Background: Acarbose has been deemed contraindicated in diabetic patients with chronic kidney disease (CKD) or end-stage renal disease (ESRD), but such use is not uncommon. We tested whether this concept hold true in this population with different background hepatic diseases.Methods: All incident diabetic patients  with stage 5 CKD/ESRD were enrolled from Taiwan between 2017 and 2013 and divided into those without chronic liver disease (CLD), with CLD but without cirrhosis, and those with cirrhosis. Among each group, acarbose users, defined as cumulative use >30 days within the preceding year, were propensity-score matched 1:2 to non-users. Our main outcome was the development of liver injury events during follow-up.Results: Acarbose users did not exhibit an increased incidence of liver injury during follow-up compared to non-users , after adjusting for demographic profiles, comorbidities, potentially hepatotoxic medication use, and diabetic severity.Conclusions: The incidence of liver injury did not increase significantly among diabetic acarbose users with severe renal insufficiency than non-users, regardless of the presence or absence of chronic liver disease. Our findings support the renaissance of acarbose as a useful adjunct in diabetic patients with stage 5 and 5D chronic kidney disease. Options of oral anti-diabetic agents (OADs) for diabetic patients with advanced CKD are thus limited; according to the updated guideline from the American Diabetic Association, only meglitinides, thiazolidinediones, and dipeptidyl peptidase-4 inhibitors (DPP4i) are suitable for oral glucose reduction in patients with an eGFR of <30 ml/min/1.73 m2  has continuously increased worldwide, and the number of affected patients is expected to exceed 600 million by 2,040 , and its protocol adheres to the declaration of Helsinki. Informed consent was waived due to data anonymity.All adults (≥20 years old) diagnosed with DM according to the International classification of disease 9th revision—clinical modification [ICD-9-CM] codes 250.x and defined as having ≥2 discrete out-patient clinics or ≥1 hospitalization with a diagnosis of DM were systemically identified from the National Health Insurance Research Database between the study period, i.e., January 1, 2007 to December 31, 2013  those with missing data, with an index date before January 1, 2008 (to reassure incident DM cases) or after June 30, 2013 ; (2) those with a history of hepatotoxic injuries (the outcome of interest) before the index date; (3) or those subsequently assigned to non-users but started acarbose during follow-up. We used criteria validated by the existing literature to screen for those with non-dialysis stage 5 CKD, consisting of those having a primary diagnosis of CKD  in ≥2 discrete out-patient clinics or ≥1 hospitalization, and receiving erythropoiesis-stimulating agent (ESA) treatment concurrently with the CKD diagnosis  cirrhosis, defined by the presence of codes 571.2, 571.5, or 571.6 in their healthcare records and the accompanying catastrophic illness certificates; (2) with chronic liver disease (CLD), including hepatitis B , hepatitis C , or alcoholic liver disease ) but without cirrhosis; and (3) those without CLD (without any of the above codes)  during follow-up. According to international consensus, DILI should be diagnosed based on clinical chemistry criteria, after excluding other diseases and being implicated as plausible candidates , 47,643 patients with incident DM and stage 5 CKD or ESRD were identified and subsequently divided into those without CLD (84.2%), with CLD but without cirrhosis (10.4%), and with cirrhosis (5.4%) , with CLD without cirrhosis (p = 0.94), and with cirrhosis  between 1 year prior to index date and the end of follow-up, to estimate the dose-response relationship. Categorizing the daily dose into three tertiles, we found that none of the acarbose users from each DDD tertile had an increased risk of liver injury event compared to non-users Table . We alsoWe performed several sensitivity analyses to verify our findings. We first excluded acarbose users who discontinued acarbose after enrollment, and the results were essentially the same Table . After fBased on a nation-wide cohort followed up prospectively, we found that among diabetic patients with stage 5 CKD or ESRD, the use of acarbose was not associated with an increased risk of liver injury. This finding clearly illustrates that the theoretical pharmacokinetic concerns of acarbose use in these patients do not occur in the real-world setting, and it is likely that we might be able to administer acarbose safely even in diabetic patients with both renal and hepatic dysfunction, a subgroup in need of more therapeutic options against hyperglycemia.According to past reports, a latency period from 2 to 6 months can exist between the initiation of acarbose and liver toxicity events among affected diabetic patients (Hsiao et al., In our study, we used Taiwan National Health Insurance Research database, an administrative healthcare database covering nearly all citizens in Taiwan, suitable for identifying rare cases or events such as DILI. Prior attempts to test for the utility of ICD-9-CM codes in identifying DILI cases based upon ICD-9-CM codes combination suggesting acute liver injury with or without codes suggesting drug poisoning/overdose (Jinjuvadia et al., In this study, we found that acarbose use in diabetic individuals with advanced CKD/ESRD and liver cirrhosis was not associated with an increased incidence of liver injury Table . This isThe incidence of liver injury in this study was comparable to those in the literature (Chen et al., Our study has several important limitations. First, laboratory data were unavailable in the current research database, which constitutes significant barrier for fully characterizing the nature of DILI events identified. DILI is a diagnosis of exclusion, and other alternative diagnoses should be ruled out, which can be difficult to be achieved using this database due to its retrospective nature and the possibility of missing information. Using ICD-9-CM codes to identify DILI cases has been deemed problematic by some researchers (Jinjuvadia et al., C-TC, K-LC: Study design; C-TC, JW, J-WH, and K-LC: Data analysis; C-TC, JW, J-WH, and K-LC: Article drafting. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study was to explore the clinical benefit and tolerability of IQP-AO-101 in healthy subjects with sleep complaints. This double-blind, randomized, placebo-controlled trial involved fifty subjects with sleep complaints. Subjects with a Pittsburgh Sleep Quality Index (PSQI) score between 6 and 15 were randomized to receive either IQP-AO-101 or placebo for 6 weeks, following a run-in period of one week. Sleep parameters were assessed at baseline and after 1, 4, and 6 weeks using the modified Athens Insomnia Scale (mAIS). Subjects were also instructed to wear an activity tracker and keep a sleep diary during the study. Other questionnaires administered were the Frankfurt Attention Inventory (FAIR-2) and the Profile of Mood States (POMS-65). Blood samples for safety laboratory parameters were taken before and at the end of the study. p < 0.001); night parameters composite score ; and day parameters composite score . All individual parameters (Items 1 to 8) were also significantly improved from baseline after 6 weeks of IQP-AO-101 intake. Analysis of variance with baseline values as covariates showed statistically significant improvements across all individual parameters for IQP-AO-101 when compared to placebo. The measurements using the activity tracker, sleep diary, FAIR-2, and POMS did not reveal any significant differences between groups. No adverse effects related to the intake of IQP-AO-101 were reported. Tolerability was rated as very good by all the subjects and by the investigator for all cases. After 6 weeks, subjects who consumed IQP-AO-101 reported significant improvements in mAIS scores compared with placebo, including mAIS total score (11.76 ± 6.85 vs 4.00 ± 4.80; In this study, IQP-AO-101 was well tolerated and efficacious for promoting sleep and enhancing daytime performance in subjects with moderate sleep disturbances.NCT03114696.This trial is registered with ClinicalTrials.gov, no.  Sleep disorders, characterised by a frequent difficulty to fall asleep or maintain sleep, are the most prevalent health conditions amongst adults. In Europe, prevalence rates vary between countries from 16.5% in Denmark and Italy to 31.2% in Poland [Sleep disorders and sleep disturbances affect nightly rest causing distress during daytime and impacting negatively on quality of life and work productivity , 4. LongAreceptor. However, for both benzodiazepines and the “z drugs”, extensive evidence for harmful side effects and drug interactions can be found in the literature. Reported side effects include morning drowsiness, impairment of cognition and psychomotor performance, amnesia, hallucinations, and even suicidality [Apart from lifestyle changes such as improving sleep habits and creating a sleep-friendly environment, prescription drugs such as benzodiazepines and the “z drugs”, particularly zolpidem, zopiclone and zaleplon are frequently used for sleep disturbances. Both groups enhance the effects of the gamma-aminobutyric acid (GABA) at a site on the GABAcidality –14. Furtcidality . For allValeriana officinalis L.), hops (Humulus lupulus L.), passion flower (Passiflora incarnata L.), lavender  or lemon balm  have been used by healers to reduce anxiety, induce calmness, and promote sleep. Since medicinal plants are made up of a variety of constituents that exert different beneficial properties that may help to combat sleep problems, different herbs are often combined to achieve synergistic effects. However, solid evidence of their efficacy is lacking for many natural products available on the market. To date, clinical trials in humans provide preliminary evidence for only a few herbal sleep aids, such as valerian, passion flower or lemon balm [The history of plant-derived sleep aids goes back as far as ancient times. Plants such as valerian  is a proprietary formulation that contains asparagus extract, saffron extract, lemon balm extract, vitamin C, vitamin E and zinc. The formulation is designed to improve restorative sleep and rejuvenate body cells for optimal physical and mental performance. A range of studies support the efficacy of individual ingredients used in the formulation to promote sleep. In clinical trials, asparagus extract was found to decrease fatigue and improve quality of life, stress resistance, and the ability to sleep , 21, an Therefore, the aim of our study was to investigate the potential of IQP-AO-101 to promote sleep in subjects with sleep disturbances and to evaluate its tolerability.2. Exclusion criteria included hypersensitivity to any of the ingredients, pregnancy or nursing, sleeping less than 5 hours per night on average (self-reported), insomnia, and substantial daily sleepiness as well as any medical condition associated with sleep disorder  or which could interfere with the trial results and/or subject safety, as judged by the investigator. Further exclusion criteria included use of drugs and/or supplements which could interfere with the results of the study ; behavioral intervention for sleep difficulties; any lifestyle factors that might be potentially associated with sleep problems such as excessive caffeine intake, shift work, and long distance traveling, significant stressors such as active grieving; alcohol or drug abuse and participation in another study. Subjects also committed to adhere to the study requirements and female subjects committed to use contraception throughout the study duration. All subjects gave their written informed consent voluntarily. The clinical trial was approved by the ethics committee