Applying new approach methodologies to assess next-generation tobacco and nicotine products.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and "roadmaps" to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

Is the intention to vaccinate enough? Systematic variation in the value of timely vaccinations and preferences for monetary vs non-monetary incentives among pregnant women in southern Tanzania.

Background: Globally, approximately 19.7 million children remain under-vaccinated; many more receive delayed vaccinations. Sustained progress towards global vaccination targets requires overcoming, or compensating for, incrementally greater barriers to vaccinating hard-to-reach and hard-to-vaccinate children. We prospectively assessed pregnant women's valuations of routine childhood vaccinations and preferences for alternative incentives to inform interventions aiming to increase vaccination coverage and timeliness in southern Tanzania. Methods: Between August and December 2017, 406 women in their last trimester of pregnancy were enrolled from health facilities and communities in the Mtwara region of Tanzania and asked contingent valuation questions about their willingness to vaccinate their child if they were (a) given an incentive, or (b) facing a cost for each vaccination. Interval censored regressions assessed correlates of women's willingness to pay (WTP) for timely vaccinations. Participants were asked to rank monetary and non-monetary incentive options for the timely vaccination of their children. Findings: All women expected to get their children vaccinated according to the recommended schedule, even without incentives. Nearly all women (393; 96.8 %) were willing to pay for vaccinations. The average WTP was Tanzania Shilling (Tsh) 3,066 (95 % confidence interval Tsh 2,523-3,610; 1 USD âˆ¼ Tsh 2,200) for each vaccination. Women's valuations of timely vaccinations varied significantly with vaccine-related knowledge and attitudes, economic status, and rural vs urban residence. Women tended to prefer non-monetary over monetary incentives for the timely vaccination of their children. Interpretation: Women placed a high value on timely childhood vaccinations, suggesting that unexpected system-level barriers rather than individual-level demand factors are likely to be the primary drivers of missed vaccinations. Systematic variation in the value of vaccinations across women reflects variation in perceived benefits and opportunity costs. In this setting, nonmonetary incentives and other interventions to increase demand and compensate for system-level barriers hold significant potential for improving vaccination coverage and timeliness. ClinicalTrialsgov Protocol: NCT03252288.

Parental concerns and uptake of childhood vaccines in rural Tanzania - a mixed methods study.

BACKGROUND: Vaccine hesitancy has been recognized as an important barrier to timely vaccinations around the world, including in sub-Saharan Africa. In Tanzania, 1 in 4 children is not fully vaccinated. The objective of this mixed methods study was to describe and contextualize parental concerns towards vaccines in Tanzania. METHODS: Between 2016 and 2017, we conducted a cross-sectional survey (n = 134) and four focus group discussions (FGDs, n = 38) with mothers of children under 2 years of age residing in Mtwara region in Southern Tanzania. The survey and FGDs assessed vaccination knowledge and concerns and barriers to timely vaccinations. Vaccination information was obtained from government-issued vaccination cards. RESULTS: In the cross-sectional survey, 72% of mothers reported missed or delayed receipt of vaccines for their child. Although vaccine coverage was high, timeliness of vaccinations was lower and varied by vaccine. Rural mothers reported more vaccine-related concerns compared to urban mothers; literacy and access to information were identified as key drivers of the difference. Mothers participating in FGDs indicated high perceived risk of vaccine-preventable illnesses, but expressed concerns related to poor geographic accessibility, unreliability of services, and missed opportunities for vaccinations resulting from provider efforts to minimize vaccine wastage. CONCLUSIONS: Findings from our cross-sectional survey indicate the presence of vaccination delays and maternal concerns related to childhood vaccines in Tanzania. In FGDs, mothers raised issues related to convenience more often than issues related to vaccine confidence or complacency. Further research is necessary to understand how these issues may contribute to the emergence and persistence of vaccine hesitancy and to identify effective mitigation strategies.

Patterns of Mobile Phone Ownership and Use Among Pregnant Women in Southern Tanzania: Cross-Sectional Survey.

