Investigation of Dry Powder Inhaler (DPI) Resistance and Aerosol Dispersion Timing on Emitted Aerosol Aerodynamic Particle Sizing by Multistage Cascade Impactor when Sampled Volume Is Reduced from Compendial Value of 4 L

Compendial methods determining dry powder inhaler (DPI)-emitted aerosol aerodynamic particle size distribution (APSD) collect a 4-L air sample containing the aerosol bolus, where the flow, which propagates through the cascade impactor (CI) measurement system from the vacuum source, is used to actuate the inhaler. A previous article described outcomes with two CIs (Andersen eight-stage cascade impactor (ACI) and Next-Generation Pharmaceutical Impactor (NGI)) when the air sample volume was ≤4 L with moderate-resistance DPIs. This article extends that work, examining the hypothesis that DPI flow resistance may be a factor in determining outcomes. APSD measurements were made using the same CI systems with inhalers representing low and high flow resistance extremes (Cyclohaler® and HandiHaler® DPIs, respectively). The ratio of sample volume to internal dead space (normalized volume (V*)) was varied from 0.25 to 1.98 (NGI) and from 0.43 to 3.46 (ACI). Inhaler resistance was a contributing factor to the rate of bolus transfer; the higher resistance DPI completing bolus relocation to the NGI pre-separator via the inlet when V* was as small as 0.25, whereas only ca. 50% of the bolus mass was collected at this condition with the Cyclohaler® DPI. Size fractionation of the bolus from either DPI was completed within the ACI at smaller values of V* than within the NGI. Bolus transfer from the Cyclohaler® capsule and from the HandiHaler® to the ACI system were unaffected by the different flow rise time observed in the two different flow controller systems, and the effects the ACI-based on APSD measurements were marginal.     

Comparison of Aerodynamic Particle Size Distribution Between a Next Generation Impactor and a Cascade Impactor at a Range of Flow Rates

Wide variation in respiratory flow rates between patients emphasizes the importance of evaluating the aerodynamic particle size distribution (APSD) of dry powder inhaler (DPI) using a multi-stage impactor at different flow rates. US Pharmacopeia recently listed modified configurations of the Andersen cascade impactor (ACI) and new sets of cut-off diameter specifications for the operation at flow rates of 60 and 90 L/min. The purpose of this study was to clarify the effect of these changes on the APSD of DPI products at varied flow rates. We obtained APSD profiles of four DPIs and device combinations, Relenza®-Diskhaler® (GlaxoSmithKline Co.), Seebri®-Breezhaler® (Novartis Pharma Co.), Pulmicort®-Turbuhaler® (Astrazeneca Co.), and Spiriva®-Handihaler® (Nippon Boehringer Ingelheim Co.) using Next Generation Impactors (NGIs) and ACIs at flow rates from 28.3 to 90 L/min to evaluate the difference in the use of previous and new sets of cut-off diameter specifications. Processing the data using the new specifications for ACI apparently reduced large differences in APSD obtained by NGI and ACI with the previous specifications at low and high flow rates in all the DPIs. Selecting the appropriate configuration of ACI corresponding to the flow rate provided comparable APSD profiles of Pulmicort®-Turbuhaler® to those using NGIs at varied flow rates. The results confirmed the relevance of the current US Pharmacopeia specifications for ACI analysis in obtaining APSD profiles of DPI products at wide flow rates.     

The Abbreviated Impactor Measurement (AIM) Concept: Part II—Influence of Evaporation of a Volatile Component—Evaluation with a “Droplet-Producing” Pressurized Metered Dose Inhaler (pMDI)-Based Formulation Containing Ethanol as Cosolvent

