Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters.

The robust control of genotoxic N-nitrosamine (NA) impurities is an important safety consideration for the pharmaceutical industry, especially considering recent drug product withdrawals. NAs belong to the 'cohort of concern' list of genotoxic impurities (ICH M7) because of the mutagenic and carcinogenic potency of this chemical class. In addition, regulatory concerns exist regarding the capacity of the Ames test to predict the carcinogenic potential of NAs because of historically discordant results. The reasons postulated to explain these discordant data generally point to aspects of Ames test study design. These include vehicle solvent choice, liver S9 species, bacterial strain, compound concentration, and use of pre-incubation versus plate incorporation methods. Many of these concerns have their roots in historical data generated prior to the harmonization of Ames test guidelines. Therefore, we investigated various Ames test assay parameters and used qualitative analysis and quantitative benchmark dose modelling to identify which combinations provided the most sensitive conditions in terms of mutagenic potency. Two alkyl-nitrosamines, N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) were studied. NDMA and NDEA mutagenicity was readily detected in the Ames test and key assay parameters were identified that contributed to assay sensitivity rankings. The pre-incubation method (30-min incubation), appropriate vehicle (water or methanol), and hamster-induced liver S9, alongside Salmonella typhimurium strains TA100 and TA1535 and Escherichia coli strain WP2uvrA(pKM101) provide the most sensitive combination of assay parameters in terms of NDMA and NDEA mutagenic potency in the Ames test. Using these parameters and further quantitative benchmark dose modelling, we show that N-nitrosomethylethylamine (NMEA) is positive in Ames test and therefore should no longer be considered a historically discordant NA. The results presented herein define a sensitive Ames test design that can be deployed for the assessment of NAs to support robust impurity qualifications.

Reaction Kinetics using a Chemputable Framework for Data Collection and Analysis.

Automated chemistry platforms have been widely explored, but many focus on fixed tasks for chemical synthesis or analysis. However, a typical synthetic chemistry workflow utilizes both, such as kinetic measurements for reaction development and optimization. Due to their repetitive and time-consuming nature, kinetic measurements are often omitted, which limits the mechanistic investigation of reactions. Herein, we present a "Chemputer" platform with on-line analytics (UV/Vis, NMR) which automates routine kinetic measurements. The system's capabilities are showcased by exploring an inverse electron-demand Diels-Alder using initial rate measurements, a metal complexation using variable time normalization analysis (VTNA), and formation of a series of tosylamide derivatives using Hammett analysis. Over 60 individual experiments are presented which required minimal intervention, highlighting the significant time savings of automation. Owing to the modular design of the platform, which facilitates rapid integration of commercial analytical tools, our approach is widely accessible and adjustable to the reaction under investigation. The platform is operated using the chemical programming language, XDL, hence experimental procedures and results are stored in a precise, computer-readable format. We propose that widespread adoption of this reporting protocol in the chemical community could build a database of validated kinetic data beneficial for Machine Learning.

Switched "On" Transient Fluorescence Output from a Pulsed-Fuel Molecular Ratchet.

We report the synthesis and operation of a molecular energy ratchet that transports a crown ether from solution onto a thread, along the axle, over a fluorophore, and off the other end of the thread back into bulk solution, all in response to a single pulse of a chemical fuel (CCl3CO2H). The fluorophore is a pyrene residue whose fluorescence is normally prevented by photoinduced electron transfer (PET) to a nearby N-methyltriazolium group. However, crown ether binding to the N-methyltriazolium site inhibits the PET, switching on pyrene fluorescence under UV irradiation. Each pulse of fuel results in a single ratchet cycle of transient fluorescence (encompassing threading, transport to the N-methyltriazolium site, and then dethreading), with the onset of the fluorescent time period determined by the amount of fuel in each pulse and the end-point determined by the concentration of the reagents for the disulfide exchange reaction. The system provides a potential alternative signaling approach for artificial molecular machines that read symbols from sequence-encoded molecular tapes.

Use of the bacterial reverse mutation assay to predict carcinogenicity of N-nitrosamines.

