The eco-evolutionary importance of reproductive system variation in the macroalgae: Freshwater reds as a case study.

The relative frequency of sexual versus asexual reproduction governs the distribution of genetic diversity within and among populations. Most studies on the consequences of reproductive variation focus on the mating system (i.e., selfing vs. outcrossing) of diploid-dominant taxa (e.g., angiosperms), often ignoring asexual reproduction. Although reproductive systems are hypothesized to be correlated with life-cycle types, variation in the relative rates of sexual and asexual reproduction remains poorly characterized across eukaryotes. This is particularly true among the three major lineages of macroalgae (green, brown, and red). The Rhodophyta are particularly interesting, as many taxa have complex haploid-diploid life cycles that influence genetic structure. Though most marine reds have separate sexes, we show that freshwater red macroalgae exhibit patterns of switching between monoicy and dioicy in sister taxa that rival those recently shown in brown macroalgae and in angiosperms. We advocate for the investigation of reproductive system evolution using freshwater reds, as this will expand the life-cycle types for which these data exist, enabling comparative analyses broadly across eukaryotes. Unlike their marine cousins, species in the Batrachospermales have macroscopic gametophytes attached to filamentous, often microscopic sporophytes. While asexual reproduction through monospores may occur in all freshwater reds, the Compsopogonales are thought to be exclusively asexual. Understanding the evolutionary consequences of selfing and asexual reproduction will aid in our understanding of the evolutionary ecology of all algae and of eukaryotic evolution generally.

Evaluation of SARS-CoV-2 RNA Rebound After Nirmatrelvir/Ritonavir Treatment in Randomized, Double-Blind, Placebo-Controlled Trials - United States and International Sites, 2021-2022.

Rebound of SARS-CoV-2 shedding or COVID-19 signs and symptoms has been described after treatment with nirmatrelvir/ritonavir (Paxlovid). The direct association of nirmatrelvir/ritonavir to COVID-19 rebound remains unclear because most reports are based on individual cases or nonrandomized studies. Viral RNA shedding data from two phase 2/3, randomized, double-blind, placebo-controlled clinical trials of nirmatrelvir/ritonavir (Evaluation of Protease Inhibition for COVID-19 in High-Risk Patients [EPIC-HR] and Evaluation of Protease Inhibition for COVID-19 in Standard-Risk Patients [EPIC-SR]) were analyzed to investigate the role of nirmatrelvir/ritonavir treatment in COVID-19 rebound. Rates of rebound of SARS-CoV-2 RNA shedding, identified based on an increase in nasopharyngeal viral RNA levels from day 5 (end-of-treatment) to day 10 or day 14, were similar between nirmatrelvir/ritonavir and placebo recipients. Among subjects with a virologic response through day 5, viral RNA rebound occurred in 6.4%-8.4% of nirmatrelvir/ritonavir recipients and 5.9%-6.5% of placebo recipients across EPIC-HR and the 2021/pre-Omicron and 2022/Omicron enrollment periods of EPIC-SR. Viral RNA rebound after nirmatrelvir/ritonavir treatment was not associated with COVID-19-related hospitalization or death. Data from randomized trials demonstrated that SARS-CoV-2 rebound can occur with or without antiviral treatment, supporting the Food and Drug Administration's determination of safety and efficacy of nirmatrelvir/ritonavir in eligible patients at high risk for severe COVID-19.

Prognostic accuracy of emergency department triage tools for adults with suspected COVID-19: the PRIEST observational cohort study.

