation\nSimulated gastric fluid sine pepsin (SGFsp) pH 1.2 and simulated intestinal fluid (SIFsp) were prepared according to the USP recipes (27). Double concentrated SIFsp containing an additional amount of sodium hydroxide (to neutralise the acid present on the first step) was prepared for the two-stage dissolution studies performed.\n\nSample Preparation\nSquashes and formula were prepared as per manufacturer’s instructions (formula: 1 scoop of powder (approximately 4.5 g) was added to 30 mL of boiled cooled water; squashes: 25 (orange) or 50 (blackcurrant) mL of concentrated product were diluted in 250 mL of water).\nFor the direct administration scenario, the formulations were introduced in the media without being mixed with a vehicle. For the scenario of mixing the formulations with vehicles, each sample was prepared by addition of the formulations (corresponding to the ‘dose tested’ in Table I) to 25 mL of drink or 10 mL (approximately 10 g) of soft food, at room temperature. All samples were then manually mixed with a stainless-steel spatula, for 30 s. Actavis® chewable tablets were crushed prior to being mixed with the vehicles or tested.\nTo test different administration practices, samples with vehicles were prepared as described above and set aside (at room temperature and protected from direct light), and after 4 h, they were remixed with a stainless-steel spatula, prior to performing the study.\n\nIn Vitro Dissolution Studies\nDissolution studies were performed with a mini-paddle apparatus (Agilent Technologies 708-DS apparatus configured with TruAlign 200-mL vessels and electropolished stainless steel mini-paddles; Agilent, USA). Experiments were conducted in a two-stage approach: in SGFsp pH 1.2 (total volume with sample: 100 mL), for 1 h, followed by SIFsp pH 6.8 (final volume: 200 mL), for 3 h. A dissolution study with a sequential media change mimics the passage of oral dosage forms through the GI tract, providing an understanding of the in vivo drug performance (28). Experiments were conducted at 37 ± 0.5°C and the agitation rate of the mini-paddle was set to 50 revolutions per minute (rpm). Sample collection took place at 5, 15, 30, 45, 60, 75, 90, 120, 180 and 240 min. Two-millilitre samples were withdrawn (with volume replacement with the corresponding media), using a 2-mL glass syringe (Fortuna Optima® fitted with a stainless tubing) through a cannula fitted with a full flow filter (10 μm). All experiments were performed without direct light exposure to avoid photodegradation of the drugs (29,30). After collection, samples were filtered through a GF/D (2.7 μm) filter and treated. Sample treatment was as follows: 1000 μL of acetonitrile (montelukast) or 10% (v/v) TFA/water (mesalazine) were added to 500 μL of sample. This mixture was vortexed (HTZ, UK) for 1 min and centrifuged (8000 rpm, 15 min, 4°C) (Beckman Coulter J2-MC centrifuge, UK). The supernatant was filtered through a RC (montelukast) or PTFE (mesalazine) (0.45 μm) filter, placed in an HPLC amber vial and analysed. The pH of the media was measured at the end of each experiment.\nThe effect of different administration scenarios and testing conditions was investigated by varying the dissolution test parameters, as described in Table II. These were as follows: (1) effect of co-administration of formulation with selected vehicles in comparison to direct administration of formulation; (2) effect of different mixing patterns (i.e. time between preparation and administration/testing of the formulation-vehicle mixture); and (3) effect of hydrodynamics (50 vs 100 rpm, in selected studies of Pentasa® and Singulair® granules).\nTable II Dosing Scenarios and Testing Conditions Investigated\nSetup Agitation speed (rpm) Scenario: direct introduction Scenario: mixing with vehicles Formulations Mixing pattern (h)\n1 50 ✓ M, OJ, BLS, PY, APSUK, F, OS, GY, APSDE All 0\n2 50 N/A M, OJ, BLS, PY, APSUK All 4\n3 100 ✓ M, OJ, BLS, PY, APSUK Singulair®, Pentasa® 0\nBLS, blackcurrant squash; OS, orange squash; M, milk; F, formula; OJ, orange juice; PY, plain yoghurt; GY, Greek yoghurt; APSUK, applesauce UK; APSDE, applesauce DE; N/A, not applicable\nAll experiments were performed in triplicate. Fresh calibration curves (concentration range: 0.5–100 μg/mL (montelukast) and 0.5–200 μg/mL (mesalazine)) were prepared in the corresponding media, by appropriate dilution of a 1000 μg/mL stock solution of the analytical standard in methanol (montelukast) or 0.05% TFA/water (mesalazine); the same treatment process was applied as described for the samples. Results were expressed as mean percentage of drug dissolved ± standard deviation (S.D.), at the given sampling time.