Synthesis and Evaluation of diaryl ether modulators of the leukotriene A4 hydrolase aminopeptidase activity.

Activation of the aminopeptidase (AP) activity of leukotriene A4 hydrolase (LTA4H) presents a potential therapeutic strategy for resolving chronic inflammation. Previously, ARM1 and derivatives were found to activate the AP activity using the alanine-p-nitroanilide (Ala-pNA) as a reporter group in an enzyme kinetics assay. As an extension of this previous work, novel ARM1 derivatives were synthesized using a palladium-catalyzed Ullmann coupling reaction and screened using the same assay. Analogue 5, an aminopyrazole (AMP) analogue of ARM1, was found to be a potent AP activator with an AC50 of 0.12 µM. An X-ray crystal structure of LTA4H in complex with AMP was refined at 2.7 Å. Despite its AP activity with Ala-pNA substrate, AMP did not affect hydrolysis of the previously proposed natural ligand of LTA4H, Pro-Gly-Pro (PGP). This result highlights a discrepancy between the hydrolysis of more conveniently monitored chromogenic synthetic peptides typically employed in assays and endogenous peptides. The epoxide hydrolase (EH) activity of AMP was measured in vivo and the compound significantly reduced leukotriene B4 (LTB4) levels in a murine bacterial pneumonia model. However, AMP did not enhance survival in the murine pneumonia model over a 14-day period. A liver microsome stability assay showed metabolic stability of AMP. The results suggested that accelerated Ala-pNA cleavage is not sufficient for predicting therapeutic potential, even when the full mechanism of activation is known.

Efficient removal of tetracycline hydrochloride through novel Fe/BiOBr/Bi2WO6 photocatalyst prepared by dual-strategy under visible-light irradiation.

It is important to investigate whether combining two modification strategies has a synergistic effect on the activity of photocatalysts. In this manuscript, Fe-doped BiOBr/Bi2WO6 heterojunctions were synthesized by a one-pot solvothermal method, and excellent photocatalytic performance was obtained for the degradation of tetracycline hydrochloride (TCH) in water without the addition of surfactant. Combining experiments and characterization, the synergistic effect between Fe ion doping and the BiOBr/Bi2WO6 heterojunction was elucidated. The Fe/BiOBr/Bi2WO6 composite photocatalyst had a beneficial void structure, enhanced visible light response, and could inhibit the recombination of photogenerated support well, which improved the photocatalytic activity. The presented experiments demonstrate that Fe/BiOBr/Bi2WO6 removes 97% of TCH from aqueous solution, while pure BiOBr and Bi2WO6 only remove 56% and 65% of TCH, respectively. Finally, the separation and transfer mechanisms of photoexcited carriers were determined in conjunction with the experimental results. This study provides a new direction for the design of efficient photocatalysts through the use of a dual co-modification strategy.

Metabolomic Profiling of Human Urine Samples Using LC-TIMS-QTOF Mass Spectrometry.

The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m/z, isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.

Glycemic Control Following GLP-1 RA or Basal Insulin Initiation in Real-World Practice: A Retrospective, Observational, Longitudinal Cohort Study.

INTRODUCTION: Injectable therapies such as glucagon-like peptide-1 receptor agonists (GLP-1 RAs) and basal insulin (BI) are well-established agents for people with type 2 diabetes (T2D). This study aimed to investigate real-world effectiveness of GLP-1 RAs or BI in adults with T2D poorly controlled on oral antihyperglycemic drugs (OADs). METHODS: This was a retrospective, observational, longitudinal cohort study of adults with T2D from the US Optum Humedica® database and UK Clinical Practice Research Datalink, who initiated either injectable between January 1, 2010, and June 30, 2016. Baseline characteristics, glycated hemoglobin (HbA1c) change, and cumulative percentage reaching HbA1c < 7% in 24 months after initiation were analyzed in four patient cohorts. RESULTS: In the US and UK databases, respectively, 20,836 and 5508 patients initiated GLP-1 RAs and 60,598 and 5083 initiated BI. Baseline mean HbA1c at initiation ranged between 8.8% and 10.3% across all cohorts. In all cohorts, a decrease of HbA1c occurred 3-6 months after initiation. The cumulative percentage of patients reaching HbA1c < 7% showed the greatest probability in the first 12 months (15-40% of patients across cohorts at 12 months), particularly in the first 6 months after initiation. The probability of reaching glycemic control diminished after the second quarter. The proportion of patients reaching HbA1c < 7% in both GLP-1 RA and BI cohorts at 12 months was < 25% if baseline HbA1c was ≥ 9%. CONCLUSIONS: For adults with T2D inadequately controlled on OADs, this analysis reveals an unmet clinical need. Initiation of first injectable therapy did not occur until HbA1c was considerably above target, when control is harder to achieve. Results suggest that in individuals with baseline HbA1c ≥ 9.0%, only a minority are likely to achieve an HbA1c < 7% with a GLP-1 RA or BI alone.

