Preclinical trial of a MAP4K4 inhibitor to reduce infarct size in the pig: does cardioprotection in human stem cell-derived myocytes predict success in large mammals?

Reducing infarct size (IS) by interfering with mechanisms for cardiomyocyte death remains an elusive goal. DMX-5804, a selective inhibitor of the stress-activated kinase MAP4K4, suppresses cell death in mouse myocardial infarction (MI), human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), and 3D human engineered heart tissue, whose fidelity to human biology is hoped to strengthen the route to clinical success. Here, DMX-10001, a soluble, rapidly cleaved pro-drug of DMX-5804, was developed for i.v. testing in large-mammal MI. Following pharmacodynamic studies, a randomized, blinded efficacy study was performed in swine subjected to LAD balloon occlusion (60 min) and reperfusion (24 h). Thirty-six animals were enrolled; 12 were excluded by pre-defined criteria, death before infusion, or technical issues. DMX-10001 was begun 20 min before reperfusion (30 min, 60 mg/kg/h; 23.5 h, 17 mg/kg/h). At all times tested, beginning 30 min after the start of infusion, DMX-5804 concentrations exceeded > fivefold the levels that rescued hPSC-CMs and reduced IS in mice after oral dosing with DMX-5804 itself. No significant reduction occurred in IS or no-reflow corrected for the area at ischemic risk, even though DMX-10001 reduced IS, expressed in grams or % of LV mass, by 27%. In summary, a rapidly cleaved pro-drug of DMX-5804 failed to reduce IS in large-mammal MI, despite exceeding the concentrations for proven success in both mice and hPSC-CMs.

IKKε and TBK1 in diffuse large B-cell lymphoma: A possible mechanism of action of an IKKε/TBK1 inhibitor to repress NF-κB and IL-10 signalling.

The IKK-related kinases, IKKε and TBK1, have essential roles in innate immunity in part through modifying MYD88 signalling from the Toll-like receptors to regulate NF-κB signalling. We investigated the expression and function of IKKε and TBK1, in diffuse large B-cell lymphoma (DLBCL). DLBCL cell lines and patient-derived xenografts were used to determine their sensitivity to IKKε and TBK1 inhibitors. To understand the function of IKKε and TBK1 secreted factors were determined following administration of inhibitors. Gene expression microarrays were used to determine the transcriptional effects of inhibitors. Higher TBK1 mRNA levels associated with poorer clinical outcome but IKKε and TBK1 were expressed in both germinal centre and non-germinal centre types of DLBCL. Survival of cell lines Ly10, Ly03 and Pfeiffer, and of some primary human lymphoma cells, was suppressed by a small molecule IKKε/TBK1 inhibitor, DMX3433. DMX3433 reduced IL-10 production from Ly10 and repressed NF-κB mediated transcription. Inhibition of IKKε and TBK1 warrants further investigation as a potential therapeutic route to suppress NF-κB signalling in lymphoma.

Testing Chemical Safety: What Is Needed to Ensure the Widespread Application of Non-animal Approaches?

Scientists face growing pressure to move away from using traditional animal toxicity tests to determine whether manufactured chemicals are safe. Numerous ethical, scientific, business, and legislative incentives will help to drive this shift. However, a number of hurdles must be overcome in the coming years before non-animal methods are adopted into widespread practice, particularly from regulatory, scientific, and global perspectives. Several initiatives are nevertheless underway that promise to increase the confidence in newer alternative methods, which will support the move towards a future in which less data from animal tests is required in the assessment of chemical safety.

Advertising to children initiatives have not reduced unhealthy food advertising on Australian television.

Background: In response to rising childhood obesity rates, the Australian food industry implemented two initiatives in 2009 to reduce the marketing of unhealthy food to children. This study evaluated the efficacy of these initiatives on the rate of unhealthy food advertising to children on Australian television. Methods: The rates of food advertisements on three free-to-air commercial television channels and a youth-oriented digital channel in Sydney, Australia were analysed over 2 weekdays (16 h) and two weekend days (22 h). Advertisements were categorized according to the healthiness of foods advertised (non-core, core, miscellaneous) and signatory status to the food industry advertising initiatives. Results: Total food advertising rates for the three channels increased from 5.5/h in 2011 to 7.3/h in 2015, due to an increase of 0.8/h for both core and miscellaneous foods. The rate of non-core food advertisements in 2015 (3.1/h) was similar to 2011 (3.0/h). The youth-oriented channel had fewer total food advertisements (3.7/h versus 7.3/h) but similar fast-food advertisement rates (1.3/h versus 1.3/h). Conclusions: There was no change in the rate of unhealthy food advertising since 2011, suggesting minimal impact of the current food industry initiatives on reducing children's exposure to unhealthy food advertising.