BACKGROUND: The WHO and National Institute for Health and Care Excellence recommend various triage tools to assist decision-making for patients with suspected COVID-19. We aimed to compare the accuracy of triage tools for predicting severe illness in adults presenting to the ED with suspected COVID-19. METHODS: We undertook a mixed prospective and retrospective observational cohort study in 70 EDs across the UK. We collected data from people attending with suspected COVID-19 and used presenting data to determine the results of assessment with the WHO algorithm, National Early Warning Score version 2 (NEWS2), CURB-65, CRB-65, Pandemic Modified Early Warning Score (PMEWS) and the swine flu adult hospital pathway (SFAHP). We used 30-day outcome data (death or receipt of respiratory, cardiovascular or renal support) to determine prognostic accuracy for adverse outcome. RESULTS: We analysed data from 20 891 adults, of whom 4611 (22.1%) died or received organ support (primary outcome), with 2058 (9.9%) receiving organ support and 2553 (12.2%) dying without organ support (secondary outcomes). C-statistics for the primary outcome were: CURB-65 0.75; CRB-65 0.70; PMEWS 0.77; NEWS2 (score) 0.77; NEWS2 (rule) 0.69; SFAHP (6-point rule) 0.70; SFAHP (7-point rule) 0.68; WHO algorithm 0.61. All triage tools showed worse prediction for receipt of organ support and better prediction for death without organ support. At the recommended threshold, PMEWS and the WHO criteria showed good sensitivity (0.97 and 0.95, respectively) at the expense of specificity (0.30 and 0.27, respectively). The NEWS2 score showed similar sensitivity (0.96) and specificity (0.28) when a lower threshold than recommended was used. CONCLUSION: CURB-65, PMEWS and the NEWS2 score provide good but not excellent prediction for adverse outcome in suspected COVID-19, and predicted death without organ support better than receipt of organ support. PMEWS, the WHO criteria and NEWS2 (using a lower threshold than usually recommended) provide good sensitivity at the expense of specificity. TRIAL REGISTRATION NUMBER: ISRCTN56149622.

Do not attempt cardiopulmonary resuscitation (DNACPR) decisions in people admitted with suspected COVID-19: Secondary analysis of the PRIEST observational cohort study.

AIMS: We aimed to describe the characteristics and outcomes of adults admitted to hospital with suspected COVID-19 according to their DNACPR decisions, and identify factors associated with DNACPR decisions. METHODS: We undertook a secondary analysis of 13,977 adults admitted to hospital with suspected COVID-19 and included in the Pandemic Respiratory Infection Emergency System Triage (PRIEST) study. We recorded presenting characteristics and outcomes (death or organ support) up to 30 days. We categorised patients as early DNACPR (before or on the day of admission) or late/no DNACPR (no DNACPR or occurring after the day of admission). We undertook descriptive analysis comparing these groups and multivariable analysis to identify independent predictors of early DNACPR. RESULTS: We excluded 1249 with missing DNACPR data, and identified 3929/12748 (31%) with an early DNACPR decision. They had higher mortality (40.7% v 13.1%) and lower use of any organ support (11.6% v 15.7%), but received a range of organ support interventions, with some being used at rates comparable to those with late or no DNACPR (e.g. non-invasive ventilation 4.4% v 3.5%). On multivariable analysis, older age (p < 0.001), active malignancy (p < 0.001), chronic lung disease (p < 0.001), limited performance status (p < 0.001), and abnormal physiological variables were associated with increased recording of early DNACPR. Asian ethnicity was associated with reduced recording of early DNACPR (p = 0.001). CONCLUSIONS: Early DNACPR decisions were associated with recognised predictors of adverse outcome, and were inversely associated with Asian ethnicity. Most people with an early DNACPR decision survived to 30 days and many received potentially life-saving interventions. REGISTRATION: ISRCTN registry, ISRCTN28342533, http://www.isrctn.com/ISRCTN28342533.

Derivation and validation of a clinical severity score for acutely ill adults with suspected COVID-19: The PRIEST observational cohort study.

