Use of PBPK Modeling To Evaluate the Performance of Dissolv It, a Biorelevant Dissolution Assay for Orally Inhaled Drug Products.

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 Dissolv It apparatus for measurement of dissolution. Different media were used in the Dissolv It 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 Dissolv It 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 Dissolv It 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.

Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles.

Thermostable dry powder inhaler (DPI) formulations with high aerosol performance are attractive inhalable solid dosage forms for local treatment of inflammatory lung diseases. We recently demonstrated that lipidoid-polymer hybrid nanoparticles (LPNs) loaded with small interfering RNA (siRNA) directed against tumor necrosis factor alpha (TNF-α) mediate efficient intracellular siRNA delivery and reduce inflammation in vivo. Here, we show that mixtures of the stabilizing excipients trehalose (Tre) and dextran (Dex), in combination with the shell-forming dispersion enhancer leucine (Leu), stabilize TNF-α siRNA-loaded LPNs during spray drying into nanocomposite microparticles, and result in DPI formulations with high aerosol performance. At low Leu content (0 to 10%, w/w), the DPI formulations were amorphous, and exhibited poor aerosol performance. When the Leu content was increased from 20 to 60% (w/w), the surface content of Leu increased from 39.2 to 68.1 mol%, and the flowability was significantly improved. Microscopy analysis suggest that the improved powder dispersibility is the result of a wrinkled surface morphology, which reduces the surface area available for interparticle interactions. Increasing the Leu content further (to above 10%, w/w) did not influence the aerosol performance, and the aerosol yield was maximal at 30-40% Leu (w/w). Formulations containing 40% Leu and a Tre:Dex ratio of 10:90 (w/w) displayed a high fine particle fraction and aerosol properties suitable for inhalation. The chemical integrity of TNF-α siRNA was preserved in the solid state, and biodistribution studies in mice showed that pulmonary administration of DPI formulations with high aerosol performance resulted in homogenous deep lung deposition. Our results demonstrate that at optimal ratios, ternary excipient mixtures of Leu, Tre and Dex protect TNF-α siRNA-loaded LPNs during spray drying. Hence, this study shows that microparticles with an amorphous Tre/Dex matrix and a crystalline Leu shell efficiently stabilize the nanocomposite LPNs in the solid state, and ensure aerosol properties suitable for inhalation.

Adapting the Aerogen Mesh Nebulizer for Dried Aerosol Exposures Using the PreciseInhale Platform.

Background: Many substances used in inhalation research are water soluble and can be administered as nebulized solutions. Typical examples are therapeutic, small-molecular agents, or macromolecules. Another category is a number of water-soluble agents used for airway diagnostics or disease modeling. Mesh nebulizers have facilitated well-controlled liquid aerosol exposures. Meanwhile, a benchtop inhalation platform, PreciseInhale, was developed for providing small-scale, well-controlled aerosol exposures in preclinical configurations. The purpose of the current research was to adapt the Aerogen mesh nebulizer to work within the PreciseInhale system for both cell culture and rodent exposures. Methods: The wet aerosols produced with the Aerogen Pro nebulizer were dried out in an aerosol holding chamber by supplying dry carrier air, which was provided by passing the incoming ambient air through a column with silica gel. The nebulizer was installed in an aerosol holding chamber between an upstream flow-rate pneumotach and a downstream aerosol monitor. By pulsing, the nebulizer output was reduced to 1%-10% of continuous operation to better match the exposure ventilation requirements. Additional drying was obtained by mantling the holding chamber with dried paper. Results and Conclusions: The nebulizer output was reduced to 3-30 µL/min and dried out before reaching the in vitro or in vivo exposure modules. Using solute concentrations in the range of 0.5%-2% (w/w), dried aerosols were produced with a mass median aerodynamic diameter of 1.5-2.0 µm, compared to the 4-5 µm droplets emitted by the nebulizer. Controlling the Aerogen nebulizer under a reduced output scheme within the PreciseInhale platform gave two major advantages: (i) by reducing aerosol output to better match exposure flow rates of single rodents, increased airway deposition yields were obtained in a range of 1%-10% relative to the nebulized amount of test substance and (ii) shrinking aerosol particle sizes through drying improved the peripheral lung deposition of test aerosols.

