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ObjectivesThe optimal timing and modality of surveillance after endovascular intervention for peripheral arterial disease is controversial, and no randomised trial to assess the value of peripheral endovascular intervention has ever been performed. The aim of this systematic review was to examine the practice of surveillance following peripheral endovascular intervention in randomised trials.Data SourcesMedline, Embase, Cochrane Library, and WHO trial registry databases.MethodsSystematic review of the literature was performed to capture surveillance strategies used in randomised trials comparing endovascular interventions. Surveillance protocols were assessed for completeness, modalities used, duration, and intensity.ResultsNinety-six different surveillance protocols were reported in 103 trials comparing endovascular interventions. Protocol specification was incomplete in 32% of trials.The majority of trials used multiple surveillance modalities (mean 3.46 modalities), most commonly clinical examination (96%), ankle-brachial index (80%), duplex ultrasound (75%), and digital subtraction angiography (51%). Trials involving infrapopliteal lesions used more angiographic surveillance than trials with femoropopliteal lesions (p=0.006).The median number of surveillance visits in the first 12 months after intervention was three and the mean surveillance duration was 21 months. Trials treating infrapopliteal vessels had a higher surveillance intensity compared to those treating femoropopliteal lesions in the first 12 months after endovascular intervention (mean 5 vs. 3 surveillance visits; p=0.017). Trials with drug-eluting devices had longer surveillance duration compared to those without (mean 26 vs. 19 months; p=0.020).ConclusionsThere is a high level of variation in the modality, duration, and intensity of surveillance protocols used in randomised trials comparing different types of peripheral endovascular arterial intervention. Further research is required to determine the value and impact of post-procedural surveillance on patient outcomes.

Background Detailed prevalence estimates of BRAFV600 mutations and BRAF inhibitor (BRAFi) treatment responses in V600-mutant glioma will inform trial development. Methods Our systematic review analyzed overall prevalence of BRAFV600 mutations in glioma and BRAFi treatment response. Results Based on 13 682 patients in 182 publications, the prevalence of BRAFV600 in epithelioid glioblastoma (eGBM) was 69% [95% CI: 45–89%]; pleomorphic xanthoastrocytoma (PXA): 56% [48–64%] anaplastic pleomorphic xanthoastrocytoma (aPXA): 38% [23–54%], ganglioglioma (GG): 40% [33–46%], and anaplastic ganglioglioma (aGG): 46% [18–76%]. Prevalence in astroblastoma was 24% [8–43%], desmoplastic infantile astrocytoma (DIA): 16% [0–57%], subependymal giant cell astrocytoma (SEGA): 8% [0–37%], dysembryoplastic neuroepithelial tumor (DNET): 3% [0–11%], diffuse astrocytoma (DA): 3% [0–9%], and pilocytic astrocytoma (PA): 3% [2–5%]. We reviewed 394 V600-mutant gliomas treated with BRAFi from 130 publications. One hundred and twenty-nine pediatric low-grade gliomas showed 4 (3.1%) complete response (CR); 53 (41.1%) partial response (PR); 64 (49.6%) stable disease (SD) and 8 (6.2%) progressive disease (PD). 25 pediatric high-grade gliomas showed CR; PR; SD; PD in 4 (16.0%); 10 (40.0%), 4 (16.0%); and 7 (28.0%) respectively. Thirty-nine adult low-grade gliomas showed CR; PR; SD; PD of 4 (10.3%); 17 (43.6%); 16 (41.0%) and 2 (5.1%) respectively. Ninety-seven adult high-grade gliomas showed CR; PR; SD; PD of 6 (6.2%); 31 (32.0%); 27 (27.8%); and 33 (34.0%) respectively. Conclusions BRAFV600 prevalence is highest in eGBM, PXA, aPXA, GG, aGG, and lower in astroblastoma, DIA, SEGA, DNET, DA, and PA. Our data provide the rationale for adjuvant clinical trials of BRAFi in V600-mutant glioma.

BackgroundThe DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of tumor cells to alkylating agents. It is a predictive biomarker in high-grade gliomas treated with temozolomide, however, there is no consensus on which test method, methylation sites, and cutoff values to use.MethodsWe performed a Cochrane Review to examine studies using different techniques to measure MGMT and predict survival in glioblastoma patients treated with temozolomide. Eligible longitudinal studies included (i) adults with glioblastoma treated with temozolomide with or without radiotherapy, or surgery; (ii) where MGMT status was determined in tumor tissue, and assessed by 1 or more technique; and (iii) where overall survival was an outcome parameter, with sufficient information to estimate hazard ratios (HRs). Two or more methods were compared in 32 independent cohorts with 3474 patients.ResultsMethylation-specific PCR (MSP) and pyrosequencing (PSQ) techniques were more prognostic than immunohistochemistry for MGMT protein, and PSQ is a slightly better predictor than MSP.ConclusionsWe cannot draw strong conclusions about use of frozen tissue vs formalin-fixed paraffin-embedded in MSP and PSQ. Also, our meta-analysis does not provide strong evidence about the best CpG sites or threshold. MSP has been studied mainly for CpG sites 76-80 and 84-87 and PSQ at CpG sites ranging from 72 to 95. A cutoff threshold of 9% for CpG sites 74-78 performed better than higher thresholds of 28% or 29% in 2 of the 3 good-quality studies. About 190 studies were identified presenting HRs from survival analysis in patients in which MGMT methylation was measured by 1 technique only.

