An Apparatus to Deliver Mannitol Powder for Bronchial Provocation in Children Under Six Years Old.

BACKGROUND: Currently bronchial provocation testing (BPT) using mannitol powder cannot be performed in children under 6 years. A primary reason is it is challenging for children at this age to generate a consistent inspiratory effort to inhale mannitol efficiently from a dry powder inhaler. A prototype system, which does not require any inhalation training from the pediatric subject, is reported here. It uses an external source of compressed air to disperse mannitol powder into a commercial holding chamber. Then the subject uses tidal breathing to inhale the aerosol. METHOD: The setup consists of a commercially available powder disperser and Volumatic™ holding chamber. Taguchi experimental design was used to identify the effect of dispersion parameters (flow rate of compressed air, time compressed air is applied, mass of powder, and the time between dispersion and inhalation) on the fine particle dose (FPD). The prototype was tested in vitro using a USP throat connected to a next generation impactor. The aerosols from the holding chamber were drawn at 10 L/min. A scaling factor for estimating the provoking dose to induce a 15% reduction in forced expiratory volume in 1 second (FEV1) (PD15) was calculated using anatomical dimensions of the human respiratory tract at various ages combined with known dosing values from the adult BPT. RESULTS: Consistent and doubling FPDs were successfully generated based on the Taguchi experimental design. The FPD was reliable over a range of 0.8 (±0.09) mg to 14 (±0.94) mg. The calculated PD15 for children aged 1-6 years ranged from 7.1-30 mg. The FPDs generated from the proposed set up are lower than the calculated PD15 and therefore are not expected to cause sudden bronchoconstriction. CONCLUSION: A prototype aerosol delivery system has been developed that is consistently able to deliver doubling doses suitable for bronchial provocation testing in young children.

Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl amides as potent and long acting muscarinic antagonists.

Novel quaternary ammonium derivatives of (3R)-quinuclidinyl amides have been identified as potent M3 muscarinic antagonists with a long duration of action in an in vivo model of bronchoconstriction. The synthesis, structure-activity relationships and biological evaluation of this series of compounds are reported.

Effects of sodium metabisulfite on the expression of BK(Ca), K(ATP), and L-Ca(2+) channels in rat aortas in vivo and in vitro.

Sodium metabisulfite (SMB) is most commonly used as the preservative in many food preparations and drugs. So far, few studies about its negative effects were reported. The purpose of this study was to investigate the effect of SMB on the expression of big-conductance Ca(2+)-activated K(+) (BKCa), ATP-sensitive K(+) (KATP), and L-type calcium (L-Ca(2+)) channels in rat aorta in vivo and in vitro. The results showed that the mRNA and protein levels of the BKCa channel subunits α and ß1 of aorta in rats were increased by SMB in vivo and in vitro. Similarly, the expression of the KATP channel subunits Kir6.1, Kir6.2, and SUR2B were increased by SMB. However, SMB at the highest concentration significantly decreased the expression of the L-Ca(2+) channel subunits Cav1.2 and Cav1.3. These results suggest that SMB can activate BKCa and KATP channels by increasing the expression of α, ß1, and Kir6.1, Kir6.2, SUR2B respectively, while also inhibit L-Ca(2+) channels by decreasing the expression of Cav1.2 and Cav1.3 of aorta in rats. The molecular mechanism of SMB-induced vasorelaxant effect might be related to the expression changes of BKCa, KATP, and L-Ca(2+) channels subunits. Further work is needed to determine the relative contribution of each channel in SMB-mediated vasorelaxant effect.

Prenatal retinoid deficiency leads to airway hyperresponsiveness in adult mice.

There is increasing evidence that vitamin A deficiency in utero correlates with abnormal airway smooth muscle (SM) function in postnatal life. The bioactive vitamin A metabolite retinoic acid (RA) is essential for formation of the lung primordium; however, little is known about the impact of early fetal RA deficiency on postnatal lung structure and function. Here, we provide evidence that during murine lung development, endogenous RA has a key role in restricting the airway SM differentiation program during airway formation. Using murine models of pharmacological, genetic, and dietary vitamin A/RA deficiency, we found that disruption of RA signaling during embryonic development consistently resulted in an altered airway SM phenotype with markedly increased expression of SM markers. The aberrant phenotype persisted postnatally regardless of the adult vitamin A status and manifested as structural changes in the bronchial SM and hyperresponsiveness of the airway without evidence of inflammation. Our data reveal a role for endogenous RA signaling in restricting SM differentiation and preventing precocious and excessive SM differentiation when airways are forming.