Real-world evidence of the effectiveness on glycaemic control of early simultaneous versus later sequential initiation of basal insulin and glucagon-like peptide-1 receptor agonists.

AIM: To assess the impact of the timing of initiating both basal insulin and glucagon-like peptide-1 receptor agonists (GLP-1 RAs) on reaching glycaemic control targets over 6 and 12 months in people with type 2 diabetes (T2D) uncontrolled on oral antihyperglycaemic drugs with an HbA1c of 9% or higher. METHODS: This retrospective cohort study assessed the impact of the timing of initiating both basal insulin and GLP-1 RA therapies on reaching glycaemic targets (HbA1c < 7% and <8%, and ≥1% and ≥2% HbA1c reduction) over 12 months in people with markedly uncontrolled T2D (HbA1c ≥ 9%) on oral antihyperglycaemic drugs identified on the Optum Humedica database (electronic medical records; 1 January 2011 to 30 June 2017). Study cohorts were defined by the days between initiating each injectable: cohort A, 30 days or less (simultaneous initiation) and cohorts B, 31-90, C, 91-180, D, 181-270 and E, 271-360 days (sequential initiation). RESULTS: Cohort A had the best glycaemic outcomes at 6 and 12 months for all four endpoints, followed by cohort B. The likelihood of achieving an HbA1c of less than 7% did not significantly differ between cohorts A and B (hazard ratio [95% confidence interval]: 0.87 [0.76-1.01]); cohorts C, D and E were significantly less likely to achieve an HbA1c of less than 7% than cohort A (0.62 [0.53-0.72]; 0.62 [0.53-0.72]; 0.63 [0.54-0.73]). CONCLUSIONS: In people with uncontrolled T2D requiring treatment with a GLP-1 RA and basal insulin, greater improvements in glycaemic control were observed when both therapies were initiated within close proximity of one another (≤90 days) compared with initiation 91-360 days apart.

A Real-World Observational Study Evaluating the Probability of Glycemic Control with Basal Insulin or Glucagon-Like Peptide-1 Receptor Agonist in Japanese Patients with Type 2 Diabetes.