OBJECTIVES: We aimed to derive and validate a triage tool, based on clinical assessment alone, for predicting adverse outcome in acutely ill adults with suspected COVID-19 infection. METHODS: We undertook a mixed prospective and retrospective observational cohort study in 70 emergency departments across the United Kingdom (UK). We collected presenting data from 22445 people attending with suspected COVID-19 between 26 March 2020 and 28 May 2020. The primary outcome was death or organ support (respiratory, cardiovascular, or renal) by record review at 30 days. We split the cohort into derivation and validation sets, developed a clinical score based on the coefficients from multivariable analysis using the derivation set, and the estimated discriminant performance using the validation set. RESULTS: We analysed 11773 derivation and 9118 validation cases. Multivariable analysis identified that age, sex, respiratory rate, systolic blood pressure, oxygen saturation/inspired oxygen ratio, performance status, consciousness, history of renal impairment, and respiratory distress were retained in analyses restricted to the ten or fewer predictors. We used findings from multivariable analysis and clinical judgement to develop a score based on the NEWS2 score, age, sex, and performance status. This had a c-statistic of 0.80 (95% confidence interval 0.79-0.81) in the validation cohort and predicted adverse outcome with sensitivity 0.98 (0.97-0.98) and specificity 0.34 (0.34-0.35) for scores above four points. CONCLUSION: A clinical score based on NEWS2, age, sex, and performance status predicts adverse outcome with good discrimination in adults with suspected COVID-19 and can be used to support decision-making in emergency care. REGISTRATION: ISRCTN registry, ISRCTN28342533, http://www.isrctn.com/ISRCTN28342533.

Post-exertion oxygen saturation as a prognostic factor for adverse outcome in patients attending the emergency department with suspected COVID-19: a substudy of the PRIEST observational cohort study.

BACKGROUND: Measurement of post-exertion oxygen saturation has been proposed to assess illness severity in suspected COVID-19 infection. We aimed to determine the accuracy of post-exertional oxygen saturation for predicting adverse outcome in suspected COVID-19. METHODS: We undertook a substudy of an observational cohort study across 70 emergency departments during the first wave of the COVID-19 pandemic in the UK. We collected data prospectively, using a standardised assessment form, and retrospectively, using hospital records, from patients with suspected COVID-19, and reviewed hospital records at 30 days for adverse outcome (death or receiving organ support). Patients with post-exertion oxygen saturation recorded were selected for this analysis. We constructed receiver-operating characteristic curves, calculated diagnostic parameters, and developed a multivariable model for predicting adverse outcome. RESULTS: We analysed data from 817 patients with post-exertion oxygen saturation recorded after excluding 54 in whom measurement appeared unfeasible. The c-statistic for post-exertion change in oxygen saturation was 0.589 (95% CI 0.465 to 0.713), and the positive and negative likelihood ratios of a 3% or more desaturation were, respectively, 1.78 (1.25 to 2.53) and 0.67 (0.46 to 0.98). Multivariable analysis showed that post-exertion oxygen saturation was not a significant predictor of adverse outcome when baseline clinical assessment was taken into account (p=0.368). Secondary analysis excluding patients in whom post-exertion measurement appeared inappropriate resulted in a c-statistic of 0.699 (0.581 to 0.817), likelihood ratios of 1.98 (1.26 to 3.10) and 0.61 (0.35 to 1.07), and some evidence of additional prognostic value on multivariable analysis (p=0.019). CONCLUSIONS: Post-exertion oxygen saturation provides modest prognostic information in the assessment of selected patients attending the emergency department with suspected COVID-19. TRIAL REGISTRATION NUMBER: ISRCTN Registry (ISRCTN56149622) http://www.isrctn.com/ISRCTN28342533.

The Integrated Review: FDA Modernizes the Review of New Drug Marketing Applications.

New Drug Applications and Biologics Licensing Applications submitted to the US Food and Drug Administration (FDA) are reviewed by an interdisciplinary team of regulatory scientists that includes medical officers, clinical pharmacologists, toxicologists, statisticians, and drug labeling experts. Upon review of an applicant's submitted evidence from nonclinical studies, clinical trials, and manufacturing capabilities, the review team evaluates the benefits and risks of the drug and makes a scientifically-informed decision. As part of a multi-year, multi-phase New Drugs Regulatory Program Modernization effort, the FDA has recently redesigned how it reviews and documents its decisions with regard to marketing applications. This article describes the origins and rationale of the new Integrated Assessment process and Integrated Review document, summarizes how these differ from the FDA's traditional review of marketing applications, and discusses what industry can expect from a modernized drug review.

Characterisation of 22445 patients attending UK emergency departments with suspected COVID-19 infection: Observational cohort study.

