Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 1.

The alarming reduction in drug effectiveness against bacterial infections has created an urgent need for the development of new antibacterial agents that circumvent bacterial resistance mechanisms. We report here a series of DNA gyrase and topoisomerase IV inhibitors that demonstrate potent activity against a range of Gram-positive and selected Gram-negative organisms, including clinically-relevant and drug-resistant strains. In part 1, we present a detailed structure activity relationship (SAR) analysis that led to the discovery of our previously disclosed compound, REDX05931, which has a minimum inhibitory concentration (MIC) of 0.06 µg mL-1 against fluoroquinolone-resistant Staphylococcus aureus. Although in vitro hERG and CYP inhibition precluded further development, it validates a rational design approach to address this urgent unmet medical need and provides a scaffold for further optimisation, which is presented in part 2.

Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 2.

Building on our previously-reported novel tricyclic topoisomerase inhibitors (NTTIs), we disclose the discovery of REDX07965, which has an MIC90 of 0.5 µg mL-1 against Staphylococcus aureus, favourable in vitro pharmacokinetic properties, selectivity versus human topoisomerase II and an acceptable toxicity profile. The results herein validate a rational design approach to address the urgent unmet medical need for novel antibiotics.

Development of a rapid screening method to determine primary aromatic amines in kitchen utensils using direct analysis in real time mass spectrometry (DART-MS).

Primary aromatic amines (PAAs) are a group of substances with undesirable health effects, that are used in a variety of commercial products. Several recent studies, using a number of screening and confirmatory methods, have reported the migration of PAAs from some kitchen utensils into acetic acid 3% (w/v). Many of these methods require significant sample preparation, therefore the aim of this work was to determine if direct analysis in real time mass spectrometry (DART-MS) could be utilised as a rapid screening tool for the determination of PAAs in kitchen utensils. DART-MS results from direct analysis of the utensil have been compared with results of PAA migration by ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method. The UPLC-MS/MS method had excellent linearity, appropriate sensitivity (LOD ≤ 1.5 µg L(-1); LOQ ≤ 4.5 µg L(-1)), repeatability from 2.4 to 13.2% and acceptable recoveries. DART-MS results were in good agreement with UPLC-MS/MS data, with 100% of non-compliant (PAA positive) samples successfully identified by DART-MS.

Migration of perfluoroalkyl acids from food packaging to food simulants.

A broad range of fluorochemicals is used to impart oil and water barrier properties to paper and paperboard food packaging. Many of the fluorochemicals are applied to paper and paperboard as complex mixtures containing reaction products and by-products and unreacted starting materials. This work primarily focussed on the determination of seven perfluorocarboxylic acids (PFCAs) in two commercially available food contact papers: a di-perfluoro-alkyloxy-amino-acid and a perfluoroalkyl phosphate surfactant. In addition, the migration of the PFCAs into five food simulants from two commercial packages was evaluated. All seven PFCAs were detected in the range of 700-2220 µg kg⁻¹ of paper, while three perfluoroalkyl sulphonates were under the LOD. Results from migration tests showed that migration depends on paper characteristics, time and food simulant. The percentage of migration after 10 days at 40°C ranged from 4.8% to 100% for the two papers and different food simulants.

Formaldehyde levels in FEMA-supplied travel trailers, park models, and mobile homes in Louisiana and Mississippi.

In 2006, area physicians reported increases in upper respiratory symptoms in patients living in U.S. Federal Emergency Management Agency (FEMA)-supplied trailers following Hurricanes Katrina and Rita. One potential etiology to explain their symptoms included formaldehyde; however, formaldehyde levels in these occupied trailers were unknown. The objectives of our study were to identify formaldehyde levels in occupied trailers and to determine factors or characteristics of occupied trailers that could affect formaldehyde levels. A disproportionate random sample of 519 FEMA-supplied trailers was identified in Louisiana and Mississippi in November 2007. We collected and tested an air sample from each trailer for formaldehyde levels and administered a survey. Formaldehyde levels among all trailers in this study ranged from 3 parts per billion (ppb) to 590 ppb, with a geometric mean (GM) of 77 ppb [95% confidence interval (CI): 70-85; range: 3-590 ppb]. There were statistically significant differences in formaldehyde levels between trailer types (P < 0.01). The GM formaldehyde level was 81 ppb (95% CI: 72-92) among travel trailers (N = 360), 57 ppb (95% CI: 49-65) among mobile homes (N = 57), and 44 ppb (95% CI: 38-53) among park models (N = 44). Among travel trailers, formaldehyde levels varied significantly by brand. While formaldehyde levels varied by trailer type, all types tested had some levels ≥ 100 ppb.