INTRODUCTION: The effectiveness of basal insulin (BI) or glucagon-like peptide-1 receptor agonists (GLP-1 RAs) in providing glycemic control in patients with type 2 diabetes (T2D) in Japanese routine practice is not well known. This real-world observational study evaluated the probability of achieving glycemic control in Japanese patients with T2D uncontrolled by oral antidiabetic drugs (OADs) who initiated BI or GLP-1 RA therapy. METHODS: Patients with T2D aged ≥ 18 years initiating BI or GLP-1 RA therapy following treatment with OADs were selected from real-world data (RWD) retrieved from a large electronic medical record database in Japan, using data from 01 January 2010 to 30 June 2019. Patients were required to have glycated hemoglobin (HbA1c) ≥ 7% within 90 days prior to the first prescription of BI or GLP-1 RA. The probability of reaching first HbA1c < 7% was assessed over a 24-month period in cohorts of patients who initiated BI (n = 3477) or GLP-1 RA (n = 780) and in subcohorts by number of OADs at baseline (1, 2, or ≥ 3), HbA1c at baseline (≥ 7 to < 8%, ≥ 8 to < 9%, or ≥ 9%), and age (< 65 or ≥ 65 years). RESULTS: Mean (standard deviation) baseline HbA1c was 9.4% (1.8%) and 8.8% (1.4%) in patients initiating BI or GLP-1 RA therapy, respectively. The cumulative probability of achieving glycemic control was 50.1% with BI and 60.3% with GLP-1 RA therapy, respectively, at 12 months, and 60.8% and 66.6%, respectively, at 24 months. Quarterly (3-month intervals) conditional probabilities of achieving glycemic control decreased over time and were < 10% after 12 months. Patients with more OADs or higher HbA1c at baseline had a lower probability of achieving glycemic control. CONCLUSION: Among Japanese patients with T2D who initiated BI or GLP-1 RA therapy after treatment with OADs, the probability of reaching first glycemic control diminished over time. Further therapy intensification is warranted in patients who do not achieve glycemic control within 6-12 months with BI or GLP-1 RA, particularly those with high HbA1c or taking multiple OADs.

Patients with type 2 diabetes (T2D) who are taking oral antidiabetic drugs (OADs) but still have high blood glucose often require injectable drugs, such as basal insulin (BI) or glucagon-like peptide-1 receptor agonists (GLP-1 RAs). While BI and GLP-1 RAs have been shown to be effective in controlled clinical trials, it is unclear how well they improve blood glucose in real-world routine practice. Here, we report the results of an observational study that used data retrieved from a large electronic medical records database in Japan to explore how well BI and GLP-1 RAs allow patients to achieve glycemic control [glycated hemoglobin (HbA1c) < 7%].In Japanese patients with T2D receiving treatment with OADs and initiating BI or GLP-1 RA therapy, the probability of achieving glycemic control in the first quarter (3 months) after initiation was 20.3% with BI and 38.6% with GLP-1 RA. Among those patients who had not previously reached glycemic control, the probability of achieving first glycemic control declined over time, as evidenced in each quarterly assessment, and it was < 10% after the first year. Patients who had higher HbA1c levels or were taking multiple OADs were less likely to achieve glycemic control compared with those with lower HbA1c or taking fewer OADs. Our findings suggest that patients who have not achieved their glycemic goals within the first 6­12 months after starting BI or GLP-1 RA therapy have a low likelihood of achieving their target by maintaining the same therapy. For such patients, intensification with additional medication (e.g., combined BI and GLP-1 RA therapy) should be considered early in treatment.

Impact of Simultaneous Versus Sequential Initiation of Basal Insulin and Glucagon-like Peptide-1 Receptor Agonists on HbA1c in Type 2 Diabetes: A Retrospective Observational Study.

INTRODUCTION: When and how to intensify treatment in patients with type 2 diabetes (T2D) not achieving glycated hemoglobin (HbA1c) targets with oral antidiabetic drugs (OADs) in clinical practice remains a matter of clinical preference. This pilot study was conducted using the retrospective observational data from such patients to evaluate the impact on HbA1c of three treatment sequences: simultaneous initiation of basal insulin (BI) and a glucagon-like peptide-1 receptor agonist (GLP-1 RA; Cohort 1); BI followed by GLP-1 RA initiation within a 90-day timeframe (Cohort 2); or BI followed by GLP-1 RA initiation beyond 90 days (Cohort 3). METHODS: Data from the regional US electronic medical records database, Research Action for Health Network (REACHnet), were extracted for all patients with T2D aged ≥ 18 years who had encounter dates between January 2011 and August 2017 and ≥ 1 HbA1c laboratory value(s) < 90 days before BI initiation and ≥ 2 HbA1c laboratory values within 1 year after BI initiation and who met the inclusion criteria for GLP-1 RA initiation set for Cohorts 1, 2, or 3. The primary endpoints were the proportion of patients achieving HbA1c < 7.0%, which was estimated via Kaplan-Meier analysis, and change in HbA1c within 12 months. RESULTS: Overall, 869 patients were analyzed, of whom 109 were in Cohort 1, 301 in Cohort 2, and 459 in Cohort 3. Baseline HbA1c was 10.3 ± 2.1, 10.3 ± 2.0, and 10.2 ± 2.1% for these three cohorts, respectively. Statistically significantly more patients in Cohort 1 than in Cohort 3 achieved HbA1c < 7.0% (33.4 vs. 20.9%, respectively; p = 0.0186). Mean observed reductions in HbA1c at 12 months were - 1.7% (Cohort 1), - 1.5% (Cohort 2), and - 1.3% (Cohort 3). CONCLUSIONS: Simultaneous initiation of BI and GLP-1 RA achieves glycemic control more effectively than sequential initiation of BI with GLP-1 RA added beyond 90 days.