The abbreviated impactor measurement (AIM) concept is a potential solution to the labor-intensive full-resolution cascade impactor (CI) methodology for inhaler aerosol aerodynamic particle size measurement. In this validation study, the effect of increasing the internal dead volume on determined mass fractions relating to aerodynamic particle size was explored with two abbreviated impactors both based on the Andersen nonviable cascade impactor (ACI) operating principle (Copley fast screening Andersen impactor [C-FSA] and Trudell fast screening Andersen impactor [T-FSA]). A pressurized metered dose inhaler-delivered aerosol producing liquid ethanol droplets after propellant evaporation was chosen to characterize these systems. Measures of extrafine, fine, and coarse particle mass fractions from the abbreviated systems were compared with corresponding data obtained by a full-resolution ACI. The use of liquid ethanol-sensitive filter paper provided insight by rendering locations visible where partly evaporated droplets were still present when the “droplet-producing” aerosol was sampled. Extrafine particle fractions based on impactor-sized mass were near equivalent in the range 48.6% to 54%, comparing either abbreviated system with the benchmark ACI-measured data. The fine particle fraction of the impactor-sized mass determined by the T-FSA (94.4 ± 1.7%) was greater than using the C-FSA (90.5 ± 1.4%) and almost identical with the ACI-measured value (95.3 ± 0.4%). The improved agreement between T-FSA and ACI is likely the result of increasing the dead space between the entry to the induction port and the uppermost impaction stage, compared with that for the C-FSA. This dead space is needed to provide comparable conditions for ethanol evaporation in the uppermost parts of these impactors.     

Evaluation of Abbreviated Impactor Measurements (AIM) and Efficient Data Analysis (EDA) for Dry Powder Inhalers (DPIs) Against the Full-Resolution Next Generation Impactor (NGI)

The full-resolution next generation impactor (NGI) and three abbreviated impactor systems were used to obtain the apparent aerodynamic particle size distribution (APSD) and other quality measures for marketed dry powder inhalers (DPIs) using the compendial method and efficient data analysis (EDA). APSD for the active pharmaceutical ingredient (API) in Spiriva^® Handihaler^®, Foradil^® Aerolizer^®, and Relenza^® Diskhaler^® was obtained using a full-resolution NGI at 39, 60, and 90 L/min, respectively. Two reduced NGI (rNGI) configurations, the filter-only configuration (rNGI-f) and the modified-cup configuration (rNGI-mc), and the fast-screening impactor (FSI) with appropriate inserts to provide a 5-μm cut size were evaluated. The fine particle dose (FPD) obtained using the FSI for Spiriva was statistically similar to that obtained using the full NGI. However, the FPD for both Foradil and Relenza obtained using the FSI was significantly different from that obtained using the full NGI. Despite this, no significant differences were observed for the fine particle fraction (FPF) obtained using the FSI relative to that obtained from the full NGI for any of the DPIs. The use of abbreviated impactor systems appears promising with good agreement observed with the full-resolution NGI, except for small differences observed for the rNGI-mc configuration. These small differences may be product- and/or flow rate-specific, and further evaluation will be required to resolve these differences.     

A Multi-laboratory in Vitro Study to Compare Data from Abbreviated and Pharmacopeial Impactor Measurements for Orally Inhaled Products: a Report of the European Aerosol Group (EPAG)

Fine particle dose (FPD) is a critical quality attribute for orally inhaled products (OIPs). The abbreviated impactor measurement (AIM) concept simplifies its measurement, provided there is a validated understanding of the relationship with the full resolution pharmacopoeial impactor (PIM) data for a given product. This multi-center study compared fine particle dose determined using AIM and PIM for five dry powder inhaler (DPIs) and two pressurized metered-dose inhaler (pMDI) products, one of which included a valved holding chamber (VHC). Reference measurements of FPD_PIM were made by each organization using either the full-resolution Andersen 8-stage non-viable impactor (ACI) or Next Generation Impactor (NGI). FPD_AIM was determined for the same OIP(s) with their choice of abbreviated impactor (fast screening impactor (FSI), fast screening Andersen (FSA), or reduced NGI (rNGI)). Each organization used its validated assay method(s) for the active pharmaceutical ingredient(s) (APIs) involved. Ten replicate measurements were made by each procedure. The upper size limit for FPD_AIM varied from 4.4 to 5.0 μm aerodynamic diameter, depending upon flow rate and AIM apparatus; the corresponding size limit for FPD_PIM was fixed at 5 μm in accordance with the European Pharmacopoeia. The 90% confidence interval for the ratio [FPD_AIM/FPD_PIM], expressed as a percentage, was contained in the predetermined 85–118% acceptance interval for nine of the ten comparisons of FPD. The average value of this ratio was 105% across all OIPs and apparatuses. The findings from this investigation support the equivalence of AIM and PIM for determination of FPD across a wide range of OIP platforms and measurement techniques.     