Exploring perceptions and beliefs about the cost of fruit and vegetables and whether they are barriers to higher consumption.

BACKGROUND: Fruit and vegetable (F&V) consumption is below recommendations, and cost may be a barrier to meeting recommendations. Limited evidence exists on individual perceptions about the cost, actual spending and consumption of F&V. This study investigated perceptions and beliefs about cost of F&V and whether this is a barrier to higher consumption. METHODS: An online survey of Australian adults (n = 2474) measured F&V consumption; expenditure on F&V and food; and perceived barriers to consumption. Multivariable logistic regression examined associations between participants' responses about cost of F&V and demographic factors, and with actual consumption and expenditure on F&V. RESULTS: Cost was identified as a barrier for 29% of people not meeting recommended fruit servings and for 14% of people not meeting recommendations for vegetables. Cost was a more common barrier for those on lower incomes (fruit aOR 1.89; 95% CI 1.20-2.98 and vegetables aOR 2.94; 95% CI 1.97-4.39) and less common for older participants (fruit aOR 0.33; 95% CI 0.17-0.62 and vegetables aOR 0.31; 95% CI 0.18-0.52). There was no association between the perceived barriers and actual F&V spending. Twenty percent of participants said F&V were not affordable; 39% said cost made it difficult to buy F&V, and for 23% the cost of F&V meant they bought less than desired. CONCLUSIONS: A minority reported F&V were not affordable where they shopped and that cost was a barrier to higher consumption. However, it is apparent that young adults and those on low incomes eat less than they would like because of cost. Strategies that remove financial impediments to consumption are indicated for these population sub-groups.

Assessing the predictive value of the rodent neurofunctional assessment for commonly reported adverse events in phase I clinical trials.

Central Nervous System (CNS)-related safety concerns are major contributors to delays and failure during the development of new candidate drugs (CDs). CNS-related safety data on 141 small molecule CDs from five pharmaceutical companies were analyzed to identify the concordance between rodent multi-parameter neurofunctional assessments (Functional Observational Battery: FOB, or Irwin test: IT) and the five most common adverse events (AEs) in Phase I clinical trials, namely headache, nausea, dizziness, fatigue/somnolence and pain. In the context of this analysis, the FOB/IT did not predict the occurrence of these particular AEs in man. For AEs such as headache, nausea, dizziness and pain the results are perhaps unsurprising, as the FOB/IT were not originally designed to predict these AEs. More unexpected was that the FOB/IT are not adequate for predicting 'somnolence/fatigue' nonclinically. In drug development, these five most prevalent AEs are rarely responsible for delaying or stopping further progression of CDs. More serious AEs that might stop CD development occurred at too low an incidence rate in our clinical dataset to enable translational analysis.

Time to address continued poor vegetable intake in Australia for prevention of chronic disease.