Probability of Achieving Glycemic Control with Basal Insulin in Patients with Type 2 Diabetes in Real-World Practice in the USA.

INTRODUCTION: Basal insulin (BI) plays an important role in treating type 2 diabetes (T2D), especially when oral antidiabetic (OAD) medications are insufficient for glycemic control. We conducted a retrospective, observational study using electronic medical records (EMR) data from the IBM® Explorys database to evaluate the probability of achieving glycemic control over 24 months after BI initiation in patients with T2D in the USA. METHODS: A cohort of 6597 patients with T2D who started BI following OAD(s) and had at least one valid glycated hemoglobin (HbA1c) result recorded both within 90 days before and 720 days after BI initiation were selected. We estimated the changes from baseline in HbA1c every 6 months, the quarterly conditional probabilities of reaching HbA1c < 7% if a patient had not achieved glycemic control prior to each quarter (Q), and the cumulative probability of reaching glycemic control over 24 months. RESULTS: Our cohort was representative of patients with T2D who initiated BI from OADs in the USA. The average HbA1c was 9.1% at BI initiation, and decreased robustly (1.5%) in the first 6 months after initiation with no further reductions thereafter. The conditional probability of reaching glycemic control decreased rapidly in the first year (26.6% in Q2; 17.6% in Q3; 8.6% in Q4), and then remained low (≤ 6.1%) for each quarter in the second year. Cumulatively, about 38% of patients reached HbA1c < 7% in the first year; only approximately 8% more did so in the second year. CONCLUSION: Our study of real-world data from a large US EMR database suggested that among patients with T2D who initiated BI after OADs, the likelihood of reaching glycemic control diminished over time, and remained low from 12 months onwards. Additional treatment options should be considered if patients do not reach glycemic control within 12 months of BI initiation. FUNDING: Sanofi Corporation.

The application of 3D printed surgical guides in resection and reconstruction of malignant bone tumor.

The clinical value of 3D printed surgical guides in resection and reconstruction of malignant bone tumor around the knee joint were studied. For this purpose, a sample of 66 patients from October 2013 to October 2015 were randomly selected and further divided into control group and observation group, each group consisted of 33 cases. The control group was treated by conventional tumor resection whereas, in the observation group, the tumor was resected with 3D printed surgical guide. However, reconstruction of tumor-type hinge prosthesis was performed in both groups and then the clinical effect was compared. Results show that there was no significant difference in the operation time between the two groups (p>0.05). However, the blood loss, resection length and complication rate were found significantly lower in the observation group than in the control group (p<0.05). The rate of negative margin and the recurrence rate in the 12-month follow-up (p>0.05) between two groups were statistically the same (p>0.05), whereas the Musculoskeletal Tumor Society (MSTS) score of the knee joint in the observation group was significantly better than that of the control group (p<0.05) after 1, 3, 6 and 12 months of the operation. Consequently, the 3D printed surgical guides can significantly improve the postoperative joint function after resection and reconstruction of malignant bone tumor around the knee joint and can reduce the incidence of complications.

The value of computer-assisted navigation for bone reconstruction after tumor resection.