The Comparison of Fluid Dynamics Parameters in an Andersen Cascade Impactor Equipped With and Without a Preseparator

The fluid dynamic data in Andersen cascade impactor (ACI) are still lacking. Airflows and those affected parameters can be predicted in a preseparator and Andersen cascade impactor (ACI) by computational modeling. This study developed a validated computational fluid dynamic (CFD) model of an ACI and investigated the effects of the preseparator on the CFD parameters. Validation of the computational nozzle velocity for each of the stage 0 to stage 5 of the ACI stages was found to be within a 3.56% error. The flow field indicated that the preseparator accelerated the airflow velocity at the induction tube from 1.13 to 3.71 ± 0.09 m/s and 2.40 to 8.68 ± 0.16 m/s (at 28.3 and 60 L/min of flow rate, respectively). The preseparator produced a nozzle''s wall shear stress ranged from 0.08 to 0.34 Pa on a collection plate, while the ex-preseparator spread wall shear from the plate''s center was in a range of 0.11 to 0.37 Pa (at 28.3 L/min of flow rate). Moreover, the nozzle velocities increased along the distance from the middle of the collection plate to the periphery. The CFD explained the airflow of the preseparator equipped model by accelerating the airflow along the inlet port to maximize the trapping of desirable particles and the generation of a smooth wall shear stress at the collection plate to reduce the particle re-entrainment. While, the ex-preseparator generated an airflow that resulted in a higher wall shear stress occurring on the lower stages.Key words: ACI, flow field, preseparator, wall shear stress     

Evaluation of an Abbreviated Impactor for Fine Particle Fraction (FPF) Determination of Metered Dose Inhalers (MDI)

Abbreviated impactors have been developed recently to allow more rapid evaluation of inhalation products as alternates to the eight-stage Andersen Cascade Impactor (ACI) which has been widely used in the pharmaceutical industry for assessing aerodynamic particle size distribution. In this paper, a two-stage abbreviated impactor, Westech Fine Particle Dose Impactor (WFPD), was used to characterize the aerodynamic particle size of metered dose inhaler (MDI) products, and the results were compared with those obtained using the standard eight-stage ACI. Seven commercial MDI products, with different propellants (chlorofluorocarbon/hydrofluoroalkane) and formulation types (suspension/solution, dry/normal/wet), were tested in this study by both WFPD and ACI. Substantially equivalent measures of fine particle fraction were obtained for most of the tested MDI products, but larger coarse particle fraction and extra-fine particle fraction values were measured from WFPD relative to those measured using the ACI. Use of the WFPD also produced more wall loss than the ACI. Therefore, it is recommended that the system suitability be evaluated on a product-by-product basis to establish substantial equivalency before implementing an abbreviated impactor measurement methodology for routine use in inhaler product characterization.     

Cascade Impactor (CI) Mensuration—An Assessment of the Accuracy and Precision of Commercially Available Optical Measurement Systems

Multi-stage cascade impactors (CIs) are the preferred measurement technique for characterizing the aerodynamic particle size distribution of an inhalable aerosol. Stage mensuration is the recommended pharmacopeial method for monitoring CI “fitness for purpose” within a GxP environment. The Impactor Sub-Team of the European Pharmaceutical Aerosol Group has undertaken an inter-laboratory study to assess both the precision and accuracy of a range of makes and models of instruments currently used for optical inspection of impactor stages. Measurement of two Andersen 8-stage ‘non-viable’ cascade impactor “reference” stages that were representative of jet sizes for this instrument type (stages 2 and 7) confirmed that all instruments evaluated were capable of reproducible jet measurement, with the overall capability being within the current pharmacopeial stage specifications for both stages. In the assessment of absolute accuracy, small, but consistent differences (ca. 0.6% of the certified value) observed between ‘dots’ and ‘spots’ of a calibrated chromium-plated reticule were observed, most likely the result of treatment of partially lit pixels along the circumference of this calibration standard. Measurements of three certified ring gauges, the smallest having a nominal diameter of 1.0 mm, were consistent with the observation where treatment of partially illuminated pixels at the periphery of the projected image can result in undersizing. However, the bias was less than 1% of the certified diameter. The optical inspection instruments evaluated are fully capable of confirming cascade impactor suitability in accordance with pharmacopeial practice.     