Background: Glioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine–DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide.Objectives: To assess which way of measuring methylation of the MGMT promoter best predicts survival when people with glioblastoma are treated with temozolomide.Search methods: We searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies we additionally searched NHS Economic Evaluation Database (EED) up to December 2014.Selection criteria: Eligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiation therapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with su*icient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focus mainly on studies comparing two or more methods, and list brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies, and cost analysis.Data collection and analysis: All steps of the identification and data extraction process for multiple-method studies were undertaken by two reviewers independently. We assessed risk of bias and applicability using our own modified and extended version of the QUIPS tool. Comparisons of different techniques, exact promoter regions (CpG sites) and thresholds for interpretation were compared within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios, using an imputed value of the correlation between results based on the same individuals.Main results: We included 32 independent cohorts involving 3474 people in which two or more methods were compared. In meta-analyses of ratios of hazard ratios (RHR), we found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds: (RHR = 1.31; 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR = 1.36; 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR = 1.14; 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarized 190 articles describing a single method for measuring MGMT promoter methylation status.Authors' conclusions: Pyrosequencing and methylation-specific PCR appear to be more prognostic for overall survival than immunohistochemistry. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 was found to perform better than higher thresholds of 28% or 29% in two of three good quality studies making such comparisons.

Background: The COVID-19 pandemic has caused considerable morbidity, mortality and disruption to people’s lives around the world. There are concerns that rates of suicide and suicidal behaviour may rise during and in its aftermath. Our living systematic review synthesises findings from emerging literature on incidence and prevalence of suicidal behaviour as well as suicide prevention efforts in relation to COVID-19, with this iteration synthesising relevant evidence up to 19th October 2020. Method: Automated daily searches feed into a web-based database with screening and data extraction functionalities. Eligibility criteria include incidence/prevalence of suicidal behaviour, exposure-outcome relationships and effects of interventions in relation to the COVID-19 pandemic. Outcomes of interest are suicide, self-harm or attempted suicide and suicidal thoughts. No restrictions are placed on language or study type, except for single-person case reports. We exclude one-off cross-sectional studies without either pre-pandemic measures or comparisons of COVID-19 positive vs. unaffected individuals. Results: Searches identified 6,226 articles. Seventy-eight articles met our inclusion criteria. We identified a further 64 relevant cross-sectional studies that did not meet our revised inclusion criteria. Thirty-four articles were not peer-reviewed (e.g. research letters, pre-prints). All articles were based on observational studies. There was no consistent evidence of a rise in suicide but many studies noted adverse economic effects were evolving. There was evidence of a rise in community distress, fall in hospital presentation for suicidal behaviour and early evidence of an increased frequency of suicidal thoughts in those who had become infected with COVID-19. Conclusions: Research evidence of the impact of COVID-19 on suicidal behaviour is accumulating rapidly. This living review provides a regular synthesis of the most up-to-date research evidence to guide public health and clinical policy to mitigate the impact of COVID-19 on suicide risk as the longer term impacts of the pandemic on suicide risk are researched.

ObjectiveTo determine the most effective interventions in recently detoxified, alcohol dependent patients for implementation in primary care.DesignSystematic review and network meta-analysis.Data sourcesMedline, Embase, PsycINFO, Cochrane CENTRAL, ClinicalTrials.gov, and the World Health Organization’s International Clinical Trials Registry Platform.Study selectionRandomised controlled trials comparing two or more interventions that could be used in primary care. The population was patients with alcohol dependency diagnosed by standardised clinical tools and who became detoxified within four weeks.Data extractionOutcomes of interest were continuous abstinence from alcohol (effectiveness) and all cause dropouts (as a proxy for acceptability) at least 12 weeks after start of intervention.Results64 trials (43 interventions) were included. The median probability of abstinence across placebo arms was 25%. Compared with placebo, the only intervention associated with increased probability of abstinence and moderate certainty evidence was acamprosate (odds ratio 1.86, 95% confidence interval 1.49 to 2.33, corresponding to an absolute probability of 38%). Of the 62 included trials that reported all cause dropouts, interventions associated with a reduced number of dropouts compared with placebo (probability 50%) and moderate certainty of evidence were acamprosate (0.73, 0.62 to 0.86; 42%), naltrexone (0.70, 0.50 to 0.98; 41%), and acamprosate-naltrexone (0.30, 0.13 to 0.67; 17%). Acamprosate was the only intervention associated with moderate confidence in the evidence of effectiveness and acceptability up to 12 months. It is uncertain whether other interventions can help maintain abstinence and reduce dropouts because of low confidence in the evidence.ConclusionsEvidence is lacking for benefit from interventions that could be implemented in primary care settings for alcohol abstinence, other than for acamprosate. More evidence from high quality randomised controlled trials is needed, as are strategies using combined interventions (combinations of drug interventions or drug and psychosocial interventions) to improve treatment of alcohol dependency in primary care.Systematic review registrationPROSPERO CRD42016049779.