This study was designed to evaluate the use of computer-assisted navigation with computed tomography (CT) images for bone reconstruction after resection in malignant bone tumor treatment. Forty-five patients with malignant bone tumors were recruited for this study. CT scan images in a computer-assisted navigation system were used to assist during the osteotomy, the pairing with allografts, and the monitoring of the allograft and joint lines to perform joint reconstruction. Our results show that osteotomy and allograft pairing were successful in all patients. The average duration of the osteotomy procedures was 46.8±12.3 min; and the average pairing time was 32.5±9.8 min. The anatomical registration points and the three-dimensional virtual CT images were successfully matched. The average error of registration was 0.36±0.09 mm. Also, the range of tumor resection and allograft osteotomy were successfully paired, with an average error of 0.11±0.03 mm. No complications such as unequal limbs length or joint deformities occurred after reconstruction. The average follow-up time was 11.6±3.9 months. The tumor recurrence rate was 11.1% (5/45) and the survival rate 95.6% (43/45). The average healing time for the allograft and host bone was 5.5±1.2 months and no unexpected internal fixations, fractures or joint collapses occurred. The average knee joint functionality MSTS score was 25.5±6.6 points. No significant differences were found in the length of tumor resection, rate of negative incision margin, duration of osteotomy or of pairing, registration error or allogeneic bone and defect matching error averages between those patients with tumor recurrence and those without it (p>0.05). Based on our results, the computer-assisted navigation system for bone reconstruction after malignant tumor resection allows for high precision during osteotomy, delivers a high success rate of pairing, results in great limb function and low complication rates, and is thus a highly successful and safe approach benefiting bone cancer patients.

Combining the G-protein-coupled receptor 40 agonist fasiglifam with sitagliptin improves glycaemic control in patients with type 2 diabetes with or without metformin: A randomized, 12-week trial.

AIMS: To evaluate the efficacy and safety of fasiglifam, an orally active G-protein-coupled receptor 40 agonist, in combination with the dipeptidyl peptidase-4 inhibitor sitagliptin, in patients with type 2 diabetes inadequately controlled with diet/exercise (± metformin). MATERIALS AND METHODS: In this randomized, double-blind, phase II study, 368 patients received once-daily placebo, sitagliptin 100 mg, fasiglifam 25 or 50 mg, or the combination of sitagliptin 100 mg plus fasiglifam 25 or 50 mg. The primary endpoint was change from baseline glycated haemoglobin (HbA1c) at 12 weeks; a key secondary endpoint was change in fasting plasma glucose (FPG). RESULTS: The fasiglifam 25 and 50 mg combination regimens produced significantly greater HbA1c reductions than sitagliptin (treatment differences of -0.45% and -0.61%; P < .01, respectively) or respective doses of fasiglifam monotherapy (-0.43% and -0.48%; P < .01) and significantly greater FPG reductions than sitagliptin (-1.1 mmol/L for both combination regimens; P < .01). Improved glycaemic control occurred by week 1 for FPG and week 4 for HbA1c in all groups. Hypoglycaemia rates were low (≤3.3%) and similar across treatments. Liver enzymes >3 × upper limit of normal occurred in four patients (fasiglifam 25 mg, n = 1; fasiglifam 50 mg, n = 2; 1 fasiglifam/sitagliptin 50/100 mg, n = 1). CONCLUSIONS: Combination of fasiglifam and sitagliptin provided significant additional effects on glycaemic control, with hypoglycaemia rates similar to placebo with or without metformin. This study provides supportive clinical evidence for the complementary mechanism of actions of this GPR40 agonist and DPP-4 inhibitor.

Evaluation of the pharmacokinetics and safety of a single oral dose of fasiglifam in subjects with normal or varying degrees of impaired renal function.