Under ICH M7, impurities are assessed using the bacterial reverse mutation assay (i.e., Ames test) when predicted positive using in silico methodologies followed by expert review. N-Nitrosamines (NAs) have been of recent concern as impurities in pharmaceuticals, mainly because of their potential to be highly potent mutagenic carcinogens in rodent bioassays. The purpose of this analysis was to determine the sensitivity of the Ames assay to predict the carcinogenic outcome with curated proprietary Vitic (n = 131) and Leadscope (n = 70) databases. NAs were selected if they had corresponding rodent carcinogenicity assays. Overall, the sensitivity/specificity of the Ames assay was 93-97% and 55-86%, respectively. The sensitivity of the Ames assay was not significantly impacted by plate incorporation (84-89%) versus preincubation (82-89%). Sensitivity was not significantly different between use of rat and hamster liver induced S9 (80-93% versus 77-96%). The sensitivity of the Ames is high when using DMSO as a solvent (87-88%). Based on the analysis of these databases, the Ames assay conducted under OECD 471 guidelines is highly sensitive for detecting the carcinogenic hazards of NAs.

Pumping between phases with a pulsed-fuel molecular ratchet.

The sorption of species from a solution into and onto solids underpins the sequestering of waste and pollutants, precious metal recovery, heterogeneous catalysis, analysis and separation science, and other technologies1,2. The transfer between phases tends to proceed spontaneously in the direction of equilibrium. For example, alkyl ammonium groups mounted on silica nanoparticles are used to chemisorb cucurbituril macrocycles from solution through host-guest binding3,4. Molecular ratchet mechanisms5-7, in which kinetic gating8-12 inhibits or accelerates particular steps, makes it possible to progressively drive dynamic systems13-16 away from equilibrium17-21. Here we report on molecular pumps22 immobilized on polymer beads23-25 that use an energy ratchet mechanism5,9,19-21,26-30 to directionally transport substrates from solution onto the beads. On the addition of trichloroacetic acid (CCl3CO2H)19,31-33 fuel19,34-37, micrometre-diameter polystyrene beads functionalized38 with solvent-accessible molecular pumps sequester from the solution crown ethers appended with fluorescent tags. After fuel consumption, the rings are mechanically trapped in a higher-energy, out-of-equilibrium state on the beads and cannot be removed by dilution or exhaustive washing. This differs from dissipative assembled materials11,13-16, which require a continuous supply of energy to persist, and from conventional host-guest complexes. The addition of a second fuel pulse causes the uptake of more macrocycles, which drives the system further away from equilibrium. The second macrocycle can be labelled with a different fluorescent tag, which confers sequence information39 on the absorbed structure. The polymer-bound substrates can be released back to the bulk either one compartment at a time or all at once. Non-equilibrium40 sorption by immobilized artificial molecular machines41-45 enables the transduction of energy from chemical fuels for the use, storage and release of energy and information.

Primary care physician's (PCP) perceived value of patient-reported outcomes (PROs) in clinical practice: a mixed methods study.

BACKGROUND: Patient-reported outcomes (PROs) can promote patient engagement, shared-decision making and improve the overall experience of care. However, PRO integration in the primary care clinical setting is limited. Exploring the perspectives of primary care physicians (PCPs) on PROs is key to understanding how they are being used in the clinical setting. We sought to elucidate this clinical perspective at one of the largest US health systems that has integrated a wide range of PROs into routine primary care. METHODS: Mixed methods study with both anonymous online surveys and in-person qualitative semistructured interviews conducted with PCPs to understand their clinical perspectives on the applications of the existing PROs. PCPs from the 19 affiliated clinics were prompted to complete the survey. Interviewed PCPs were selected via a combination of random and purposive selection from the PCP directory. RESULTS: Of 172 PCPs, 117 (68%) completed the online survey and 28 completed semistructured interviews. Most PCPs (77%) reviewed PRO responses with their patients. PCPs endorsed that PROs improve clinic efficiency and clinical management. However, PCPs have heterogeneous perspectives on the relevance of PROs in clinical practice, likely due to variations in clinic practice. For specific PRO instruments, PCPs reported anxiety and depression screening PROs to be most helpful. PCPs felt that PROs assisted with completing screening questions that are required by regulatory bodies. Barriers to using PROs include poor user-interface for both clinicians and patients and inadequate training. CONCLUSIONS: Most PCPs regularly use PRO data though there are mixed opinions about their clinical relevance. An adaptable, user-friendly PRO system has the potential to have meaningful clinical applications in primary care.

Decorating polymer beads with 1014 inorganic-organic [2]rotaxanes as shown by spin counting.

Polymer beads have been used as the core of magnetic particles for around twenty years. Here we report studies to attach polymetallic complexes to polymer beads for the first time, producing beads of around 115 microns diameter that are attached to 1014 hybrid inorganic-organic [2]rotaxanes. The bead is then formally a [1014] rotaxane. The number of complexes attached is counted by EPR spectroscopy after including TEMPO radicals within the thread of the hybrid [2]rotaxanes.