OBJECTIVE: Randomised trials of new devices for peripheral arterial endovascular intervention are published regularly. The evidence for which antiplatelet and/or anticoagulant (antithrombotic) therapy to use after an intervention is lacking. The aim of this systematic review was to examine the antithrombotic regimens in randomised trials for peripheral arterial endovascular intervention to understand choices made and trends with time or type of device.METHODS: Data sources were the Medline, Embase, and Cochrane Library databases. Randomised trials including participants with peripheral arterial disease undergoing any endovascular arterial intervention were included. Trial methods were assessed to determine whether an antithrombotic protocol had been specified, its completeness, and the agent(s) prescribed. Antithrombotic therapy protocols were classed as peri-procedural (preceding and during intervention), immediate post-procedural (up to 30 days following intervention), and maintenance post-procedural (therapy continuing beyond 30 days).RESULTS: Ninety-four trials were included in narrative synthesis. Study quality was low. None of the trials justified their antithrombotic therapy protocol. Only 29% of trials had complete peri-procedural antithrombotic protocols, and 34% had complete post-procedural protocols. In total, 64 different peri-procedural protocols, and 51 separate post-procedural protocols were specified. Antiplatelet monotherapy and unfractionated heparin were the most common regimen choices in the peri-procedural setting, and dual antiplatelet therapy (55%) was most commonly utilised post procedure. Over time there has been an increasing tendency to use dual therapy (p < .001). This corresponds with the introduction of newer technologies and trials focussed on below knee intervention.CONCLUSION: Randomised trials comparing different types of peripheral endovascular arterial intervention have a high level of heterogeneity in their antithrombotic regimens. Antiplatelet therapy needs to be standardised in trials comparing endovascular technologies to reduce potential confounding. To do this, an independent randomised trial specifically examining antiplatelet therapy following peripheral arterial endovascular intervention is needed.

Assessment of risk of bias is regarded as an essential component of a systematic review on the effects of an intervention. The most commonly used tool for randomised trials is the Cochrane risk-of-bias tool. We updated the tool to respond to developments in understanding how bias arises in randomised trials, and to address user feedback on and limitations of the original tool.

In older individuals, pulmonary artery pressure rises markedly during exercise, probably due in part to increased pulmonary vascular resistance and in part to an increase in left-heart filling pressure. Older individuals also show more marked pulmonary vascular response to hypoxia at rest. Treatment with intravenous iron reduces the rise in pulmonary artery pressure observed during hypoxia. Here, we test the hypothesis that intravenous iron administration may also attenuate the rise in pulmonary artery pressure with exercise in older individuals. In a randomized double-blind placebo-controlled physiology study in 32 healthy participants aged 50-80 years, we explored the hypothesis that iron administration would deliver a fall in systolic pulmonary artery pressure (SPAP) during moderate cycling exercise (20 min duration; increase in heart rate of 30 min-1 ) and a change in maximal cycling exercise capacity ( V ˙ O 2 m a x ). Participants were studied before, and at 3 h to 8 weeks after, infusion. SPAP was measured using Doppler echocardiography. Iron administration resulted in marked changes in indices of iron homeostasis over 8 weeks, but no significant change in hemoglobin concentration or inflammatory markers. Resting SPAP was also unchanged, but SPAP during exercise was lower by ~3 mmHg in those receiving iron (P V ˙ O 2 m a x remained unaffected in the iron-replete healthy participants studied here, this study demonstrates for the first time the ability of intravenous iron supplementation to reduce systolic pulmonary artery pressure during exercise.