BACKGROUND: There is a paucity of subnational data on patterns of mobile phone ownership and use in Tanzania to inform the development of digital health interventions. OBJECTIVE: The aim of this study is to assess patterns of mobile phone ownership and use in pregnant women to inform the feasibility and design of digital health interventions for promoting timely uptake of childhood vaccines in southern Tanzania. METHODS: Between August and November 2017, pregnant women in their third trimester were enrolled at health facilities and from surrounding communities, and asked about their patterns of mobile phone ownership and use in an interviewer administered survey. RESULTS: Of 406 women, only 3 had never used a phone. Most women (>98%) could make and receive phone calls. Compared to urban women, rural women reported higher mobile phone use rates but were less likely to be sole owners of phones, and less likely to send or receive SMS, transact money, browse the internet, or use social media via mobile phones. CONCLUSIONS: The findings suggest high feasibility for digital health interventions delivered via mobile phones to pregnant women in southern Tanzania. The feasibility of smartphone-based interventions or strategies relying on the use of social media or the internet is limited.

Spatial and Ontogenetic Variation in Mercury in Lake Superior Basin Sea Lamprey (Petromyzon marinus).

Mercury concentrations were measured in eggs, larvae, and adult spawning-phase sea lampreys (Petromyzon marinus) collected in tributaries of Lake Superior to investigate spatial and ontogenetic variation. There were significant differences in mercury concentrations between all three life stages, with levels highest in adults (mean = 3.01 µg/g), followed by eggs (mean = 0.942 µg/g), and lowest in larvae (mean = 0.455 µg/g). There were no significant differences in mercury concentrations by location for any life stage or by sex in adults. Mercury was not correlated with adult or larval lamprey length or mass. Mercury levels in adult lampreys exceeded U.S. and Canadian federal guidelines for human consumption. Mercury concentrations in all life stages exceeded criteria for the protection of piscivorous wildlife, posing a threat to local fish, birds, and mammals. High mercury levels in adult lampreys combined with their semelparous life history make them a potential source of lake-derived mercury to spawning streams.

Variation in bioaccumulation of persistent organic pollutants based on octanol-air partitioning: Influence of respiratory elimination in marine species.

Risk assessments of persistent organic pollutants (POPs) are often based on octanol-water (KOW) partitioning dynamics and may not adequately reflect bioaccumulation in air-breathing organisms. It has been suggested that compounds with low KOW and high octanol-air partitioning (KOA) coefficients have the potential to bioaccumulate in air-breathing organisms, including marine mammals. Here we evaluate differences in concentrations of POPs for two trophically matched Arctic species, spotted seal (Phoca largha) and sheefish (Stenodus leucichthys). We compared concentrations of 108 POPs in matched tissues (liver and muscle) across three ranges of KOW. We found a significant positive correlation between POP concentration and log KOA in spotted seal tissues for low log KOW compounds (log KOW <5.5, p<0.05). This provides further evidence for empirical models and observed bioaccumulation patterns in air-breathing organisms, and highlights the potential for bioaccumulation of these compounds in Arctic marine mammals.

Discovery of AZD6642, an inhibitor of 5-lipoxygenase activating protein (FLAP) for the treatment of inflammatory diseases.

A drug discovery program in search of novel 5-lipoxygenase activating protein (FLAP) inhibitors focused on driving a reduction in lipophilicity with maintained or increased ligand lipophilic efficiency (LLE) compared to previously reported compounds led to the discovery of AZD6642 (15b). Introduction of a hydrophilic tetrahydrofuran (THF) ring at the stereogenic central carbon atom led to a significant shift in physicochemical property space. The structure-activity relationship exploration and optimization of DMPK properties leading to this compound are described in addition to pharmacokinetic analysis and an investigation of the pharmacokinetic (PK)-pharmacodynamic (PD) relationship based on ex vivo leukotriene B4 (LTB4) levels in dog. AZD6642 shows high specific potency and low lipophilicity, resulting in a selective and metabolically stable profile. On the basis of initial PK/PD relation measured, a low dose to human was predicted.

Mercury in gray wolves (Canis lupus) in Alaska: increased exposure through consumption of marine prey.

Mercury (Hg) bioaccumulates in the tissues of organisms and biomagnifies within food-webs. Gray wolves (Canis lupus) in Alaska primarily acquire Hg through diet; therefore, comparing the extent of Hg exposure in wolves, in conjunction with stable isotopes, from interior and coastal regions of Alaska offers important insight into their feeding ecology. Liver, kidney, and skeletal muscle samples from 162 gray wolves were analyzed for total mercury (THg) concentrations and stable isotopic signatures (δ(13)C, δ(15)N, and δ(34)S). Median hepatic THg concentrations were significantly higher in wolves with coastal access compared to wolves from interior Alaska. Stable isotope ratios, in conjunction with THg concentrations, provide strong evidence that coastal wolves are utilizing marine prey representing several trophic levels. The utilization of cross-ecosystem food resources by coastal wolves is clearly contributing to increased THg exposure, and may ultimately have negative health implications for these animals.

