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.     

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.     

Improved Quality Control Metrics for Cascade Impaction Measurements of Orally Inhaled Drug Products (OIPs)

This study of aerodynamic mass-weighted particle size distribution (APSD) data from orally inhaled products (OIPs) investigated whether a set of simpler (than currently used) metrics may be adequate to detect changes in APSD for quality control (QC) purposes. A range of OIPs was examined, and correlations between mass median aerodynamic diameter and the ratio of large particle mass (LPM) to small particle mass (SPM) were calculated. For an Andersen cascade impactor, the LPM combines the mass associated with particle sizes from impactor stage 1 to a product-specific boundary size; SPM combines the mass of particles from that boundary through to terminal filter. The LPM–SPM boundary should be chosen during development based on the full-resolution impactor results so as to maximize the sensitivity of the LPM/SPM ratio to meaningful changes in quality. The LPM/SPM ratio along with the impactor-sized mass (ISM) are by themselves sufficient to detect changes in central tendency and area under the APSD curve, which are key in vitro quality attributes for OIPs. Compared to stage groupings, this two-metric approach provides better intrinsic precision, in part due to having adequate mass and consequently better ability to detect changes in APSD and ISM, suggesting that this approach should be a preferred QC tool. Another advantage is the possibility to obtain these metrics from the abbreviated impactor measurements (AIM) rather than from full-resolution multistage impactors. Although the boundary is product specific, the testing could be accomplished with a basic AIM system which can meet the needs of most or all OIPs.     

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 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.     

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.     

Product Lifecycle Approach to Cascade Impaction Measurements

Over the lifecycle of an orally inhaled product (OIP), multi-stage cascade impactor (CI) measurements are used for different purposes and to address different questions. Full-resolution CIs can provide important information during product development and are widely used but are time- and resource-intensive, highly variable, and suboptimal for OIP quality control (QC) testing. By contrast, Efficient Data Analysis (EDA) combined with Abbreviated Impactor Measurement (AIM) systems pertinent either for QC and-possibly-for adult Human Respiratory Tract (pHRT) has been introduced for OIP performance assessment during and post-development. This article summarizes available evidence and discusses a strategy for using either abbreviated or full-resolution CI systems depending on the purpose of the measurement, such that adequate, accurate, and efficient testing of aerodynamic particle size distribution (APSD) of OIPs can be achieved throughout the lifecycle of a product. Under these proposals, a comprehensive testing program should initially be conducted by full-resolution CI in OIP development to ascertain the product's APSD. Subsequently, correlations should be established from the selected AIM CIs to the corresponding full-resolution system, ideally developing specifications common to both techniques. In the commercial phase, it should be possible to release product using AIM/EDA, keeping the full-resolution CI for investigations, change control, and trouble-shooting, thus optimizing resources for APSD characterization throughout the product lifecycle. If an in vitro-in vivo relationship is established and clinically relevant sizes are known, an AIM-pHRT could serve as a quick indicator that clinically relevant fractions have not changed and also, in the management of post-approval changes.     

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.     

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.     

Effect of Sampling Volume on Dry Powder Inhaler (DPI)-Emitted Aerosol Aerodynamic Particle Size Distributions (APSDs) Measured by the Next-Generation Pharmaceutical Impactor (NGI) and the Andersen Eight-Stage Cascade Impactor (ACI)

Current pharmacopeial methods for testing dry powder inhalers (DPIs) require that 4.0 L be drawn through the inhaler to quantify aerodynamic particle size distribution of “inhaled” particles. This volume comfortably exceeds the internal dead volume of the Andersen eight-stage cascade impactor (ACI) and Next Generation pharmaceutical Impactor (NGI) as designated multistage cascade impactors. Two DPIs, the second (DPI-B) having similar resistance than the first (DPI-A) were used to evaluate ACI and NGI performance at 60 L/min following the methodology described in the European and United States Pharmacopeias. At sampling times ≥2 s (equivalent to volumes ≥2.0 L), both impactors provided consistent measures of therapeutically important fine particle mass (FPM) from both DPIs, independent of sample duration. At shorter sample times, FPM decreased substantially with the NGI, indicative of incomplete aerosol bolus transfer through the system whose dead space was 2.025 L. However, the ACI provided consistent measures of both variables across the range of sampled volumes evaluated, even when this volume was less than 50% of its internal dead space of 1.155 L. Such behavior may be indicative of maldistribution of the flow profile from the relatively narrow exit of the induction port to the uppermost stage of the impactor at start-up. An explanation of the ACI anomalous behavior from first principles requires resolution of the rapidly changing unsteady flow and pressure conditions at start up, and is the subject of ongoing research by the European Pharmaceutical Aerosol Group. Meanwhile, these experimental findings are provided to advocate a prudent approach by retaining the current pharmacopeial methodology.KEY WORDS: cascade impactor, compendial method, dry powder inhaler, sample volume     