BACKGROUND: Australian and most international Dietary Guidelines recommend people consume more fruits and vegetables (F&V) to maintain a healthy weight and reduce chronic disease risk. Previous Australian and international surveys have shown sub-optimal consumption of F&V. OBJECTIVES: This study aimed to assess adults' F&V consumption, knowledge of recommended servings, readiness to change, barriers/enabling factors, so that this knowledge might be used for campaigns that support improved consumption. MATERIAL AND METHODS: An online survey of a representative sample of adults living in New South Wales, Australia (n = 2474) measuring self-reported F&V consumption; attitudes towards F&V consumption; stage of change for increasing F&V; barriers to consumption; and knowledge of cancer-health benefits. RESULTS: F&V consumption was below recommendations, with vegetable consumption notably low. Only 10% of participants ate at least five servings of vegetables/day (median intake was two daily servings), and 57% consumed two servings fruit/day. There was poor recognition that intake of vegetables was inadequate and this was a barrier to improving vegetable consumption; with preferences for other foods, habit and cost also important barriers. Key barriers to increasing fruit intake were habit, preferences for other foods, perishability, and cost. For vegetable consumption, 49% of participants were in the pre-contemplation stage of change, whereas for fruits 56% were in the action/maintenance stage. Sixty-four percent of respondents believed that eating F&V would protect against cancer, with 56% reporting they thought not eating enough F&V would cause cancer. IMPLICATIONS: Understanding what motivates and prevents people from consuming F&V is important for developing effective health promotion programs. Similar to previous surveys, there has been little shift in F&V consumption. Social marketing campaigns have been shown to improve health-related behaviours, and this study may assist in identifying audience segmentation for better targeted campaigns.

The melanocortin 4 receptor: oligomer formation, interaction sites and functional significance.

This study involves the structural and functional properties of the recombinant melanocortin 4 receptor (MC(4)R) expressed in the HEK-293 cell line. Using co-immuno-purification approaches, the receptor appears to be an oligomer, which can be crosslinked through disulphide bonds involving a native cysteine residue (84) to give a dimeric species. This position is located near the cytosolic region of transmembrane segment 2 and it is suggested that this is an interacting interface between MC(4)R monomers. Using co-expression of the native protein and a C84A mutant, it appears that the receptor also forms higher order oligomers via alternative interfaces. Interestingly, disulphide crosslink formation does not occur if the receptor is uncoupled from its G-protein, even though the oligomeric state is preserved. This suggests that the conformational changes, which occur on activation, affect the TM2 interface. The pharmacology of the agonist, NDP-MSH, indicates that the MC(4)R retains high affinity for the ligand in the absence of the G-protein but occupancy for the ligand is increased. The data can be interpreted to suggest that the G-protein exerts a negative allosteric effect on the receptor. Co-expression of one receptor lacking the ability to signal with another, which cannot bind the agonist, restored ligand-dependent activation of the G-protein to situations in which neither receptor on its own could activate the G-protein. Such transactivation suggests meaningful cross talk between the receptor subunits in the oligomeric complex. These studies demonstrate further unique features of the MC(4)R.

Recommendations from a global cross-company data sharing initiative on the incorporation of recovery phase animals in safety assessment studies to support first-in-human clinical trials.

An international expert group which includes 30 organisations (pharmaceutical companies, contract research organisations, academic institutions and regulatory bodies) has shared data on the use of recovery animals in the assessment of pharmaceutical safety for early development. These data have been used as an evidence-base to make recommendations on the inclusion of recovery animals in toxicology studies to achieve scientific objectives, while reducing animal use. Recovery animals are used in pharmaceutical development to provide information on the potential for a toxic effect to translate into long-term human risk. They are included on toxicology studies to assess whether effects observed during dosing persist or reverse once treatment ends. The group devised a questionnaire to collect information on the use of recovery animals in general regulatory toxicology studies to support first-in-human studies. Questions focused on study design, the rationale behind inclusion or exclusion and the impact this had on internal and regulatory decisions. Data on 137 compounds (including 53 biologicals and 78 small molecules) from 259 studies showed wide variation in where, when and why recovery animals were included. An analysis of individual study and programme design shows that there are opportunities to reduce the use of recovery animals without impacting drug development.

Transient Receptor Potential Melastatin 2 (TRPM2) ion channel is required for innate immunity against Listeria monocytogenes.