BACKGROUND: Hospital emergency departments play a crucial role in the initial assessment and management of suspected COVID-19 infection. This needs to be guided by studies of people presenting with suspected COVID-19, including those admitted and discharged, and those who do not ultimately have COVID-19 confirmed. We aimed to characterise patients attending emergency departments with suspected COVID-19, including subgroups based on sex, ethnicity and COVID-19 test results. METHODS AND FINDINGS: We undertook a mixed prospective and retrospective observational cohort study in 70 emergency departments across the United Kingdom (UK). We collected presenting data from 22445 people attending with suspected COVID-19 between 26 March 2020 and 28 May 2020. Outcomes were admission to hospital, COVID-19 result, organ support (respiratory, cardiovascular or renal), and death, by record review at 30 days. Mean age was 58.4 years, 11200 (50.4%) were female and 11034 (49.6%) male. Adults (age >16 years) were acutely unwell (median NEWS2 score of 4), frequently had limited performance status (46.9%) and had high rates of admission (67.1%), COVID-19 positivity (31.2%), organ support (9.8%) and death (15.5%). Children had much lower rates of admission (27.4%), COVID-19 positivity (1.2%), organ support (1.4%) and death (0.3%). Similar numbers of men and women presented to the ED, but men were more likely to be admitted (72.9% v 61.4%), require organ support (12.2% v 7.7%) and die (18.2% v 13.0%). Black or Asian adults tended to be younger than White adults (median age 54, 50 and 67 years), were less likely to have impaired performance status (43.1%, 26.8% and 51.6%), be admitted to hospital (60.8%, 57.3%, 69.6%) or die (11.6%, 11.2%, 16.4%), but were more likely to require organ support (15.9%, 14.3%, 8.9%) or have a positive COVID-19 test (40.8%, 42.1%, 30.0%). Adults admitted with suspected and confirmed COVID-19 had similar age, performance status and comorbidities (except chronic lung disease) to those who did not have COVID-19 confirmed, but were much more likely to need organ support (22.2% v 8.9%) or die (32.1% v 15.5%). CONCLUSIONS: Important differences exist between patient groups presenting to the emergency department with suspected COVID-19. Adults and children differ markedly and require different approaches to emergency triage. Admission and adverse outcome rates among adults suggest that policies to avoid unnecessary ED attendance achieved their aim. Subsequent COVID-19 confirmation confers a worse prognosis and greater need for organ support. REGISTRATION: ISRCTN registry, ISRCTN56149622, http://www.isrctn.com/ISRCTN28342533.

Factors influencing the uptake and use of nicotine replacement therapy and e-cigarettes in pregnant women who smoke: a qualitative evidence synthesis.