Relative Precision of Inhaler Aerodynamic Particle Size Distribution (APSD) Metrics by Full Resolution and Abbreviated Andersen Cascade Impactors (ACIs): Part 2—Investigation of Bias in Extra-Fine Mass Fraction with AIM-HRT Impactor

The purpose of this study was to resolve an anomalously high measure of extra-fine particle fraction (EPF) determined by the abbreviated cascade impactor possibly relevant for human respiratory tract (AIM-HRT) in the experiment described in Part 1 of this two-part series, in which the relative precision of abbreviated impactors was evaluated in comparison with a full resolution Andersen eight-stage cascade impactor (ACI). Evidence that the surface coating used to mitigate particle bounce was laterally displaced by the flow emerging from the jets of the lower stage was apparent upon microscopic examination of the associated collection plate of the AIM-HRT impactor whose cut point size defines EPF. A filter soaked in surfactant was floated on top of this collection plate, and further measurements were made using the same pressurized metered-dose inhaler-based formulation and following the same procedure as in Part 1. Measures of EPF, fine particle, and coarse particle fractions were comparable with those obtained with the ACI, indicating that the cause of the bias had been identified and removed. When working with abbreviated impactors, this precaution is advised whenever there is evidence that surface coating displacement has occurred, a task that can be readily accomplished by microscopic inspection of all collection plates after allowing the impactor to sample ambient air for a few minutes.     

Relative Precision of Inhaler Aerodynamic Particle Size Distribution (APSD) Metrics by Full Resolution and Abbreviated Andersen Cascade Impactors (ACIs): Part 1

The purpose of this study was to compare relative precision of two different abbreviated impactor measurement (AIM) systems and a traditional multi-stage cascade impactor (CI). The experimental design was chosen to provide separate estimates of variability for each impactor type. Full-resolution CIs are useful for characterizing the aerosol aerodynamic particle size distribution of orally inhaled products during development but are too cumbersome, time-consuming, and resource-intensive for other applications, such as routine quality control (QC). This article presents a proof-of-concept experiment, where two AIM systems configured to provide metrics pertinent to QC (QC-system) and human respiratory tract (HRT-system) were evaluated using a hydrofluoroalkane-albuterol pressurized metered dose inhaler. The Andersen eight-stage CI (ACI) served as the benchmark apparatus. The statistical design allowed estimation of precision with each CI configuration. Apart from one source of systematic error affecting extra-fine particle fraction from the HRT-system, no other bias was detected with either abbreviated system. The observed bias was shown to be caused by particle bounce following the displacement of surfactant by the shear force of the airflow diverging above the collection plate of the second impaction stage. A procedure was subsequently developed that eliminated this source of error, as described in the second article of this series (submitted to AAPS PharmSciTech). Measurements obtained with both abbreviated impactors were very similar in precision to the ACI for all measures of in vitro performance evaluated. Such abbreviated impactors can therefore be substituted for the ACI in certain situations, such as inhaler QC or add-on device testing.     