of Charité – Universitätsmedizin Berlin and was performed in compliance with principles of the World Health Organization Declaration of Helsinki [Fifty (50) generally healthy, male and female volunteers between 23 and 64 years of age were screened for eligibility to participate in the study. All 50 (25 per group) subjects were successfully enrolled. None of the subjects were excluded from the study. Subjects had to meet inclusion criteria such as suffering from non-organic moderate sleep complaints in the past year, as per clinical assessment by the investigator. Sleeping difficulties were assessed at screening with a 19-item self-report questionnaire, the Pittsburgh Sleep Quality Index (PSQI) . SubjectHelsinki  and the  Asparagus officinalis, standardized in cyclo-(L-Ile-L-Pro) content, supplied by Amino Up, Japan; 80 mg lemon balm extract ; 30 mg saffron extract ; 60 mg vitamin C ; 30 IU vitamin E (as dl-tocopheryl acetate) and 10 mg zinc (as zinc gluconate). Standard excipients such as filler, coloring, and flavoring were also added. Placebo sachets contained a similar formulation with the active ingredients replaced by excipient. Both verum and placebo powders were identical in appearance and flavour. Additionally, the subjects were asked to maintain habitual diet, level of physical activity and smoking habits throughout the study.Eligible subjects entered a 1-week run-in period to assess their ability to adhere to requirements to wear an activity tracker (at least 5 times during the week) and completion of a sleep diary as scheduled. Thereafter, subjects were randomly allocated to either IQP-AO-101 or placebo group at a ratio of 1:1 according to the randomization code provided by an independent statistician. Subjects were instructed to mix one sachet of the investigational product (either IQP-AO-101 or placebo) in a glass of water (100ml) and consume 30 minutes before bedtime, daily for 6 weeks. Each sachet of the IQP-AO-101 contained the following active ingredients: 300 mg aqueous extract from stem ofAt each study visit , subjectAdditional sleep data was recorded by an activity-tracking device  during the run-in week, the week preceding visit 3, and the week preceding visit 5. Parameters measured included total sleep time, wake time, number of awakenings, and time in bed. All subjects were also asked to record the time they went to sleep and woke up, along with any stressful events in a sleep diary on a daily basis.At study visits 2 to 5, subjects also completed the Frankfurt Attention Inventory 2 (FAIR-2)  and ProfSafety assessment included measurement of vital signs (blood pressure and pulse rate) during each visit. Full blood count, clinical chemistry, liver and renal functions, and blood lipid parameters as well as urine analysis were performed at visit 1 and at the end of the study. Adverse events were recorded regardless of causality at every visit.At the end of the study, subjects and investigator evaluated the benefits and tolerability of the investigational products.The present study was designed as an exploratory pilot study to generate initial data for information regarding benefits and tolerability of IQP-AO-101. The main objective of this study was to evaluate the effect of IQP-AO-101 on the improvement of sleep in healthy subjects experiencing sleep complaints. Therefore, the main endpoint of this pilot study was comparison of changes in mAIS scores from baseline to week 6 between IQP-AO-101 and placebo. Additionally, the effects of IQP-AO-101 on mental performance and mood and the tracker and diary sleep parameters were explored.The variables were evaluated primarily by non-parametric procedures: Mann-U test for independent groups (comparison of groups) and Wilcoxon test for dependent groups (pre-post comparison within groups). Fisher's exact test was used for comparison of percentages. The influences of baseline values were explored using analysis of variance with baseline values as covariates. Changes in clinical parameters over time (repeated measurements) were analyzed using analysis of variance with respect to differences in groups and systematic changes over time within each group, respectively. All tests were performed with a significance level (type I error) of 5.0% (two-tailed test). The 95% confidence interval was performed. Multiple tests were conducted without correction of significance level in the explorative analysis. Efficacy and safety data were analyzed based on Full Analysis Set (FAS) population. All statistical analyses were done using the SPSS Statistic software, V22.0 .p = 0.706) indicating that the level of sleep disturbances was similar for both groups at baseline.A total of fifty (50) subjects were enrolled in this double-blind, placebo-controlled study between April and July 2017. All subjects completed the study at visit 5. There were no statistically significant differences between the study groups in age, gender distribution, body height, body weight and BMI at baseline. Mean subject age was 44.8 ± 12.7 years, with a higher percentage of female subjects than male subjects in each study group. The baseline characteristics of both study groups are given in  p = 0.001 and 11.76 ± 6.85 vs. 4.00 ± 4.80, p < 0.001, respectively). The mean mAIS total scores throughout the study are shown in The verum and placebo groups did not differ significantly at baseline on any of the mAIS parameters measured. However, significant changes were observed after 4 and 6 weeks of IQP-AO-101 intake. The mean increase in mAIS total score was significantly larger from baseline to both week 4 and week 6 in the IQP-AO-101 group than in the placebo group  to the week before v3 (week 1) as well as between baseline and the week before v5 (week 6).Data retrieved from the sleep diary also did not show any significant difference between groups in total time in bed measured during the study.The FAIR-2 assessment showed no statistically significant differences between the study groups in the attention quality, global attention performance and continuity in attention at any visits. p = 0.025) and 4  but not at visit 5.The POMS-65 assessment showed no significant differences between the study groups in anger, confusion, depression, fatigue, tension and total mood disturbances at any of the visits. A significant difference was reported between groups for factor vigor at visit 3 . The benefit assessment by investigator showed similar results   .A total of 4 adverse events were reported during the course of the study , including back pain, menstrual cramps, heartburn and common cold. None of the reported adverse events was related to the administration of IQP-AO-101. Additionally, no clinically relevant changes were observed for the safety laboratory parameters  at the end of the study for both groups. For IQP-AO-101, all subjects and the investigator rated the tolerability as “very good”.IQP-AO-101 is a product containing asparagus extract, saffron extract, lemon balm extract, vitamin C, vitamin E and zinc that could potentially alleviate sleep disturbances. It has been developed based on the current scientific literature evidence. The primary objective of this randomized, double-blind, placebo-controlled clinical study was to evaluate safety and efficacy of IQP-AO-101 on sleep parameters over a period of 6 weeks in healthy subjects experiencing sleep disturbances. In the present trial, it was demonstrated that subjects who consumed IQP-AO-101 showed a statistically significant improvement in the mean mAIS total score after 6 weeks. All individual mAIS night parameters related to sleep  and all parameters related to daytime performance that may be affected by sleep quality  showed a statistically significant improvement after consumption of IQP-AO-101 for 6 weeks. These findings indicate that IQP-AO-101 may improve nighttime sleep and, hence, as a result improve daytime performance.The assessment of the sleep data obtained with the activity tracker did not show any significant difference between the IQP-AO-101 or placebo group with regard to tracked total time in bed, sleep time, wake time and number of night awakenings. Similarly, no significant differences between both study groups were observed in total time in bed as reported by subjects in a sleep diary. One explanation for the discrepancy between the findings is the different underlying approach of the mAIS questionnaire and activity tracker to assessing sleep parameters. An activity tracker is a wearable device worn on the wrist that measures every motion caused by wrist or arm. It works based on the assumption that people move most during wake states with a progressive reduction in motion as they approach the deepest stages of sleep, such that sleep/wake states may be discriminated by measuring and analyzing these movements to quantify the sleep patterns . CompariWe investigated the potential effect of IQP-AO-101 on mood states by administering the POMS-65 questionnaire which measures six different dimensions of mood swing, including tension, depression, anger, fatigue, confusion and vigor. However, our results revealed that IQP-AO-101 had not significantly impacted the mood states of the subjects in the current study. This could have been due to a small sample size, large individual variability, and/or generally low level of mood disturbances reported by the subjects at baseline thereby making significant changes difficult to detect.One important part of this study is that IQP-AO-101 showed an excellent tolerability profile as per evaluation by both participants and investigator. There were no clinically significant changes in subjects' safety parameters and no investigational product-related adverse effects were reported, indicating that the administration of IQP-AO-101 is tolerable for short-term use. Melissa officinalis L. was reported to modulate serotonergic pathways in rats in a forced swimming test [ in vitro study, thus possibly increasing GABA levels [ in vitro cell study by Ogasawara et al. 2014, asparagus extract was shown to scavenge the negative effect of free radicals at the cellular level [γ-glutamylcysteine synthetase (γ-GCS) [IQP-AO-101 contains ingredients that have previously been shown to alleviate sleep disturbances in animal or human clinical trials. Possible mechanisms of action for this effect may relate to modulation of the hypothalamus-pituitary-adrenal axis, interaction between neurohormonal systems of the brain and protection of neuronal cells from oxidative stress , 36–38. ing test . Furthering test . Active A levels . Yoo et A levels . Zinc miA levels . Neuroprar level . In anotar level . There iar level . Moreove (γ-GCS) . In addi (γ-GCS) . FurtherA limitation of this study is that the evaluation of safety and efficacy was performed over a relatively short period of 6 weeks, and no follow-up assessment was performed after IQP-AO-101 had been discontinued. Hence, no information about the benefits on sleep and safety of long-term use is currently available. Furthermore, the study was conducted as a pilot clinical trial without prior estimation of sample size and statistical power, thus it may be difficult to draw firm conclusions from its results. Lastly, it is unknown if the use of IQP-AO-101 would produce similar results in individuals with more pronounced sleep difficulties. Future studies should validate the efficacy of IQP-AO-101 for alleviating sleep problems in a sufficiently powered study with morelarger subject numbers. Its safety and tolerability should also be evaluated over a longer period of time.This is the first trial to investigate the clinical benefit and tolerability of IQP-AO-101 in humans. The clinical study results provide initial evidence that IQP-AO-101 results in improvements in sleep quality, daytime performance, and well-being in subjects with moderate sleep disturbances and is well-tolerated.