BACKGROUND: Nicotine replacement therapy (NRT) delivers nicotine without the toxic chemicals present in tobacco smoke. It is an effective smoking cessation aid in non-pregnant smokers, but there is less evidence of effectiveness in pregnancy. Systematic review evidence suggests that pregnant women do not adhere to NRT as prescribed, which might undermine effectiveness. Electronic cigarettes (e-cigarettes) have grown in popularity, but effectiveness and safety in pregnancy are not yet established. The determinants of uptake and use of NRT and e-cigarettes in pregnancy are unknown. OBJECTIVES: To explore factors affecting uptake and use of NRT and e-cigarettes in pregnancy. SEARCH METHODS: We searched MEDLINE(R), CINAHL and PsycINFO on 1 February 2019. We manually searched OpenGrey database and screened references of included studies and relevant reviews. We also conducted forward citation searches of included studies. SELECTION CRITERIA: We selected studies that used qualitative methods of data collection and analysis, included women who had smoked in pregnancy, and elicited participants' views about using NRT/e-cigarettes for smoking cessation or harm reduction (i.e. to smoke fewer cigarettes) during pregnancy. DATA COLLECTION AND ANALYSIS: We identified determinants of uptake and use of NRT/e-cigarettes in pregnancy using a thematic synthesis approach. Two review authors assessed the quality of included studies with the Wallace tool. Two review authors used the CERQual approach to assess confidence in review findings. The contexts of studies from this review and the relevant Cochrane effectiveness review were not similar enough to fully integrate findings; however, we created a matrix to juxtapose findings from this review with the descriptions of behavioural support from trials in the effectiveness review. MAIN RESULTS: We included 21 studies: 15 focused on NRT, 3 on e-cigarettes, and 3 on both. Studies took place in five high-income countries. Most studies contributed few relevant data; substantially fewer data were available on determinants of e-cigarettes. Many studies focused predominantly on issues relating to smoking cessation, and determinants of NRT/e-cigarette use was often presented as one of the themes. We identified six descriptive themes and 18 findings within those themes; from these we developed three overarching analytical themes representing key determinants of uptake and adherence to NRT and/or e-cigarettes in pregnancy. The analytical themes show that women's desire to protect their unborn babies from harm is one of the main reasons they use these products. Furthermore, women consider advice from health professionals when deciding whether to use NRT or e-cigarettes; when health professionals tell women that NRT or e-cigarettes are safer than smoking and that it is okay for them to use these in pregnancy, women report feeling more confident about using them. Conversely, women who are told that NRT or e-cigarettes are as dangerous or more dangerous than smoking and that they should not use them during pregnancy feel less confident about using them. Women's past experiences with NRT can also affect their willingness to use NRT in pregnancy; women who feel that NRT had worked for them (or someone they know) in the past were more confident about using it again. However, women who had negative experiences were more reluctant to use NRT. No trials on e-cigarette use in pregnancy were included in the Cochrane effectiveness review, so we considered only NRT findings when integrating results from this review and the effectiveness review. No qualitative studies were conducted alongside trials, making full integration of the findings challenging. Women enrolled in trials would have agreed to being allocated to NRT or control group and would have received standardised information on NRT at the start of the trial. Overall, the findings of this synthesis are less relevant to women's decisions about starting NRT in trials and more likely to help explain trial participants' adherence to NRT after starting it. We considered most findings to be of moderate certainty; we assessed findings on NRT use as being of higher certainty than those on e-cigarette use. This was mainly due to the limited data from fewer studies (only in the UK and USA) that contributed to e-cigarette findings. Overall, we judged studies to be of acceptable quality with only minor methodological issues. AUTHORS' CONCLUSIONS: Consistent messages from health professionals, based on high-quality evidence and clearly explaining the safety of NRT and e-cigarettes compared to smoking in pregnancy, could help women use NRT and e-cigarettes more consistently/as recommended. This may improve their attitudes towards NRT or e-cigarettes, increase their willingness to use these in their attempt to quit, and subsequently encourage them to stay smoke-free.

Sustained virological response rates with direct-acting antivirals in black subjects with HCV genotype 1 infection: systematic analysis of clinical trials.

OBJECTIVES: Under representation of black subjects in trials of hepatitis C virus (HCV) direct-acting antivirals (DAAs) complicates assessment of differential outcomes for black individuals vs non-black individuals. HCV trials submitted to the Food and Drug Administration (2013-2017) to support approval or to expand an indication of 12-week interferon-free DAA regimens with or without ribavirin to treat HCV genotype 1 (GT1) infection were pooled to explore efficacy comparisons by ethnicity. METHODS: Twenty-six trials were pooled and included 2869 individuals with HCV GT1 alone and 742 individuals with both HCV GT1 and HIV. RESULTS: Of the 2869 HCV GT1-mono-infected subjects, 408 (14.2%) were black. Sustained virological response assessed 12 weeks following cessation of treatment (SVR12) was 92%-100% in black individuals and 87.5%-100.0% in non-black individuals. In pooled analyses, SVR12 was numerically similar between black and non-black subjects (97.1% vs 97.3%). Baseline characteristics did not affect SVR12 for the two groups. Of the 742 subjects with both HCV GT1 and HIV, 243 (32.7%) were black: SVR12 was 89.5%-100% in black individuals and 94.4%-100% in non-black individuals. In pooled analyses for HCV GT1/HIV co-infection, black individuals had a 4% (95% confidence interval -7.7% to 0.3%) lower SVR12 than non-black individuals (93.4% vs 97.0%). This difference was driven by ION-4 in which study SVR12 was approximately 10% lower for black than for non-black individuals (89.5% vs 99.1%). Baseline characteristics did not affect SVR12 for the two groups. CONCLUSION: No notable SVR12 differences were seen in between black and non-black individuals with HCV GT1 alone. Although a numerical difference was observed between black and non-black individuals with both HCV GT1 and HIV, this finding was driven by results from a single trial and may be due to reasons other than ethnicity: 19 subgroup analyses showed baseline characteristics did not affect SVR12 for black and non-black individuals with both HCV GT1 and HIV.

