HPLC-UV/VIS for Determination of Ipratropium Bromide Mixed with Salbuterol, Beclomethasone Propionate and Budesonide using Dual Wavelength-Detection Method.

BACKGROUND: Inhalation preparation involves liquid or solid raw materials for delivering to lungs as aerosol or vapor. The liquid preparation for nebulizer is effective for convenient use and patient compliance and it has been extensively used in the treatment of clinical lung diseases. Clinical staff often mixes the compound ipratropium bromide with beclomethasone propionate and budesonide inhaler but reference values of inhalants for clinical use need to be established for simplifying the operation procedure. The high-performance liquid chromatography (HPLC) method of compound ipratropium bromide solution, beclomethasone propionate suspension and budesonide suspension after mixed atomization was studied. METHODS: The specificity, linearity, recovery (accuracy), precision and stability of compound ipratropium bromide, beclomethasone propionate and budesonide were tested to verify the developed liquid phase method. RESULTS: The developed liquid phase method had high specificity, linear R2≥0,999, recovery (accuracy) RSD (relative standard deviation) less than 2%, precision RSD less than 2,0%, and stability RSD less than 2,0%. CONCLUSION: The liquid phase methodology developed in this study can be used for the determination of compound ipratropium bromide mixed with beclomethasone propionate and budesonide. The current methodology can also be used to provide a reference for the determination of its content after mixing, and further data support for its clinical medication.

Drug distribution transients in solution and suspension-based pressurised metered dose inhaler sprays.

This paper presents in situ time-resolved drug mass fraction measurements in pressurised metered dose inhaler (PMDI) sprays, using a novel combination of synchrotron X-ray fluorescence and scattering. Equivalent suspension and solution formulations of ipratropium bromide in HFA-134a propellant were considered. Measurements were made both inside the expansion chamber behind the nozzle orifice, and in the first few millimeters of the spray where droplet and particle formation occur. We observed a consistent spike in drug mass fraction at the beginning of the spray when the first fluid exits the nozzle orifice. Approximately 20% of the total delivered dose exits the nozzle in the first 0.1 s of the spray. The drug mass fraction in the droplets immediately upon exiting the nozzle peaked at approximately 50% of the canister mass fraction, asymptoting to approximately 20% of the canister concentration. The effect is due to a change in the drug mass fraction inside the droplets, rather than changes in droplet size or distribution. The transient was found to originate inside the expansion chamber. We propose that this effect may be a major contributor to low delivery efficiency in PMDIs, and have important implications for oropharyngeal deposition and inhalation technique. This highlights the importance of expansion chamber and nozzle design on the structure of PMDI sprays, and indicates areas of focus that may lead to improvement in drug delivery outcomes.

The Effect of Active Pharmaceutical Ingredients on Aerosol Electrostatic Charges from Pressurized Metered Dose Inhalers.

PURPOSE: This study investigated the effect of different active pharmaceutical ingredients (API) on aerosol electrostatic charges and aerosol performances for pressurized metered dose inhalers (pMDIs), using both insulating and conducting actuators. METHODS: Five solution-based pMDIs containing different API ingredients including: beclomethasone dipropionate (BDP), budesonide (BUD), flunisolide (FS), salbutamol base (SB) and ipratropium bromide (IPBr) were prepared using pressure filling technique. Actuator blocks made from nylon, polytetrafluoroethylene (PTFE) and aluminium were manufactured with 0.3 mm nominal orifice diameter and cone nozzle shape. Aerosol electrostatics for each pMDI formulation and actuator were evaluated using the electrical low-pressure impactor (ELPI) and drug depositions were analysed using high performance liquid chromatography (HPLC). RESULTS: All three actuator materials showed the same net charge trend across the five active drug ingredients, with BDP, BUD and FS showing positive net charges for both nylon and PTFE actuators, respectively. While SB and IPBr had significantly negative net charges across the three different actuators, which correlates to the ionic functional groups present on the drug molecule structures. CONCLUSIONS: The API present in a pMDI has a dominant effect on the electrostatic properties of the formulation, overcoming the charge effect arising from the actuator materials. Results have shown that the electrostatic charges for a solution-based pMDI could be related to the interactions of the chemical ingredients and change in the work function for the overall formulation.

[Effectiveness of inhaled hypertonic saline in children with bronchiolitis].