The generation of reactive oxygen species (ROS) is inherent to immune responses. ROS are crucially involved in host defense against pathogens by promoting bacterial killing, but also as signaling agents coordinating the production of cytokines. Transient Receptor Potential Melastatin 2 (TRPM2) is a Ca(2+)-permeable channel gated via binding of ADP-ribose, a metabolite formed under conditions of cellular exposure to ROS. Here, we show that TRPM2-deficient mice are extremely susceptible to infection with Listeria monocytogenes (Lm), exhibiting an inefficient innate immune response. In a comparison with IFNγR-deficient mice, TRPM2(-/-) mice shared similar features of uncontrolled bacterial replication and reduced levels of inducible (i)NOS-expressing monocytes, but had intact IFNγ responsiveness. In contrast, we found that levels of cytokines IL-12 and IFNγ were diminished in TRPM2(-/-) mice following Lm infection, which correlated with their reduced innate activation. Moreover, TRPM2(-/-) mice displayed a higher degree of susceptibility than IL-12-unresponsive mice, and supplementation with recombinant IFNγ was sufficient to reverse the unrestrained bacterial growth and ultimately the lethal phenotype of Lm-infected TRPM2(-/-) mice. The severity of listeriosis we observed in TRPM2(-/-) mice has not been reported for any other ion channel. These findings establish an unsuspected role for ADP-ribose and ROS-mediated cation flux for innate immunity, opening up unique possibilities for immunomodulatory intervention through TRPM2.

Sustainability in a state comprehensive cancer control coalition: lessons learned.

The Alabama Comprehensive Cancer Control Coalition (ACCCC) has developed an integrated and coordinated approach to reducing cancer incidence, morbidity, and mortality, and to improving the quality of life for cancer survivors, their families, and their caregivers. The ACCCC is currently in a maintenance phase and a formal plan for sustainability of the coalition was needed to keep the members engaged and productive. A training session in coalition sustainability conducted in 2013 identified the following elements as essential to success: (1) increased marketing of the coalition by simplifying its mission; (2) improved networking including flexibility in coalition meeting location and attendance; (3) increased membership satisfaction through transformational leadership; (4) revision of the working structure of committees and improved accountability; and (5) enhancement of partner satisfaction with coalition activities designed to recruit and retain new partners. A self-administered membership satisfaction survey was given to assess coalition mission, meeting logistics, organization, capacity building, and coalition goals. Results indicated that the subcategories of communication, mission, and meeting logistics were rated satisfied to very satisfied on a five-point scale. Although the ACCCC had clearly written goals, improvement could be made in leadership participation and new member orientation could be improved. Most members rated their parent organization as highly involved with the ACCCC and many offered suggestions on capacity building. Results of the sustainability training have clarified the ACCCC's plans to ensure coalition viability and improve strategies to inform stakeholders of the benefits of participation in the coalition.

A comparison of the cost of generic and branded food products in Australian supermarkets.

OBJECTIVE: Food cost is an important factor influencing the consumption of nutritious foods and subsequent chronic disease risk. The present study compared the cost of branded food products with their generic equivalents across a range of food categories. SETTING: The survey was conducted within two major supermarket chains across six locations in Sydney, Australia (n 12). DESIGN: Price differences were calculated for 'core' (nutrient dense and low in energy) and 'extra' (high in undesirable nutrients and/or energy) packaged foods (n 22) between generic and branded items. RESULTS: A cost saving of 44 % was found by purchasing generic over branded products across all food categories. The most significant savings were for core foods, such as bread and cereals, and the smallest cost savings were seen for fruit products. There was little variation in cost saving between branded and generic products by socio-economic status of the supermarket location. CONCLUSIONS: The large price differential between branded and generic food products implies that consumers, particularly those on lower incomes, could benefit financially from purchasing generic items. The promotion of core generic products may be an effective strategy to assist people on lower incomes to meet dietary guidelines.

Pharmaceutical toxicology: designing studies to reduce animal use, while maximizing human translation.

Evaluation of the safety of new chemicals and pharmaceuticals requires the combination of information from various sources (e.g. in vitro, in silico and in vivo) to provide an assessment of risk to human health and the environment. The authors have identified opportunities to maximize the predictivity of this information to humans while reducing animal use in four key areas; (i) accelerating the uptake of in vitro methods; (ii) incorporating the latest science into safety pharmacology assessments; (iii) optimizing rodent study design in biological development and (iv) consolidating approaches in developmental and reproductive toxicology. Through providing a forum for open discussion of novel proposals, reviewing current research and obtaining expert opinion in each of the four areas, the authors have developed recommendations on good practice and future strategy.

A global pharmaceutical company initiative: an evidence-based approach to define the upper limit of body weight loss in short term toxicity studies.