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

Dissipative Catalysis with a Molecular Machine.

We report on catalysis by a fuel-induced transient state of a synthetic molecular machine. A [2]rotaxane molecular shuttle containing secondary ammonium/amine and thiourea stations is converted between catalytically inactive and active states by pulses of a chemical fuel (trichloroacetic acid), which is itself decomposed by the machine and/or the presence of additional base. The ON-state of the rotaxane catalyzes the reduction of a nitrostyrene by transfer hydrogenation. By varying the amount of fuel added, the lifetime of the rotaxane ON-state can be regulated and temporal control of catalysis achieved. The system can be pulsed with chemical fuel several times in succession, with each pulse activating catalysis for a time period determined by the amount of fuel added. Dissipative catalysis by synthetic molecular machines has implications for the future design of networks that feature communication and signaling between the components.

Dosage uniformity problems which occur due to technological errors in extemporaneously prepared suppositories in hospitals and pharmacies.

The availability of suppositories in Hungary, especially in clinical pharmacy practice, is usually provided by extemporaneous preparations. Due to the known advantages of rectal drug administration, its benefits are frequently utilized in pediatrics. However, errors during the extemporaneous manufacturing process can lead to non-homogenous drug distribution within the dosage units. To determine the root cause of these errors and provide corrective actions, we studied suppository samples prepared with exactly known errors using both cerimetric titration and HPLC technique. Our results show that the most frequent technological error occurs when the pharmacist fails to use the correct displacement factor in the calculations which could lead to a 4.6% increase/decrease in the assay in individual dosage units. The second most important source of error can occur when the molding excess is calculated solely for the suppository base. This can further dilute the final suppository drug concentration causing the assay to be as low as 80%. As a conclusion we emphasize that the application of predetermined displacement factors in calculations for the formulation of suppositories is highly important, which enables the pharmacist to produce a final product containing exactly the determined dose of an active substance despite the different densities of the components.

Whole Farm Net Greenhouse Gas Abatement from Establishing Kikuyu-Based Perennial Pastures in South-Western Australia.

On-farm activities that reduce GHG emissions or sequester carbon from the atmosphere to compensate for anthropogenic emissions are currently being evaluated by the Australian Government as carbon offset opportunities. The aim of this study was to examine the implications of establishing and grazing Kikuyu pastures, integrated as part of a mixed Merino sheep and cropping system, as a carbon offset mechanism. For the assessment of changes in net greenhouse gas emissions, results from a combination of whole farm economic and livestock models were used (MIDAS and GrassGro). Net GHG emissions were determined by deducting increased emissions from introducing this practice change (increased methane and nitrous oxide emissions due to higher stocking rates) from the soil carbon sequestered from growing the Kikuyu pasture. Our results indicate that livestock systems using perennial pastures may have substantially lower net GHG emissions, and reduced GHG intensity of production, compared with annual plant-based production systems. Soil carbon accumulation by converting 45% of arable land within a farm enterprise to Kikuyu-based pasture was determined to be 0.80 t CO2-e farm ha(-1) yr(-1) and increased GHG emissions (leakage) was 0.19 t CO2-e farm ha(-1) yr(-1). The net benefit of this practice change was 0.61 t CO2-e farm ha(-1) yr(-1) while the rate of soil carbon accumulation remains constant. The use of perennial pastures improved the efficiency of animal production almost eight fold when expressed as carbon dioxide equivalent emissions per unit of animal product. The strategy of using perennial pasture to improve production levels and store additional carbon in the soil demonstrates how livestock should be considered in farming systems as both sources and sinks for GHG abatement.

Seventy-two-hour returns may not be a good indicator of safety in the emergency department: a national study.

OBJECTIVES: The objective was to measure the association between returns to an emergency department (ED) within 72 hours and resource utilization, severity of illness, mortality, and admission rate. METHODS: This was a retrospective, cross-sectional analysis of ED visits using data from the National Hospital Ambulatory Medical Care Survey (NHAMCS) from 1998 to 2006. Cohorts were patients who had been seen in the ED within the past 72 hours versus those without the prior visit. A multivariate model was created to predict adjusted-resource utilization and mortality or admission rate. RESULTS: During the study period, there were 218,179 ED patient visits and a 3.2% 72-hour return rate. Patients with Medicare (3.5%) and without insurance (3.5%) were more likely to return within 72 hours. Visits associated with alcohol (4.1%), low triage acuity (4.0%), or dermatologic conditions (5.9%) were more likely to return. Seventy-two-hour return visits used fewer resources (5.0 [±0.1] vs. 5.5 [±0.1] tests, medications, procedures), were less likely to be Level I triage acuity (17% vs. 20%), and had a similar admission rate (13% vs. 13%) as those not seen within 72 hours. The sample size was too small to evaluate mortality. CONCLUSIONS: Patients who return to the ED within 72 hours do not use more resources, are not more severely ill, and do not have a higher hospital admission rate than those who had not been previously seen. These findings do not support the use of 72-hour returns as a quality or safety indicator. A more refined variation such as 72-hour returns resulting in admission may have more value.