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.     

Mycobacterial Phosphatidylinositol Mannosides Negatively Regulate Host Toll-like Receptor 4, MyD88-dependent Proinflammatory Cytokines, and TRIF-dependent Co-stimulatory Molecule Expression

Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol anchor phosphatidyl-myo-inositol hexamannosides PIM₆ or PIM₂ exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12 (IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing adaptor protein inducing interferon β (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced NFκB translocation. Synthetic PIM₁ and a PIM₂ mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM analogs represent powerful anti-inflammatory leads.Multifold interactions between Mycobacterium tuberculosis and host phagocytes determine immune responses to M. tuberculosis and tuberculosis pathogenesis (for review, see Refs. 1 and 2). Alveolar macrophages, the primary host cells for M. tuberculosis, and dendritic cells that carry mycobacterial antigens from the infection site to the draining lymph nodes to establish a T cell-mediated immune response contribute to modulate the innate immune response by secreting cytokines after recognition of microbial motives. Among them, TNF² is an essential mediator for granuloma formation and containment of M. tuberculosis infection. Similarly, IL-12, interferon γ, but also IL-1, IL-18, IL-23, and nitric oxide are required for host defense (1–6). Phagocytes are also a source of immuno-modulatory cytokines, such as IL-10 and transforming growth factor-β, which dampen the immune response and inflammation. Mycobacteria-derived molecules down-modulating the immune system have been described, including the protein ESAT-6, mannose-capped lipoarabinomannan (ManLAM), and lipomannans (LM) (7–12). Here, we report that phosphatidyl-myo-inositol mannosides (PIM), the glycosylphosphatidylinositol (GPI) anchor structure of LAM and LM, exert strong anti-inflammatory activities.Mycobacterial cell wall LAM, LM, and PIM are recognized by macrophages and dendritic cells through various pattern recognition receptors, including Toll-like receptors (TLRs) (13–16) and C-type lectins such as mannose receptor (MR/CD206) and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN/CD209), central to M. tuberculosis binding and internalization by human dendritic cells (17–20). DC-SIGN and mannose receptor were proposed to mediate ManLAM inhibition of LPS-induced IL-12 production in dendritic cells, an activity ascribed to the mannosylated cap (8, 9). We showed recently that mycobacterial LM have a dual potential for pro-inflammatory and anti-inflammatory effects (11), tri- and tetra-acylated LM fractions exerting stimulatory effects through TLR2, TLR4, and MyD88 (21), whereas diacylated LM inhibit LPS-induced cytokine response independently of TLR2, SIGN-R1, and mannose receptor (12).PIM are biosynthetic precursors of LM and LAM (22–25). Dimannoside (PIM₂) and hexamannoside (PIM₆) PIM are the two most abundant classes of PIM found in M. tuberculosis H37Rv and Mycobacterium bovis BCG (see Fig. 1). PIM purification and molecular chemical characterization revealed four major acyl forms, mono- to tetra-acylated (lyso-PIM for one acyl, PIM for two acyl, Ac₁PIM for three acyl, and Ac₂PIM for four acyl, respectively; see Fig. 1) for both PIM₂ and PIM₆ (26–29). Higher order PIM with mannose cap-like structures were found to preferentially associate with human MR and to contribute to phagosome-lysosome fusion (20). The degree of acylation influenced higher order PIM association with the MR, whereas PIM₂ was recognized by DC-SIGN independently of its acylation degree. The complete synthesis of the different PIM has recently been reported (30–33).Open in a separate windowFIGURE 1.Natural PIM and synthetic PIM₁ and PIM₂ mimetics used in the study. Shown is a schematic representation of natural lyso-PIM₆, PIM₆, Ac₁PIM₆, Ac₂PIM₆, and PIM₂ (A) and synthetic PIM₁ (B) showing the C16 and C18 acyl groups on glycerol chain positions sn-2 and sn-1, the precursor PI, a synthetic mimetic of PIM₂ (PIM₂ mimetic) bearing C16 and C18 acyl chains, the de-acylated control molecule precursor of the PIM₂ mimetic (de-AcPIM₂ mimetic), and a PIM₂ mimetic with replacement of the phosphodiester moiety by a carbonate.Here, we analyzed isolated acyl forms of PIM and identified PIM₂ and PIM₆ but also synthetic PIM₁ and a mimetic of PIM₂ as strong inhibitors of endotoxin-induced proinflammatory responses in vitro and in vivo. Using macrophages from genetically modified mice, we characterized PIM inhibitory effects on MyD88, TRIF, and NFκB signaling pathways. Hence, not only complex mycobacterial lipoglycans like ManLAM and LM but also small molecular weight PIM analogues are potent inhibitors of host inflammatory responses.     