INTRODUCTION: Approximately one-third of patients with type 2 diabetes mellitus (T2DM) have concurrent renal impairment. There are limited therapeutic options for these patients. Fasiglifam is a G protein-coupled receptor 40 agonist that was under investigation for the treatment of T2DM. The objective of this study was to evaluate the potential effect of renal impairment on the pharmacokinetics and safety of a single dose of fasiglifam and its metabolite M-1. METHODS: This was a phase I, open-label, parallel-group study. Subjects with varying degrees of renal function received a single oral dose of fasiglifam 50 mg. Blood and urine samples were collected through 168 h postdose. Study endpoints were pharmacokinetic and safety variables. RESULTS: Fifty-three subjects were enrolled. Mean fasiglifam plasma concentrations were higher in subjects with mild renal impairment compared with other groups, but within each renal function cohort, plasma concentrations tended to decrease with decreasing renal function. Regression analyses indicated that fasiglifam exposure decreased and M-1 exposure increased with decreasing renal function. Predicted exposure values at about the midpoint of creatinine clearance for each renal impairment group differed by up to 21% (fasiglifam) and 87% (M-1) from that of the normal renal function group. Hemodialysis had no effect on fasiglifam or M-1 exposure. Fasiglifam renal clearance (CLR) was not affected, but M-1 CLR decreased with increasing impairment. No incidences of hypoglycemia were reported during the study. CONCLUSION: Varying renal function status did not have a significant impact on the clearance of fasiglifam in this study.

Effects of ramelteon on insomnia symptoms induced by rapid, eastward travel.

OBJECTIVE: Ramelteon, an MT(1)/MT(2) melatonin receptor agonist, was evaluated for its ability to reduce sleep-onset difficulties associated with eastward jet travel. METHODS: Healthy adults (n=110) with a history of jet lag sleep disturbances were flown eastward across five time zones from Hawaii to the east coast of the US. Ramelteon 1, 4, or 8 mg or placebo was administered 5 min before bedtime (local time) for four nights. Sleep parameters were measured using polysomnography (PSG) on Nights 2, 3, and 4. Next-day residual effects were assessed using psychomotor and memory function tests. RESULTS: Compared to placebo, there was a significant decrease in mean latency to persistent sleep (LPS) on Nights 2-4 with ramelteon 1mg (-10.64 min, P=0.030). No consistent significant differences were observed with ramelteon vs. placebo on measures of next-day residual effects except on Day 4 where participants in all ramelteon groups performed significantly worse on the immediate memory recall test compared with placebo (P < or = 0.05). The incidence of adverse events was similar for ramelteon and placebo. CONCLUSION: After a 5-h phase advance due to eastward jet travel, ramelteon 1mg taken before bedtime for four nights reduced mean LPS relative to placebo in healthy adults.

Effect of ramelteon on middle-of-the-night balance in older adults with chronic insomnia.

STUDY OBJECTIVES: To evaluate the effect of ramelteon on middle-of-the-night balance, mobility, and memory in older insomniacs. METHODS: Thirty-three older adults (age > or = 65 years) with insomnia were enrolled in a single-dose, 3-way crossover study of balance after bedtime administration of ramelteon, 8 mg; zolpidem, 10 mg (positive control); or placebo. Subjects were administered study medication 30 minutes before bedtime and were awakened 2 hours after dosing to evaluate balance (Sensory Organization Test), turning speed and stability, memory (immediate and delayed word recall), and adverse events. There was a 4- to 10-day washout between treatments. RESULTS: Ramelteon or zolpidem (positive control) was compared with placebo. There were no differences between placebo and ramelteon on the Sensory Organization Test (p = 0.837), turn time (p = 0.776), or turn sway (p = 0.982). The positive control (zolpidem) did reveal significant impairments on the Sensory Organization Test, turn time, and turn sway (p < 0.001, all). Immediate and delayed memory recall were not significantly different with ramelteon (p = 0.683 and p = 0.650, respectively). Immediate recall declined significantly with zolpidem (p = 0.002). Adverse events were infrequent (ramelteon, n = 7; placebo, n = 7; zolpidem, n = 13); none were serious. CONCLUSION: In older adults, ramelteon did not impair middle-of-the night balance, mobility, or memory relative to placebo.

Circadian phase-shifting effects of repeated ramelteon administration in healthy adults.