The effects of particle size on measurement of airway hyperresponsiveness to methacholine.

BACKGROUND: The effect of particle size on methacholine provocation concentration causing a decrease in forced expiratory volume of 1 second (FEV1) of 20% (PC20) is debatable. OBJECTIVE: To evaluate the functional effects of 3 different particle size nebulizers on methacholine PC20. METHODS: Participants were randomly assigned to have 3 methacholine challenges on 3 separate days. Nebulizer mass median aerodynamic diameter (MMAD) was provided by manufacturers. The Wright nebulizer (MMAD, 1.0 µm), Aeroneb (MMAD, 3 µm), and Aeroneb (MMAD, 5 µm) were calibrated, and the nebulizer outputs were calculated to administer 0.26 mL of methacholine over 120, 112, and 83 seconds, respectively. After each inhalation, spirometry was performed and the test was terminated when the PC20 was achieved. RESULTS: Eight nonsmoking patients with mild asthma (4 male and 4 female) completed the study. The mean (SD) age was 25 (13.9) years, and the mean (SD) baseline FEV1 was 88% (11.3%). Patients using the Aeroneb (MMAD, 5 µm) nebulizer had the lowest PC20 (bronchoconstricted at lowest methacholine concentration), with a PC20 geometric mean of 0.62 mg/mL compared with patients using the Aeroneb (MMAD, 3.0 µm), who had a PC20 of 1.76 mg/mL, and patients using the Wright nebulizer (MMAD, 1.0 µm), who had a PC20 of 6.32 mg/mL. There was a significant difference in PC20 across all particle sizes (P < .001). The pairwise differences revealed a P < .001 between 3 µm and 1 µm and between 5 µm and 1 µm and a P = .008 between 5 µm and 3 µm. CONCLUSION: Our results reveal a variability in methacholine PC20 using 3 different nebulizers, despite adjusting the nebulizers' outputs. Our results are consistent with the previous reports, which recommended using larger particle size nebulizers in the assessment of airway hyperresponsiveness in asthma. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00529477.

Challenging the two-minute tidal breathing challenge test.

BACKGROUND: In the adenosine 5'-monophosphate (AMP) bronchial challenge test, AMP is usually administered according to dosing protocols for methacholine. We investigated whether the 2-min tidal breathing challenge test for methacholine is applicable to AMP. Parameters known to affect nebulizer output were studied. Our aim was to determine whether control of additional parameters is needed for currently standardized protocols. METHODS: The study was performed with the Sidestream nebulizer from the APS Pro Aerosol Provocation System (CareFusion Respiratory). The effects of AMP concentration, jet pressure, and suction flow rate on nebulizer output rate and aerosol droplet size distribution were determined. RESULTS: The volume median diameter for water increased from 5.10 µm to 8.49 µm when the jet pressure was reduced to obtain the prescribed output rate of 0.13 mL/min. The output rate was increased when a suction flow rate was used to remove the aerosol. Increasing the AMP concentration resulted in smaller droplets and a lower output rate when a suction flow was applied. CONCLUSIONS: The effects of AMP concentration on nebulizer performance may result in changes in the administered dose and site of deposition of AMP at dose escalation. All of the investigated parameters influence nebulizer performance, hence the outcome of a bronchial challenge test. Therefore, these parameters should not only be specified in challenge testing, but be actively controlled as well.

Sodium metabisulfite as a contact allergen--an example of a rare chemical mechanism for protein modification.