Background: The COVID-19 pandemic has caused morbidity and mortality, as well as, widespread disruption to people’s lives and livelihoods around the world. Given the health and economic threats posed by the pandemic to the global community, there are concerns that rates of suicide and suicidal behaviour may rise during and in its aftermath. Our living systematic review (LSR) focuses on suicide prevention in relation to COVID-19, with this iteration synthesising relevant evidence up to June 7th 2020. Method: Automated daily searches feed into a web-based database with screening and data extraction functionalities. Eligibility criteria include incidence/prevalence of suicidal behaviour, exposure-outcome relationships and effects of interventions in relation to the COVID-19 pandemic. Outcomes of interest are suicide, self-harm or attempted suicide and suicidal thoughts. No restrictions are placed on language or study type, except for single-person case reports. Results: Searches identified 2070 articles, 29 (28 studies) met our inclusion criteria, of which 14 articles were research letters or pre-prints awaiting peer review. All articles reported observational data: 12 cross-sectional; eight case series; five modelling; and three service utilisation studies. No studies reported on changes in rates of suicidal behaviour. Case series were largely drawn from news reporting in low/middle income countries and factors associated with suicide included fear of infection, social isolation and economic concerns. Conclusions: A marked improvement in the quality of design, methods, and reporting in future studies is needed. There is thus far no clear evidence of an increase in suicide, self-harm, suicidal behaviour, or suicidal thoughts associated with the pandemic. However, suicide data are challenging to collect in real time and economic effects are evolving. Our LSR will provide a regular synthesis of the most up-to-date research evidence to guide public health and clinical policy to mitigate the impact of COVID-19 on suicide. PROSPERO registration: CRD42020183326 01/05/2020

BACKGROUND: Surgical site infection (SSI) rates vary from 1% to 5% in the month following surgery. Due to the large number of surgical procedures conducted annually, the costs of these SSIs can be considerable in financial and social terms. Many interventions are used with the aim of reducing the risk of SSI in people undergoing surgery. These interventions can be broadly delivered at three stages: preoperatively, intraoperatively and postoperatively. The intraoperative interventions are largely focused on decontamination of skin using soap and antiseptics; the use of barriers to prevent movement of micro‐organisms into incisions; and optimising the patient's own bodily functions to promote best recovery. Both decontamination and barrier methods can be aimed at people undergoing surgery and operating staff. Other interventions focused on SSI prevention may be aimed at the surgical environment and include methods of theatre cleansing and approaches to managing theatre traffic. OBJECTIVES: To present an overview of Cochrane Reviews of the effectiveness and safety of interventions, delivered during the intraoperative period, aimed at preventing SSIs in all populations undergoing surgery in an operating theatre. METHODS: Published Cochrane systematic reviews reporting the effectiveness of interventions delivered during the intraoperative period in terms of SSI prevention were eligible for inclusion in this overview. We also identified Cochrane protocols and title registrations for future inclusion into the overview. We searched the Cochrane Library on 01 July 2017. Two review authors independently screened search results and undertook data extraction and 'Risk of bias' and certainty assessment. We used the ROBIS (risk of bias in systematic reviews) tool to assess the quality of included reviews, and we used GRADE methods to assess the certainty of the evidence for each outcome. We summarised the characteristics of included reviews in the text and in additional tables. MAIN RESULTS: We included 32 Cochrane Reviews in this overview: we judged 30 reviews as being at low risk of bias and two at unclear risk of bias. Thirteen reviews had not been updated in the past three years. Two reviews had no relevant data to extract. We extracted data from 30 reviews with 349 included trials, totaling 73,053 participants. Interventions assessed included gloving, use of disposable face masks, patient oxygenation protocols, use of skin antiseptics for hand washing and patient skin preparation, vaginal preparation, microbial sealants, methods of surgical incision, antibiotic prophylaxis and methods of skin closure. Overall, the GRADE certainty of evidence for outcomes was low or very low. Of the 77 comparisons providing evidence for the outcome of SSI, seven provided high‐ or moderate‐certainty evidence, 39 provided low‐certainty evidence and 31 very low‐certainty evidence. Of the nine comparisons that provided evidence for the outcome of mortality, five provided low‐certainty evidence and four very low‐certainty evidence. There is high‐ or moderate‐certainty evidence for the following outcomes for these intraoperative interventions. (1) Prophylactic intravenous antibiotics administered before caesarean incision reduce SSI risk compared with administration after cord clamping (10 trials, 5041 participants; risk ratio (RR) 0.59, 95% confidence interval (CI) 0.44 to 0.81; high‐certainty evidence ‐ assessed by review authors). (2) Preoperative antibiotics reduce SSI risk compared with placebo after breast cancer surgery (6 trials, 1708 participants; RR 0.74, 95% CI 0.56 to 0.98; high‐certainty evidence ‐ assessed by overview authors). (3) Antibiotic prophylaxis probably reduce SSI risk in caesarean sections compared with no antibiotics (82 relevant trials, 14,407 participants; RR 0.40, 95% CI 0.35 to 0.46; moderate‐certainty evidence; downgraded once for risk of bias ‐ assessed by review authors). (4) Antibiotic prophylaxis probably reduces SSI risk for hernia repair compared with placebo or no treatment (17 trials, 7843 participants; RR 0.67, 95% CI 0.54 to 0.84; moderate‐certainty evidence; downgraded once for risk of bias ‐ assessed by overview authors); (5) There is currently no clear difference in the risk of SSI between iodine‐impregnated adhesive drapes compared with no adhesive drapes (2 trials, 1113 participants; RR 1.03, 95% CI 0.66 to 1.60; moderate‐certainty evidence; downgraded once for imprecision ‐ assessed by review authors); (6) There is currently no clear difference in SSI risk between short‐term compared with long‐term duration antibiotics in colorectal surgery (7 trials; 1484 participants; RR 1.05 95% CI 0.78 to 1.40; moderate‐certainty evidence; downgraded once for imprecision ‐ assessed by overview authors). There was only one comparison showing negative effects associated with the intervention: adhesive drapes increase the risk of SSI compared with no drapes (5 trials; 3082 participants; RR 1.23, 95% CI 1.02 to 1.48; high‐certainty evidence ‐ rated by review authors). AUTHORS' CONCLUSIONS: This overview provides the most up‐to‐date evidence on use of intraoperative treatments for the prevention of SSIs from all currently published Cochrane Reviews. There is evidence that some interventions are useful in reducing SSI risk for people undergoing surgery, such as antibiotic prophylaxis for caesarean section and hernia repair, and also the timing of prophylactic intravenous antibiotics administered before caesarean incision. Also, there is evidence that adhesive drapes increase SSI risk. Evidence for the many other treatment choices is largely of low or very low certainty and no quality‐of‐life or cost‐effectiveness data were reported. Future trials should elucidate the relative effects of some treatments. These studies should focus on increasing participant numbers, using robust methodology and being of sufficient duration to adequately assess SSI. Assessment of other outcomes such as mortality might also be investigated as part of non‐experimental prospective follow‐up of people with SSI of different severity, so the risk of death for different subgroups can be better understood.