The identification of biomarkers of response might speed drug development and set the premises to assist clinical practice in psychiatry. In this work, we evaluated a panel of peripheral biomarkers  in depressed patients receiving paroxetine, venlafaxine, or placebo. Samples were obtained from two randomised placebo-controlled studies evaluating the efficacy and tolerability of a novel drug candidate, using either paroxetine or venlafaxine as active comparators. In both studies, the biomarker candidates were analysed in plasma collected at randomization and after 10 weeks of treatment with either placebo or active comparator . Data were obtained by multiplexing sandwich-ELISA system. Data were subjected to statistical analysis to assess their correlation with baseline severity and with response outcome. Increases in biomarker levels were correlated with reduction in depression severity for TNF-α, IL-6 IL-10 and CRP. Response to paroxetine treatment correlated with baseline IL-10, IL-6 and TNF-α levels, with the strongest signal being observed in males. In the venlafaxine study, a correlation was observed only between CRP level at randomisation and response, suggesting differences between the two active treatments and the two studies. Our investigations suggest that a combination of pro- and anti-inflammatory cytokines may predict response outcome in patients treated with paroxetine. The potential for IL-10, IL-6 and TNF-α as response biomarkers for a wider range of antidepressants warrants further investigations in clinical trials with other monoamine reuptake inhibitors. MDD is a severe psychiatric disorder with lifetime prevalence in excess of 15%, which is the fourth leading cause of disability worldwide4. Notwithstanding extensive biological research, the pathophysiology of depression remains elusive. At present, the diagnosis and treatment of MDD is still based on the subjective assessment of symptoms. Biomarkers in MDD could help identify homogeneous sets of patients who will benefit most from a particular treatment. Biomarkers could also complement clinical assessment by highlighting changes in the levels of biomarkers that occur in parallel or ahead of changes in clinical symptoms, allowing physicians to make adjustments in therapy quickly. Identification of biomarkers for likely placebo responders could provide a means to reduce the size (and therefore costs) of pivotal clinical studies. Finally, biomarkers could eventually lead to the selection of more efficacious and tolerated treatments in clinical practice5.The application of biomarkers in drug development and clinical decision-making has the potential to change the delivery of healthcare dramatically. Biomarkers could have a profound impact on the diagnosis and treatment of psychiatric disorders, such as major depressive disorder (MDD)8. It is known that inflammatory stimuli can elicit depressive-like symptoms both in preclinical species and in humans, and several data indicate an altered immunologic state in depression. For example, pro-inflammatory cytokines and bacterial endotoxins elicit sickness behaviours and symptoms observed in anxiety and depressive disorders that can be attenuated by chronic antidepressant treatment8. Similarly, the therapeutic use of IL-2 or INF-α induces depressive symptoms that are similar to those seen in MDD. In addition, it has been demonstrated that anti-cytokine treatment in chronic inflammatory conditions leads to improved depressive symptoms9. The potential application of anti-inflammatory agents in depression was tested in a proof-of-principle study with a TNF antagonist which showed that while TNF antagonism did not exhibit generalized efficacy, it improved depressive symptoms in a subgroup of patients with increased inflammation10. Accumulating evidence supports the hypothesis that peripheral immune activation plays a role in the pathophysiology of MDD, as documented by many studies observing altered levels of cytokines, chemokines and other inflammatory markers in depressed patients15. Data also show that antidepressant treatment may reduce peripheral inflammatory markers20. In particular, it has been investigated whether components of the inflammatory response could be used as predictive biomarkers able to direct or aid the selection of the best antidepressant agent. It has been proposed that elevated levels of inflammatory markers contribute to treatment resistance26. However, other studies did not consistently detect this increase and a recent meta-analysis could not detect statistically significant differences in cytokine levels between treatment responders and non-responders27. It should be remarked that a large degree of heterogeneity is evident in this literature, and that the above biomarkers might be affected by the concomitant presence of inflammatory conditions, or concomitant anti-inflammatory treatments, in MDD patients. Moreover, only a small number of studies investigated biomarkers of prognostic efficacy28.A number of biological hypotheses have been exploited in the search for MDD biomarkers. The immuno-inflammatory hypothesis of MDD is based on the discovery of reciprocal communication between the immune and nervous systems29. We selected protein markers  or a serotonin-norepinephrine reuptake inhibitor  and placeboThe studies were 10-week randomized, multi-centre, double-blind, parallel-group, placebo- and active-controlled, flexible-dose studies in male and female outpatients (18–64 years of age) with a psychiatric diagnosis of a major depressive episode associated with major depressive disorder according to DSM-IV-TR. The paroxetine study population consisted of 493 male and female subjects ; 171 of them were randomized to GSK372475, 166 to paroxetine and 156 to placebo treatment group. 343 (70%) subjects completed the trial. The venlafaxine study population consisted of 393 male and female subjects ; 134 of them were randomized to GSK372475, 133 to venlafaxine and 126 to placebo. 233 (59%) subjects completed the trial. For both studies, treatment efficacy was assessed by trained clinicians using the Inventory of Depressive Symptomatology (IDS), the 17-item version of the Hamilton Rating Scale for Depression (HAMD) and the 6-item Bech subscale derived from the HAMD. For the paroxetine study only, the Montgomery Asberg Depression Rating Scale (MADRS) was additionally used to assess treatment efficacy.Both multi-centre study protocols were reviewed and approved by national, regional, or investigational centre Ethics Committees or Institutional Review Boards, and were conducted in accordance with the International Conference on Harmonisation (ICH) Good Clinical Practice guidelines, applicable country-specific requirements, and ethical principles outlined in the World Medical Association Declaration of Helsinki on the Ethical Principles for Medical Research Involving Human Subjects. Written informed consent was obtained from all subjects before their involvement in any study-related procedure.a priori criteria to limit the potential heterogeneity of the study population, as follows. Blood samples were submitted to biomarker analysis for patients: (i) of Caucasian origin; (ii) completing the week 10 assessment; (iii) not coded as protocol violators; iv) from centres where at least 10 patients completed the study. While these additional criteria may have impacted the generalizability of the findings, we felt that they were needed to minimize heterogeneity in biomarkers data (which might be sensitive to ethnicity and country-specific effects) and treatment compliance, thus increasing the chance of detecting biomarker-response associations.Since no efficacy was seen with GSK372475, the biomarker analysis was restricted to the paroxetine, venlafaxine and placebo groups. We applied a number of additional Circulating biomarkers were analysed in blood samples collected at randomization (week 0), and week 10.g for 15 min) within 1 h of collection. The resultant plasma samples were removed, transferred to an EDTA tube and stored frozen at −20 °C pending shipment for analysis. Plasma profile for the selected protein panel of candidates  was obtained by multiplexing sandwich-ELISA system based on chemiluminescent detection at Aushon, Inc. Single samples were randomized by GSK statisticians and shipped to Aushon, Inc. Each assay consisted of a 96-well plate custom arrayed with target protein-specific anti-human antibodies, a lyophilized recombinant standard for each assay, sample diluent containing 0.1% sodium azide, biotinylated antibody reagent, streptavidin-horseradish peroxidase (SA-HRP) reagent, SuperSignal stable peroxide solution, and SuperSignal luminol/enhancer solution and Wash Buffer. Plates were read using the SearchLight Black Ice Cooled CCD Camera System serial #200201. Images were analysed using Array Analyst software, and data analysis were completed and summarized using Microsoft® Excel. Diluted samples, standards and controls were incubated for one hour on the arrayed plates. All incubations were performed at room temperature with shaking at 200 rpm. Plates were decanted and washed six times before adding a cocktail of biotinylated detection antibodies to each well. After incubating with detection antibodies for 30 min, plates were washed three times and incubated for 30 min with SA-HRP. Plates were again washed before adding SuperSignal Femto Chemiluminescent substrate. The plates were immediately imaged using the SearchLight Black Ice imaging system, and data was analysed using Array Analyst Software. Concentrations of all unknown samples were back-calculated using results extrapolated from the corresponding standard curve and expressed as pg/ml blood volume. A four-parameter curve fit was used for all standard curves. A concentrated lyophilized standard for each protein