The Abbreviated Impactor Measurement (AIM) Concept: Part 1—Influence of Particle Bounce and Re-Entrainment—Evaluation with a “Dry” Pressurized Metered Dose Inhaler (pMDI)-Based Formulation

The abbreviated impactor measurement concept is a potential improvement to the labor-intensive full-resolution cascade impactor methodology for inhaler aerosol aerodynamic particle size distribution (APSD) measurement by virtue of being simpler and therefore quicker to execute. At the same time, improved measurement precision should be possible by eliminating stages upon which little or no drug mass is collected. Although several designs of abbreviated impactor systems have been developed in recent years, experimental work is lacking to validate the technique with aerosols produced by currently available inhalers. In part 1 of this two-part article that focuses on aerosols produced by pressurized metered dose inhalers (pMDIs), the evaluation of two abbreviated impactor systems (Copley fast screening Andersen impactor and Trudell fast screening Andersen impactor), based on the full-resolution eight-stage Andersen nonviable cascade impactor (ACI) operating principle, is reported with a formulation producing dry particles. The purpose was to investigate the potential for non-ideal collection behavior associated with particle bounce in relation to internal losses to surfaces from which particles containing active pharmaceutical ingredient are not normally recovered. Both abbreviated impactors were found to be substantially equivalent to the full-resolution ACI in terms of extra-fine and fine particle and coarse mass fractions used as metrics to characterize the APSD of these pMDI-produced aerosols when sampled at 28.3 L/min, provided that precautions are taken to coat collection plates to minimize bounce and entrainment.     

The Effect of Nonideal Cascade Impactor Stage Collection Efficiency Curves on the Interpretation of the Size of Inhaler-Generated Aerosols

Cascade impactors, operating on the principle of inertial size separation in (ideally) laminar flow, are used to determine aerodynamic particle size distributions (APSDs) of orally inhaled product (OIP) aerosols because aerodynamic diameter can be related to respiratory tract deposition. Each stage is assumed typically to be an ideal size fractionator. Thus, all particles larger than a certain size are considered collected and all finer particles are treated as penetrating to the next stage (a step function stage efficiency curve). In reality, the collection efficiency of a stage smoothly increases with particle size as an “S-shaped” curve, from approximately 0% to 100%. Consequently, in some cases substantial overlap occurs between neighboring stages. The potential for bias associated with the step-function assumption has been explored, taking full resolution and two-stage abbreviated forms of the Andersen eight-stage nonviable impactor (ACI) and the next-generation pharmaceutical impactor (NGI) as example apparatuses. The behavior of unimodal, log-normal APSDs typical of OIP-generated aerosols has been investigated, comparing known input values to calculated values of central tendency (mass median aerodynamic diameter) and spread (geometric standard deviation, GSD). These calculations show that the error introduced by the step change assumption is larger for the ACI than for the NGI. However, the error is sufficiently small to be inconsequential unless the APSD in nearly monodisperse (GSD ≤1.2), a condition that is unlikely to occur with realistic OIPs. Account may need to be taken of this source of bias only for the most accurate work with abbreviated ACI systems.     

Aerodynamic particle size analysis of aerosols from pressurized metered-dose inhalers: Comparison of andersen 8-stage cascade impactor,next generation pharmaceutical impactor,and model 3321 aerodynamic particle sizer aerosol spectrometer

The purpose of this research was to compare three different methods for the aerodynamic assessment of (1) chloroflurocarbon (CFC)-fluticasone propionate (Flovent), (2) CFC-sodium cromoglycate (Intal), and (3) hydrofluoroalkane (HFA)-beclomethasone dipropionate (Qvar) delivered by pressurized metered dose inhaler. Particle size distributions were compared determining mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), and fine particle fraction <4.7 μm aerodynamic diameter (FPF_{<4.7 μm}). Next Generation Pharmaceutical Impactor (NGI)-size distributions for Flovent comprised finer particles than determined by Andersen 8-stage impactor (ACI) (MMAD=2.0±0.05 μm [NGI]; 2.8±0.07 μm [ACI]); however FPF_{<4.7 μm} by both impactors was in the narrow range 88% to 93%. Size distribution agreement for Intal was better (MMAD=4.3±0.19 μm (NGI), 4.2±0.13 μm (ACI), with FPF_{<4.7 μm} ranging from 52% to 60%. The Aerodynamic Particle Sizer (APS) undersized aerosols produced with either formulation (MMAD=1.8±0.07 μm and 3.2±0.02 μm for Flovent and Intal, respectively), but values of FPF_{<4.7 μm} from the single-stage impactor (SSI) located at the inlet to the APS (82.9%±2.1% [Flovent], 46.4%±2.4% [Intal]) were fairly close to corresponding data from the multi-stage impactors. APS-measured size distributions for Qvar (MMAD=1.0±0.03 μm; FPF_{<4.7 μm}=96.4% ±2.5%), were in fair agreement with both NGI (MMAD=0.9±0.03 μm; FPF_{<4.7 μm}=96.7%±0.7%), and ACI (MMAD=1.2±0.02 μm, FPF_{<4.7 μm}=98%±0.5%), but FPF_{<4.7 μm} from the SSI (67.1%±4.1%) was lower than expected, based on equivalent data obtained by the other techniques. Particle bounce, incomplete evaporation of volatile constituents and the presence of surfactant particles are factors that may be responsible for discrepancies between the techniques.     