OBJECTIVE: To assess the efficacy and safety of inhaled nebulized hypertonic saline (HS) solution in infants with acute bronchiolitis. METHOD: Totally 129 patients with acute bronchiolitis (clinical severity score ≥ 4, aged 2-18 months) admitted to the Capital Institute of Pediatrics from November 2012 to January 2013 were enrolled. All the subjects were assigned to receive 1.5 ml compound ipratropium bromide solution for inhalation and 1 ml budesonide firstly, twice a day. Then, the subjects were randomized to receive 2 ml doses of nebulized 5% HS (Group A), 3% HS (Group B) or 0.9% NS (Group C), twice a day. The treatment lasted for 3 days. Clinical severity scores before treatment and 24, 48, 72 h after treatment were documented. Bronchospasm, nausea and emesis were recorded to assess safety. RESULT: A total of 124 patients completed this research.Group A included 40 cases, Group B included 42 cases, Group C included 42 cases. Demographic characteristics, pre-treatment duration and clinical severity score before treatment were similar among the 3 group.Seventy-two hours after treatment, the clinical severity score of Group A, B, and C were 3.5 (1.0) , 4.0 (1.0) and 5.0 (0) . At 24, 48, and 72 h after treatment, the clinical severity score were significantly different among the three groups (χ(2) = 36.000, 51.200, 50.800, P < 0.05) .One patient in Group A got paroxysmal cough everytime as soon as he received 5% HS (6 times).Other 3 patients in Group A got paroxysmal cough once. The incidence of adverse effect of Group A was 3.75% (9/240); no adverse event occurred in other group. The incidence of adverse effect among this three group was significantly different (χ(2) = 19.13, P < 0.01). CONCLUSION: Inhalation of nebulized 5% and 3% hypertonic saline could decrease clinical symptoms of patient with acute bronchiolitis; 5% HS was superior to 3% HS. But 2 ml dose of 5% HS may induce paroxysmal cough.

Efficacy and safety of eco-friendly inhalers: focus on combination ipratropium bromide and albuterol in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality and its treatment is critical to improve quality of life, reduce symptoms, and diminish the frequency of COPD exacerbations. Due to the harmful environmental effects of pressurized metered-dose inhalers (pMDIs) containing chlorofluorocarbons (CFCs), newer systems for delivering respiratory medications have been developed. METHODS: A search of the literature in the PubMed database was undertaken using the keywords "COPD," "albuterol," "ipratropium bromide," and "Respimat® Soft Mist Inhaler™"; pertinent references within the identified citations were included. The environmental effect of CFC-pMDIs, the invention of the Respimat® Soft Mist Inhaler™ (SMI) (Boehringer Ingelheim, Ingelheim, Germany), and its use to deliver the combination of albuterol and ipratropium bromide for the treatment of COPD were reviewed. RESULTS: The adverse environmental effects of CFC-pMDIs stimulated the invention of novel delivery systems including the Respimat SMI. This review presents its development, internal mechanism, and use to deliver the combination of albuterol and ipratropium bromide. CONCLUSION: CFC-pMDIs contributed to the depletion of the ozone layer and the surge in disorders caused by harmful ultraviolet B radiation. The banning of CFCs spurred the development of novel delivery systems for respiratory medications. The Respimat SMI is an innovative device that produces a vapor of inhalable droplets with reduced velocity and prolonged aerosol duration that enhance deposition within the lower airway and is associated with improved patient satisfaction. Clinical trials have demonstrated that the Respimat SMI can achieve effects equivalent to pMDIs but with lower medication doses. The long-term safety and efficacy remain to be determined. The Respimat SMI delivery device is a novel, efficient, and well-received system for the delivery of aerosolized albuterol and ipratropium bromide to patients with COPD; however, the presence of longer-acting, less frequently dosed respiratory medications provide patients and providers with other therapeutic options.

Co-deposition of a triple therapy drug formulation for the treatment of chronic obstructive pulmonary disease using solution-based pressurised metered dose inhalers.