Exposure to sustained hypoxia of 8 h duration increases the sensitivity of the pulmonary vasculature to acute hypoxia, but it is not known whether exposure to sustained hyperoxia affects human pulmonary vascular control. We hypothesized that exposure to 8 h of hyperoxia would diminish the hypoxic pulmonary vasoconstriction (HPV) that occurs in response to a brief exposure to hypoxia. Eleven healthy volunteers were studied in a crossover protocol with randomization of order. Each volunteer was exposed to acute isocapnic hypoxia (end-tidal PO2 = 50 mmHg for 10 min) before and after 8 h of hyperoxia (end-tidal PO2 = 420 mmHg) or euoxia (end-tidal PO2 = 100 mmHg). After at least three days, each volunteer returned and was exposed to the other condition. Systolic pulmonary artery pressure (an index of HPV) and cardiac output were measured using Doppler echocardiography. Eight hours of hyperoxia had no effect on HPV or the response of cardiac output to acute hypoxia.

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The introduction of a functionalised amido substituent into a series of 1-(biphenylmethylacetamido)-pyrimidones has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency and very encouraging developability properties. Diethylaminoethyl derivative 32, SB-435495, was selected for progression to man.

Oral bioavailability measurements in rats for over 1100 drug candidates studied at SmithKline Beecham Pharmaceuticals (now GlaxoSmithKline) have allowed us to analyze the relative importance of molecular properties considered to influence that drug property. Reduced molecular flexibility, as measured by the number of rotatable bonds, and low polar surface area or total hydrogen bond count (sum of donors and acceptors) are found to be important predictors of good oral bioavailability, independent of molecular weight. That on average both the number of rotatable bonds and polar surface area or hydrogen bond count tend to increase with molecular weight may in part explain the success of the molecular weight parameter in predicting oral bioavailability. The commonly applied molecular weight cutoff at 500 does not itself significantly separate compounds with poor oral bioavailability from those with acceptable values in this extensive data set. Our observations suggest that compounds which meet only the two criteria of (1) 10 or fewer rotatable bonds and (2) polar surface area equal to or less than 140 A(2) (or 12 or fewer H-bond donors and acceptors) will have a high probability of good oral bioavailability in the rat. Data sets for the artificial membrane permeation rate and for clearance in the rat were also examined. Reduced polar surface area correlates better with increased permeation rate than does lipophilicity (C log P), and increased rotatable bond count has a negative effect on the permeation rate. A threshold permeation rate is a prerequisite of oral bioavailability. The rotatable bond count does not correlate with the data examined here for the in vivo clearance rate in the rat.