analysed was reconstituted each day to prepare the standard curve. The top standard for each array was prepared by adding an equal volume of each reconstituted standard included in the array and sample diluent to a final concentration of 1:16 of the lyophilized vial concentration. The top standard was serially diluted 1:2, then 1:4 four times, and then 1:2. The assay standard curve range was selected to optimize sensitivity and linearity. Standard curve precision profiles and control performances are included in Supplementary Table Approximately 2 ml of blood for biomarker analysis were obtained from a forearm vein at the study site. Blood samples were collected into tubes containing EDTA, immediately chilled on crushed water ice. Plasma was separated by refrigerated centrifugation  curve analysis was performed. Furthermore, a PLS-DA analysis was performed to assess the accuracy of the marker panel as a classifier.All testing was done at a nominal significance level of 0.05. Statistical analyses were performed using the SAS software  and R 3.5.3. For data visualisation and data mining the Spotfire software (TIBCO) was used.The selection of the patients in which biomarkers were measured, resulted in a subset of 106 male (32%) and female (68%) patients from the paroxetine study Table  and 104 The biomarker population was compared with the “intention to treat” (ITT) population of the SND103288 and SND103285 studies (Supplementary Table p < 0.001)29.The active comparators paroxetine (study SND103288) and venlafaxine (study SND103285) demonstrated superiority compared with placebo across all primary and secondary efficacy endpoints  showed a reduction in HAMD scores of at least 50% at the end of the 10-week treatment period. The remaining 16 (31%) were non-responders to paroxetine treatment. Of 54 patients in the placebo treatment group, 19 (35%) showed a reduction in HAMD scores of at least 50% at the end of the 10-week treatment period. In the venlafaxine study, of 51 patients in the venlafaxine treatment group, 36 (71%) showed a reduction in HAMD scores of at least 50% at the end of the 10-week treatment period, whereas 15 (29%) were non-responders. Of 53 patients in the placebo treatment group, 29 (55%) showed a reduction in HAMD scores of at least 50% at the end of the 10-week treatment period. At baseline, the severity of depression based on HAMD did not importantly differ between responder and non-responder groups.Finally, to address potential gender effect, we tested for differences in response between males and females in the biomarker population, by looking at drug treatment and placebo arms separately. No significant differences were observed neither in the paroxetine nor in the venlafaxine study   and IL-10  levels and severity of depression evaluated by HAMD. A negative correlation trend was seen for TNFRII . In order to identify possible gender-related differential responses, the analysis of the association of cytokine levels with HAMD was conducted separately for gender. At baseline, the positive correlation found for IL-6 in the full population was still present in males . In addition, significant negative correlations were found for TNFRII  and CRP , respectively. Trend to correlation could be observed for TNF-α . Remarkably, no associations were found in females, except for a correlation trend in MMP9 .In the paroxetine study, at randomisation we observed a positive correlation between IL-6  and severity of depression evaluated by HAMD. No correlations were detected for the other biomarkers. Also for the venlafaxine study, the analysis of the association of biomarker levels with HAMD was conducted separately for gender. At baseline, the trend for positive correlation for BDNF levels found in the full population was significant in females , whereas no significant correlations were found in males.In the venlafaxine study, at randomisation, we observed a trend for positive correlation between BDNF levels , IL-6 , IL-10  and CRP   and change in CRP  in male patients. Changes in TNF-α (p = 0.078) demonstrated trend to correlation with HAMD reduction. Female subjects showed similar but weaker correlations in changes over time. A significant correlation with HAMD reduction was observed in females for CRP  with a trend towards correlation for IL-6 (p = 0.063) and BDNF increase (p = 0.080). No correlations were observed for MMP9 and TNFRII either for males or for females. In the venlafaxine study, there were significant correlations for IL-6 changes with changes in HAMD score in both male and females, but in opposite directions  and MMP-9 , while males showed a significant correlation between TNFRII increase and HAMD reduction .We also observed a gender effect Table . In the Subsequently, we investigated the association between baseline biomarker profile and changes in efficacy endpoints, with the objective of identifying biomarkers able to predict treatment response.In the paroxetine study, we discovered an association between higher biomarker baseline levels and better response for TNF-α and IL-10 in the paroxetine group Table , which wp = 0.0016), and IL-10    Fig. . The sig42) Fig. .Fig. 1Scp = 0.0099 based on logistic regression in the paroxetine group, see Supplementary Fig. We then examined differences in the baseline levels of all markers between responder and non-responder groups separately for drug treatment (either paroxetine or venlafaxine) and placebo  could identify patient subgroups represented by subsequent responders and non-responders to paroxetine, but this was not the case for venlafaxine. IL-10 is a multifunctional cytokine playing a key role in regulating the innate and adaptive immune responses to infections and the Th1/Th2 cytokine balance31. Its function appears to be to limit and ultimately terminate inflammatory responses. IL-10 is produced by Th2 cells, as well as by a subset of regulatory T cells, by monocytes and dendritic cells, and can inhibit activation of pro-inflammatory cytokine production by macrophages and Th1 cells32. Higher levels of IL-10 have been shown in late-onset MDD33, which was earlier suggested to have better prognosis with respect to early-onset depression34. On the above premises, one might speculate that high IL-10 levels identify a sub-set of patients characterised by increased anti-inflammatory response with prevailing Th2 component. Of note, whilst IL-10 peripheral levels are reduced in mouse chronic stress models, administration of IL-10 is able to revert stress-induced depressive-like behaviours36. Similarly, IL-10 deletion in mice results with depressive-like behaviours, which are also reversed by IL-10 administration37.This study aimed at investigating the association between the peripheral levels of eight putative biomarkers with MDD and with response to antidepressant or placebo treatment. We observed significant association with HAMD levels for IL-6, TNF-α, TNFRII, IL-10, CRP, BDNF, PAI1-active and MMP9, although these correlations were not consistently observed across studies and genders. Focussing on the most-investigated pro- and anti-inflammatory cytokines, in the paroxetine study IL-6 and IL-10 had higher baseline values in patients with higher HAMD scores, in good agreement with available evidence that MDD patients express higher levels of these cytokines38. A high degree of data heterogeneity between studies was observed, possibly due to differences in assay methods, medication status, length of disease, potential confounders such as body mass index and smoking, and endophenotypic heterogeneity of MDD11. A subset of studies addressed the correlation between peripheral inflammatory marker levels and MDD severity. Significant high correlations between IL1-β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, GM-CSF, IFN-γ and TNF-α and BDI or HAMD scores were detected in unmedicated MDD patients44. On the other hand, other studies found no significant associations between plasma or serum concentrations of several cytokines and cytokine receptors and depression severity scores48.Several groups have investigated the interactions between the clinical course of MDD, treatment conditions and immunological parameters. Recent meta-analyses of studies comparing depressed patients to healthy controls provided evidence that peripheral levels of IL-6, TNF-α, IL-10, TNFRII, and CRP were significantly higher in the MDD group, whereas IFN-γ were slightly lower50. In particular, stronger evidence is available for IL-6, TNF-α, IL-10 and CCL-220. The correlation with the reduction of depression symptomatology we observed for TNF-α, IL-6, and IL-10 levels is not fully consistent with the reported alterations of these cytokines after antidepressant treatment according to most recent meta-analyses20. However, these inconsistencies could be justified by the heterogeneity of the studies in terms of classes of antidepressants. Based on the complexity of the interaction between MDD, antidepressants and the immune system, a broader quantification of the Th1/Th2 cytokines balance would be required51.Several investigations have looked at antidepressants and their effect on the inflammatory response; however, again results are not consistent and a high degree of heterogeneity is observed. Nevertheless, recent meta-analyses provide overall evidence that antidepressant treatment may decrease peripheral cytokine levels52. The same group was able to identify an absolute cut-off level of IL-1β, MIF mRNA for prediction of non-response on an absolute basis53. Higher baseline plasma IL-6, TNF-α and CRP proteins have been reported in non-responders to antidepressant treatment54, even though in other studies the opposite was found for IL-655 and TNF-α39. Other studies showed no association between baseline IL-6, IFN-γ, TNF-α, IL-10 and CRP levels and treatment