OBJECTIVES: The formulation of multi-drug pressurised metered dose inhalers (pMDIs) opens up exciting therapeutic opportunities for the treatment of asthma and chronic obstructive pulmonary disease (COPD). We have investigated the formulation of a solution-based triple therapy pMDI containing ipratropium, formoterol, budesonide and ethanol as co-solvent. METHODS: This system was characterised for in-vitro performance and compared with marketed pMDIs (Atrovent and Symbicort). KEY FINDINGS: No significant difference was found in the stage deposition of each drug from the triple therapy formulation, suggesting that the droplets contained a fixed ratio of the three components used. Stage deposition of formoterol and budesonide from the suspension-based marketed Symbicort were significantly different, suggesting that the two drugs were deposited as separate entities. Calculation of the mass median aerodynamic diameter (MMAD) of each formulation suggested Atrovent (ipratropium, MMAD = 0.9 ± 0.0 µm) to have a small particle size, similar to the triple therapy formulation. Atrovent, like the triple therapy formulation was solution based and it contained ethanol as a co-solvent (triple therapy formulation, MMAD = 1.3 ± 0.0 µm). CONCLUSIONS: This study demonstrated the feasibility of formulating a solution-based pMDI containing a triple therapy with identical deposition pattern for the treatment of several respiratory diseases where multi-drug cell targeting is required.

Influence of crystal form of ipratropium bromide on micronisation and aerosolisation behaviour in dry powder inhaler formulations.

OBJECTIVES: This study aimed to investigate the relationship between the mechanical properties of anhydrous and monohydrate ipratropium bromide (IB) crystals, their processing behaviour upon air-jet micronisation and aerosolisation performance in dry powder inhaler (DPI) formulations. METHODS: IB monohydrate and anhydrous crystals were produced from seed crystals and supercritical carbon dioxide crystallisation, respectively. Young's modulus of anhydrous and monohydrate IB crystals was determined using nanoindentation. For air-jet micronised crystals, the physicochemical and surface interfacial properties via the cohesive-adhesive balance (CAB) approach were investigated. These data were correlated to in-vitro aerosolisation performance of carrier-based DPI formulations containing either anhydrous or monohydrate IB. KEY FINDINGS: Particle size and Young's modulus of both crystals were similar and this was reflected in their similar processing upon micronisation. Particle size of micronised anhydrous and monohydrate crystals were similar. CAB measurements of the micronised particles of monohydrate or anhydrous forms of IB with respect to lactose were 0.70 (R² = 0.998) and 0.77 (R² = 0.999), respectively. These data suggested that both samples had similar adhesion to lactose, which correlated with their similar in-vitro aerosolisation performance in DPI formulations. CONCLUSIONS: Monohydrate and anhydrous crystals of IB exhibited similar mechanical properties and interfacial properties upon secondary processing. As a result, the performance of the DPI formulations were similar.

Electrostatic charge characteristics of jet nebulized aerosols.

BACKGROUND: Liquid droplets can be spontaneously charged in the absence of applied electric fields by spraying. It has been shown by computational simulation that charges may influence particle deposition in the airways. The electrostatic properties of jet nebulized aerosols and their potential effects on lung deposition have hardly been studied. A modified electrical low pressure impactor (ELPI) was employed to characterize the aerosol charges generated from jet nebulized commercial products. METHODS: The charge and size measurements were conducted at 50% RH and 22 degrees C with a modified ELPI. Ventolin, Bricanyl, and Atrovent were nebulized using PARI LC Plus jet nebulizers coupled to a DeVilbiss Pulmo-Aide compressor. The aerosols were sampled in 30-sec durations. The drug deposits on the impactor stages were assayed chemically using high-performance liquid chromatography (HPLC). The charges of nebulized deionized water, isotonic saline, and the three commercial products diluted with saline were also measured to analyze the contributions of the major nebule ingredients on charging. No mass assays were performed on these runs. RESULTS: All three commercial nebules generated net negative charges. The magnitude of the charges reduced over the period of nebulization. Ventolin and Bricanyl yielded similar charge profiles. Highly variable charges were produced from deionized water. On the other hand, nebulized saline reproducibly generated net positive charges. Diluted commercial nebules showed charge polarity inversion. The charge profiles of diluted salbutamol and terbutaline solutions resembled those of saline, while the charges from diluted ipratropium solutions fluctuated near neutrality. CONCLUSIONS: The charge profiles were shown to be influenced by the concentration and physicochemical properties of the drugs, as well as the history of nebulization. The drugs may have unique isoelectric concentrations in saline at which the nebulized droplets would carry near-zero charges. According to results from computational simulation models in the literature, the numbers of elementary charges per droplet estimated from the data were not high enough to potentially affect lung deposition.

Dry powder aerosols generated by standardized entrainment tubes from drug blends with lactose monohydrate: 2. Ipratropium bromide monohydrate and fluticasone propionate.