Defining the Critical Material Attributes of Lactose Monohydrate in Carrier Based Dry Powder Inhaler Formulations Using Artificial Neural Networks

The study aimed to establish a function-based relationship between the physical and bulk properties of pre-blended mixtures of fine and coarse lactose grades with the in vitro performance of an adhesive active pharmaceutical ingredient (API). Different grades of micronised and milled lactose (Lactohale (LH) LH300, LH230, LH210 and Sorbolac 400) were pre-blended with coarse grades of lactose (LH100, LH206 and Respitose SV010) at concentrations of 2.5, 5, 10 and 20 wt.%. The bulk and rheological properties and particle size distributions were characterised. The pre-blends were formulated with micronised budesonide and in vitro performance in a Cyclohaler device tested using a next-generation impactor (NGI) at 90 l/min. Correlations between the lactose properties and in vitro performance were established using linear regression and artificial neural network (ANN) analyses. The addition of milled and micronised lactose fines with the coarse lactose had a significant influence on physical and rheological properties of the bulk lactose. Formulations of the different pre-blends with budesonide directly influenced in vitro performance attributes including fine particle fraction, mass median aerodynamic diameter and pre-separator deposition. While linear regression suggested a number of physical and bulk properties may influence in vitro performance, ANN analysis suggested the critical parameters in describing in vitro deposition patterns were the relative concentrations of lactose fines % < 4.5 μm and % < 15 μm. These data suggest that, for an adhesive API, the proportion of fine particles below % < 4.5 μm and % < 15 μm could be used in rational dry powder inhaler formulation design.KEY WORDS: critical material attributes, cohesive-adhesive balance, dry powder inhaler, lactose, quality-by-design     

Novel Simvastatin Inhalation Formulation and Characterisation

Simvastatin (SV), a drug of the statin class currently used orally as an anti-cholesterolemic via the inhibition of the 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase, has been found not only to reduce cholesterol but also to have several other pharmacological actions that might be beneficial in airway inflammatory diseases. Currently, there is no inhalable formulation that could deliver SV to the lungs. In this study, a pressurised metered-dose inhaler (pMDI) solution formulation of SV was manufactured, with ethanol as a co-solvent, and its aerosol performance and physico-chemical properties investigated. A pMDI solution formulation containing SV and 6% w/w ethanol was prepared. This formulation was assessed visually and quantitatively for SV solubility. Furthermore, the aerosol performance (using Andersen Cascade impactor at 28.3 L/min) and active ingredient chemical stability up to 6 months at different storage temperatures, 4 and 25°C, were also evaluated. The physico-chemical properties of the SV solution pMDI were also characterised by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and laser diffraction. The aerosol particles, determined using scanning electron microscopy (SEM), presented a smooth surface morphology and were spherical in shape. The aerosol produced had a fine particle fraction of 30.77 ± 2.44% and a particle size distribution suitable for inhalation drug delivery. Furthermore, the short-term chemical stability showed the formulation to be stable at 4°C for up to 6 months, whilst at 25°C, the formulation was stable up to 3 months. In this study, a respirable and stable SV solution pMDI formulation for inhalation has been presented that could potentially be used clinically as an anti-inflammatory therapy for the treatment of several lung diseases.Key Words: lung inflammation, pMDI, pressurised metered dose inhaler, simvastatin     