Clinically Relevant <Emphasis Type="Italic">In Vitro</Emphasis> Testing of Orally Inhaled Products—Bridging the Gap Between the Lab and the Patient

Current pharmacopeial methods for in vitro orally inhaled product (OIP) performance testing were developed primarily to support requirements for drug product registration and quality control. In addition, separate clinical studies are undertaken in order to quantify safety and efficacy in the hands of the patient. However, both laboratory and clinical studies are time-consuming and expensive and generally do not investigate either the effects of misuse or the severity of the respiratory disease being treated. The following modifications to laboratory evaluation methodologies can be incorporated without difficulty to provide a better linkage from in vitro testing to clinical reality: (1) examine all types of OIP with patient-representative breathing profiles which represent normal inhaler operation in accordance with the instructions for use (IFU); (2) evaluate OIP misuse, prioritizing the importance of such testing on the basis of (a) probability of occurrence and (b) consequential impact in terms of drug delivery in accordance with the label claim; and (3) use age-appropriate patient-simulated face and upper airway models for the evaluation of OIPs with a facemask. Although it is not necessarily foreseen that these suggestions would form part of future routine quality control testing of inhalers, they should provide a closer approximation to the clinical setting and therefore be useful in the preparation for in vivo studies and in improving guidance for correct use.     

Effect of interactive ternary mixtures on dispersion characteristics of ipratropium bromide in dry powder inhaler formulations

The purpose of this investigation was to evaluate the effect of mixing order and the influence of adding fines on in vitro performance of ipratropium bromide (ITB) dry powder inhaler formulations. Coarse lactose (CL) in varying mass ratio with or without addition of micronized lactose (ML) and ITB in different mixing sequences was used to formulate ternary mixtures. A binary mixture composed of CL and ITP served as control. The in vitro deposition of ITB from these formulations was measured using an Andersen cascade impactor (aerosolization at 39 L/min) employing a HandiHaler as the delivery device. It was observed that mixing order has a significant effect (P<.05) on in vitro deposition of ITB. Formulations with preblending of CL and ITB produced similar deposition profiles as the control, regardless of the added ML. In contrast, formulations without preblending resulted in significantly higher fine particle dose (FPD) as compared with the control. In addition, an increased quantity of ML generally resulted in an increase in drug deposition. The results show that the effect of ML on dispersion of ITB is highly dependent upon the mixing order. The evaluation of atomic force measurement (AFM) to forecast drug detachment and predict the aerodynamic characteristics resulted in similar attraction forces for the different pairs lactose/lactose (42.66±25.01 nN) and lactose/ITB (46.77±17.04 nN). Published: April 20, 2007     

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.     

Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) Kinases Promote Human T Helper 1 Cell Differentiation

The differentiation of human primary T helper 1 (Th1) cells from naïve precursor cells is regulated by a complex, interrelated signaling network. The identification of factors regulating the early steps of Th1 cell polarization can provide important insight in the development of therapeutics for many inflammatory and autoimmune diseases. The serine/threonine-specific proviral integration site for Moloney murine leukemia virus (PIM) kinases PIM1 and PIM2 have been implicated in the cytokine-dependent proliferation and survival of lymphocytes. We have established that the third member of this family, PIM3, is also expressed in human primary Th cells and identified a new function for the entire PIM kinase family in T lymphocytes. Although PIM kinases are expressed more in Th1 than Th2 cells, we demonstrate here that these kinases positively influence Th1 cell differentiation. Our RNA interference results from human primary Th cells also suggest that PIM kinases promote the production of IFNγ, the hallmark cytokine produced by Th1 cells. Consistent with this, they also seem to be important for the up-regulation of the critical Th1-driving factor, T box expressed in T cells (T-BET), and the IL-12/STAT4 signaling pathway during the early Th1 differentiation process. In summary, we have identified PIM kinases as new regulators of human primary Th1 cell differentiation, thus providing new insights into the mechanisms controlling the selective development of human Th cell subsets.     

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.     

Biosynthesis of hexamannophosphoinositides inMycobacterium smegmatis ATCC 607

The biosynthesis of hexamannophosphoinositides inMycobacterium smegmatis ATCC 607 was examined using labelled tri- and tetraacylated dimannophosphoinositides (PIM₂-3F and PIM₂-4F) as precursors, byin vivo andin vitro incorporation. Tetraacylated dimannoside was metabolically more active as compared to triacylated dimannoside and seems to be the precursor for the synthesis of hexamannophosphoinositides.Abbreviations M. 607 Mycobacterium smegmatis ATCC 607 - PIMs Mannophosphoinositides - PIM₂-2F diacylated dimannoside - PIM₂-3F triacylated dimannoside - PIM₂-4F tetraacylated dimannoside - PIM₃-3F triacylated trimannoside - PIM₆-3F triacylated hexamannoside - PIM₆-4F tetraacylated hexamannoside - GDP-mannose Guanosine diphosphomannose     

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan

Based on chemical analysis, we have previously concluded thatthe biologically important lipoarabinomannan (LAM) and lipomannan(LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositolmannosides (PIMs), the characteristic cell envelope mannophosphoinositidesof mycobacteria Using definitive analytical techniques, we havenow re-examined the reported multiacylated nature of PIMs inorder to gain a better insight into their possible roles asbiosynthethic precursors of LM and LAM. High-sensitivity fastatom bombardment-mass spectrometry analyses of the perdeuteroacetyland permethyl derivatives of PIMs from Mycobacterium tuberculosisand Mycobacterium leprae enabled us to define the exact fattyacyl compositions of the multiacylated, heterogeneous PIM families,notably the dimannoside (PIM₂) and the hexamannoside (PIM₆).Specifically, in conjunction with other chemical and gas chromatography-massspectrometry (GCMS) analyses, the additional C16 fatty acylsubstituent on PIM₂ and its lyso form were defined as attachedto the C6 position of mannose. We also present evidence fortriacylated mannophosphoinositide as a common lipid anchor forboth LM and LAM, and further postulate that acylation of PIM₂may constitute a key regulatory step in their biosynthesis. FAB-MS lipoarabinomannan lipomannan Mycobacterium tuberculosis phosphatidylinositol mannosides     

Evaluation of Four Aerobiological Sampling Methods for the Retrieval of Aerosolized Pseudomonas syringae

The Andersen six-stage impactor, the SAS (Surface Air System) impactor, the AGI-30 impinger, and gravity plates were evaluated for the retrieval of aerosol-released Pseudomonas syringae. The upper limits of the impactor samplers were exceeded at a spray concentration of 10⁷ CFU/ml, indicating that these samplers are not appropriate for monitoring high airborne concentrations. Decreased cell concentrations were retrieved with increased sampling time for the Andersen and AGI samplers, indicating that a minimum sampling time is preferable for monitoring aerosolized vegetative cells.