The objectives of this study were: systematic investigation of dry powder aerosol performance using standardized entrainment tubes (SETs) and lactose-based formulations with two model drugs; mechanistic evaluation of performance data by powder aerosol deaggregation equation (PADE). The drugs (IPB and FP) were prepared in sieved and milled lactose carriers (2% w/w). Aerosol studies were performed using SETs (shear stresses tau(s) = 0.624-13.143 N/m(2)) by twin-stage liquid impinger, operated at 60 L/min. PADE was applied for formulation screening. Excellent correlation was observed when PADE was adopted correlating FPF to tau(s). Higher tau(s) corresponded to higher FPF values followed by a plateau representing invariance of FPF with increasing tau(s). The R(2) values for PADE linear regression were 0.9905-0.9999. Performance described in terms of the maximum FPF (FPF(max): 15.0-37.6%) resulted in a rank order of ML-B/IPB > ML-A/IPB > SV-A/IPB > SV-B/IPB > ML-B/FP > ML-A/FP > SV-B/FP > SV-A/FP. The performance of IPB was superior to FP in all formulations. The difference in lactose monohydrate carriers was less pronounced for the FPF in IPB than in FP formulations. The novel PADE offers a robust method for evaluating aerodynamic performance of dry powder formulations within a defined tau(s) range.

Inhaled albuterol/salbutamol and ipratropium bromide and their combination in the treatment of chronic obstructive pulmonary disease.

IMPORTANCE OF THE FIELD: Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality throughout the world. Combination therapy with albuterol and ipratropium bromide was approved > 15 years ago for the treatment of COPD. We review the mechanism of action, clinical efficacy, and safety of albuterol, ipratropium and combined albuterol-ipratropium therapy. AREAS COVERED IN THIS REVIEW: We conducted a PubMed literature search using the keywords COPD, albuterol, ipratropium bromide and Combivent (Boehringer Ingelheim Corp., Ridgefield, CT, USA); pertinent references within the identified citations are included in the review. Data from the manufacturers are also evaluated. WHAT THE READER WILL GAIN: At the time of its approval, albuterol/ipratropium bromide was an innovative combination of existing medications for the treatment of COPD. The combined formulation provides better improvement in airflow than either component alone and, by reducing the number of separate inhalers, simplifies therapy and improves compliance compared with the individual components. TAKE HOME MESSAGE: The recent development and approval of longer acting and more potent beta agonists, anticholinergics and newer combination treatments have surpassed many of the advantages of combined albuterol-ipratropium for the treatment of patients with stable COPD.

[The compatibility between packing material and ipratropium bromide aerosol].

With the establishment of HPLC and LC-MS methods to determine the related substances and the content of active pharmaceutical ingredient (API) in ipratropium bromide aerosol products, several packing material-related impurities were identified, including antioxygen BHT and antioxygen 2246. Results showed that these leachable additives from the packing materials may present at a relative high level in the drug solution, and the low content of API in the drug products is usually due to the adsorption of the packing material as well as the leaking of contents. The current available assay methods for the control of ipratropium bromide aerosol products are often lack of specificity and unable to assure the drug quality effectively. To meet the increasing attention on the regulations of drug packing materials, our research would be a pilot study, indicating that the inappropriate packing materials could cause the migration and adsorption of the active ingredients, and the importance to have compatibility studies between packing materials and drugs.

A new approach to characterise pharmaceutical aerosols: measurement of aerosol from a single dose aqueous inhaler with an optical particle counter.

An in-line sampling system with dilution units for aqueous droplet aerosols from single dose inhalers (Berodual Respimat, Boehringer Ingelheim Pharma GmbH & Co. KG, Germany) for an optical particle counter is described. The device has been designed to interface with a white light aerosol spectrometer (welas digital 2100, Palas GmbH, Germany) that allows the time-resolved measurement of highly concentrated aerosols. Performance of the sampling system with regard to the measured particle size distribution (PSD) is compared to Next Generation Impactor (NGI) and to laser diffraction measurements (Sympatec Inhaler and open bench). Optimal settings of the sampling system lead to PSDs that correspond well to those measured by the evaporation minimising NGI approach (15 L/min, cooled) and laser diffraction. The better accuracy of the new dilution unit in presence of an additional aerosol sampling filter in comparison to a previously described aerosol sampling system is shown for different settings of the sampling system. This allows a more precise quantification of the delivered drug amount which is also well correlated to the aerosol volume measured by the welas system. In addition, using time-resolved welas measurements provides insight into droplet size, evaporation and size changes of aerosol clouds delivered by liquid inhalers.