Short term toxicity studies are conducted in animals to provide information on major adverse effects typically at the maximum tolerated dose (MTD). Such studies are important from a scientific and ethical perspective as they are used to make decisions on progression of potential candidate drugs, and to set dose levels for subsequent regulatory studies. The MTD is usually determined by parameters such as clinical signs, reductions in body weight and food consumption. However, these assessments are often subjective and there are no published criteria to guide the selection of an appropriate MTD. Even where an objective measurement exists, such as body weight loss (BWL), there is no agreement on what level constitutes an MTD. A global initiative including 15 companies, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), has shared data on BWL in toxicity studies to assess the impact on the animal and the study outcome. Information on 151 studies has been used to develop an alert/warning system for BWL in short term toxicity studies. The data analysis supports BWL limits for short term dosing (up to 7days) of 10% for rat and dog and 6% for non-human primates (NHPs).

Artificial flowers as a tool for investigating multimodal flower choice in wild insects.

Flowers come in a variety of colours, shapes, sizes and odours. Flowers also differ in the quality and quantity of nutritional reward they provide to entice potential pollinators to visit. Given this diversity, generalist flower-visiting insects face the considerable challenge of deciding which flowers to feed on and which to ignore. Working with real flowers poses logistical challenges due to correlations between flower traits, maintenance costs and uncontrolled variables. Here, we overcome this challenge by designing multimodal artificial flowers that varied in visual, olfactory and reward attributes. We used artificial flowers to investigate the impact of seven floral attributes (three visual cues, two olfactory cues and two rewarding attributes) on flower visitation and species richness. We investigated how flower attributes influenced two phases of the decision-making process: the decision to land on a flower, and the decision to feed on a flower. Artificial flowers attracted 890 individual insects representing 15 morphospecies spanning seven arthropod orders. Honeybees were the most common visitors accounting for 46% of visitors. Higher visitation rates were driven by the presence of nectar, the presence of linalool, flower shape and flower colour and was negatively impacted by the presence of citral. Species richness was driven by the presence of nectar, the presence of linalool and flower colour. For hymenopterans, the probability of landing on the artificial flowers was influenced by the presence of nectar or pollen, shape and the presence of citral and/or linalool. The probability of feeding increased when flowers contained nectar. For dipterans, the probability of landing on artificial flowers increased when the flower was yellow and contained linalool. The probability of feeding increased when flowers contained pollen, nectar and linalool. Our results demonstrate the multi-attribute nature of flower preferences and highlight the usefulness of artificial flowers as tools for studying flower visitation in wild insects.

Differential effects of mutations of POPDC proteins on heteromeric interaction and membrane trafficking.

The Popeye domain containing (POPDC) genes encode sarcolemma-localized cAMP effector proteins. Mutations in blood vessel epicardial substance (BVES) also known as POPDC1 and POPDC2 have been associated with limb-girdle muscular dystrophy and cardiac arrhythmia. Muscle biopsies of affected patients display impaired membrane trafficking of both POPDC isoforms. Biopsy material of patients carrying mutations in BVES were immunostained with POPDC antibodies. The interaction of POPDC proteins was investigated by co-precipitation, proximity ligation, bioluminescence resonance energy transfer and bimolecular fluorescence complementation. Site-directed mutagenesis was utilised to map the domains involved in protein-protein interaction. Patients carrying a novel homozygous variant, BVES (c.547G > T, p.V183F) displayed only a skeletal muscle pathology and a mild impairment of membrane trafficking of both POPDC isoforms. In contrast, variants such as BVES p.Q153X or POPDC2 p.W188X were associated with a greater impairment of membrane trafficking. Co-transfection analysis in HEK293 cells revealed that POPDC proteins interact with each other through a helix-helix interface located at the C-terminus of the Popeye domain. Site-directed mutagenesis of an array of ultra-conserved hydrophobic residues demonstrated that some of them are required for membrane trafficking of the POPDC1-POPDC2 complex. Mutations in POPDC proteins that cause an impairment in membrane localization affect POPDC complex formation while mutations which leave protein-protein interaction intact likely affect some other essential function of POPDC proteins.

TRPM2 Ca2+ channel regulates energy balance and glucose metabolism.