Effect of Helicobacter pylori infection on symptoms of gastroenteritis due to enteropathogenic Escherichia coli in adults.

BACKGROUND: Helicobacter pylori can cause hypochlorhydria in some hosts and predispose to diarrheal infections. AIMS: We tested the hypothesis that chronic H. pylori infection increases the risk of diarrheal illness due to an acid-sensitive organism: enteropathogenic Escherichia coli (EPEC). METHODS: After testing healthy adult volunteers for H. pylori, 19 infected and 26 uninfected subjects had gastric pH probes placed and were given 5-10 × 10(9) EPEC organisms; six had previously received a proton pump inhibitor. We measured diarrhea and created a composite gastroenteritis severity score based on symptoms in the 48 h following exposure. Outcomes were compared using logistic regression and analysis of covariance. RESULTS: More H. pylori-infected (36.8%) than H. pylori-uninfected subjects (7.7%) were hypochlorhydric (P = 0.02). Six (31.6%) H. pylori-infected and five H. pylori-uninfected subjects (19.2%) developed diarrhea (P = 0.34). Hypochlorhydria was a strong risk factor for diarrhea [odds ratio (OR) 6.25, confidence interval (CI): 1.29-30.35]. After adjusting for hypochlorhydria and EPEC dose, H. pylori was not associated with diarrhea (OR 0.89, CI: 0.17-4.58). Among those with symptoms, H. pylori-infected subjects had lower gastroenteritis severity score than did H. pylori-uninfected subjects (2.6, CI: 1.9-3.4 versus 1.5, CI: 1.1-1.9, P = 0.01), particularly if they were also hypochlorhydric (3.8, CI: 2.3-5.3 versus 1.9, CI: 1.3-2.5, P = 0.02). CONCLUSIONS: In adults, H. pylori infection was associated with hypochlorhydria but had no detectable effect on occurrence of diarrhea. Among symptomatic subjects, H. pylori infection decreased severity of gastroenteritis.

Use of advanced radiology during visits to US emergency departments for injury-related conditions, 1998-2007.

CONTEXT: Excessive use of medical imaging increases health care costs and exposure to ionizing radiation (a potential carcinogen) without yielding significant benefits to all patients. OBJECTIVE: To determine whether there has been a change in the prevalence of emergency department visits for injury-related conditions for which computed tomography (CT) or magnetic resonance imaging (MRI) was obtained and whether there has been a change in the diagnosis of life-threatening conditions and patient disposition. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cross-sectional analysis of emergency department visits using data from the National Hospital Ambulatory Medical Care Survey (1998-2007). Sampled visits were weighted to produce estimates for the United States. MAIN OUTCOMES MEASURES: Proportion of visits for injury-related conditions during which a CT or MRI was obtained, a life-threatening condition was diagnosed (eg, cervical spine fracture, skull fracture, intracranial bleeding, liver and spleen laceration), and which resulted in hospital and intensive care unit admission. RESULTS: The prevalence of CT or MRI use during emergency department visits for injury-related conditions increased from 6% (95% confidence interval [CI], 5%-7%) (257 of 5237 visits) in 1998 to 15% (95% CI, 14%-17%) (981 of 6567 visits) in 2007 (P < .001 for trend). There was a small increase in the prevalence of life-threatening conditions (1.7% [95% CI, 1.2%-2.2%; 89 of 5237 visits] in 1998 and 2.0% [95% CI, 1.6%-2.5%; 142 of 6567 visits] in 2007; P=.04 for trend) [corrected].There was no change in prevalence of visits during which patients were either admitted to the hospital (5.9% [95% CI, 4.9%-6.9%] in 1998 and 5.5% [95% CI, 4.7%-6.5%] in 2007; P = .50 for trend) or to an intensive care unit (0.62% [95% CI, 0.40%-1.00%] in 1998 and 0.80% [95% CI, 0.53%-1.21%] in 2007; P = .14 for trend). Visits during which CT or MRI was obtained lasted 126 minutes (95% CI, 123-131 minutes) longer than those for which CT or MRI was not obtained. CONCLUSION: From 1998 to 2007, the prevalence of CT or MRI use during emergency department visits for injury-related conditions increased significantly, without an equal increase in the prevalence of life-threatening conditions.