Computational and vibrational spectroscopic studies of ipratropium bromide.

In this study, ipratropium bromide is investigated using vibrational spectroscopy and quantum chemical calculations. The structure of ipratropium bromide was optimised using density functional theory calculations and the geometry optimisation has been carried out on two conformations with and without intramolecular hydrogen bonding. Infrared and Raman spectra were calculated from the optimised structures. Many modes in the calculated spectra could be matched with the experimental spectra and a description of the modes is given. By analysis of the theoretical vibrational modes, it is shown that ipratropium bromide specimens are likely to be a mixture of the two conformations with and without intramolecular hydrogen bonding. In addition, several spectral features and band intensities in the CH and OH stretching regions are explained. Quantum mechanical calculations allowed improved understanding of ipratropium bromide and its vibrational spectra.

Chemical and physical compatibility of levalbuterol inhalation solution concentrate mixed with budesonide, ipratropium bromide, cromolyn sodium, or acetylcysteine sodium.

BACKGROUND: Medications are frequently combined in the nebulizer cup, so it is important to determine their chemical and physical compatibility. OBJECTIVE: To determine the chemical and physical compatibility of levalbuterol with ipratropium bromide, cromolyn sodium, acetylcysteine sodium, and budesonide. METHODS: We mixed one dose of levalbuterol inhalation solution concentrate (1.25 mg/0.5 mL) with one dose of ipratropium bromide (0.5 mg/2.5 mL), cromolyn sodium (20 mg/2 mL), acetylcysteine sodium (1,000 mg/5 mL), or budesonide (0.5 mg/2 mL). Immediately after mixing the 2 drugs (time zero [T(0)]), and again after 30 min at room temperature (T(30)), we visually inspected the admixtures, measured their pH, and conducted high-pressure liquid chromatography (HPLC). RESULTS: There was no evidence of physical incompatibility with these drugs combinations. With all the admixtures, both drugs were chemically stable for at least 30-min. Admixture pH had not changed significantly at T(30). Drug recovery was 93.2-102.6% of the initial or control values. CONCLUSIONS: The 2-drug admixtures we studied were compatible for at least 30 min at room temperature.

Compatibility and aerosol characteristics of formoterol fumarate mixed with other nebulizing solutions.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) are often given admixtures of nebulizable drugs to minimize the time of administration in treatment regimens. OBJECTIVE: To evaluate the physicochemical compatibility and aerodynamic characteristics of formoterol fumarate 20 microg/2 mL when mixed or sequentially nebulized with budesonide inhalation suspension 0.5 mg/2 mL, ipratropium bromide 0.5 mg/2.5 mL, cromolyn sodium 20 mg/2 mL, or acetylcysteine 10% (100 mg/mL). METHODS: The admixtures were prepared in triplicate and analyzed for physicochemical compatibility at 0, 15, 30, and 60 minutes after mixing at room temperature. Physical compatibility was determined by visual examination and measurements of pH, osmolality, and turbidity. Chemical stability was evaluated using compendial or in-house-validated high-performance liquid chromatography (HPLC) assay methods. The aerodynamic characteristics of the admixtures or sequentially nebulized drugs were determined from aerosols generated from a Pari LC Plus nebulizer, using an 8-stage cascade impactor followed by HPLC analysis of the deposited drug. RESULTS: The admixtures remained clear, colorless solutions with no precipitation, except for cloudiness observed in the formoterol/budesonide combination due to budesonide suspension. The pH, osmolality, and turbidity for all admixtures were within the initial values (< or = 3%), and there were no significant changes (< or = 2%) in potency of the active components throughout the 1-hour study period. Due to increased drug volume or reconcentration in the nebulizer cup, the respirable fraction/delivered dose increased significantly (p < 0.05) for the mixed or sequentially nebulized drug. However, the fine particle fraction (FPF), mass median aerodynamic diameter, and geometric standard deviation generally remained unchanged for all admixtures, with the exception of FPF for the formoterol/budesonide combination. CONCLUSIONS: Our results indicate that admixtures of formoterol with budesonide, ipratropium, cromolyn, or acetylcysteine are physically and chemically compatible. However, admixing or sequential nebulization significantly increased the amount of drug delivered compared with single drug nebulization. The clinical implications of the in vitro data in patients with COPD have not been determined.

PVC membrane electrode for the potentiometric determination of Ipratropium bromide using batch and flow injection techniques.