response58. A meta-analysis of the data suggested overall no significant differences in levels of baseline TNF-α, IL-6 and CRP between patients subsequently responding or not responding to antidepressant treatment, although with large heterogeneity18.The correlation between baseline biomarker levels and the response to antidepressant treatment was also investigated. Cattaneo et al. assessed the leukocyte mRNA expression of inflammatory-related genes in antidepressant-treated patients, showing that higher levels of the three inflammation-related genes  could predict lack of response to antidepressants51. In partial agreement with our results, Chen et al.59 discovered that a different peripheral cytokine response was elicited by paroxetine vs. venlafaxine treatments in MDD patients, suggesting that these antidepressants have different immunomodulatory properties. Moreover, recent findings show that different baseline immune-inflammatory biomarkers are associated with response to different antidepressant treatments, in agreement with the present study, implying the possibility of matching the appropriate therapy to a given patient based on baseline biomarkers61.We discovered higher IL-10 values in responders; in particular, high IL-10 levels could predict response to paroxetine treatment. However, the same pattern could not be reproduced in the venlafaxine study. These findings suggest that predictive biomarkers specific for each antidepressant class will need to be identified in order to be able to effectively predict efficacy. The difference in biomarker effects we observed between paroxetine and venlafaxine might be explained by different mechanisms of action, and in particular, different effects on the inflammatory pathway and on the Th1/Th2 balance62. However, available evidence supports the notion of gender-related differential responses, more than elevated inflammation specifically apparent in depressed women. Consistent with our results, Penninx et al.63 observed a significant gender difference in the association between depressed mood and IL-6 levels, with a stronger effect detected in men. Moreover, in an epidemiological study in psychiatric patients, it was reported that age and gender significantly affect plasma cytokine levels64. Kim et al.42 have previously reported gender differences in the levels of stimulated cytokine production in MDD patients: IL-6, TNF-α, and IFN-γ levels were significantly lower in female patients at baseline and after antidepressant treatment. Myin et al. found gender differences in changes of plasma IFN-γ levels after antidepressant therapy65. In a general population-based sample, significantly higher IL-1Ra and lower IL-1β levels were detected in males with depressive symptomatology, and not in females66. Other population studies showed that higher CRP levels69 and higher IL-6 levels69 were associated to higher levels of depressive symptoms specifically in men. In adolescent patients, Pallavi et al.70 discovered that only females had higher IL-6 as compared to the respective healthy controls. More recently, Majd et al.71 investigated gender difference in response in stimulated cytokine production from depressed patients. Interestingly, the study showed a significant positive association between depressive symptoms and stimulated TNF-α in men, whereas a negative association between depressive symptoms and stimulated TNF-α and IL-10 was observed in women. Overall, this collection of partially contradictory findings suggests that further investigations are needed to address gender-related differences in peripheral biomarkers.An additional finding of our study was the difference in male and female biomarker profiles, and in particular, pro- and anti-inflammatory cytokine profiles, which were found to correlate both with depression severity and with antidepressant response. Effects of gender on cytokines and other circulating biomarker levels have not been extensively studied in MDD. Because the immune response in women can be affected by oestrogen, it would be necessary to control for menstrual phases and oral contraceptive use to precisely evaluate the gender differences in cytokine production. The observation that women experience higher rates of depression than men, and also show higher rates of autoimmune diseases, has suggested inflammation as a key contributor to MDD especially for women3, did not show any association with treatment response in our study. Differences in the study design, in the clinical population and in biomarker assays may explain this lack of replication. In addition, literature findings mainly suggest a negative treatment outcome based on baseline elevation of pro-inflammatory cytokines, whilst our study indicates a wider activation of immunological pathways in responders, including pro- and anti-inflammatory cytokines, with a predominant and facilitating role of IL-10 in the response to paroxetine. A possible explanation for different results may lie in the differences in sampling schedules and assay procedures. In our investigations, we used a plasma assay for cytokine concentration, whereas other groups measured cytokine concentrations from isolated (and stimulated) blood cells or in serum. Plasma samples have the advantage of determining cytokine production of leukocytes in a more physiological setting, and are not affected by coagulation-induced interferences and technical issues related to fibrin formation typical of serum. Artifactual changes in blood analytes may prevent accurate diagnosis of disease states or other physiological conditions. Some changes appear to occur rapidly in serum, even when the blood is handled in a manner deemed appropriate by NCCLS. Although proteins are the ultimate effectors of most cellular processes, It should be pointed out that investigations of cytokine mRNA levels appear to be more consistent across studies, and they have resulted with more reliable and accurate predictors of antidepressant response with respect to protein levels72. The same applies to BDNF, for which studies on peripheral blood mRNA have resulted with more consistent results with respect to serum or protein level analyses73. Indeed, mRNA profiles of peripheral blood cells might be less dependable on analytical factors and they could serve as a good proxy for CNS expression74. As observed by other investigators11, irrespective from the modality, the lack of complete agreement between findings from different studies may be further explained by the fact that MDD is a heterogeneous disorder, and different subtypes may have different physiological profiles.The above literature findings share a reasonable consistency with our results. However, there are a number of conflicting findings with the literature, and within literature data. It should be noticed that some markers previously associated with depression severity or with antidepressant response outcome, such as BDNF29. Therefore, it is to be expected that the population characteristics do not mirror the wider clinical population of MDD patients and the external validity/translatability to the very heterogeneous MDD population at large may be partial. Moreover, a relatively small patient number was investigated in each study, thus replication in independent samples will be needed to validate our findings. Also, the applicability to other SSRIs or to antidepressants with different mechanism of action will need to be further tested and validated in additional clinical trials.A limitation of our investigation is the lack of control for potential confounders such as cigarette smoking, BMI, comorbid diseases, or socioeconomic status. In addition, most of the markers tested could be affected by concomitant inflammatory conditions or by anti-inflammatory treatment. Furthermore, samples were not run with replicates, and the results here reported are not adjusted for multiple testing and might be partly false positives. An additional limitation of the present study is that strict inclusion criteria were applied60. Our data indicate that increased baseline levels of IL-10, IL-6 and TNF-α in MDD patients may be associated with better response to treatment with paroxetine. Therefore, immune dysregulation in depressive disorders does not involve exclusively pro-inflammatory cytokines, such as IL-6 and TNF-α, but also activation of anti-inflammatory cytokines, which may result in positive treatment outcomes. In addition, we have shown a different pattern of immune system activation during depression and antidepressant treatment in male and female patients.In conclusion, our study suggests that the assessment of a combination of anti- and pro-inflammatory cytokines may be valuable for informing response to antidepressant therapy, as previously proposedAlthough the above data are preliminary, the potential for IL-10, IL-6 and TNF-α as biomarkers for paroxetine response warrants further investigation and replication in additional trials.Supplementary TablesSupplementary Figures