Tibetan natives have lived on the Tibetan plateau (altitude ∼ 4,000 m) for at least 25,000 years, and as such they are adapted to life and reproduction in a hypoxic environment. Recent studies have identified two genetic loci, EGLN1 and EPAS1, that have undergone natural selection in Tibetans, and further demonstrated an association of EGLN1/EPAS1 genotype with hemoglobin concentration. Both genes encode major components of the hypoxia-inducible factor (HIF) transcriptional pathway, which coordinates an organism's response to hypoxia. Patients living at sea level with genetic disease of the HIF pathway have characteristic phenotypes at both the integrative-physiology and cellular level. We sought to test the hypothesis that natural selection to hypoxia within Tibetans results in related phenotypic differences. We compared Tibetans living at sea level with Han Chinese, who are Tibetans' most closely related major ethnic group. We found that Tibetans had a lower hemoglobin concentration, a higher pulmonary ventilation relative to metabolism, and blunted pulmonary vascular responses to both acute (minutes) and sustained (8 h) hypoxia. At the cellular level, the relative expression and hypoxic induction of HIF-regulated genes were significantly lower in peripheral blood lymphocytes from Tibetans compared with Han Chinese. Within the Tibetans, we found a significant correlation between both EPAS1 and EGLN1 genotype and the induction of erythropoietin by hypoxia. In conclusion, this study provides further evidence that Tibetans respond less vigorously to hypoxic challenge. This is evident at sea level and, at least in part, appears to arise from a hyporesponsive HIF transcriptional system.

A series of novel, fluorescent pyrido[1,2-a]quinoxalines have been synthesized with crystal structures obtained for several analogues. These heterocycles exhibit characteristic pH-dependent UV-visible absorption and fluorescence properties that may be utilized for in-situ pH-sensing in biological experiments. The pKa values for this series of compounds, ranging from 4.5 to 7.5, can be systematically varied by manipulating the substitutions on the quinoxaline ring. These novel fluorescent pH indicators are readily available in one synthetic step from commercially available starting materials therefore being significantly more straightforward and less expensive to synthesize than the current standard fluorescent indicators.

In this study, we demonstrate transdermal iontophoretic delivery of radiolabeled growth hormone-releasing peptide ([ 3 H]GHRP, [ 3 H]SK&F 110 679) from an iontophoretic patch delivery device in rats. [ 3 H]SK&F 110 679 was adsorbed on a hydrophilic microporous membrane, which was positioned on a support in the device and sealed in place. A silver/silver chloride electrode system was used for iontophoresis and a potential of 4.5 V (rectangular pulse, 40 kHz, 30% duty cycle) was applied using an Advance depolarizing pulse iontophoresis system (ADIS-4030). Current-dependent appearance of [ 3 H]SK&F 110 679 equivalents in blood, bile and urine did not depend on membrane loading between 1.8 and 8 mg adsorbed on the membrane. Blood levels of [ 3 H]SK&F 110 679 equivalents persisted for at least 2 h after the current was turned off, indicative of depot formation in the skin. Fractions of bile and urine were analyzed by reversed-phase high-performance liquid chromatography (HPLC). The radiochemical profiles were dominated by a single species, coeluting with intact [ 3 H]SK&F 110 679. A flux of at least 0.8–1.2 ug/h per cm 2 was achieved, indicating that, with appropriate optimization of the patch device, therapeutic levels of SK&F 110 679 in man may be attainable by transdermal iontophoresis.

In vitro intestinal permeability screening identified a compound, SB 217242, in the indane carboxylic acid series of endothelin receptor antagonists, with greatly improved permeability over the initial lead compound in this series. Based on mucosal-to-serosal and serosal-to-mucosal permeability studies, and on the fact that SB 217242 transport does not correlate with changes in mannitol permeability, it was concluded that SB 217242 follows a passive transcellular pathway. Permeability studies at different pH values demonstrate that the permeating species is the unionized form of SB 217242. This compound is well absorbed upon intraduodenal dosing in the rat (Ohlstein et al. (1996) J. Pharm. Exp. Ther. 276, 609–615).

Mycobacterium tuberculosis remains the leading cause of death by a bacterial pathogen worldwide. Increasing prevalence of multidrug-resistant organisms means prioritizing identification of targets for antituberculars. 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD), part of the cholesterol metabolism operon, is vital for survival within macrophage. The C-C bond hydrolase, HsaD, has a serine protease-like catalytic triad. We tested a range of serine protease and esterase inhibitors for their effects on HsaD activity. As well as providing a potential starting point for drug development, the data provides evidence for the mechanism of C-C bond hydrolysis. This screen also provides a route to initiate development of fragment-based inhibitors.