Ipratropium (IP(+)) ion-selective electrode (ISE) has been constructed from poly(vinyl chloride) matrix membrane containing Ipratropium-tetraphenylborate (IP-TPB) as the electroactive component using 2-nitrophenyloctylether as plasticizer. The electrode exhibits near Nernstian response to Ipratropium bromide (IPBr) over the concentration range 10(-5) to 10(-2) mol L(-1) and detection limit 5.1x10(-6) mol L(-1). The electrode offers significant advantages including long lifetime (>2 months), excellent stability and reproducibility, fast response time (<10 s), wide pH working range (pH 2-9), high thermal stability (isothermal coefficient 0.37 mV/degrees C) and superior selectivity for IPBr over a large number of inorganic and organic substances. The electrode was successfully used as indicator electrode in the potentiometric titration of IPBr versus sodium tetraphenylborate (NaTPB) and in the determination of IPBr in Atrovent vials and spiked urine samples applying batch and flow injection techniques, with satisfactory results.

Type I hypersensitivity in an asthmatic child allergic to peanuts: was soy lecithin to blame?

BACKGROUND: Soy lecithin is widely used as an emulsifier, not only in topical skin care products but also in various drugs administered either topically, orally, or intravenously or by inhalation. Patients strongly allergic to soy and/or peanuts can develop an anaphylactic reaction when exposed to soy lecithin. METHOD: We report a 3-year-old asthmatic boy, allergic to peanuts, who was treated at the emergency department for an exacerbation of asthma following an upper respiratory tract infection. Within an hour after receiving the second of two inhalations of an ipratropium bromide (Atrovent) metered dose inhaler, he developed respiratory distress and generalized urticaria, an adverse event that regressed within 48 hours of withdrawal of the suspected drug. Soy lecithin, contained as an excipient in the metered dose inhaler, was strongly suspected of being responsible for this reaction. CONCLUSION: Drug products containing soy lecithin can cause severe allergic reactions in patients allergic to peanuts or soy. Physicians should be aware that adverse drug reactions can be due to both the active medical component and the excipient ingredients.

The effect of relative humidity on electrostatic charge decay of drugs and excipient used in dry powder inhaler formulation.

Electrostatic forces arising from charge accumulation on drug and excipient powders cause agglomeration and adhesion of particles to solid surfaces and problems during the manufacture and use of many pharmaceutical dosage forms, including dry powder inhalers (DPIs). The ability of materials to dissipate the acquired charge is therefore important and the aim of this work was to investigate the charge decay of salbutamol sulfate, ipratropium bromide monohydrate and alpha-lactose monohydrate. Differences in tri-phasic charge decay rates of the three materials in the order ipratropium bromide > lactose > salbutamol sulfate were demonstrated after corona charging and all materials showed an increased decay rate as the relative humidity was increased up to 86%. Preformulation knowledge of charge accumulation and decay in such materials will contribute to formulation, manufacture and performance of pharmaceutical dosage forms in general, and in particular DPIs.

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).

Inhibition of pneumococcal choline-binding proteins and cell growth by esters of bicyclic amines.

Streptococcus pneumoniae is one of the major pathogens worldwide. The use of currently available antibiotics to treat pneumococcal diseases is hampered by increasing resistance levels; also, capsular polysaccharide-based vaccination is of limited efficacy. Therefore, it is desirable to find targets for the development of new antimicrobial drugs specifically designed to fight pneumococcal infections. Choline-binding proteins are a family of polypeptides, found in all S. pneumoniae strains, that take part in important physiologic processes of this bacterium. Among them are several murein hydrolases whose enzymatic activity is usually inhibited by an excess of choline. Using a simple chromatographic procedure, we have identified several choline analogs able to strongly interact with the choline-binding module (C-LytA) of the major autolysin of S. pneumoniae. Two of these compounds (atropine and ipratropium) display a higher binding affinity to C-LytA than choline, and also increase the stability of the protein. CD and fluorescence spectroscopy analyses revealed that the conformational changes of C-LytA upon binding of these alkaloids are different to those induced by choline, suggesting a different mode of binding. In vitro inhibition assays of three pneumococcal, choline-dependent cell wall lytic enzymes also demonstrated a greater inhibitory efficiency of those molecules. Moreover, atropine and ipratropium strongly inhibited in vitro pneumococcal growth, altering cell morphology and reducing cell viability, a very different response than that observed upon addition of an excess of choline. These results may open up the possibility of the development of bicyclic amines as new antimicrobials for use against pneumococcal pathologies.