We identified 12 best hits characterized by different chemical scaffolds and conformational and physicochemical properties. All of them were associated to an improved theoretical binding affinity with respect to that of known selective G-binders. Among these hits there is a chalcone derivative; structurally very similar to the polyphenol butein; known to remarkably inhibit the telomerase activity.The G-quadruplex DNA structures are mainly present at the terminal portion of telomeres and can be stabilized by ligands able to recognize them in a specific manner. The recognition process is usually related to the inhibition of the enzyme telomerase indirectly involved and over-expressed in a high percentage of human tumors. There are several ligands, characterized by different chemical structures, already reported in the literature for their ability to bind and stabilize the G-quadruplex structures. Using the structural and biological information available on these structures; we performed a high throughput  Twelve best candidates, associated to an improved theoretical binding affinity with respect to that of known selective G-binders, were identified and their biophysical and biological assays are actually in progress.Effectively, also our research group carried out several studies focused on the G-quadruplex target by analyzing both selective synthetic and natural G-binders ,53,54,55eriments . Conversversus false-positive rates for all compounds. The area under the ROC (AU-ROC) curve is the probability of active compounds being ranked earlier than decoy compounds.For each analyzed complex, we generated several pharmacophore hypotheses based on the structural information reported in literature. In order to validate our models, we used datasets of active, inactive and decoys compounds and we evaluated the ROC curve. Receiver operating characteristic (ROC) curves have been widely used to evaluate VS methods ,58. An Rd(TAGGGTTAGGGT) in parallel conformation, the first pharmacophore model generated by LigandScout [For the 3CE5  PDB 59]59] modelandScout  was reprAfter validation, we selected H_2 , composeThe ROC curve related to this hypothesis indicated a sensitivity equal to 39.4%, as shown in For the 3SC8  PDB struAfter validation, H_3 , based oThe sensitivity obtained by this hypothesis, able to find 73 active and four inactive compounds, was equal to 40.5%, as indicated by the ROC curve .As regards the 4FXM  PDB struAmong them, H_9, shown in The ROC curve related to this hypothesis indicated a sensitivity equal to 50.0%, as shown in The three best pharmacophore hypotheses obtained after the validation procedure were used for virtually screening the library of 31,041 natural compounds. In particular, as reported in After the virtual screening procedure, we analyzed the LigandScout fit scores for all the identified ligands, but we observed no significant differences among them. Thus we decided to perform Glide HTVS  docking Within this cut-off value, from the initial 4759 compounds obtained after the virtual screening of the natural database, only 230 were finally selected, and among them 47, seven and 176 were from 3CE5, 3SC8 and 4FXM screening, respectively. In order to further filter them, we considered only those ligands whose binding affinity towards 3CE5 model was better if compared to that of BRACO-19 .As shown in The twelve theoretically most promising hits are reported in Analyzing the best poses of our hits, we observed that all of them were able to better recognize the bottom site  of the GAs reported in Finally, with the aim to theoretically evaluate the chemical and physical properties of the 12 best hits, we used the Canvas software , that alAnalyzing the chemical scaffolds of the best hits identified by our virtual screening approach, we observed that ZINC79190432 and ZINC32124244 belong to the quinazoline alkaloids, while ZINC12377179 is characterized by a quinoline moiety. In particular this class of ligands was associated to antiviral and anticancer activities .By contrast, ZINC77031588 and ZINC03985155 are based on a chromene structure, already reported in several G-binders related to the Green tea catechins , known aMoreover, anthraquinone-containing extracts from different plant sources have been found to have wide variety of pharmacological activities such as antiinflammatory, wound healing, analgesic, antipyretic, antimicrobial, and antitumor activities . In partS)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, an amino acid isolated from C. endivia. L., was found for the first time to show cytotoxic activity in colorectal tumor cell line HCT-8 [On the other hand, ZINC20760949 is related to a tetrahydro-β-carboline chemical structure, present in several fruits and vegetables ,72 and wne HCT-8 , and thuT. vernicifluum medicinal plant and known for its antioxidant properties [One of the most interesting hit resulted the chalcone ZINC04252698, structurally very similar to the polyphenol butein. This compound, extracted from operties , was fouoperties .Our results focused some chemical scaffolds already known for their selectivity against DNA G-quadruplex conformation. In particular, quinazoline, quinoline, anthraquinone and bis-indole derivatives were tested for their ability to inhibit the telomerase enzyme and for their cytotoxic effect against some tumor cell lines . These o50 values were lower than 10 μM, obtaining an active test set of 180 compounds. For the selection of the inactive molecules, we applied the same approach, thus filtering 37 compounds characterized by IC50 values higher than 10 μM. Our choice was based on the same experimental assay and conditions. In 50 value of the active compounds are reported.The active compounds used in this study were obtained from the ChEMBL database . We firsUnfortunately, we have not any decoys set for the telomerase target. However, we used a focused library already reported in our recent publication  that ref−1·mol−1.All the molecules used as test sets were submitted to 2000 iterations of full energy minimization adopting the Polake-Ribiere Conjugated Gradient (PRCG) algorithm and the “all atoms” notation of the MMFF force field . Solventin silico approach was conducted by virtually screening ~210,000 natural compounds publicly available in the ZINC [The high throughput the ZINC  repositothe ZINC , Analytithe ZINC , IBScreethe ZINC , Indofinthe ZINC , Moleculthe ZINC , Nubbe Nthe ZINC , Princetthe ZINC , Selleckthe ZINC , Specs Nthe ZINC , TCM Datthe ZINC  and UEFSthe ZINC ). Instanthe ZINC  allowed the ZINC ,97 usingN-methyl-piperazine side-chains. The NDI molecules in both structures are stacked effectively over the terminal 3' G-quartet surfaces, with extensive π-π contacts, resulting in a 1:1 quadruplex:ligand stoichiometry. There are significant differences between the complexes in terms of ligand mobility and interactions within G-quadruplex grooves. One of the two ligands is markedly less mobile in the crystal complex and is more G-quadruplex-stabilizing, forming multiple electrostatic/hydrogen bond contacts with quadruplex phosphate groups [For building the pharmacophore models used in this study and for performing the virtual screening we adopted LigandScout v. 3.1 , a softwe groups .4FXM is the X-ray crystal structure of a complex between the N-methyl mesoporphyrin IX (NMM) and human telomeric DNA sequence. NMM is able to adjust its macrocycle geometry to closely match that of the terminal G-tetrad required for efficient π-π stacking. The out-of-plane N-methyl group of NMM fits perfectly into the center of the parallel guanine core, where it aligns with potassium ions and the molecule results well stabilized by means of hydrophobic contacts. In contrast, the interaction of the N-methyl group with duplex DNA or antiparallel G-quadruplex would lead to steric clashes that prevent NMM from binding to these structures, thus explaining its unique selectivity towards the parallel conformation .d(TAGGGTTAGGGT) in parallel conformation and BRACO-19. Each bimolecular quadruplex in this structure contains three planar stacked G-tetrads with a BRACO-19 molecule stacking directly onto the 3' end G-tetrad face. The drug is asymmetrically stacked on the G-tetrad at the end of one quadruplex, showing π-π overlap with just two guanine bases and the cationic ring nitrogen atom of the acridine ring in-line with the K+ ion channel that runs through the quadruplex [3CE5 is a complex between a bimolecular human telomeric G-quadruplex of sequence adruplex .During the initial validation step, the first generated pharmacophore models, obtained from each of the three PDB complexes, were associated to poor performance. Therefore, we manually modified them by including or removing some features, by increasing or reducing the tolerance and by adjusting the exclusion volume spheres, with the aim to better characterize the binding site. In this way, we obtained optimized pharmacophore hypotheses, subsequently evaluated by their Receiver Operating Characteristic (ROC) curve profile, and used for the virtual screening of the natural database.3, centered onto the centroid of the receptor. All default settings were used for docking.All docking and scoring calculations were performed using Glide version 5.8 HTVS mode . DNA modThe filtered 31,041 ligands were docked flexibly, generating 10 poses per ligand. Since full force-field minimization was not performed during the docking stage, a post-docking minimization was applied. In this energy minimization the ligand geometries were optimized and re-scored.The binding pocket characteristics and the protein-ligand interactions were identified with automatically-generated Maestro  2D liganIn this manuscript we developed some pharmacophore models starting from three experimental DNA G-quadruplex complexes. The best hypotheses, validated by the ROC analysis, were used for the virtual screening of natural compounds database extracted from ZINC. A first cutoff value was developed using a large set of active/inactive compounds and adopted for the selection of the screened ligands. A second filtering criterion was based on the re-docked BRACO-19 reference compound score and applied to reduce the first identified molecules. Finally, twelve compounds satisfied all criteria and, interestingly, most of them contain chemical scaffolds already associated with G-quadruplex binding properties and antiproliferative effects.These hits are currently under experimental investigations in order to complete their biophysical and biological profiles. The most promising ligands will be object of further chemical optimization against the G-quadruplex targets.