Growth hormone releasing peptide (GHRP, sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH 2 ) is a synthetic hexapeptide under consideration for transdermal iontophoretic drug delivery. Cyclic voltammetry, controlled-potential electrolysis, HPLC/UV analysis, LC/MS/MS analysis, and EPR spin-trapping studies indicate that the electrolysis-induced oxidative degradation of GHRP is likely to be mediated by electrogenerated oxygen radicals from the electrolysis of water. Within 2 h and up to 2.5 V versus an Ag/AgCl reference electrode, the peptide backbone remains largely intact. The chemical modifications are selectively on imidazole (histidine) and indole (tryptophan). Strategies for alleviating the electrolysis-induced degradation of GHRP are proposed.

Microdialysis was utilized to determine blood and brain distribution of spin-trapping nitrone compounds in the rat following intraperitoneal administration. In vivo quantitation by high-pressure liquid chromatography (HPLC) analysis, in vitro calibration of microdialysis probes, optimum perfusion rate, and the relationship of microdialysis sample recovery to tissue levels were evaluated in detail. The microdialysis sampling and HPLC analysis provided on-line, within-animal pharmacokinetic time-course determinations. At equimolar concentrations, 150 mg/kg alpha-phenyl-N-tert-butyl nitrone (PBN) or 165 mg/kg alpha-4-pyridyl-N-oxide N-tert-butyl nitrone (POBN) reached a similar, steady-state venous blood concentration of 224 +/- 21 microM and 210 +/- 10 microM, respectively. The POBN steady-state brain concentration was 149 +/- 9 microM, a significantly (p.05) lower concentration than in the blood. In contrast, the brain concentration of PBN was 331 +/- 25 microM, significantly (p.05) higher than its concentration in the blood. The increased brain distribution/penetration of PBN was attributed to its greater lipophilicity as measured by its octanol/water partition coefficient. All microdialysis results were validated by direct measurement of blood and brain levels at steady-state using conventional extraction procedures and assays. Also, the amount of tissue/cell bound versus unbound nitrones was determined by comparing the microdialysis "dialyzable" fraction with the total amount from whole tissue extracts. These data demonstrate that on-line determinations of nitrone spin-trap brain penetration/levels can be carried out accurately using in vivo microdialysis. The implication of these results for potential use of the microdialysis technique for detection of free radical products in in vivo animal models is discussed.

A series of novel, fluorescent pyrido[1,2-a]quinoxalines have been synthesized with crystal structures obtained for several analogues. These heterocycles exhibit characteristic pH-dependent UV-visible absorption and fluorescence properties that may be utilized for in-situ pH-sensing in biological experiments. The pKa values for this series of compounds, ranging from 4.5 to 7.5, can be systematically varied by manipulating the substitutions on the quinoxaline ring. These novel fluorescent pH indicators are readily available in one synthetic step from commercially available starting materials therefore being significantly more straightforward and less expensive to synthesize than the current standard fluorescent indicators.

Carvedilol, a beta-adrenergic blocker used to treat cardiovascular diseases, protects cell membranes from lipid peroxidative damage. Previous studies suggested the drug resides in a non-polar environment and partitions into cell membranes, perturbing their fluidity. Here differential scanning calorimetry (DSC) and fluorescence spectroscopy were applied to further investigate interactions of carvedilol with a liposome model. Results indicate the association is relatively unaffected by pH or temperature, but could be sensitive to liposome composition. The drug's carbazole group plays the dominant role in bilayer perturbation. Compared with other beta-blockers examined, carvedilol produced the strongest liposome DSC perturbation. Locations of carbazole and carvedilol in the liposome were determined using depth-dependent fluorescent probes. Both compounds are situated in the middle of the bilayer, consistent with strong hydrophobic interactions. This combination of high lipophilicity and specific chemical structure appear required for carvedilol's novel antioxidant activity, and may enhance cardioprotection.

Modification of the pyrimidone 5-substituent in clinical candidate SB-435495 has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A 2 with sub-nanomolar potency. Cyclopentyl fused derivative 21 , SB-480848, showed an enhanced in vitro and in vivo profile versus SB-435495 and has been selected for progression to man.

The introduction of a functionalised amido substituent into a series of 1-(biphenylmethylacetamido)-pyrimidones has given a series of inhibitors of recombinant lipoprotein-associated phospholipase A(2) with sub-nanomolar potency and very encouraging developability properties. Diethylaminoethyl derivative 32, SB-435495, was selected for progression to man.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRp), the primary catalytic enzyme of the HCV replicase complex. We established a biochemical RNA synthesis assay, using purified recombinant NS5B lacking the C-terminal 21 amino acid residues, to identify potential polymerase inhibitors from a high throughput screen of the GlaxoSmithKline proprietary compound collection. The benzo-1,2,4-thiadiazine compound 1 was found to be a potent, highly specific inhibitor of NS5B. This agent interacts directly with the viral polymerase and inhibits RNA synthesis in a manner noncompetitive with respect to GTP. Furthermore, in the absence of an in vitro-reconstituted HCV replicase assay employing viral and host proteins, the ability of compound 1 to inhibit NS5B-directed viral RNA replication was determined using the Huh7 cell-based HCV replicon system. Compound 1 reduced viral RNA in replicon cells with an IC(50) of approximately 0.5 microm, suggesting that the inhibitor was able to access the perinuclear membrane and inhibit the polymerase activity in the context of a replicase complex. Preliminary structure-activity studies on compound 1 led to the identification of a modified inhibitor, compound 4, showing an improvement in both biochemical and cell-based potency. Lastly, data are presented suggesting that these compounds interfere with the formation of negative and positive strand progeny RNA by a similar mode of action. Investigations are ongoing to assess the potential utility of such agents in the treatment of chronic HCV disease.