Assessing direct analysis in real-time-mass spectrometry (DART-MS) for the rapid identification of additives in food packaging.

The ambient ionization technique direct analysis in real time (DART) was characterized and evaluated for the screening of food packaging for the presence of packaging additives using a benchtop mass spectrometer (MS). Approximate optimum conditions were determined for 13 common food-packaging additives, including plasticizers, anti-oxidants, colorants, grease-proofers, and ultraviolet light stabilizers. Method sensitivity and linearity were evaluated using solutions and characterized polymer samples. Additionally, the response of a model additive (di-ethyl-hexyl-phthalate) was examined across a range of sample positions, DART, and MS conditions (temperature, voltage and helium flow). Under optimal conditions, molecular ion (M+H+) was the major ion for most additives. Additive responses were highly sensitive to sample and DART source orientation, as well as to DART flow rates, temperatures, and MS inlet voltages, respectively. DART-MS response was neither consistently linear nor quantitative in this setting, and sensitivity varied by additive. All additives studied were rapidly identified in multiple food-packaging materials by DART-MS/MS, suggesting this technique can be used to screen food packaging rapidly. However, method sensitivity and quantitation requires further study and improvement.

Migration of fluorochemical paper additives from food-contact paper into foods and food simulants.

Fluorochemical-treated paper was tested to determine the amount of migration that occurs into foods and food-simulating liquids and the characteristics of the migration. Migration characteristics of fluorochemicals from paper were examined in Miglyol, butter, water, vinegar, water-ethanol solutions, emulsions and pure oil containing small amounts of emulsifiers. Additionally, microwave popcorn and chocolate spread were used to investigate migration. Results indicate that fluorochemicals paper additives do migrate to food during actual package use. For example, we found that microwave popcorn contained 3.2 fluorochemical mg kg(-1) popcorn after popping and butter contained 0.1 mg kg(-1) after 40 days at 4 degrees C. Tests also indicate that common food-simulating liquids for migration testing and package material evaluation might not provide an accurate indication of the amount of fluorochemical that actually migrates to food. Tests show that oil containing small amounts of an emulsifier can significantly enhance migration of a fluorochemical from paper.

Determination of 2,6-diisopropylnaphthalene (DIPN) and n-dibutylphthalate (DBP) in food and paper packaging materials from US marketplaces.

A gas chromatography-ion-trap tandem mass spectrometry procedure was developed for the determination of 2,6-diisopropylnaphthalene (DIPN) and n-dibutylphthalate (DBP) in domestic and imported paper packages and food sold in US marketplaces. The procedure involved ultrasonic extraction with dichloromethane, followed by analysis with the gas chromatography-ion-trap tandem mass spectrometry. Calibration curves for DIPN and DBP were achieved with concentrations ranging from 0.01 to 10 microg ml(-1) and the corresponding r(2) values were 0.9976 and 0.9956, respectively. In most of the fortified samples the recoveries were higher than 80% with a relative standard deviation (RSD) <10%. Using this procedure, it was found that less than 20% of the tested domestic packages and more than 60% of the tested imported food packages contained both DIPN and DBP. The concentrations of DIPN and DBP ranged from 0.09 to 20 mg kg(-1) and 0.14 to 55 mg kg(-1), respectively, with most of the DINP and DBP levels lower than 20 mg kg(-1). DIPN was not detected (<0.01 mg kg(-1)) in 41 food samples and DBP was only detected in two domestic and four imported food samples with concentrations ranging from <0.01 to 0.81 mg kg(-1).

Rapid quantitative and qualitative confirmatory method for the determination of monofluoroacetic acid in foods by liquid chromatography-mass spectrometry.