TRPM2 Ca(2+)-permeable cation channel is widely expressed and activated by markers of cellular stress. Since inflammation and stress play a major role in insulin resistance, we examined the role of TRPM2 Ca(2+) channel in glucose metabolism. A 2-h hyperinsulinemic euglycemic clamp was performed in TRPM2-deficient (KO) and wild-type mice to assess insulin sensitivity. To examine the effects of diet-induced obesity, mice were fed a high-fat diet for 4-10 mo, and metabolic cage and clamp studies were conducted in conscious mice. TRPM2-KO mice were more insulin sensitive partly because of increased glucose metabolism in peripheral organs. After 4 mo of high-fat feeding, TRPM2-KO mice were resistant to diet-induced obesity, and this was associated with increased energy expenditure and elevated expressions of PGC-1α, PGC-1ß, PPARα, ERRα, TFAM, and MCAD in white adipose tissue. Hyperinsulinemic euglycemic clamps showed that TRPM2-KO mice were more insulin sensitive, with increased Akt and GSK-3ß phosphorylation in heart. Obesity-mediated inflammation in adipose tissue and liver was attenuated in TRPM2-KO mice. Overall, TRPM2 deletion protected mice from developing diet-induced obesity and insulin resistance. Our findings identify a novel role of TRPM2 Ca(2+) channel in the regulation of energy expenditure, inflammation, and insulin resistance.

The design of chronic toxicology studies of monoclonal antibodies: implications for the reduction in use of non-human primates.

The changing environment of monoclonal antibody (mAb) development is impacting on the cost of drug development and the use of experimental animals, particularly non-human primates (NHPs). The drive to reduce these costs is huge and involves rethinking and improving nonclinical studies to make them more efficient and more predictive of man. While NHP use might be unavoidable in many cases because of the exquisite specificity and consequent species selectivity of mAbs, our increasing knowledge base can be used to improve drug development and maximise the output of experimental data. Data on GLP regulatory toxicology studies for 58mAbs were obtained from 10 companies across a wide range of therapeutic indications. These data have been used to investigate current practice and identify study designs that minimise NHP use. Our analysis shows that there is variation in the number of animals used for similar studies. This information has been used to develop practical guidance and make recommendations on the use of science-based rationale to design studies using fewer animals taking into account the current regulatory guidance. There are eight recommendations intended to highlight areas for consideration. They include guidance on the main group size, the inclusion of recovery groups and the number of dose groups used in short and long term chronic toxicology studies.

Colorectal cancer screening practices in Alabama: a survey of primary care physicians.

In order to inform efforts to increase screening rates for colorectal cancer (CRC), we conducted a survey of Alabama primary care physicians regarding CRC screening practices, educational preferences, and perceptions of obstacles to screening. A mail survey of 2,378 Alabama physicians in Family Medicine, Internal Medicine, and Obstetrics & Gynecology was conducted. Many physicians are not fully up-to-date with current CRC screening practices that could improve patient compliance with screening guidelines. One example is the potential use of high-sensitivity stool tests, such as the fecal immunochemical test, instead of the no longer recommended low-sensitivity guaiac fecal occult blood tests. In addition, enhanced multimedia and web-based approaches to educating physicians and patients could be more fully utilized. Further, greater use of health information technologies could increase screening rates. Enhancing primary care physicians' knowledge of screening modalities and increasing their use of electronic technology could significantly improve colorectal cancer screening outcomes.

An achievement of professional, public, and patient education: the design and evaluation of a comprehensive cancer control plan for Alabama.

This Alabama statewide cancer control plan for 2011-2015 seeks to build on the successes of two previous 5-year plans while developing new objectives that address cancer disparities and cancer prevention over the entire lifespan. The approach to defining objectives for this Plan was systematic and sought input from all members of the Alabama Comprehensive Cancer Control Coalition (ACCCC). The Plan that was fashioned is based on input from academic medical centers, private physicians, government agencies, regulatory agencies, health societies, private citizens, and cancer survivors, all of whom are active Coalition members who exchange information, opinions, and knowledge from their respective points of view. The Plan could not have taken shape without the full input of health professionals, statisticians, graduate students, former patients, and concerned citizens; it is truly an example of the synergy of professional, public, and patient education.