Two different mechanisms for modulation of bronchoconstriction in guinea-pigs by cyclooxygenase metabolites.

Leukotriene D(4) (LTD(4))-induced bronchoconstriction in guinea-pig airways has a cyclooxygenase (COX)-dependent component. The main objective of this study was to establish if prostaglandin (PG) D(2)-induced bronchoconstriction also was modulated by COX products. The effects of non-selective and selective COX-1 and COX-2 inhibitors on bronchoconstriction induced by LTD(4) and PGD(2) were investigated in the perfused and ventilated guinea-pig lung (IPL). Both LTD(4)-induced bronchoconstriction and thromboxane (TX) A(2) release was suppressed by COX inhibitors or by TX synthesis inhibition. The release of additional COX products following CysLT(1) receptor activation by LTD(4) was established by measurements of immunoreactive 6-keto PGF(1alpha) (a stable metabolite of PGI(2)) and PGE(2). In contrast, TP receptor-mediated bronchoconstriction by PGD(2) was somewhat enhanced by COX inhibitors, and there was no measurable release of COX products after TP receptor activation with U-46619. PGE(2) was bronchoprotective in IPL as it inhibited the histamine-induced bronchoconstriction. In the isolated guinea-pig trachea, neither PGD(2) nor U-46619 actively released PGE(2), but continuous production of PGE(2) and PGI(2) was established, and the response to PGD(2) was enhanced also in the trachea by COX inhibition. The study documented that bronchoconstriction induced by LTD(4) and PGD(2) in IPL was modulated differently by COX products. Whereas bronchoconstriction induced by LTD(4) was amplified predominantly by secondarily released TXA(2), that induced by PGD(2) was attenuated by bronchoprotective PGE(2) and PGI(2), presumably tonically produced in the airways.

Effect of particle deposition density of dry powders on the results produced by an in vitro test system simulating dissolution- and absorption rates in the lungs.

The surface area of the air/liquid interface in the lungs is substantial, so deposited doses of aerosol medicines per interface surface area when administered via the inhalation route is always quite low. However, in most in vitro systems used for dissolution testing of dry powder inhalables, the dose per surface area is generally much higher. The aim of this study was to investigate in one in vitro lung dissolution system, the DissolvIt, the manner in which the deposited dose per test surface area of drug particles influences the simulated dissolution- and absorption rate. Here we used the dissolution test method DissolvIt to investigate the influence on dissolution behavior by varying the deposited surface density of tested drugs. Dry powders of three different active pharmaceutical ingredients with different solubilities were used; salmeterol, budesonide and fluticasone propionate. It was found that by varying the dose density from 0.23 to 29 µg/cm2 the dissolution- and absorption rate of test particles was affected for all three substances, with decreasing relative dissolution rates above certain dose limits. The effect was much more prominent with the least soluble fluticasone propionate. In contrast, in a real lung it has been shown that a tenfold increase of the even less soluble fluticasone furoate did not affect the pulmonary dissolution- and absorption as measured in the ex vivo isolated perfused rat lung. This indicates that the deposited particle dose on the test surface used must be carefully considered in all in vitro dissolution testing apparatuses used for inhalation drugs, especially when aiming for in vitro-in vivo correlations. Conclusive data show that in the DissolvIt system consistent normalized dissolution- and absorption data can be obtained if the deposition density of test substance are kept below 1 µg/cm2 and the variability between the initial drug doses is smaller than 10-15% expressed as standard deviation.

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.

Investigation of Lung Pharmacokinetic of the Novel PDE4 Inhibitor CHF6001 in Preclinical Models: Evaluation of the PreciseInhale Technology.