A rapid quantitative method and a qualitative confirmatory method for the determination of monofluoroacetic acid (MFA) in complex food matrices are presented. The quantitative method utilizes a water extraction, solid phase extraction clean-up and liquid chromatography-mass spectrometry (LC-MS) for determination of MFA. This method showed a high degree of specificity, detecting MFA in all of the spiked samples, while none of the unfortified samples tested positive for MFA. Spike recoveries were high in all matrices analyzed, varying from 85 to 110%, and comparable at low (2mg/L) and high (20mg/L) spiking levels. Repeatability tests at the low spiking levels yielded RSDs of less than 5% for all matrices analyzed. The qualitative confirmatory method developed is conceptually different from the quantitative method, ensuring that both methods would not be subject to the same interferences. The method uses the formation of the hydrazide of MFA through derivatization with 2-nitrophenylhydrazine. This derivatization is well established for the determination of carboxylic acids, but this is the first application to the determination of MFA. The derivatization yield was matrix dependent, however the limit of detection (LOD) (0.8microg/L) was sufficient to confirm the presence of MFA in all spiked matrices. Repeatability tests at the low spiking levels yielded RSDs of approximately 7% for all matrices analyzed.

Use of oral montelukast in the treatment of asthma.

Montelukast, a new leukotriene modifier, has several benefits in the treatment of asthma in adults and children including improved relief of asthma symptoms, rapid onset, a safety profile comparable with placebo, and oral, once-daily dosing means excellent adherence.

Long-term asthma control with oral montelukast and inhaled beclomethasone for adults and children 6 years and older.

BACKGROUND: Leukotriene receptor antagonists have demonstrated clinical benefits in chronic asthma studies of up to 3 months in duration. The effects of these agents over extended periods of time have not been reported. OBJECTIVE: To describe the long-term effect of oral montelukast, a potent and specific cysteinyl leukotriene receptor antagonist, compared with inhaled corticosteroids in both adult and paediatric patients with chronic asthma. METHODS: Male and female patients with chronic, stable asthma (adults aged 15-85 years, children aged 6-14 years), who had completed double-blind, placebo-controlled clinical studies, participated in three extension studies with oral montelukast taken once daily (10 mg tablet for adults, 5 mg chewable tablet for paediatric patients) or inhaled corticosteroids (beclomethasone 200 microg twice daily for adults, beclomethasone 100 microg or equivalent three times daily for children). A double-blind adult extension study was 37 weeks in duration; open-label adult extension studies were 156 (adults) and 112 (paediatric) weeks in duration. A total of 436, 374, and 245 patients entered these extension studies, respectively. RESULTS: Treatment with both montelukast and inhaled corticosteroids resulted in improvement in multiple parameters of asthma control. Improvements in daytime symptom scores were generally comparable among treatment groups. No tachyphylaxis to the effects of montelukast was evident. In the adult open-label study, however, the effect of beclomethasone on mean forced expiratory volume in 1 second (FEV1) gradually decreased from start of the study to the end of the follow-up treatment period. CONCLUSION: Both montelukast and inhaled corticosteroids were effective in controlling mild to moderate chronic asthma; the relative effectiveness of montelukast and beclomethasone were similar in open-label conditions. The hypothesis, that clinical practice conditions (e.g., adherence) may have a significant impact on the effectiveness of these therapies, should be tested in future clinical trials.

Clinical safety and tolerability of montelukast, a leukotriene receptor antagonist, in controlled clinical trials in patients aged > or = 6 years.

OBJECTIVE: Montelukast is a leukotriene receptor antagonist administered orally once daily for treatment of chronic asthma in adults and children. A comprehensive analysis of safety data from double-blind, randomized, placebo-controlled trials with montelukast has not been previously reported. PATIENTS AND METHODS: A pooled analysis of safety data from 11 multicentre, randomized, controlled montelukast Phase IIb and III trials and five long-term extension studies was performed. A total of 3386 adult patients (aged 15-85 years) and 336 paediatric patients (aged 6-14 years) were enrolled in the trials; 2031 adults received montelukast for up to 4.1 years, and 257 children received montelukast for up to 1.8 years. Summary statistics comparing incidences of adverse events among treatment groups were calculated. RESULTS: The overall incidence of clinical and laboratory adverse events among montelukast-treated patients, both adult and paediatric, was similar to that among patients receiving placebo. There were no clinically relevant differences in individual adverse events, including infectious upper respiratory conditions and transaminase elevations, between montelukast and placebo groups. Discontinuations due to adverse events occurred with similar frequencies during placebo, montelukast and inhaled beclomethasone therapy. No dose-related adverse effects of montelukast were observed in adults treated with dosages as high as 200 mg per day (20 times the recommended dose) for 5 months. This tolerability profile montelukast observed in clinical trials has been generally reflected in the post-marketing safety experience seen to date. CONCLUSIONS: These data indicate a tolerability profile for montelukast similar to placebo during both short-term and long-term administration, even at doses substantially higher than the recommended clinical dose of 10 mg once daily for adults and 5 mg once daily for children aged 6-14 years.