Heat-Stable Dry Powder Oxytocin Formulations for Delivery by Oral Inhalation

In this work, heat stable dry powders of oxytocin (OT) suitable for delivery by oral inhalation were prepared. The OT dry powders were prepared by spray drying using excipients chosen to promote OT stability including trehalose, isoleucine, polyvinylpyrrolidone, citrate (sodium citrate and citric acid), and zinc salts (zinc chloride and zinc citrate). Characterization by laser diffraction indicated that the OT dry powders had a median particle size of 2 μm, making them suitable for delivery by inhalation. Aerodynamic performance upon discharge from proprietary dry powder inhalers was evaluated by Andersen cascade impaction (ACI) and in an anatomically correct airway (ACA) model, and confirmed that the powders had excellent aerodynamic performance, with respirable fractions up to 77% (ACI, 30 L/min). Physicochemical characterization demonstrated that the powders were amorphous (X-ray diffraction) with high glass transition temperature (modulated differential scanning calorimetry, MDSC), suggesting the potential for stabilization of the OT in a glassy amorphous matrix. OT assay and impurity profile were conducted by reverse phase HPLC and liquid chromatography-mass spectrometry (LC-MS) after storage up to 32 weeks at 40°C/75%RH. Analysis demonstrated that OT dry powders containing a mixture of citrate and zinc salts retained more than 90% of initial assay after 32 weeks storage and showed significant reduction in dimers and trisulfide formation (up to threefold reduction compared to control).

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-015-0314-0) contains supplementary material, which is available to authorized users.KEY WORDS: dry powder, oral inhalation, oxytocin, peptide delivery, postpartum hemorrhage     

Swimming against the flow—Environmental DNA can detect bull sharks (Carcharhinus leucas) across a dynamic deltaic interface

Human activities in coastal areas are accelerating ecosystem changes at an unprecedented pace, resulting in habitat loss, hydrological modifications, and predatory species declines. Understanding how these changes potentially cascade across marine and freshwater ecosystems requires knowing how mobile euryhaline species link these seemingly disparate systems. As upper trophic level predators, bull sharks (Carcharhinus leucas) play a crucial role in marine and freshwater ecosystem health. Telemetry studies in Mobile Bay, Alabama, suggest that bull sharks extensively use the northern portions of the bay, an estuarine–freshwater interface known as the Mobile‐Tensaw Delta. To assess whether bull sharks use freshwater habitats in this region, environmental DNA surveys were conducted during the dry summer and wet winter seasons in 2018. In each season, 5 × 1 L water samples were collected at each of 21 sites: five sites in Mobile Bay, six sites in the Mobile‐Tensaw Delta, and ten sites throughout the Mobile‐Tombigbee and Tensaw‐Alabama Rivers. Water samples were vacuum‐filtered, DNA extractions were performed on the particulate, and DNA extracts were analyzed with Droplet Digital™ Polymerase Chain Reaction using species‐specific primers and an internal probe to amplify a 237‐base pair fragment of the mitochondrial NADH dehydrogenase subunit 2 gene in bull sharks. One water sample collected during the summer in the Alabama River met the criteria for a positive detection, thereby confirming the presence of bull shark DNA. While preliminary, this finding suggests that bull sharks use less‐urbanized, riverine habitats up to 120 km upriver during Alabama''s dry summer season.     

Reassessing the folding of the KIX domain: Evidence for a two‐state mechanism

The debate about the presence and role of intermediates in the folding of proteins has been a critical issue, especially for fast folders. One of the classical methodologies to identify such metastable species is the “burst-phase analysis,” whereby the observed signal amplitude from stopped-flow traces is determined as a function of denaturant concentration. However, a complication may arise when folding is sufficiently fast to jeopardize the reliability of the stopped-flow technique. In this study, we reassessed the folding of the KIX domain from cAMP Response Element-Binding (CREB)-binding protein, which has been proposed to involve the formation of an intermediate that accumulates in the dead time of the stopped flow. By using an in-house-built capillary continuous flow with a 50-μs dead time, we demonstrate that this intermediate is not present; the problem arose because of the instrumental limitation of the standard stopped flow to assess very fast refolding rate constants (e.g., ≥500 s⁻¹).     