Toxicokinetics of mercury in blood compartments and hair of fish-fed sled dogs.

BACKGROUND: Understanding mercury (Hg) distribution in blood and the importance of hair as an excretory pathway is critical for evaluating risk from long term dietary Hg exposure. The major objective of this study was to characterize changes in total Hg concentrations in specific blood compartments and hair over time due to long term piscivory. METHODS: Eight sled dogs (Canis lupus familiaris) were fed either a fish and kibble diet (n = 4), or a fish-free control diet (n = 4) for 12 weeks. Concentrations of Hg were monitored throughout the exposure period, and for 10 weeks post exposure, until Hg concentrations in all blood compartments of one of the exposed dogs dropped below detection limit. Additionally, foreleg hair was sampled during acclimation and weeks 0 and 12. RESULTS: Hg was detected primarily in whole blood and packed cells, although it was sporadically detected at low concentrations in plasma and serum in two of the fish fed dogs. Dogs ingested an estimated average of 13.4 ± 0.58 µg Hg per kg body weight per day. Hg was detectable in whole blood and packed cells within a week of exposure. Detected concentrations continued to rise until plateauing at approximately 3-6 weeks of exposure at a mean of 9.2 ± 1.97 ng/g (ppb) in whole blood. Hg concentration decreased post exposure following 1st order elimination. The mean half-life (t1/2) in whole blood for Hg was 7 weeks. Mean Hg in hair for the fish-fed dogs at week 12 was 540 ± 111 ppb and was significantly greater (about 7-fold) than the Hg hair concentration for the control dogs. The hair to blood ratio for Hg in fish-fed dogs was 59.0 ± 7.6:1. CONCLUSIONS: This study found the sled dog model to be an effective method for investigating and characterizing blood Hg distribution (whole blood, serum, plasma, packed cells) and toxicokinetics associated with a piscivorous diet, especially for Hg-exposed fur bearing mammals (such as polar bears). Although hair excretion and hair to blood Hg ratios were not similar to human concentrations and ratios, the sled dog toxicokinetics of Hg in blood, was more similar to that of humans than traditional laboratory animals (such as the rat).

Spatiotemporal trends of mercury in walleye and largemouth bass from the Laurentian Great Lakes region.

The risk of mercury (Hg) exposure to humans and wildlife from fish consumption has driven extensive mercury analysis throughout the Great Lakes Region since the 1970s. This study compiled fish-Hg data from multiple sources in the region and assessed spatiotemporal trends of Hg concentrations in two representative top predator fish species. Walleye (Sander vitreus) and largemouth bass (Micropterus salmoides) were chosen for the trend analysis because they had more Hg records (63,872) than other fish species that had been sampled from waters throughout the region. Waterbody types were inland lakes (70%), the Great Lakes, impoundments, and rivers. The compiled datasets were analyzed with a mixed effects statistical model having random effects of station, year, and fish length; and fixed effects of year, tissue type, fish length, habitat, and season. The results showed a generally declining temporal trend in fish-Hg for the region (1970-2009), with spatial trends of increasing Hg concentration from south to north and from west to east across the region. Nonlinearity was evident in the general downward trends of Ontario walleye, with a shift to an upward trend beginning in the 1990s. Only ongoing monitoring can reveal if this upward shift is an oscillation in a long-term decline, a statistical anomaly, or a sustained declining temporal trend in regional fish-Hg concentrations.

Organic nutrients and contaminants in subsistence species of Alaska: concentrations and relationship to food preparation method.

OBJECTIVES: To determine nutrient and contaminant concentrations, document concentration changes related to common preparation methods and provide a basic risk-benefit analysis for select subsistence foods consumed by residents of Kotzebue, Alaska. STUDY DESIGN: Eleven organic nutrients and 156 persistent organic pollutants (POPs) were measured in foods derived from spotted seals and sheefish. METHODS: Nutrients in foodstuffs were compared to Daily Recommended Intake criteria. POPs were compared to Tolerable Daily Intake Limits (TDIL). RESULTS: Cooking, as well as absence/presence of skin during sheefish processing, altered nutrient and contaminant concentrations in seals and fish. Sheefish muscle and seal blubber were particularly rich in omega-3 fatty acids and seal liver in vitamin A. Seal liver exceeded the recommended upper limit for vitamin A. POP contribution to TDIL was >25% in all tissues except blubber, in which 4 POPS were present at >25% TDIL. No POPs exceeded TDIL in a serving of any tissue studied. The most prominent concerns identified were levels of vitamin A in spotted seal liver and certain POPs in blubber, warranting consideration when determining how much and how often these foods should be consumed. CONCLUSIONS: Preparation methods altering tissues from their raw state significantly affect nutrient and contaminant concentrations, thus direct evaluation of actual food items is highly recommended to determine risk-benefits ratios of traditional diets. Traditional foods provide essential nutrients with very limited risk from contaminants. We encourage the consumption of traditional foods and urge public health agencies to develop applicable models to assess overall food safety and quality.

