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Lung cancer is a major cause of cancer-related deaths. Alectinib is the first line of treatment for patients with ALK-positive lung cancer, but the survival rate beyond 2-3 years is low. Co-targeting secondary oncogenic drivers such as SHP2 is a potential strategy for improving drug efficacy. This is because SHP2 is expressed ubiquitously, but ALK expression is largely restricted to cancer cells. Thus, the combination of ALK and SHP2 inhibitors may provide a way to restrict synergistic cytotoxicity to cancer cells only, by reducing the dose of SHP2 inhibitors required for anticancer action and minimising SHP2-dependent systemic toxicity. The objective of this study was to investigate whether the combination of a SHP2 inhibitor (SHP099) with alectinib would synergistically suppress the growth of ALK-positive lung cancer cells. Our results demonstrated that the drug combination significantly and synergistically decreased cell viability at relatively low concentrations in ALK-positive H3122 and H2228 cells, due to G1 cell cycle arrest and increased apoptosis because of suppressed downstream RAS/MAPK signalling. The drug combination also induced the expression of mediators of the intrinsic apoptotic pathway, Bim and cleaved caspase-3, and modulated the expression of cell cycle mediators cyclin D1, cyclin B1, and phosphorylated CDK1., (© 2023. The Author(s).)

Ageing and urbanisation pose significant challenges for public health and urban planning. Ageing populations are at particular risk from hazards arising from urbanisation processes, some of which are in turn exacerbated by climate change. One approach for mitigating the negative effects of urbanisation on ageing populations is the leveraging of the beneficial effects of urban green infrastructure as a public health intervention in the planning process. We assessed the potential of available theoretical frameworks to provide the context for such leverage. This involved active engagement with academics and practitioners specialising in ageing, green infrastructure and health and well-being through a knowledge-brokering approach. We concluded that an integrated and comprehensive framework on the socio-cultural-ecological determinants of health is lacking. To address this, we present a set of principles for overcoming challenges to knowledge integration when working at the intersection of green infrastructure, ageing, health and well-being. Our findings-and the co-production process used to generate them-have wider significance for trans-disciplinary research into the benefits of the natural environment to human health and well-being as well as other complex and interconnected topics associated with global grand challenges., (© 2022. The Author(s).)

Background: Lung cancer is the leading cause of cancer death in Aotearoa New Zealand, killing over 1,700 people each year. Despite the burden of lung cancer in Aotearoa New Zealand, the popular press has referred to it as the cancer type that no one talks about. Here, we investigate one factor that may contribute to this state of affairs: lung cancer stigma., Methods: Participants were university students and members of the general public. University students were recruited via an online experiment participation system in 2021. Members of the public were recruited via social media. All participants completed the Cancer Stigma Scale (CSS) for one of five cancer types (lung, cervical, breast, skin, or bowel). The CSS is a 25-item scale with six subscales: awkwardness, avoidance, severity, policy opposition, personal responsibility, and financial discrimination., Results: The mean age of participants was 24.3 (Standard Deviation = 10.4). Data from each subscale were submitted to an analysis of covariance (ANCOVA), with cancer type as a between-participant factor (5: lung, cervical, breast, skin, or bowel) and stigma as the dependent variable. Relative to most other cancer types, people were more likely to avoid someone with lung cancer, view interacting with someone with lung cancer as more awkward, and view people with lung cancer as being responsible for their condition., Conclusion: The Health Research Council of New Zealand recently funded the very first trial of lung cancer screening in Aotearoa New Zealand. The current study suggests that addressing stigma will be essential for the success of such programs, with stigma likely influencing those who engage in such trials., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2022 Jess Godward et al.)