Reinventing HCV Treatment: Past and Future Perspectives.

This review paper summarizes the epidemiology of hepatitis C virus (HCV) and chronic HCV infection, including HCV virology and treatment regimens. Specifically, we focus on the evolution of past, current, and future HCV treatment options, the reasons for treatment failure, and the impact of resistance-associated variants on treatment success.

Small molecule tolfenamic acid and dietary spice curcumin treatment enhances antiproliferative effect in pancreatic cancer cells via suppressing Sp1, disrupting NF-kB translocation to nucleus and cell cycle phase distribution.

Combination of dietary/herbal spice curcumin (Cur) and COX inhibitors has been tested for improving therapeutic efficacy in pancreatic cancer (PC). The objective of this study was to identify agent with low toxicity and COX-independent mechanism to induce PC cell growth inhibition when used along with Cur. Anticancer NSAID, tolfenamic acid (TA) and Cur combination were evaluated using PC cell lines. L3.6pl and MIA PaCa-2 cells were treated with Cur (5-25µM) or TA (25-100µM) or combination of Cur (7.5µM) and TA (50µM). Cell viability was measured at 24-72h posttreatment using CellTiter-Glo kit. While both agents showed a steady/consistent effect, Cur+TA caused higher growth inhibition. Antiproliferative effect was compared with COX inhibitors, Ibuprofen and Celebrex. Cardiotoxicity was assessed using cordiomyocytes (H9C2). The expression of Sp proteins, survivin and apoptotic markers (western blot), caspase 3/7 (caspase-Glo kit), Annexin-V staining (flow cytometry), reactive oxygen species (ROS) and cell cycle phase distribution (flow cytometry) was measured. Cells were treated with TNF-α, and NF-kB translocation from cytoplasm to nucleus was evaluated (immunofluorescence). When compared to individual agents, combination of Cur+TA caused significant increase in apoptotic markers, ROS levels and inhibited NF-kB translocation to nucleus. TA caused cell cycle arrest in G0/G1, and the combination treatment showed mostly DNA synthesis phase arrest. These results suggest that combination of Cur+TA is less toxic and effectively enhance the therapeutic efficacy in PC cells via COX-independent mechanisms.

Randomized double-blind safety comparison of intravenous iron dextran versus iron sucrose in an adult non-hemodialysis outpatient population: A feasibility study.

BACKGROUND: Intravenous iron therapy is a treatment option for iron deficient patients who are intolerant to oral iron or where oral iron is ineffective, but with possible adverse effects. Currently, prospective studies comparing different intravenous iron formulations are needed to determine safety and efficacy of these agents. METHODS: We conducted a prospective, double-blind, randomized controlled trial (RCT) to assess the feasibility of a trial comparing the safety of high molecular weight intravenous iron dextran, Infufer®, with intravenous iron sucrose, Venofer®, in non-hemodialysis adult outpatients. Primary outcome was the occurrence of immediate severe drug reactions. RESULTS: We enrolled 143 patients in a one-year period. Overall, 45/143 (31.5 %) patients (20 iron dextran, 25 iron sucrose) developed 48 infusion reactions (14 immediate, 28 delayed, and 3 both). The risk of an immediate reaction was similar in both groups, 9/73 (12.3 %) iron dextran versus 8/70 (11.4 %) iron sucrose, RR = 0.93 (95 % CI; 0.38 to 2.27). The risk of a delayed reaction was significantly higher in the iron sucrose group 22/70 (31.4 %) versus the iron dextran group 9/73 (12.3 %), RR = 2.55 (95 % CI; 1.26 to 5.15; p = 0.0078). CONCLUSION: In this limited feasibility study, no major differences in immediate reactions were seen, but a significantly higher number of delayed reactions were seen in the iron sucrose group. Further, under our assumptions and design a full RCT to evaluate the safety of different intravenous iron preparations is not feasible. Future studies should consider modifying the clinical outcomes, utilize multiple centers, and consider other emerging parenteral iron formulations. (ClinicalTrials.gov NCT005936197 January 3, 2008).