\n\nChromatographic Conditions for Drug Analysis\nThe chromatographic methods used for drug analysis were modifications of published methods (31,32). Drug quantification was performed with HPLC with ultraviolet (UV) detection (Agilent HPLC system 1100/1200 series; Agilent, USA). A RP Agilent Eclipse XDB C18 column (250 mm × 4.6 mm, 5-μm particle size) was used for both drugs. For montelukast, the mobile phase was composed of ammonium acetate buffer pH 5.5 and methanol (solvents A and B, respectively) delivered at a flow rate of 1 mL min−1, on a linear gradient. The selected gradient started with 10% of solvent B, which was increased to 50% within 2 min, and 90% within 4 min; at 11.30 min, the initial conditions of analysis were re-established. Injection volume was 100 μL. Analysis was performed at 20°C and the detection wavelength was 284 nm. Elution time for montelukast was 8.9 min. For analysis of mesalazine, the mobile phase was composed of 0.05% TFA/water and methanol (95:5), delivered at a flow rate of 1 mL min−1. Injection volume was 20 μL. Analysis was performed at 40°C and the detection wavelength was 304 nm. Elution time for mesalazine was 4.6 min.\n\nStatistical Analysis of Dissolution Data\nTo describe and compare the dissolution profiles obtained, linear trapezoidal method was used to calculate the area under the curve of each profile over 4 h (AUC0–4h). This allowed the use of one value representative of drug dissolution to compare the different scenarios tested.\nOne-way analysis of variance (ANOVA) with a post hoc Tukey honest significant difference (HSD) test was conducted to investigate statistically significant differences (p \u003c 0.05 noting significance level) in the AUC0–4h between direct administration of formulation and mixing the formulations with the different vehicles. t test analysis was used to compare AUC0–4h results obtained between drug dissolution after mixing the formulations with vehicles of same subtype or drug dissolution after mixing the formulations with the same vehicle under different testing conditions (i.e. agitation rate or time between preparation and mixing) (p \u003c 0.05 noting significance). The analyses were performed with GraphPad Prism® v.7 software (San Diego, USA).\nPartial least squares regression (PLS-R) analysis was used to correlate the AUC0–4h values of the different testing scenarios (response factor) with the physicochemical properties and macronutrient composition of the vehicles (pH, buffer capacity, surface tension, viscosity, osmolality; percentage of fat, sugars and proteins), drug solubility in each vehicle, type of formulation and testing conditions (i.e. preparation time) (XLSTAT Software; an Add-In for Excel, Microsoft®). The physicochemical properties and macronutrient composition of the vehicles and well as drug solubility values in each vehicle were previously presented (11). When analysing both drugs together, drug characteristics (logP (log octanol-water partition coefficient) and ionisation percentage (obtained from ACD/Labs© 2010–2018)) were also considered as variables. The quality of the model was evaluated with the square of the coefficient of determination (R2) and goodness of prediction (Q2), with values close to 1 being indicative of good fit and prediction power, respectively (33). 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). The standardised coefficients of the factors indicated the relative effect (positive or negative) of their corresponding variables on the response. The variable importance in projection (VIP) value was used to evaluate the importance of each factor on the model (33). Model variables with VIP values \u003e 1 were evaluated as the most important in explaining the variation in the dependent variable, whilst values between 0.7 and 1 were considered moderately influential for the model. Values \u003c 0.7 were deemed not of significance for the prediction of the dependent variable (33)."}