We analyzed exosomal miRNAs differentially expressed among these three groups. MirTarBase was employed to predict potential target genes of the miRNAs specific for LA GVHD. We detected 55 miRNAs that were differentially expressed with a significant change >2.0-fold between LA GVHD and non-GVHD. Of these, we selected the 10 miRNAs  with the most significant differential expression. Using quantitative RT-PCR, we further identified that miR-128 was significantly upregulated at the onset of LA GVHD compared with that in normal controls and is a promising diagnostic marker of LA GVHD, with an area under the curve (AUC) value of 0.975. MirTarBase analysis revealed that the predicted target genes of miR-128 are involved in the immune system and inflammation. Increased expression of miR-128 may serve as a novel, noninvasive biomarker for early LA GVHD diagnosis.Recent studies have demonstrated that exosomal microRNAs (miRNAs) have the potential of facilitating molecular diagnosis. Currently, little is known about the underlying mechanism behind late-onset acute graft-versus-host disease (LA GVHD). Identifying differentially expressed miRNAs in exosomes should be useful for understanding the role of miRNAs in this disease. This study was established to investigate the relevance of miRNAs in exosomes derived from patients developing LA GVHD after allogeneic hematopoietic stem cell transplantation (HSCT). Plasma samples were collected from patients with LA GVHD ( The occurrence of severe graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) leads to a poor prognosis. Recently, with this expansion in the utilization of allogeneic HSCT, such as the introduction of reduced-intensity conditioning and donor lymphocyte infusions, late-onset acute and chronic GVHD has been reclassified based on the exact clinical manifestation instead of the time of onset . AccordiMicroRNAs (miRNAs) are small noncoding RNA molecules that regulate post-transcriptional gene expression by degrading or suppressing their target mRNA . RecentlIn recent studies, miRNA profiling identified a GVHD-specific miRNA signature that can control the regulation of classic acute GVHD in allogeneic immunity. In particular, miR-155, known as an immune-related microRNA, and miR-15a/16 and miR-17-92a were shown to be closely involved in the pathophysiology behind the development of classic acute GVHD in a xenogeneic GVHD murine model ,19. DespA summary of patients’ clinical characteristics is presented in The procedure for selecting candidate miRNAs is illustrated in p < 0.05) when compared with the levels in healthy subjects  significant difference in the expression of miRNAs is observed between LA GVHD and healthy volunteers ; or (2), no significant difference in the expression of miRNAs is observed between the non-GVHD and healthy volunteers . We firstly compared samples obtained from patients at the diagnosis of LA GVHD with those from healthy volunteers  and found that three miRNAs  were significantly overexpressed in the exosomes of those with LA GVHD . In contrast, miR-128 and miR-125b were significantly increased in LA GVHD patients  (To confirm the specific deregulated miRNAs change obtained from the TaqMan low-density array (TLDA), we quantified miR-128 and miR-125b expression by quantitative real-time PCR (qRT-PCR). There were no significant differences in miR128 A and miRctively) A,B. To ectively) A. Similactively) B. The cuTo further explore whether exosomal miRNAs can be used as predictive biomarkers of LA GVHD, we next measured miR-125b and miR128 expressions sequentially in exosomes of available serum samples taken from two transplanted patients  GVHD being established. Given the short history of this category, the pathophysiology of LA GVHD is still poorly understood. Although the diagnosis of GVHD is judged based on clinical manifestations and pathological findings of the tissue sections from the intestines or liver, it is difficult to provide an accurate pathological diagnosis of GVHD. For this reason, pathological findings have greatly affected various factors of radiation damage, drug injury, and viral infection, presenting a diagnostic dilemma. Therefore, there is an urgent need of exploring reliable, noninvasive biomarkers of GVHD. In the context of GVHD after allogeneic HSCT, biomarker research has progressed through a variety of techniques, such as immunocompetent cells , plasma (soluble IL-2R), and inflammatory cytokines . For example, Paczesny et al. identified that a panel of four biomarkers  in serum was useful for the diagnosis of acute GVHD . MoreoveBecause a number of previous studies investigated the role of exosomes and exosomal miRNAs as potential predictive biomarkers in acute GVHD ,28, we aWe perfomed ROC analysis to assess the diagnostic capacity of exosomal miRNAs. Exosomal miR-128 exhibited its potential in discriminating LA GVHD from non-GVHD groups. When the expression level of candidate miRNAs was analyzed sequentially in a subset of LA GVHD patients during allogeneic HSCT, miR-128 was shown to increase over the cut-off value earlier than the development of the disease. However, our results must be interpreted with caution, because this study was limited by the relatively small number of samples from LA GVHD patients and absence of a validation cohort of any sort. After all, molecular mechanisms on the dysregulation of exosomal miRNAs in patients with LA GVHD could not be fully provided. Also, candidate miRNAs were then examined longitudinally in only two patients over time. Thus, these results need to be further studied with large cohort.The results of the top 10 miRNAs obtained by TLDA included several characterized miRNAs  known to be dysregulated in acute GVHD ,20,21. IIn summary, this study demonstrated that the levels of several exosomal miRNAs change at the time of LA GVHD onset after HSCT. Although a longitudinal series of blood samples was taken from 2 patients only, upregulation of miR-128 expression level preceded of LA GVHD incidence. In particular, our results suggest that biological alterations of miR-128 in exosomes may serve as potent predictive biomarkers for close monitoring of the onset of LA GVHD. We consider that this work could provide a foundation for revealing the importance of miRNA in the pathophysiology of LA GVHD. GVHD remains a major cause of transplant-associated complications, which can lead not only to lethal organ damage, but also to a decreased quality of life. Inhibition of certain exosomal miRNAs, such as miR-128, might be considered as a novel potential treatment of LA GVHD to target the immune system and inflammatory signaling, to control GVHD. However, further studies are needed to elucidate the molecular mechanisms behind the development of LA GVHD.The use of patient samples was approved by the Institutional Review Board of Tokyo Medical University . Written informed consent was obtained from all of the participants before the collection of specimens, in accordance with the Declaration of Helsinki.The diagnosis of LA GVHD here was based on clinical symptoms or was histologically proven by biopsy in the target organs, as described previously . Patientn = 2; liver, n = 2; skin and gut, n = 1) and five stable patients without GVHD symptoms (non-GVHD) under immunosuppression. All samples were collected sequentially after HSCT. For patients with LA GVHD, we evaluated blood samples obtained at the onset of this disease. No treatment for LA GVHD had been given at the time of sampling. For comparison, blood samples from HSCT patients without GVHD symptoms and other complications were used. In addition, eight healthy controls were also randomly selected in our hospital. We extracted exosome fractions from 200 µL of plasma using Total Exosome Isolation Reagent  and then assessed exosomal miRNA expression in a subset of samples, as described previously [Ten patients with hematological malignancies who underwent allogeneic HSCT between February 2012 and November 2013 were included in this study, which consisted of five patients with LA GVHD  using the miRNeasy Mini kit . Exosomes were diluted with 700 μL of QiaZol. After 5 min of incubation, 1 μL of 1 nM ath-miR-159  was added to the aliquot followed by vortexing for 30 s and incubation for 5 min. Subsequent phenol extraction and centrifuge filtration were performed, in accordance with the manufacturer’s instructions . The RT reaction and pre-amplification step were set up in accordance with the manufacturer’s instructions . miRNAs were reverse-transcribed with Megaplex Prime Pools .® software  and R software . The Benjamini–Hochberg algorithm was used for the estimation of false discovery rates, as we reported previously [The miRNA expression profiles were determined with a TaqMan miRNA Array Human Card A . Quantitative RT-PCR was performed on an Applied Biosystem 7900 HT thermocycler, in accordance with the manufacturer’s recommended program . Using Seviously .−ΔΔCt method.We determined the levels of the individual miRNAs by real-time quantitative RT-PCR with a TaqMan MicroRNA Assay  and the following miRNA-specific stem-loop primers: hsa-miR-128  and hsa-miR-125b . Subsequently, quantitative real-time PCR was performed with an ABI Prism 7900 sequence detection system . The reaction was initiated by incubation at 95 °C for 2 min, followed by 50 cycles of 95 °C for 15 s and then 60 °C for 1 min. All reactions were run in duplicate. Mean (Ct) values for all miRNAs were quantified with sequence detection system software  . The exphttp://mrtarbase.mbc.nctu.edu.tw/, accessed on 11 September 2017).Following the identification of differentially expressed miRNAs, the predicted target genes of these altered miRNAs were investigated using the experimentally validated miRNA–target interactions database MirTarBase (Data are expressed as mean ± standard deviation (SD). Multiple groups were compared by one-way analysis of variance (ANOVA) with post hoc Tukey’s test. Statistical analysis was performed using GeneSpring software, and miRNAs with both a ΔCt of either >1.0 or <−1.0 and a P value of less than 0.05 were deemed to be differentially expressed. The area under the curve (AUC) of the receiver-operating characteristic (ROC) curve was calculated. GraphPad Prism software  was used for statistical analysis.