Ribavirin Reduces Absolute Lymphocyte Counts in Hepatitis C Virus-Infected Patients Treated With Interferon-Free, Direct-Acting Antiviral Regimens.

In clinical trials of interferon-free, direct-acting antiviral treatment of chronic hepatitis C, subjects who received ribavirin had reduced lymphocyte levels (median decline of approximately 0.4-0.5 × 10(9) cells/L). A modest decline in CD4(+) T cells was observed in subjects with human immunodeficiency virus type 1 coinfection without documented opportunistic infections.

Anti-leukemic response of a NSAID, tolfenamic acid.

Tolfenamic acid (TA), a non-steroidal anti-inflammatory drug, is known to inhibit human cancer cells and mouse tumor growth in some cancer models; however, its anti-leukemic response has not been evaluated. TA targets specificity protein (Sp) transcription factors that mediate the expression of several genes associated with cancer including survivin, a key member of inhibitor of apoptosis protein family. Our aim was to test the anti-leukemic efficacy of TA in pre-clinical experiments. The anti-leukemic response of TA was determined using Jurkat and Nalm-6 cell lines. Cells were treated with increasing (25/50/75 µM) concentrations of TA, and cell viability was measured at 24, 48, and 72 h post-treatment. TA showed a steady and consistent decrease in cell viability following a clear dose and time dependent response. Apoptosis and cell cycle analysis was performed using flow cytometry. Results showed a significant increase in the apoptotic fraction (annexin V positive) following TA treatment, while cell cycle phase distribution analysis showed G0/G1 arrest. TA-induced apoptosis was further confirmed by examining the activation of caspase 3/7 and the expression of cleaved PARP. TA modulated the expression of critical candidates associated with the early phases of cell cycle and validated its efficacy in causing G0/G1 arrest. The Western blot results revealed that TA significantly decreases Sp1 and survivin expression. These results demonstrate that the anti-leukemic response of TA occurs potentially through targeting Sp1 and inhibiting survivin and suggest the efficacy of TA as a novel therapeutic agent for leukemia.

Cellular and organismal toxicity of the anti-cancer small molecule, tolfenamic acid: a pre-clinical evaluation.

BACKGROUND/AIMS: The small molecule, Tolfenamic acid (TA) has shown anti-cancer activity in pre-clinical models and is currently in Phase I clinical trials at MD Anderson Cancer Center Orlando. Since specificity and toxicity are major concerns for investigational agents, we tested the effect of TA on specific targets, and assessed the cellular and organismal toxicity representing pre-clinical studies in cancer. METHODS: Panc1, L3.6pl, and MiaPaCa-2 (pancreatic cancer), hTERT-HPNE(normal), and differentiated/un-differentiated SH-SY5Y (neuroblastoma) cells were treated with increasing concentrations of TA. Cell viability and effect on specific molecular targets, Sp1 and survivin were determined. Athymic nude mice were treated with vehicle or TA (50mg/kg, 3times/week for 6 weeks) and alterations in the growth pattern, hematocrit, and histopathology of gut, liver, and stomach were monitored. RESULTS: TA treatment decreased cell proliferation and inhibited the expression of Sp1 and survivin in cancer cells while only subtle response was observed in normal (hTERT-HPNE) and differentiated SH-SY5Y cells. Mice studies revealed no effect on body weight and hematocrit. Furthermore, TA regimen did not cause signs of internal-bleeding or damage to vital tissues in mice. CONCLUSION: These results demonstrate that TA selectively inhibits malignant cell growth acting on specific targets and its chronic treatment did not cause apparent toxicity in nude mice.