Meeting report: risk assessment of tamiflu use under pandemic conditions.

On 3 October 2007, 40 participants with diverse expertise attended the workshop Tamiflu and the Environment: Implications of Use under Pandemic Conditions to assess the potential human health impact and environmental hazards associated with use of Tamiflu during an influenza pandemic. Based on the identification and risk-ranking of knowledge gaps, the consensus was that oseltamivir ethylester-phosphate (OE-P) and oseltamivir carboxylate (OC) were unlikely to pose an ecotoxicologic hazard to freshwater organisms. OC in river water might hasten the generation of OC-resistance in wildfowl, but this possibility seems less likely than the potential disruption that could be posed by OC and other pharmaceuticals to the operation of sewage treatment plants. The work-group members agreed on the following research priorities: a) available data on the ecotoxicology of OE-P and OC should be published; b) risk should be assessed for OC-contaminated river water generating OC-resistant viruses in wildfowl; c) sewage treatment plant functioning due to microbial inhibition by neuraminidase inhibitors and other antimicrobials used during a pandemic should be investigated; and d) realistic worst-case exposure scenarios should be developed. Additional modeling would be useful to identify localized areas within river catchments that might be prone to high pharmaceutical concentrations in sewage treatment plant effluent. Ongoing seasonal use of Tamiflu in Japan offers opportunities for researchers to assess how much OC enters and persists in the aquatic environment.

The effects of ambulance diversion: a comprehensive review.

OBJECTIVES: To review the current literature on the effects of ambulance diversion (AD). METHODS: The authors performed a systematic review of AD and its effects. PubMed, EMBASE, the Cochrane database, societal meeting abstracts, and references from relevant articles were searched. All articles were screened for relevance to AD. RESULTS: The authors examined 600 citations and reviewed the 107 articles relevant to AD. AD is a common occurrence that is increasing in frequency. AD is associated with periods of emergency department (ED) crowding (Mondays, mid-afternoon to early evening, influenza season, and when hospitals are at capacity). Interventions that redesign the AD process or that provide additional hospital or ED resources reduce diversion frequency. AD is associated with increased patient transport times and time to thrombolytics but not with mortality. AD is associated with loss of estimated hospital revenues. Short of anecdotal or case reports, no studies measured the effect of AD on ED crowding, morbidity, patient and provider satisfaction, or EMS resource utilization. CONCLUSIONS: Despite its common use, there is a relative paucity of studies on the effects of AD. Further research into these effects should be performed so that we may understand the role of AD in the health system.

Effect of intense lifestyle modification and cardiac rehabilitation on psychosocial cardiovascular disease risk factors and quality of life.

This study examined the effect of the Ornish Program for Reversing Heart Disease and cardiac rehabilitation (CR) on psychosocial risk factors and quality of life in patients with confirmed coronary artery disease. Participants had previously undergone a revascularization procedure. The 84 patients self-selected to participate in the Ornish Program for Reversing Heart Disease (n = 507 28), CR (n = 28), or a control group (n = 28). Twelve psychosocial risk factors and quality of life variables were collected from all three groups at baseline, 3 months, and 6 months. At 3 and 6 months, Ornish group participants demonstrated significant improvements in all 12 outcome measures. The rehabilitation group improved in 7 of the 12, and the control group showed significant improvements in 6 of the variables. Intensive lifestyle modification programs significantly affect psychosocial risk factors and quality of life.

The influence of an intense cardiovascular disease risk factor modification program.

In an effort to make intensive lifestyle modification programs more accessible to patients with cardiovascular disease, the Ornish Program was offered at eight independent medical centers located across the United States. The purpose of this study was to determine if one of these independent sites was able to replicate outcomes produced by the original Ornish Program. Fifty program participants from six different cohorts provided baseline, 3- and 12-month data consisting of blood lipids, body fat, blood pressure, anginal pain, quality of life, stress, depression, social support, and hostility. A pooled analysis showed significant reductions in almost all physiological and psychosocial variables with most reductions persisting for at least 12 months. These findings suggest that cardiovascular disease patients who choose to participate in an independent, intensive lifestyle modification program can experience significant improvements in both physiological and psychosocial cardiovascular disease risk factors.