During cardiac reperfusion after myocardial infarction, the heart is subjected to cascading cycles of ischaemia reperfusion injury (IRI). Patients presenting with this injury succumb to myocardial dysfunction resulting in myocardial cell death, which contributes to morbidity and mortality. New targeted therapies are required if the myocardium is to be protected from this injury and improve patient outcomes. Extensive research into the role of mitochondria during ischaemia and reperfusion has unveiled one of the most important sites contributing towards this injury; specifically, the opening of the mitochondrial permeability transition pore. The opening of this pore occurs during reperfusion and results in mitochondria swelling and dysfunction, promoting apoptotic cell death. Activation of mitochondrial ATP-sensitive potassium channels (mitoK ATP ) channels, uncoupling proteins, and inhibition of glycogen synthase kinase-3β (GSK3β) phosphorylation have been identified to delay mitochondrial permeability transition pore opening and reduce reactive oxygen species formation, thereby decreasing infarct size. Statins have recently been identified to provide a direct cardioprotective effect on these specific mitochondrial components, all of which reduce the severity of myocardial IRI, promoting the ability of statins to be a considerate preconditioning agent. This review will outline what has currently been shown in regard to statins cardioprotective effects on mitochondria during myocardial IRI., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mathematical modelling of tumour mutation dynamics has suggested that cancer drug targets that have different resistance mechanisms should be good candidates for combination treatment. This is because the development of mutations that cause resistance to all drugs at once should arise relatively infrequently. However, it is difficult to identify drug targets fulfilling this requirement for particular cancers. Here we present four experimental criteria that we argue are necessary (but not sufficient) conditions that drug combinations should meet in order to be considered for combination drug treatment aimed at delaying or overcoming cancer drug resistance. We present the results of our own experiments - guided by these criteria - using anaplastic lymphoma kinase mutated lung cancer cells. Each set of experiments demonstrate results for different drug combinations. We conclude that the combination of ALK and MEK inhibitors come closest to meeting all our criteria., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Histochemistry of tumor sections is a widely employed technique utilized to examine cell death in preclinical xenograft animal models of cancer. However, this is under the assumption that tumors are homogeneous, leading to practices such as automatic cell counting across the entire section. We have noted that in our experiments the core of the tumor is largely or partially necrotic, and lacks evidence of vascularization (in contrast to the outer areas of the tumor). We note that this can bias and confound immunohistochemical analyses that do not take care to sample areas of interest in a way to take this into account. Design-based stereology with image analysis techniques is an alternative process that could be used to measure the volume of the necrotic region compared to the volume of the whole tumor.

A second-generation enantiospecific synthesis of spiroleucettadine is described. The original reported antibacterial activity was not observed when the experiment was repeated on the synthetic samples; however, significant anti-proliferative activity was uncovered for both enantiomers of spiroleucettadine. Comparison of the optical rotational data and ORD-CD spectra of both enantiomers and the reported spectrum from the natural source have not provided a definitive answer regarding the absolute stereochemistry of naturally occurring spiroleucettadine. Efforts then focussed on alteration at the C-4 and C-5 positions of the slightly more active (-)-spiroleucettadine. Ten analogues were synthesised, with three analogues found to possess similar anti-proliferative profiles to spiroleucettadine against the H522 lung cancer cell line., (© 2020 Wiley-VCH GmbH.)