BACKGROUND: Skin-sensitizing chemicals that cause allergic contact dermatitis do so by reacting with self-proteins such that the modified structure becomes antigenic. The reaction chemistry involved is well characterized, but there are exceptions, such as the occasional allergen sodium metabisulfite. OBJECTIVES: To identify the potential in cutaneo reaction chemistry of sodium metabisulfite. METHODS: The established protein reaction chemistry associated with aqueous sulfite chemistry was explored in the context of the protein modification stage in allergic contact dermatitis. RESULTS: A probable mechanism for the in cutaneo modification of proteins by sodium metabisulfite involves the sulfite di-anion, acting as a nucleophile towards electrophilic centres in proteins, which is a rare mechanism, as most known skin-sensitizing chemicals behave as electrophiles. CONCLUSIONS: Sodium metabisulfite is an unusual but not infrequent contact allergen whose chemistry suggests a previously unrecognized protein modification mechanism involving nucleophilic attack by sulfite di-anions on target electrophilic centres in skin proteins. The chemical properties required for sensitization by nucleophilic attack on skin proteins are quite restrictive, so the domain of nucleophilic sensitizers is expected to be small. Thiourea derivatives are among the sensitizers likely to act by this mechanism.

Methacholine dry powder inhaler as a new tool for bronchial challenge test.

BACKGROUND: The methacholine (MCH) challenge test is performed to detect bronchial hyperresponsiveness in subjects suffering from asthma. It is conducted by inhaling spasmogen substances at increasing doses and measuring FEV1-PD20 variation following the bronchoconstriction evoked. AIM: This paper describes a new method for MCH challenge test using pre-metered respirable powders of MCH at different doses for facilitating test execution. The availability of a series of pre-metered doses gives higher control over aerosolized dose and fine particle fraction (respirable dose), improving the accuracy and repeatability of the test. Dosimetric tests with MCH solution and pre-dosed powder challenge tests were clinically compared. METHODS AND MATERIALS: The inhalation powders were prepared by spray drying of solutions of methacholine, mannitol and hydroxypropylmethylcellulose in which different concentrations of MCH were included. The methacholine powders prepared were carefully characterized in terms of aerodynamic properties. RESULTS: Inhalation powders containing methacholine from 12.5 to 200 microg per metered dose, having a fine particle fraction between 40 and 60%, were prepared using mannitol and cellulose polymer. Eighteen subjects (12 hyperresponsive and six normal) were subjected to both the MCH solution and powder tests in random sequence. No significant differences in FEV1 and PD20 values were found between the challenge tests performed with liquid and powder formulations of methacholine. CONCLUSIONS: Powders of MCH having high respirability of the delivered doses can be prepared by spray drying. They allow for the performance of a challenge test using a dry powder inhaler. The powder dose series can be an alternative to the current dosimetric test with MCH solutions.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

Stability of frozen methacholine solutions in unit-dose syringes for bronchoprovocation.

OBJECTIVE: Methacholine solutions < 0.25 mg/mL must be prepared fresh daily, while concentrations > or = 0.25 mg/mL must be prepared at 2-week intervals according to US Food and Drug Administration-required labeling. The purpose of this report was to determine whether freezing methacholine solutions in unit-dose syringes would allow less frequent preparation. DESIGN: Diluent containing 0.5% sodium chloride, 0.275% sodium bicarbonate, and 0.4% phenol was used to prepare 11 concentrations of methacholine ranging from 0.031 to 32.0 mg/mL. Three milliliters of each dilution was placed into 5-mL polypropylene syringes and immediately frozen. Methacholine concentrations were determined using a validated high-performance liquid chromatography assay after preparation (time zero) and at 0.5, 1, 1.5, 2, 3, 4, 5, and 6 months. On the day of analysis, the samples were allowed to thaw to room temperature. An additional set of each dilution was stored at room temperature for 24 h after thawing and then analyzed for methacholine. RESULTS: Samples > or = 0.062 mg/mL analyzed immediately after thawing retained > or = 90% of labeled potency for at least 6 months, while the 0.031-mg/mL sample retained 90% potency for 4 months. Most samples analyzed 24 h after thawing lost potency. CONCLUSION: If prepared and stored in unit-dose syringes frozen, methacholine solutions containing 0.062 to 32.0 mg/mL can be prepared at 6-month intervals, and solutions containing 0.031 mg/mL can be prepared at 4-month intervals. Once thawed, unused methacholine solutions should be discarded.

Stability of stored methacholine solutions: study of hydrolysis kinetic by IP-LC.