A series of C-1 oxazole isosteres of pseudomonic acid A (mupirocin) bearing a nitroheterocycle have been synthesized, and significant differences in both spectrum of activity and potency were found between these derivatives and mupirocin. Additionally, the antibacterial potency of two members of this class of compounds against mupirocin-resistant staphylococci could not be accounted for solely by inhibition of the target enzyme isoleucyl-tRNA synthetase (IRS), indicating an additional mode of action. The most potent compound, the nitrofuran 3f (SB 205952), was the most electron affinic derivative prepared and was transformed by NAD(P)H-dependent bacterial reductases at a rate similar to that for nitrofurantoin. The second mode of action of this compound may therefore arise from its reduction to a species with cellular targets other than IRS. In in vivo studies, 3f was shown to be a very effective agent by both the subcutaneous and oral routes of administration.

Objective: Oxygen radical mediated endothelial injury plays an important role in cardiovascular disease. Carvedilol, a new β blocker and antihypertensive agent, has been shown to have antioxidant activity. The aim of this study was to determine whether carvedilol protects oxygen radical induced endothelial injury. Methods: Cultured bovine pulmonary artery (BPAEC) and human umbilical vein endothelial cells (HUVEC) were used and oxygen radicals were generated by xanthine-xanthine oxidase or phorbol myristate acetate (PMA) activated human neutrophils. Cell injury was assessed by lactate dehydrogenase (LDH) release and cell death, or 51Cr release from prelabelled BPAEC. The electron paramagnetic resonance (EPR) spin trapping technique was used to detect the amount of radical spin adducts formed in cell lipids. Results: Carvedilol dose dependently inhibited xanthine-xanthine oxidase induced LDH release from BPAEC and HUVEC, with IC50 values of 3.8 μM and 2.6 μM, respectively, and significantly reduced cell death by xanthine-xanthine oxidase. Other β blockers tested (propranolol, labetalol, pindolol, and celiprolol) showed a mild effect or no effect at all. Increasing the time of pretreatment with carvedilol enhanced its cell protective effect against oxidative stress. Carvedilol also protected BPAEC dose dependently from PMA activated, neutrophil induced cell injury. Carvedilol had no effect on xanthine oxidase activity. EPR study confirmed that xanthine-xanthine oxidase induced the formation of lipid derived radicals in cell lipids and carvedilol scavenged free radicals, as indicated by the decreased EPR signal. Conclusions: Carvedilol protects endothelial cells against oxygen radical mediated cell injury and death by scavenging free radicals. The prevention of oxidative injury to endothelial cells might potentially contribute to the clinical beneficial effects of carvedilol as an antihypertensive agent. Cardiovascular Research 1994; 28 :400-406

The micellar properties of Poloxamer 213 (1), a Pluronic copolymer shown to affect lipid absorption and serum cholesterol level in experimental animals, are investigated by surface tension measurements, photon correlation spectroscopy (PCS), Reichardt's dye solubilization technique, differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR) spectrometry, and fluorescence spectroscopy. The clear inflection point at 3 x 10(-6) M (25 degrees C) observed in the surface tension-concentration curve may not represent the CMC for the formation of multimolecular aggregates. In the 10(-4) to 10(-2) M concentration range, the temperature-dependent transition (as the concentration of 1 increases) from the larger (hydrodynamic radius, Rh approximately 20-40 nm), highly hydrated aggregates to the contracted (Rh approximately 6-7 nm), less polar form occurs as a discontinuity. This process is endothermic, with an average delta H of 34.5 kcal/mol. At 25 degrees C, both the 25-(NBD-methylamino)-27-norcholesterol (fluorescence probe) and 3-Doxyl-5 alpha-cholestane (EPR spin probe) begin to show significant interaction with 1 in the 10(-3) M range. The sequestration of the fluorescent cholesterol probe by 1 aggregates begins at approximately 2 x 10(-4) M at 35 degrees C. Analysis of EPR and fluorescence data indicates that the cholesterol analogues are in a nonpolar micellar environment of low fluidity. The significance and implication of the data are discussed in the context of the hypothesized cholesterol sequestration by 1 under physiological conditions.