Cell based studies have suggested that the diabetes drug metformin may combine with the anaplastic lymphoma kinase receptor (ALK) inhibitor crizotinib to increase ALK positive lung cancer cell killing and overcome crizotinib resistance. We therefore tested metformin alone and in combination with crizotinib in vivo, by employing a xenograft mouse model of ALK positive lung cancer. We found that 14 days of daily oral metformin (100 mg/kg) alone had a moderate but statistically significant effect on tumour growth suppression, but in combination with crizotinib, produced no greater tumour suppression than crizotinib (25 mg/kg) alone. We also reassessed the effect of metformin on EML 4 -ALK positive lung cancer (H3122) cell viability. Although metformin alone did have a moderate effect on cell viability (30% suppression) this was only at a clinically irrelevant concentration (5 mM) and there was no additive effect with cytotoxic concentrations of crizotinib. Moreover, metformin did not overcome crizotinib resistance in our resistant cells. Nevertheless, we were able to show that metformin induces a G1-cell cycle arrest and apoptosis alone and in combination with crizotinib. Also, consistent with earlier work, the addition of insulin-like growth factor-1 (IGF-1) to EML 4 -ALK positive cancer cells reduced cell killing by crizotinib. We therefore hypothesised that the effect of metformin in vivo was not due to direct cytotoxicity on cancer cells, but by modulation of IGF-1 expression. We therefore measured levels of IGF-1 in plasma taken from mice treated with metformin, but found no difference between the drug treatment and control groups. We further hypothesised that the effect of metformin could be due to modulation of thrombospondin 1 (TSP-1), which metformin has been proposed to regulatein vivo, but again we found no difference between the experimental groups. Finally, we investigated the potential for liver and kidney toxicity, as well as CYP3A based interactions, from the combination of metformin with crizotinib., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The objective of this study was to compare head impact data acquired with an impact monitoring mouthguard (IMM) to the video-observed behavior of athletes' post-collision relative to their pre-collision behaviors. A total of n = 83 college and high school American football players wore the IMM and were video-recorded over 260 athlete-exposures. Ex-athletes and clinicians reviewed the video in a two-step process and categorized abnormal post-collision behaviors according to previously published Obvious Performance Decrement (OPD) definitions. Engineers qualitatively reviewed datasets to check head impact and non-head impact signal frequency and magnitude. The ex-athlete reviewers identified 2305 head impacts and 16 potential OPD impacts, 13 of which were separately categorized as Likely-OPD impacts by the clinical reviewers. All 13 Likely-OPD impacts were in the top 1% of impacts measured by the IMM (ranges 40-100 g, 3.3-7.0 m/s and 35-118 J) and 12 of the 13 impacts (92%) were to the side or rear of the head. These findings require confirmation in a larger data set before proposing any type of OPD impact magnitude or direction threshold exists. However, OPD cases in this study compare favorably with previously published impact monitoring studies in high school and college American football players that looked for OPD signs, impact magnitude and direction. Our OPD findings also compare well with NFL reconstruction studies for ranges of concussion and sub-concussive impact magnitudes in side/rear collisions, as well as prior theory, analytical models and empirical research that suggest a directional sensitivity to brain injury exists for single high-energy impacts.