Binge drinking, defined as consuming five or more standard alcoholic drinks for men (four for women) within a 2-hour period, is common among young adults and is associated with significant alcohol-related morbidity and mortality. To date, most research on this problem in young adults has relied upon retrospective questionnaires or costly laboratory-based procedures. Smartphone-based ecological momentary assessment (EMA) may address these limitations by allowing researchers to measure alcohol use and related consequences in real time and in drinkers’ natural environments. To date, however, relatively less research has systematically examined the utility of this approach in a sample of young adults targeting real-world heavy drinking episodes specifically.This study aimed to evaluate the feasibility, acceptability, and safety of a smartphone-based EMA method targeting binge drinking and related outcomes in heavy drinking young adults during real-world drinking occasions.Young adult binge drinkers in the smartphone group  years; 58% (48/83) male; bingeing on 23.2% (6.5/28) days in the past month) completed baseline measures of alcohol use and drinking-related consequences, followed by up to two smartphone-based EMA sessions of typical drinking behavior and related outcomes in their natural environments. They also completed next-day and two-week follow-up surveys further assessing alcohol use and related consequences during the EMA sessions and two weeks after study participation, respectively. A separate demographic- and drinking-matched safety comparison group (N=25) completed the baseline and two-week follow-up surveys but did not complete EMA of real-world drinking behavior.Most participants  in the smartphone group engaged in binge drinking during at least one 3-hour EMA session, consuming 7.3 (SD 3.0) standard alcoholic drinks. They completed 87.2% (507/581) system-initiated EMA prompts during the real-world drinking episode, supporting the feasibility of this approach. The procedure was acceptable, as evidenced by high participant ratings for overall satisfaction with the EMA software and study procedures and low ratings for intrusiveness of the mobile surveys. Regarding safety, participants endorsed few drinking-related consequences during or after the real-world drinking episode, with no adverse or serious adverse events reported. There were no differences between the groups in terms of changes in drinking behavior or consequences from baseline to two-week follow-up.This study provided preliminary support for the feasibility, acceptability, and safety of a smartphone-based EMA of real-time alcohol use and related outcomes in young adult heavy drinkers. The results suggest that young adults can use smartphones to safely monitor drinking even during very heavy drinking episodes. Smartphone-based EMA has strong potential to inform future research on the epidemiology of and intervention for alcohol use disorder by providing researchers with an efficient and inexpensive way to capture large amounts of data on real-world drinking behavior and consequences. Binge drinking, also known as heavy episodic drinking, is a significant public health problem in the United States and accounts for more than half of the 88,000 alcohol-related deaths in the United States each year ,2. ApproPrior research has used EMA to measure real-world light-to-moderate alcohol use  with good compliance across studies  -15. EMA Given our current era of rapid advances in mobile technology and changing user preferences, the modality of alcohol monitoring is important. Most prior EMA studies of drinking have employed either palmtop computers  or olderTherefore, the purpose of this study was to evaluate the feasibility, acceptability, and safety of a real-time smartphone-based EMA method to obtain self-reported alcohol use and related outcomes  during real-world drinking events in young adult heavy drinkers. We targeted the EMA procedure in this study toward binge drinking episodes specifically because previous work in this field has focused mainly on capturing drinking behavior generally over a period, for example, several days or weeks ,16,19, rThe Mobile Alcohol Response Study was conducted between April 2017 and June 2018 and employed a within-subject design with each participant undergoing 1 or 2 separate real-time smartphone assessments of typical drinking episodes in their natural environment. As stated above, a separate safety comparison sample completed baseline and follow-up surveys of drinking behavior and related outcomes but did not undergo real-time mobile monitoring. As part of a larger study, participants also completed a laboratory session with alcohol administration to validate the smartphone-based EMA method; those data will be reported elsewhere. All study procedures were approved by the University of Chicago Institutional Review Board.Candidates were recruited from Web-based advertisements and word-of-mouth referrals. Inclusion criteria were age 21 to 29 years, generally healthy young adults with weekly heavy alcohol consumption  for at . The surveys included the Alcohol Use Disorder Identification Test −(60β × t), where c is the number of standard drinks consumed to that point in the drinking episode, GC is a gender constant (7.5 for men and 9.0 for women), w is weight in pounds, 60β is a constant representing the average population alcohol metabolism rate (0.017 g/dl per hour), and t is the time in hours since drinking began. We assumed that participants consumed the first drink in the episode over 20 min when calculating t, as in previous studies [Feasibility was examined by (1) the percentage of nonparticipant-initiated prompts completed and (2) the estimated number of standard drinks reported at each time point. The number of estimated standard drinks consumed was used to determine the percentage of episodes that included heavy drinking. In addition, this information was used to estimate BAC  levels throughout the drinking episode according to the equation of Matthews and Miller : eBAC=[ analyses examining group, time, and their interaction. Skewed data were log-transformed before analysis, as appropriate.Examination of data from the mobile assessments revealed that a high majority of participants correctly recorded their drinking during the drinking episode . Despite training participants during screening to indicate only the number of drinks finished since the prior EMA prompt, 4 participants incorrectly reported their cumulative number of drinks at each prompt instead. Thus, their data could not be interpreted, resulting in a final study sample of 83 participants. eBAC calculated for a small number of survey responses was ≥0.30 g/dl, a level of intoxication associated with loss of consciousness or death. We assumed that those time points reflected errors in reporting  and opteAll participants completed the self-initiated baseline and post first-drink survey prompts and 87.2% (507/581) of the remaining 7 system-initiated prompts over the ensuing 3 hours of the EMA session . Most paParticipants reported consuming any alcohol on 75.4% (445/590) of 590 completed prompts from the self-initiated post first-drink survey onward . They consumed 7.3 (SD 3.0) standard drinks during the 3-hour drinking assessment, with eBAC reaching 0.13 (SD 0.07) g/dL at the final time point  had high response rates and completed all the study prompts (9/9) with an average of 8.1 and 8.3 prompts completed in the first and second episodes, respectively. There was more drinking alone during the second episode  than the first . Participants’ reported location when completing the prompts or consumption of food, nicotine, or other drugs did not differ across assessment sessions, all Ps>.07.Background characteristics and alcohol consumption during drinking episodes did not differ between participants who completed 2 smartphone drinking assessments  and those who completed 1 assessment (n=22), all P=.02), but other acceptability ratings did not differ based upon the number of smartphone sessions completed (Ps>.16).Participants rated the smartphone survey software as easy to use overall  and endorsed high satisfaction with study participation . Similarly, participants did not consider the surveys to be time consuming  or intrusive . Overall, participants agreed that they would recommend participation in the study to others . Intrusiveness ratings were slightly higher among participants who completed only 1 smartphone assessment than among those who completed 2 assessments  of participants in the smartphone group completed the next-day survey after the real-world drinking episode. Most participants  reported continued drinking after the final survey prompt for the episode see . ParticiP=.008), but binge drinking frequency and drinks consumed per drinking day did not change for either group (Ps>.12). There was a significant increase in self-reported alcohol consequences from baseline to 2-week follow-up .GEE revealed that self-reported drinking frequency decreased at follow-up for both the smartphone and control groups  completed the EMA protocol in the setting of a drinking binge. The smartphone-based approach was feasible, as evidenced by a high response rate  to system-initiated study prompts during the 3-hour assessment period, and acceptable, with participants reporting high satisfaction study procedures and low ratings on intrusiveness for the brief (1 min each) EMA surveys. Safety was evidenced by few drinking-related consequences and no increases in drinking quantity or frequency over the 2-week follow-up. The results of this study show that young adult heavy drinkers can complete mobile assessments of their drinking behavior and related outcomes even during very heavy drinking episodes at BAC well over the threshold for intoxication. The method described here is user-friendly and easy to administer on participants’ own smartphones and could be customized to facilitate future research on a variety of topics related to risky drinking. Furthermore, it may be less susceptible than retrospective or laboratory-based studies of binge drinking outcomes to limitations posed by recall bias and unclear ecological validity, respectively.typical drinking.Relative to prior EMA studies in social drinkers, our 87% response rate to survey prompts is similar to, or higher than, response rates reported in previous studies ,12-15. NRegarding feasibility and acceptability of the EMA approach, participants reported good compliance with study directions to avoid alcohol or other drug use during the assessments and high overall satisfaction with study procedures. The data from this study suggest that heavy drinkers can comply with instructions to avoid those substances even during binge episodes with high BAC and that this approach can produce data on drinking-related outcomes that are contaminated only minimally by other substance use. The high acceptability ratings reported in this study echo previous findings from EMA studies in general young adult drinker samples ,36. The Importantly, the results of this study suggest that EMA of a real-world binge drinking episode in heavy drinkers is safe. Participants reported few alcohol-related problems during the drinking episode, and those that were reported tended to be minor . Both groups reported a small decrease in their frequency of alcohol use from baseline to 2-week follow-up, with no changes in binge drinking frequency. This finding is in line with the results of previous studies indicating that EMA of alcohol use is associated with minimal or no changes in reported drinking behavior ,37-39. HThis study featured several strengths, including a well-characterized sample of young adult heavy drinkers, application of existing mobile phone technology to support EMA of real-world heavy drinking and related outcomes, and inclusion of an independent heavy drinking sample as a safety comparison group. However, there are also some limitations worth noting. First, although we recruited current binge drinkers, light drinkers and those meeting criteria for severe AUD were excluded from participation. Thus, based on the current data, we cannot infer the feasibility, acceptability, or safety of our smartphone-based EMA approach in those groups. Second, the duration of our monitoring period  was brief and fixed, in contrast to previous studies that have assessed alcohol use outcomes over several weeks  and scalIn sum, the results of this study provide preliminary support for the feasibility, acceptability, and safety of using smartphone-based EMA to assess alcohol use and related outcomes  in young adult heavy drinkers. These data suggest that young adults can use smartphones to monitor their drinking even during very heavy drinking episodes, with average consumption approximately double (or more) that of previous work in this field ,11,14,35