BACKGROUND: Preclinical evaluation of new chemical entities (NCEs) designed to be administered by inhalation route requires lung administration to rodents, especially in the discovery phase. Different administration methods have been used until now, but more efforts are required to obtain controlled and reproducible lung deposition when only small amounts of neat powder material are available. METHODS: The PreciseInhale platform used in the present study enables well-controlled powder aerosol exposures with only small amounts of micronized neat material, providing data on inhalation pharmacokinetic (PK) of NCEs at a very early stage. The DustGun aerosol technology uses compressed air to generate a respirable aerosol from milligram-amounts of powder that is delivered to one animal at a time. The new methodology was used to investigate the inhalation PK and lung retention in the rat of the novel Chiesi PDE4 inhibitor CHF6001 in three exposure models of the PreciseInhale platform: nose-only, intratracheally intubated rat, and the isolated, ventilated, and perfused rat lung. Results were compared with data from two other pulmonary delivery systems commonly used in preclinical studies: liquid instillation and powder insufflation. RESULTS: Administration of micronized CHF6001 using the PreciseInhale system yielded lung exposures in the same range as the other tested devices, but the reproducibility in lung deposition was improved. The initial amount of CHF6001 in lungs at the first sampling time point was close to the predetermined target dose. Tracheal deposition with PreciseInhale (0.36 ± 0.22 µg) was significantly less than with other tested delivery systems: PennCentury (23.7 ± 3.2 µg) and Airjet (25.6 ± 7.2 µg). CONCLUSIONS: The PreciseInhale platform enabled the administration of CHF6001 powder with good accuracy and reproducibility, with low tracheal deposition. The new platform can be used at an early discovery stage to obtain inhalatory PK data for respirable aerosols of neat NCE powder without excipients and with minimal use of dry powder formulation work.

Dry powder inhalation exposures of the endotracheally intubated rat lung, ex vivo and in vivo: the pulmonary pharmacokinetics of fluticasone furoate.

BACKGROUND: The isolated perfused rat lung (IPL) is a suitable model for studying lung-specific pharmacokinetics (PK) of inhaled drugs. So far, little has been known, however, whether the PK measured in the ex vivo organ corresponds to the PK measured in similarly exposed animals in vivo, in particular the endotracheally intubated rat (EIR). The purpose of the current research was to compare the PK of inhaled corticosteroid fluticasone furoate (FF) in the IPL and the EIR. METHOD: Aerosols of FF with mass median aerodynamic diameters ranging from 2.2 to 3.2 µm were generated with the DustGun aerosol generator. The IPL, perfused in the single-pass mode, was exposed via inhalation to 5.6 and 46 µg of FF. Following inhalation, the perfusate was repeatedly sampled for 100 min, after which the lungs were recovered for quantitation of remaining FF. Two groups of EIR were also exposed via inhalation to 7 µg of FF. One group was immediately euthanized for determination of the initial deposition of FF in the lungs. From the second group, four venous blood samples were drawn up to 4 hr after exposure. The animals were then sacrificed for determination of FF remaining in the lungs. RESULTS: Following inhalation, FF was slowly disappearing from both the IPL and the lungs of the EIR, with a half-life of pulmonary retention of 4.3-4.9 hr for all three exposure series. For the low exposure levels, the concentration curve of FF in the IPL perfusate was similar in shape to that in venous blood of the EIR, with a Cmax of 1.0 and 0.8 nM for the IPL and the EIR, respectively. CONCLUSIONS: The results indicate that the IPL and the EIR, when used jointly in PK studies, can provide a detailed characterization of inhaled drugs or toxicants.

Delivering horseradish peroxidase as a respirable powder to the isolated, perfused, and ventilated lung of the rat: the pulmonary disposition of an inhaled model biopharmaceutical.

BACKGROUND: Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). METHOD: The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 µm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. RESULTS AND CONCLUSIONS: The total amount of HRP deposited in the lungs was 335 ± 100 µg and 568 ± 47 µg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.