Background: Massage therapy's ability to mitigate breast imaging associated anxiety has not been previously studied. Anxiety is, however, often cited as a harm of screening mammography with few options offered to diminish anxiety other than not screening. Reducing anxiety may improve compliance, and reduce breast cancer mortality and morbidity. A complimentary massage therapy program evaluated patient acceptance, anxiety perception and perceived value of massage., Methods: Over 10 weeks, verbal agreement was obtained from 113 breast imaging patients who desired a hand or shoulder/neck massage. Licensed massage therapists performed massages before, and/or during, or after, or in between imaging tests. After the massage, questionnaires assessed patients' self-rated perceptions of anxiety before and after massage on a scale from 0 to 10. Participants' age-group, reason for appointment, self-rated value of massage service, and willingness to return to and willingness to refer to the facility were reported. Changes in perceived average anxiety were estimated using a linear mixed effects model. Fisher's exact test was used to evaluate associations among categorical variables., Results: A significant decrease in perceived anxiety was observed following massage (d = -3.2, p < 0.001). 107/108 (99%) of respondents reported an improved patient experience with massage. 84/106 (79%) reported willingness to pay at least $5 for massage service., Conclusion: Massage therapy improves the patient experience and decreases perceptions of anxiety. It may be associated with improved breast imaging compliance. Patients' willingness to pay for the service may defray some cost of a massage program., Competing Interests: Declaration of competing interest No competing financial interest exists., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer most commonly arises through EML4 (Echinoderm Microtuble Like 4)-ALK chromosomal fusion. We have previously demonstrated that combination of the ALK inhibitor crizotinib with the MEK inhibitor selumetinib was highly effective at reducing cell viability of ALK-positive non-small-cell lung cancer (H3122) cells. In this study, we further investigated the efficacy of crizotinib and selumetinib combination therapy in an in vivo xenograft model of ALK-positive lung cancer. Crizotinib decreased tumor volume by 52% compared with control, and the drug combination reduced tumor growth compared with crizotinib. In addition, MEK inhibition alone reduced tumor growth by 59% compared with control. Crizotinib and selumetinib alone and in combination were nontoxic at the dose of 25 mg/kg, with values for ALT (<80 U/l) and creatinine (<2 mg/dl) within the normal range. Our results support the combined use of crizotinib with selumetinib in ALK-positive lung cancer but raise the possibility that a sufficient dose of an MEK inhibitor alone may be as effective as adding an MEK inhibitor to an ALK inhibitor. SIGNIFICANCE STATEMENT: This study contains in vivo evidence supporting the use of combination MEK inhibitors in ALK+ lung cancer research, both singularly and in combination with ALK inhibitors. Contrary to previously published reports, our results suggest that it is possible to gain much of the benefit from combination treatment with an MEK inhibitor alone, at a tolerable dose., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Dexamethasone has been observed to cause night-time wakefulness without accompanying fatigue. It is proposed here that the drug interrupts a sleep signal, and that although sleep has an ultimate ecological function, this signal is a proximate mechanism but not an ultimate physiological necessity, such that sleep is not a necessity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Non-small cell lung cancer with ALK rearrangements can be targeted effectively with ALK inhibitors such as crizotinib. However, cancer progression typically occurs within a year as drug resistance develops. One strategy to overcome this drug resistance is to determine if novel cytotoxic agents retain the ability to kill lung cancer cells that have developed ALK inhibitor resistance. We therefore examined curcumin, a drug with anticancer properties, and 2 s-generation curcumin derivatives (1-methyl-3,5-bis[(E)-4-pyridyl) methylidene]-4-piperidone (RL66) and 1-isopropyl-3,5-bis[(pyridine-3-yl) methylene]piperidin-4-one (RL118)) in lung cancer cell lines. The cytotoxicity of curcumin, RL66, and RL118 were tested in both ALK + lung cancer cells (H3122), crizotinib resistant ALK + cells (CR-H3122) and ALK - lung cancer cells (A549), both alone and in combination with crizotinib. ALK + cells were 2-3x more sensitive to RL66 and RL118 than ALK - cells, with the drugs' eliciting IC 50 values in the range of 0.7-1 μM in H3122 cells. Retained cytotoxic potency of the curcumin derivatives in crizotinib resistant cells indicated that mechanisms of resistance to the two drug types are independent, with resistance to ALK inhibitors not necessarily causing cross-resistance to curcumin derivatives. This was further corroborated by drug combination analysis where the effect of the drugs in combination was consistent with Bliss additivity, consistent with independent targets for crizotinib and curcumin derivatives. Results from Western blotting showed that RL118 (2 μM) inhibited p-ALK/ALK by ~50%, which was not as potent as the 90% inhibition elicited by crizotinib (0.25 μM). Since this is the primary mechanism of crizotinib cytotoxicity this provides further evidence of independent mechanisms of toxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Anaplastic lymphoma kinase (ALK) rearrangement, a key oncogenic driver in a small subset of non-small cell lung cancers, confers sensitivity to ALK tyrosine kinase inhibitors (TKIs). Crizotinib, a first generation ALK-TKI, has superiority to standard chemotherapy with longer progression-free survival and higher objective response rate. However, clinical benefit is limited by development of resistance, typically within a year of therapy. In this study the combined effect of crizotinib and the MEK inhibitor selumetinib was investigated in both crizotinib naïve (H3122) and crizotinib resistant (CR-H3122) ALK-positive lung cancer cells. Results showed that combination treatment potently inhibited the growth of both H3122 and CR-H3122 cells, resulting from increased apoptosis and decreased cell proliferation as a consequence of suppressed downstream RAS/MAPK signalling. The drug combination also elicited a greater than 3-fold increase in Bim, a mediator of apoptosis, and p27, a cyclin dependent kinase inhibitor compared to crizotinib alone. The results support the hypothesis that combining MEK inhibitors with ALK inhibitor can overcome ALK inhibitor resistance, and identifies Bim, PARP and CDK1 as druggable targets for possible triple drug therapy.

As New Zealand considers cannabis legal reform, we ask: what exactly is medicinal cannabis, and why does this matter? Cannabis is not a single entity but comes in diverse forms with various active ingredients. This contrasts with the legal and pharmaceutical definitions of medicines, with wide-ranging implications for quality control, prescriber practice and the assessment of clinical evidence. We argue that what is considered a medicine in the legal and pharmaceutical sense should not be changed in an ad hoc way to accommodate cannabis, but that cannabis products should be held to the same standards as other medicines., Competing Interests: Dr Glass is the chair of the Medicinal Cannabis Research Collaborative, a group that was established to design and carry out research around medicinal use of cannabis, and to provide advice as appropriate to others in this area (government, policy makers or people in the medicinal cannabis industry). She is not paid for this role.

