PurposeTo characterise a biorelevant simulated lung fluid (SLF) based on the composition of human respiratory tract lining fluid. SLF was compared to other media which have been utilized as lung fluid simulants in terms of fluid structure, biocompatibility and performance in inhalation biopharmaceutical assays.MethodsThe structure of SLF was investigated using cryo-transmission electron microscopy, photon correlation spectroscopy and Langmuir isotherms. Biocompatibility with A549 alveolar epithelial cells was determined by MTT assay, morphometric observations and transcriptomic analysis. Biopharmaceutical applicability was evaluated by measuring the solubility and dissolution of beclomethasone dipropionate (BDP) and fluticasone propionate (FP), in SLF.ResultsSLF exhibited a colloidal structure, possessing vesicles similar in nature to those found in lung fluid extracts. No adverse effect on A549 cells was apparent after exposure to the SLF for 24 h, although some metabolic changes were identified consistent with the change of culture medium to a more lung-like composition. The solubility and dissolution of BDP and FP in SLF were enhanced compared to Gamble’s solution.ConclusionThe SLF reported herein constitutes a biorelevant synthetic simulant which is suitable to study biopharmaceutical properties of inhalation medicines such as those being proposed for an inhaled biopharmaceutics classification system.
Biorelevant fluids are required to enable meaningful in vitro experimental determinations of the biopharmaceutical properties of inhaled medicines, e.g. drug solubility, particle dissolution, cellular uptake. Our aim was to develop a biorelevant simulated lung fluid (SLF) with a well-defined composition and evidence-based directions for use. The SLF contained dipalmitoylphosphotidylcholine, dipalmitoylphosphatidylglycerol, cholesterol, albumin, IgG, transferrin and antioxidants. Freshly made SLF had pH 7.2, viscosity 1.138 × 10−3 Pa s, conductivity 14.5 mS/m, surface tension 54.9 mN/m and density 0.999 g/cm3. Colour, surface tension and conductivity were the most sensitive indicators of product deterioration. The simulant was stable for 24 h and 48 h at 37 °C and 21 °C, respectively, (in-use stability) and for 14 days when stored in a refrigerator (storage stability). To extend stability, the SLF was vacuum freeze-dried in batches to produce lyophilised powder that can be reconstituted readily when needed at the point of use. In conclusion, we have reported the composition and manufacture of a biorelevant, synthetic SLF, provided a detailed physico-chemical characterisation and recommendations for how to store and use a product that can be used to generate experimental data to provide inputs to computational models that predict drug bioavailability in the lungs.
This article reports on the stereochemical aspects of the chemical stability of lactose solutions stored between 25 and 60 °C. The lactose used for the preparation of the aqueous solutions was α-lactose monohydrate with an anomer purity of 96% α and 4% β based on the supplied certificate of analysis (using a GC analytical protocol), which was further confirmed here by nuclear magnetic resonance (NMR) analysis. Aliquots of lactose solutions were collected at different time points after the solutions were prepared and freeze-dried to remove water and halt epimerization for subsequent analysis by NMR. Epimerization was also monitored by polarimetry and infrared spectroscopy using a specially adapted Fourier transform infrared attenuated total reflectance (FTIR-ATR) method. Hydrolysis was analyzed by ion chromatography. The three different analytical approaches unambiguously showed that the epimerization of lactose in aqueous solution follows first order reversible kinetics between 25 to 60 °C. The overall rate constant was 4.4 × 10(-4) s(-1) ± 0.9 (± standard deviation (SD)) at 25 °C. The forward rate constant was 1.6 times greater than the reverse rate constant, leading to an equilibrium constant of 1.6 ± 0.1 (±SD) at 25 °C. The rate of epimerization for lactose increased with temperature and an Arrhenius plot yielded an activation energy of +52.3 kJ/mol supporting the hypothesis that the mechanism of lactose epimerization involves the formation of extremely short-lived intermediate structures. The main mechanism affecting lactose stability is epimerization, as no permanent hydrolysis or chemical degradation was observed. When preparing aqueous solutions of lactose, immediate storage in an ice bath at 0 °C will allow approximately 3 min (180 s) of analysis time before the anomeric ratio alters significantly (greater than 1%) from the solid state composition of the starting material. In contrast a controlled anomeric composition (~38% α and ~62% β) will be achieved if an aqueous solution is left to equilibrate for over 4 h at 25 °C, while increasing the temperature up to 60 °C rapidly reduces the required equilibration time.
The dissolution of inhaled drug particles in the lungs is a challenge to model using biorelevant methods in terms of (i) collecting a respirable emitted aerosol fraction and dose, (ii) presenting this to a small volume of medium that is representative of lung lining fluid, and (iii) measuring the low concentrations of drug released. We report developments in methodology for each of these steps and utilize mechanistic in silico modeling to evaluate the in vitro dissolution profiles in the context of plasma concentration–time profiles. The PreciseInhale aerosol delivery system was used to deliver Flixotide aerosol particles to DissolvIt apparatus for measurement of dissolution. Different media were used in the DissolvIt chamber to investigate their effect on dissolution profiles, these were (i) 1.5% poly(ethylene oxide) with 0.4% l-alphaphosphatidyl choline, (ii) Survanta, and (iii) a synthetic simulated lung lining fluid (SLF) based on human lung fluid composition. For fluticasone proprionate (FP) quantification, solid phase extraction was used for sample preparation with LC–MS/MS analysis to provide an assay that was fit for purpose with a limit of quantification for FP of 312 pg/mL. FP concentration–time profiles in the flow-past perfusate were similar irrespective of the medium used in the DissolvIt chamber (∼0.04–0.07%/min), but these were significantly lower than transfer of drug from air-to-perfusate in isolated perfused lungs (0.12%/min). This difference was attributed to the DissolvIt system representing slower dissolution in the central region of the lungs (which feature nonsink conditions) compared to the peripheral regions that are represented in the isolated lung preparation. Pharmacokinetic parameters (Cmax, Tmax, and AUC0-∞) were estimated from the profiles for dissolution in the different lung fluid simulants and were predicted by the simulation within 2-fold of the values reported for inhaled FP (1000 μg dose) administered via Flixotide Evohaler 250 μg strength inhaler in man. In conclusion, we report methods for performing biorelevant dissolution studies for orally inhaled products and illustrate how they can provide inputs parameters for physiologically based pharmacokinetic (PBPK) modeling of inhaled medicines.
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