Inorganic nutrients and contaminants in subsistence species of Alaska: linking wildlife and human health.

OBJECTIVES: To determine inorganic nutrient and contaminant concentrations in subsistence foods consumed by Alaska Natives, concentration changes related to common preparation methods and provide a basic risk-benefit analysis for these foods. STUDY DESIGN: Eleven essential and six non-essential elements were measured in foods derived from spotted seals and sheefish. METHODS: Essential nutrients in foodstuffs were compared to Daily Recommended Intake (DRI) criteria. Non-essential elements were compared to Tolerable Daily Intake Limits (TDIL). These comparisons serve as a risk-benefit analysis, not as consumption advice. RESULTS: Cooking altered nutrient and contaminant concentrations. Spotted seal muscle and kidney are rich in Fe and Se; liver in Cu, Fe, Mo and Se; and sheefish muscle in Se. TDIL was exceeded in a 100 g serving of seal for THg in raw and fried liver and boiled kidney; MeHg in dried muscle and raw and fried liver; Cd in raw and boiled kidney; and As in raw and rendered blubber. Arsenic exceeded TDIL in sheefish muscle. However, toxicity potential is likely reduced by the element form (i.e., organic As, inorganic Hg) and the presence of protective nutrients such as Se. CONCLUSIONS: Preparation methods alter wildlife tissues from their raw state, significantly affecting element concentrations. Direct evaluation of actual food items is warranted to determine risk-benefit ratios of traditional diets. Traditional foods provide many essential nutrients with a very limited risk from contaminants. We encourage continued consumption of traditional foods, and urge public health agencies to develop applicable models for providing consumption advice, incorporating food processing considerations.

Improving the content uniformity of a low-dose tablet formulation through roller compaction optimization.

In this investigation, the potency distribution of a low-dose drug in a granulation was optimized through a two-part study using statistically designed experiments. The purpose of this investigation was to minimize the segregation potential by improving content uniformity across the granule particle size distribution, thereby improving content uniformity in the tablet. Initial operating parameters on the Gerteis 3-W-Polygran 250/100/3 Roller Compactor resulted in a U-shaped potency function (potency vs. granule particle size) with superpotent fines and large granules. The roller compaction optimization study was carried out in two parts. Study I used a full factorial design with roll force (RF) and average gap width (GW) as independent variables and Study II used a D-optimal response surface design with four factors: RF, GW, granulating sieve size (SS), and granulator speed (GS). The planned response variables for Study I were bypass weight % and potency of bypass. Response variables for Study II included mean granulation potency with % relative standard deviation (% RSD), granulation particle size, sieve cut potency % RSD, tablet potency with % RSD, compression force at 7 kP crushing strength, and friability of 7-kP tablets. A constraint on GW was determined in Study I by statistical analysis. Bypass and observations of ribbon splitting were minimized when GW was less than 2.6 mm. In Study II, granulation potency, granulation uniformity, and sieve cut uniformity were optimized when the SS was 0.8 mm. Higher RF during dry granulation produced better sieve cut uniformity and tablets with improved uniformity throughout the run, as measured by stratified tablet samples taken during compression and assayed for potency. The recommended optimum roller compaction and milling operating parameters that simultaneously met all constraints were RF = 9 kN, GW = 2.3 mm, SS = 0.8 mm, and GS = 50 rpm. These parameters became the operating parameter set points during a model confirmation trial. The results from the confirmation trial proved that the new roller compaction and milling conditions reduced the potential for segregation by minimizing the granulation potency variability as a function of particle size as expressed by sieve cut potency % RSD, and thus improved content uniformity of stratified tablet samples.

P2 receptor expression profiles in human vascular smooth muscle and endothelial cells.

P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.