Lung cancer drug development requires screening in animal models. We aimed to develop orthotopic models of human non-small lung cancer using A549 and H3122 cells delivered by tail vein injection. This procedure has been used previously for a mouse lung cancer (Lewis lung carcinoma) and as a model of human breast cancer metastasis to lung. We report that the procedure led to poor animal condition 7-8 weeks after injection, and produced lesions in the lungs visible at necropsy but we were unable identify individual cancer cells using immunohistochemistry. We conclude that if this method is to produce a model that can be used in drug experiments, improvements are required for cancer cell detection post mortem, such as by using of a fluorescently tagged human lung cancer cell line., Competing Interests: No competing interests were disclosed.

Background: The degree to which cannabis use causes long term harm to mental functioning is contentious., Objective: To determine the evidence for residual and long term effects of cannabis use on mental functions., Method: Comprehensively review human studies addressing detrimental effects on human mental and life functioning., Results: Heavy use causes immediate effects on perception, mood and sedation, but also deficits in cognitive ability. But cessation following heavy use has withdrawal effects and is associated with residual effects on cognition that may last for several weeks. Heavy use also raises the risk of impoverishment of life outcomes and a decline in socioeconomic status as well as the risk of mental health problems. Young age at the start of heavy cannabis use causes a risk of lifelong detrimental effects, and as a worst case together with genetic vulnerability exacerbate a predisposition to schizophrenia., Conclusion: Heavy regular use of cannabis that begins in adolescence heightens the risk of longterm impairment of life and mental functioning., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Cannabinoid CB2 receptor agonists are under investigation for clinical use. At the same time, synthetic cannabinoids have been implicated in a number of deaths. One cause of death is thought to be cardiac arrest subsequent to extreme tachycardia. Central mechanisms are thought to play a role in this, with CB1 but not CB2 receptors thought to mediate central effects. However, the direct effects of cannabinoids on the heart are less well understood. We therefore tested the effects of cannabinoids on isolated rat atria to test whether activation of myocardial CB1 and CB2 receptors could contribute to tachycardia. Although we found a moderate effect that can be attributed to CB1 receptors, we did not find any evidence for chronotropic effects by a CB2 receptor activation. Our results indicate that cannabinoid cardiotoxicity may partially involve CB1 receptors in the myocardium, and that CB2 receptor agonists are unlikely to have significant effects on the heart.

The claim has been made that it can be proven that most published findings in medical, biological, and allied sciences are false and that the reason for this can be proven and explained with a mathematical model. It has not, however, been mathematically proven that most research findings are false, and this can be proven. The model used in the proof is incoherent and has been falsified. Furthermore, advice to researchers derived from the model is misleading and distracts from more important issues in experimental standards., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Although β2-receceptor agonists are powerful bronchodilators and are at the forefront of asthma symptom relief, patients who use them frequently develop partial resistance to them. This can be a particularly serious problem during severe attacks, where high dose β2-agonist treatment is the front line therapy. Alternative bronchodilators are urgently needed. In this article we review the evidence for the bronchodilator effects of the cannabinoid CB1 receptor tetrahydrocannabinol (THC) and suggest that the mechanisms of action for these effects are sufficiently independent of the mechanisms of standard bronchodilators to warrant clinical investigation. Specifically, clinical trials testing the bronchodilator effects of THC in β2 agonist resistant asthmatic patients would show whether THC could fill the role of rescue bronchodilator in cases of β2 agonist resistance., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