Improving consumption rate estimates by incorporating wild activity into a bioenergetics model

Consumption is the basis of metabolic and trophic ecology and is used to assess an animal''s trophic impact. The contribution of activity to an animal''s energy budget is an important parameter when estimating consumption, yet activity is usually measured in captive animals. Developments in telemetry have allowed the energetic costs of activity to be measured for wild animals; however, wild activity is seldom incorporated into estimates of consumption rates. We calculated the consumption rate of a free‐ranging marine predator (yellowtail kingfish, Seriola lalandi) by integrating the energetic cost of free‐ranging activity into a bioenergetics model. Accelerometry transmitters were used in conjunction with laboratory respirometry trials to estimate kingfish active metabolic rate in the wild. These field‐derived consumption rate estimates were compared with those estimated by two traditional bioenergetics methods. The first method derived routine swimming speed from fish morphology as an index of activity (a “morphometric” method), and the second considered activity as a fixed proportion of standard metabolic rate (a “physiological” method). The mean consumption rate for free‐ranging kingfish measured by accelerometry was 152 J·g⁻¹·day⁻¹, which lay between the estimates from the morphometric method (μ = 134 J·g⁻¹·day⁻¹) and the physiological method (μ = 181 J·g⁻¹·day⁻¹). Incorporating field‐derived activity values resulted in the smallest variance in log‐normally distributed consumption rates (σ = 0.31), compared with the morphometric (σ = 0.57) and physiological (σ = 0.78) methods. Incorporating field‐derived activity into bioenergetics models probably provided more realistic estimates of consumption rate compared with the traditional methods, which may further our understanding of trophic interactions that underpin ecosystem‐based fisheries management. The general methods used to estimate active metabolic rates of free‐ranging fish could be extended to examine ecological energetics and trophic interactions across aquatic and terrestrial ecosystems.     

The 60- and 70-kDa heat-shock proteins and their correlation with cardiovascular risk factors in postmenopausal women with metabolic syndrome

We investigated the association between circulating levels of 60 and 70 kDa heat-shock proteins (HSP60 and 70) and cardiovascular risk factors in postmenopausal women with or without metabolic syndrome (MetS). This cross-sectional study included 311 Brazilian women (age ≥45 years with amenorrhea ≥12 months). Women showing three or more of the following diagnostic criteria were diagnosed with MetS: waist circumference (WC) ≥88 cm, blood pressure ≥130/85 mmHg, triglycerides ≥150 mg/dl, high-density lipoprotein (HDL) <50 mg/dl, and glucose ≥100 mg/dl. Clinical, anthropometric, and biochemical parameters were collected. HSP60, HSP70, antibodies to HSP60 and HSP70, and C-reactive protein (CRP) levels were measured in serum. Student''s t test, Kruskal–Wallis test, chi-square test, and Pearson correlation were used for statistical analysis. Of the 311 women, 30.9 % (96/311) were diagnosed with MetS. These women were, on average, obese with abdominal fat deposition and had lower HDL values as well as higher triglycerides and glucose levels. Homeostasis model assessment-insulin resistant (HOMA-IR) test values in these women were compatible with insulin resistance (P < 0.05). CRP and HSP60 concentrations were higher in women with MetS than in women without MetS (P < 0.05). HSP60, anti-HSP70, and CRP concentrations increased with the number of features indicative of MetS (P < 0.05). There was a significant positive correlation between anti-HSP70 and WC, blood pressure and HOMA-IR, and between CRP and WC, blood pressure, glucose, HOMA-IR, and triglycerides (P < 0.05). In postmenopausal women, serum HSP60 and anti-HSP70 concentrations increased with accumulating features of the metabolic syndrome. These results suggest a greater immune activation that is associated with cardiovascular risk in postmenopausal women with metabolic syndrome.