Methacholine chloride is a powerful cholinergic bronchoconstrictor agent used during bronchial airway hyper-responsiveness diagnosis. Methacholine is susceptible to hydrolysis in aqueous solutions in acetic acid and beta-methylcholine. In the present work, kinetics of hydrolysis with different solvents (water and phosphate-buffered saline (PBS) pH 7.4) at different temperatures have been studied using a newly developed high-performance liquid chromatography. At 4 degrees C, kinetic determination of hydrolysis in methacholine chloride solutions (50 mg/ml) shows no hydrolysis in either aqueous or phosphate-buffered solutions over a 40-day period. At 30 degrees C, concentration of unbuffered methacholine chloride solutions remained unchanged, but buffered methacholine chloride solutions have degradation up to 5.5% over a 40-day period. At 40 degrees C, concentration of unbuffered methacholine chloride has degradation up to 5% and buffered methacholine chloride solutions have degradation up to 10% over a 40-day period. Methacholine chloride solutions are susceptibly to be used in hospital pharmacy at different concentrations. We have studied pH and osmolality for methacholine solutions prepared with different diluents potentially used in hospital pharmacies, i.e. deionized water, 0.9% NaCl and PBS pH 7.4. We have demonstrated that methacholine solutions prepared with deionized water at 50 mg/ml and diluted with PBS pH 7.4 from 5 to 40 mg/ml are isoosmotic and potentially available for inhalation tests to measure non-specific bronchial hyper-responsiveness.

[Pharmaceutical contribution to the non specific challenge in bronchial provocation tests]. / Contribution pharmaceutique a l'exploration de l'hyperréactivité bronchique non spécifique.

Aerosols of methacholine chloride are used in inhalation tests to measure non-specific bronchial responsiveness. Inhalation challenge solutions are not commercially available neither by pharmaceutical industry nor in France by the PCH-APHP. To reply to doctor's request, dilute concentrations of methacholine must be prepared properly in order to protect the patient and to assure doctor of test reproductibility. Osmolarity, pH and degradation of different dilute concentration of methacholine chloride have been compared with two diluents. This study allowed us to validate our preparation of solutions.

Stability of methacholine chloride in isotonic sodium chloride using a capillary electrophoresis assay.

OBJECTIVE: To investigate the stability of methacholine chloride in 0.9% sodium chloride solutions. METHOD: Methacholine powder was mixed with diluent to a final concentration of 5 and 10 mg/ml. Duplicates of each admixture were divided and stored in glass vials at 25 degrees C, 4 degrees C and -20 degrees C for 12 months. At appropriate times intervals, samples were removed from solutions and analysed. Methacholine concentrations were measured using a high performance capillary electrophoresis assay. RESULTS: No colour or other visual changes were seen in any sample. However, an additional peak was observed in some samples. CONCLUSION: Methacholine chloride solutions 5 mg/ml were stable in isotonic sodium chloride after refrigeration or freezing over a period of one year; methacholine chloride solutions 10 mg/ml were stable for one year after freezing. The solutions stored at ambient temperature were stable for 35 days and for less than 14 days, respectively, for the 5 and the 10 mg/ml solutions.

Effect of pH on the stability of methacholine chloride in solution.

Methacholine chloride bronchoprovocation challenges are performed for the diagnosis and investigation of hyperreactive airways. Over the last 20 yrs various formulations and pH values for the preparation of solutions of methacholine have been described. To determine the stability of methacholine chloride solutions prepared in a variety of buffers with differing pH values and under varying storage temperatures, we measured methacholine concentrations at intervals from 1 to 5 weeks. It was found that methacholine chloride solutions rapidly decompose if the pH is greater than 6 and that decomposition is more rapid as the pH is raised; solutions at pH 9, i.e. bicarbonate buffer, and stored at 27 degrees C have degradation up to 36% after only one week. Solutions of the same pH but prepared in different buffers can have both varied rates of deterioration and different absolute amounts of methacholine hydrolysed, e.g. solutions prepared in pH 9 borate buffer and stored at 27 degrees C have up to 60% degradation after 1 week. Solutions prepared in saline are stable probably because methacholine solutions are weakly acidic. The results emphasise the importance of preparing methacholine chloride in the proper buffers for use in the accurate assessment of airway responsiveness.