There is now a considerable body of evidence for sildenafil possessing anticancer properties. In this article, we argue the case for testing sildenafil as a lung cancer therapy chemoadjuvant. Currently, lung cancer is a disease with insufficient treatment options, with only 20% of patients responding to systemic chemotherapy, and even incremental potential improvements should be explored. We review the literature concerning the biochemical, physiological and metabolic effects on cancer cells by sildenafil alone, and when combined with chemotherapeutic agents. Most studies have shown that sildenafil is cytotoxic to cancer cells, both as a monotherapy and as a chemoadjuvant. Sildenafil enhances cancer cell apoptosis when used as a chemoadjuvant both in vitro and in vivo. In particular, in rodent experiments sildenafil has decreased tumour size compared to chemotherapy alone. Sildenafil has also been proven as an agent to decrease drug-efflux by cancer cells and increases blood perfusion to lung tissue, which can potentially increase the dosage of chemotherapeutic agents delivered to lung cancer cells compared to healthy tissue. In addition, the proven clinical effects of sildenafil on other lung diseases suggest that it could improve other patient outcomes, such as right ventricular function and quality of life. Sildenafil may also extend the half-life of docetaxel and some small molecule inhibitors used in lung cancer treatment by acting as an inhibitor of CYP3A4. We conclude that the evidence strongly warrants clinical investigation into the use of sildenafil as an agent for the treatment of lung-cancer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

ALK positive non-small cell lung cancer is highly responsive to ALK inhibitors such as crizotinib, but drug resistance typically develops within a year of treatment. In this study we investigated whether IGF-1R is an independent druggable target in ALK-positive lung cancer cells. We confirmed that combination ALK and IGF-1R inhibitor treatment is synergistically cytotoxic to ALK-positive lung cancer cells and that this remains the case for at least 12 days after initial exposure to crizotinib. ALK-positive cells with acquired resistance to crizotinib did not acquire cross-resistance to IGF-1R inhibition, though combination treatment in the resistant cells gave additive rather than synergistic cytotoxicity. We concluded that IGF-1R is an independent druggable target in ALK-positive lung cancer and support the trial of combination treatment.

Cannabinoids produce analgesia through a variety of mechanisms. It has been proposed that one mechanism is by modulating the release of CGRP in the spinal cord pain pathways. Previous studies have reported that cannabinoids, particularly CB2 receptor agonists, can modulate CGRP release in the isolated rat spinal cord. In our experiments, the TRPV1 agonist capsaicin evoked CGRP release and this was supressed by the TRPV1 antagonist capsazepine and by the opioid receptor agonist DAMGO. However, none of the cannabinoid receptor agonists that we tested were able to modulate evoked CGRP release; including WIN 55,212-2, methanandamide, and GW405833. These results question the role of spinal cord cannabinoid receptors in the regulation of CGRP signaling., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Cannabinoid receptor agonists are moderately effective at reducing neuropathic pain but are limited by psychoactivity. We developed a styrene maleic acid (SMA) based on the cannabinoid WIN 55,212-2 (WIN) and tested in a rat model of neuropathic pain and in the rotarod test. We hypothesized that miceller preparation can ensure prolonged plasma half-life being above the renal threshold of excretion. Furthermore, SMA-WIN could potentially reduce the central nervous system effects of encapsulated WIN by limiting its transport across the blood-brain barrier. Using the chronic constriction injury model of sciatic neuropathy, the SMA-WIN micelles were efficacious in the treatment of neuropathic pain for a prolonged period compared to control (base WIN). Attenuation of chronic constriction injury-induced mechanical allodynia occurred for up to 8 h at a dose of 11.5 mg/kg of SMA-WIN micelles. To evaluate central effects on motor function, the rotarod assessment was utilized. Results showed initial impairment caused by SMA-WIN micelles to be identical to WIN control for up to 1.5 h. Despite this, the SMA-WIN micelle formulation was able to produce prolonged analgesia over a time when there was decreased impairment in the rotarod test compared with base WIN.

Hypoxia-ischemia (HI) is a model of cerebral ischemia used to model neonatal hypoxia and to study brain damage. Interpreting the results of experiments that use HI rests partly on the assumption that only the brain suffers major damage. In this study, we demonstrate that HI also has adverse consequences on the heart. Both infarction scoring and measurements of troponin I indicate cardiac damage subsequent to HI. These results indicate that the effects of HI on the heart may confound the interpretation of experiments that have used HI to study neuroprotection or other aspects of brain damage.

Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues., (© The Author(s) 2014.)