Montelukast dose selection in 6- to 14-year-olds: comparison of single-dose pharmacokinetics in children and adults.

Montelukast, an oral leukotriene-receptor antagonist, has demonstrated efficacy and tolerability for the treatment of chronic asthma in adults. A once-daily 10 mg dose (film-coated tablet) was selected as the optimal adult dose based on dose-ranging studies. Asthma is a similar disease and is treated with the same medications in children and adults. These observations suggested that a dose of montelukast in children providing overall drug exposure (i.e., montelukast plasma concentrations) similar to that of the 10 mg film-coated tablet dose in adults would be efficacious, well tolerated, and obviate the need for separate dose-ranging studies in children. Therefore, the dose of montelukast for 6- to 14-year-old children was selected by identifying the chewable tablet dose of montelukast yielding a single-dose area under the plasma concentration-time curve (AUC) comparable to that achieved with the adult 10 mg film-coated tablet dose. Based on this approach, which included dose normalization of data from several pediatric pharmacokinetic studies, a 5 mg chewable tablet dose of montelukast was selected for use in clinical efficacy studies in 6- to 14-year-old children with asthma.

Hair and blood as substrates for screening children for lead poisoning.

In Russia, hair, rather than blood, is usually used as a substrate for screening children for lead poisoning. We attempted to gauge the accuracy of this method by comparing these two methods. The evaluation was done in Saratov, Russia. We collected hair and blood samples from 189 children who attended 11 kindergartens. Their mean blood lead concentration was 9.8 microg/dl (range = 3.1-35.7 microg/dl), and their mean hair lead concentration was 7.2 microg/g (range = 1.0-7.2 microg/g [i.e., 1.0 being the lowest detectable limit]). Hair lead concentration as a screening method had 57% sensitivity and resulted in 18% of the children being classified as false-negatives. We conclude, therefore, that measuring hair lead concentration is not an adequate method with which to screen children for lead poisoning.

Childhood Lead Poisoning in Russia: A Site-specific Pediatric Blood Lead Evaluation.

Lead poisoning in children is an international concern. Health effects vary according to a child's blood lead level. Historically, the problem of lead poisoning in Russia had been defined by analysis of hair samples. In Saratov, Russia, during the spring of 1996, the authors conducted the first evaluation of blood lead levels among Russian children. The mean blood lead level of 579 samples analyzed at the Centers for Disease Control and Prevention was 7.7 µg/dL (range 3.0 to 35.7 µg/dL). The sensitivity of hair analysis in identifying blood lead levels above 10 µg/dL was 50%. Most of the environmental samples evaluated (including water, dust, paint, and soil) were within acceptable U.S. remediation standards. Almost one-fourth of the Russian children evaluated during this site-specific investigation had blood lead levels capable of causing adverse health effects in children. Hair analysis is not an adequate method for identifying these children. Additional investigations in other Russian cities are necessary in order to determine the extent of lead poisoning in Russia's children.

Effects of montelukast (MK-0476), a new potent cysteinyl leukotriene (LTD4) receptor antagonist, in patients with chronic asthma.

BACKGROUND: Cysteinyl leukotrienes mediate signs and symptoms of asthma. In a double-blind, placebo-controlled, crossover study, a new potent and specific cysteinyl leukotriene (LTD4) receptor antagonist, montelukast (MK-0476), was evaluated for tolerability and clinical efficacy in patients with chronic asthma (receiving and not receiving inhaled corticosteroids). METHODS: Twenty-nine nonsmoking patients with asthma (15 treated concomitantly with inhaled corticosteroids) with FEV1 percent predicted values between 50% to 80% received MK-0476, 200 mg, or placebo three times daily for 10 1/3 days (31 doses) in a random, crossover manner, after a 2-week, open, baseline period. Comparisons in FEV1 (mean percent change from baseline after the first and last dose), mean daily daytime asthma and nocturnal awakening scores, and mean daily beta-agonist use were made between treatment periods. RESULTS: Montelukast, compared with placebo, caused improvements in FEV1 (mean percentage point difference of the percentage change from baseline) 3 and 4 hours after dosing on day 1 (hour 3, 9.0%; 95% confidence interval [CI] 0.53, 18.72; hour four, 10.9%; 95% CI -0.25, 20.20) and day 11 (hour 3, 14.0%; 95% CI 0.76, 31.43; hour 4, 13.4%; 95% CI 1.24, 28.83). Reductions were observed in mean daily beta-agonist use (1.0 puff/day [95% CI -1.61, -0.26]), mean daytime symptom scores, and nocturnal awakenings over the 10 1/3 day treatment period. There were no important differences between the groups receiving and those not receiving inhaled corticosteroids. Montelukast was well tolerated with no serious clinical adverse events reported. CONCLUSIONS: In this study Montelukast, 200 mg, administered three times daily for 10 1/3 days, compared with placebo, was generally well tolerated and resulted in significant improvement in chronic asthma, irrespective of the presence of inhaled corticosteroids.