The cannabinoid CB2 receptor has been under investigation as a potential target for neuroprotection with the suppression of neuroinflammation as the proposed mechanism of action. Several studies have now reported that CB2 agonists are neuroprotective in models of cerebral ischemia. However, these studies have tended to measure brain infarctions in rodents 1-3 days after drug administration and have not assessed behavioral outcomes. As it has been shown that apparent protection soon after injury is not necessarily correlated with improved outcome after several weeks, we tested the CB2 selective agonist GW405833 in a model of cerebral hypoxia-ischemia, and assessed histological and behavioral outcomes 15 days after injury. Many putatively neuroprotective drugs have failed to translate from promising preclinical results to clinical success. We designed our experiments to not only stringently test CB2 mediated neuroprotection, but also to test several drug administration regimens to maximize the chance of detecting any therapeutic effect. However, GW405833 failed to provide neuroprotection in any of our experiments. These results challenge how far the results of earlier studies into CB2 mediated neuroprotection as measured at early time points may be extrapolated to later time points or to other models., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Cerebral ischaemia triggers various physiological processes, some of which have been considered deleterious and others beneficial. These processes have been characterized in one influential model as being part of a transition from injury to repair processes. We argue that another important distinction is between dysregulated and regulated processes. Although intervening in the course of dysregulated processes may be neuroprotective, this is unlikely to be true for regulated processes. This is because from an evolutionary perspective, regulated complex processes that are conserved across many species are likely to be adaptive and provide a survival advantage. We argue that the neuroinflammatory cascade is an adaptive process in this sense, and contrast this with a currently popular theory which we term the maladaptive immune response theory. We review the evidence from clinical and preclinical pharmacology with respect to this theory, and deduced that the evidence is inconclusive at best, and probably falsifies the theory. We argue that this is why there are no anti-inflammatory treatments for cerebral ischaemia, despite 30 years of seemingly promising preclinical results. We therefore propose an opposing theory, which we call the adaptive immune response hypothesis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Statins reduce the risk of death from cardiovascular disease in millions of people worldwide. Recent pharmacovigilance data has suggested that people taking statins have an increased risk of psychiatric adverse events such as amnesia and anxiety. This study aimed to investigate the possibility of statin-induced amnesia through animal models of memory and learning. We conducted extracellular field recordings of synaptic transmission in area CA1 of hippocampal slices to examine the effects of acute cholesterol lowering with lipid lowering drugs. We also assessed the effect of six weeks of simvastatin (2mg/kg/d) and atorvastatin (1mg/kg/d) treatment using the Morris water maze. Long Term Potentiation (LTP) was significantly diminished in the presence of 3µM atorvastatin or simvastatin and by the cholesterol sequestering agent methyl-β-cyclodextrin (MBCD). The effects were reversed in the MBCD but not the statin treated slices by the addition of cholesterol. In the water maze, statin treatment did not cause any deficits in the first five days of reference memory testing, but statin treated guinea pigs preformed significantly worse than control animals in a working memory test. The deficits observed in our experiments in water maze performance and hippocampal LTP are suggestive of statin induced changes in hippocampal plasticity. The effects on LTP are independent of cholesterol regulation, and occur at concentrations that may be relevant to clinical use. Our results may help to explain some of the behavioural changes reported in some people after beginning statin treatment., (© 2013 Published by Elsevier B.V.)

In recent decades, growth of computing power has facilitated powerful techniques for reconstructing evolutionary relationships from online genetic and proteomic databases. These methods are useful tools for pharmacologists for analyzing relationships between receptors and associated enzymes. Phylogenetic analysis can help generate hypotheses and leads for experimentation. Reconstruction of molecular phylogenies for the nonspecialist is described in this article using the example of the orphaned g protein coupled receptor GPR18.

The delivery of some classes of drugs is challenging. Solubility, absorption, distribution, and duration of action may all be altered by combination with vehicle molecules. It has already been discovered that polyethylene glycol - which is used as a stabiliser in peptide drug formulations - has biological activity in its own right, including potential neuroprotective properties. In this article we review the evidence for confounding activity for four distinct compounds that have been used as solvents and/or carrier molecules for the delivery of lipophilic drugs under investigation for potential neuroprotective properties. We discuss the evidence that cyclodextrins, ethanol, dimethyl sulphoxide, and a castor oil derivative - Cremophor™ EL - have all been found to have mild to moderate neuroprotective effects. We argue that this has probably reduced the statistical power and increased the Type II error rates of neuroprotection experiments that have employed these vehicles, and suggest experimental design considerations to help correct the problem. However, we also note that the properties of these compounds may represent an opportunity for drug development, particularly for the newer compounds that have been subject to only limited experimental investigation.

Biomedical animal models predict clinical efficacy with varying degrees of success. An important feature of in vivo modeling is matching the age of the animals used in preclinical research to the age of peak incidence for a disease state in humans. However, growth and development are highly variable between mammalian species, and age matching is always based on assumptions about the nature of development. We propose that researchers commonly make the assumption that developmental sequences are highly conserved between mammalian species--an assumption that we argue is often incorrect. We instead argue that development is often a modular process. Consideration of the modular nature of development highlights the difficulty in matching animal ages to human ages in a one-to-one scalar manner. We illustrate this with a discussion of the problem of age matching rodents to humans for neuroprotection experiments, and argue that researchers should pay deliberate attention to the modularity of developmental processes in order to optimally match ages between species in biomedical research.

The question of whether cannabinoid CB₂ receptors are expressed on neurons in the brain and under what circumstances they are expressed is controversial in cannabinoid neuropharmacology. While some studies have reported that CB₂ receptors are not detectable on neurons under normal circumstances, other studies have reported abundant neuronal expression. One reason for these apparent discrepancies is the reliance on incompletely validated CB₂ receptor antibodies and immunohistochemical procedures. In this study, we demonstrate some of the methodological problems encountered using three different commercial CB₂ receptor antibodies. We show that (1) the commonly used antibodies that were confirmed by many of the tests used for antibody validation still failed when examined using the knockout control test; (2) the coherence between the labeling patterns provided by two antibodies for the same protein at different epitopes may be misleading and must be validated using both low- and high-magnification microscopy; and (3) although CB₂ receptor antibodies may label neurons in the brain, the protein that the antibodies are labeling is not necessarily CB₂. These results showed that great caution needs to be exercised when interpreting the results of brain immunohistochemistry using CB₂ receptor antibodies and that, in general, none of the tests for antibody validity that have been proposed, apart from the knockout control test, are reliable., (Copyright © 2013 Elsevier B.V. All rights reserved.)

O-1602 is a cannabidiol analogue that does not bind with high affinity to either the cannabinoid CB1 receptor or CB2 receptor. However, there is evidence that O-1602 has significant effects in the central nervous system as well as other parts of the body. Depending upon the model, O-1602 has anti-inflammatory or pronociceptive effects, mediated through a number of distinct receptors. This article reviews the evidence for functional effects of O-1602, particularly in the CNS, and describes its known targets as they relate to these effects. These include the abnormal cannabidiol (Abn- CBD) receptor and GPR55. The GPR18 receptor has been identified with the Abn-CBD receptor, and therefore the evidence that O-1602 also acts at GPR18 is also reviewed. Finally, the evidence that these receptor targets are expressed in the CNS and the phenotypes of cells expressing these targets is discussed, concluding with a discussion of the prospects for O-1602 as a therapeutic agent in the CNS.

In the last decade a number of products have appeared in various countries that contain synthetic cannabinoids. This article reviews the history of the sale of these drugs, and the evidence that they contain synthetic cannabinoids. The biochemistry of the synthetic cannabinoids identified thus far is discussed, including a discussion of chemical structures and biochemical targets. The cannabinoid receptor targets for these drugs are discussed, as well as other possible targets such as serotonin receptors. Evidence for the abuse potential of these drugs is reviewed. The toxicity of synthetic cannabinoids and cannabinoid products is reviewed and compared to that of the phytocannabinoid Δ9- tetrahydrocannabinol (THC). As cannabinoids are a structurally diverse class of drugs, it is concluded that synthetic cannabinoids should be classified by biological activity rather than by structure, and that if this isn't done, novel synthetic cannabinoids will continue to emerge that fall outside of current regulatory classification models.