Tail docking in dogs: a sample of attitudes of veterinarians and dog breeders in Queensland.

One hundred veterinarians and 100 breeders of traditionally docked dogs from Queensland were surveyed by telephone to determine their attitudes towards tail docking. Eighty-four percent of the breeders surveyed were in favour of docking, whereas 83% of veterinarians were opposed to the practice. Most pups were docked between 1 and 3 days of age. All veterinarians surgically amputated the tail, whereas 16% of breeders applied rubber bands to the tail. Seventy-six percent of the veterinarians, but only 18% of the breeders believed that docking caused significant to severe pain. No veterinarians, but 25% of the dog breeders believed that docking was painless. Although recent changes to the Queensland Canine Control Council's rulings allow dogs with intact tails to be shown in traditionally docked classes, the requirement of breed standard was cited as the major reason for tail docking by both breeders and veterinarians.

Estimated workplace protection factors for positive-pressure self-contained breathing apparatus.

An analytical model is presented that estimates the distribution of workplace protection factor (WPF) values for positive-pressure respirators. Input for the model is (1) the instantaneous facepiece pressure measured as a function of time and (2) the distribution of WPF values for a negative-pressure version of the respirator. As an example application, the model was applied to 57 measurements of facepiece pressure made in a previous National Institute for Occupational Safety and Health study called "Firesmoke." That study involved professional firefighters wearing positive-pressure self-contained breathing apparatus (SCBA). During Firesmoke, there were four donnings in which facepiece pressure momentarily went negative one or more times during use. The purpose of the effort described here was to assess the significance of these momentary, negative excursions in facepiece pressure. To that end, an analytical model was developed that estimates the ratio of the mass of contaminant that enters the facepiece during these negative excursions to that which would be expected to enter a negative-pressure respirator utilizing the same facepiece. Thus, the performance of a positive-pressure SCBA can be determined relative to the performance of a negative-pressure respirator with the same facepiece--either a negative-pressure SCBA or a negative pressure air-purifying respirator. The NIOSH-assigned protection factor (APF) for a negative-pressure full facepiece is 50; the APF for a positive-pressure SCBA is 10,000. The results of the application of this analytical model are consistent with the current NIOSH APF for a positive-pressure SCBA.

Environmental study of firefighters.

A study of firefighter exposures was undertaken at the request of the U.S. Fire Administration. This work was part of a larger study which included field evaluation of the performance of the self-contained breathing apparatus (SCBA) worn by firefighters during structural firefighting. Measurements were made for a variety of contaminants including CO, CO2, benzene, HCN, HCl, H2SO4, HF, acrolein, CH4, formaldehyde and PNAs. Many of the analyses were performed by collection of bag samples followed by Fourier transform infrared spectroscopy using a field mobile spectrometer. Measurements were also made using solid sorbent tubes and direct-reading meters. Sampling was done both during the knockdown and during overhaul phases of structural firefighting. Also, in order to estimate exposures including those when the SCBAs were worn, measurements were made both inside and outside the SCBA facepiece. Carbon monoxide was the most common contaminant found during knockdown, and about 10% of the samples were greater than 1500 ppm. Formaldehyde, acrolein, hydrogen chloride, hydrogen cyanide, sulphuric acid and hydrogen fluoride all exceeded their respective short-term exposure limits (STEL) on some occasions. Approximately 50% of the knockdown samples for acrolein exceeded the STEL. During overhaul, when masks were usually not worn, many of the contaminants found during knockdown were detected, but typically at much lower concentrations. Inside-mask sampling data suggest that exposure to low concentrations of a variety of compounds is occurring but this is believed to be principally the result of early mask removal or of non-use during knockdown rather than of leakage. The three basic sampling approaches (bag sampling, sorbent tubes and direct-reading meters) proved in this study to be complementary and served to maximize our ability to detect and quantify a wide range of combustion products.