STUDY OBJECTIVES: To assess the ability of repeated daily oral ramelteon to facilitate re-entrainment of human circadian rhythms after an imposed phase advance of the sleep-wake cycle. METHODS: A total of 75 healthy adult volunteers aged 18-45 years remained in a sleep laboratory for 6 days and 5 nights; a 5-h phase advance in their sleep-wake cycle was imposed under dim-light conditions. Oral ramelteon (1,2, 4, or 8 mg once daily for 4 days) or placebo was administered 30 min before bedtime. The primary endpoint was the phase of the circadian rhythm as assessed by the time at which salivary melatonin concentrations declined below 3 pg/mL after morning awakening (dim-light melatonin offset [DLMoff]). RESULTS: After 4 days of once-daily treatment, participants receiving 1, 2, or 4 mg ramelteon exhibited statistically significant phase shifts in DLMoff of -88.0 (16.6), -80.5 (14.8), and -90.5 (15.2) minutes respectively, versus -7.1 (18.6) minutes for placebo (least-squares mean(SEM), p = 0.002, p = 0.003, p = 0.001, respectively). Change in DLMoff for the 8 mg dose of ramelteon, -27.9 (16.4) minutes, was not significantly different than that for placebo (p = 0.392). CONCLUSIONS: Ramelteon (1, 2, or 4 mg per day) administered before bedtime significantly advanced the phase of the circadian rhythm after a 5-h phase advance in the sleep-wake cycle. These findings suggest that ramelteon has potential as a specific therapy for circadian rhythm sleep disorders.

Use of computerized dynamic posturography to assess balance in older adults after nighttime awakenings using zolpidem as a reference.

BACKGROUND: Computerized dynamic posturography (CDP) has been used to detect balance and stability impairments in adults of all ages. The goal of the current pilot study was to evaluate balance in healthy older adults after a middle-of-the-night awakening and to assess the ability of CDP to measure effects of bedtime zolpidem administration. METHODS: Two studies used CDP to evaluate balance in healthy older adults (> or = 65 years) during middle-of-the-night awakenings. The first study used a drug-free, single-period, within-subject, repeated measures study design. Subjects were evaluated during the day, pre-sleep, and 2 hours after bedtime for dynamic standing balance using the NeuroCom EquiTest Sensory Organization Test (SOT). Pairwise comparisons were made using one-way ANOVA. The second study was a single-blind, randomized, placebo-controlled, crossover study evaluating the ability of the SOT to measure medication-induced dynamic standing balance impairments using the commonly prescribed sleep medication, zolpidem 10 mg, as a test medication. Assessments were performed at night before zolpidem administration and then again 2 hours after bedtime. Comparisons were made between the 2 groups using an ANCOVA model. RESULTS: Twelve older adults (mean age 68.4 years) were evaluated in the first study. There was no significant difference between pre-sleep and middle-of-the-night assessments for the SOT composite score (P = 0.439). Eleven older adults (mean age 68.9 years) were evaluated in the second study. Zolpidem administration significantly decreased the SOT composite score after a middle-of-the-night awakening compared with placebo (P < 0.001). CONCLUSION: In healthy older adults, getting up in the middle of the night did not have a significant effect on dynamic standing balance; however, bedtime administration of zolpidem 10 mg did lead to significant impairments. Thus, the SOT was able to measure medication-induced dynamic standing balance impairments and may be useful for future studies comparing balance effects of medications.

Genome-wide transcriptome expression in the liver of a mouse model of high carbohydrate diet-induced liver steatosis and its significance for the disease.

PURPOSE: To perform a large-scale gene profiling of the liver in a mouse model of fatty liver induced by high carbohydrate (sucrose) diet (HCD) to gain a deeper insight into potential mechanisms of diet-induced hepatic steatosis. METHODS: C57BL/6 male mice were fed either a purified, control diet or a HCD for 16 weeks. HCD feeding led to marked liver steatosis without inflammation or necrosis. The expression of 42,500 genes/sequences was assessed. RESULTS: A number of genes (471) underwent significant expression changes in HCD- as compared to standard diet-fed mice (n = 5/group; P < 0.01). Of these genes, 211 were down- and 260 up-regulated. The latter group includes 20 genes encoding enzymes involved in carbohydrate conversion to fat. The genes that underwent expression changes perform a large variety of molecular functions, and the vast majority of these have never been tested before in non-alcoholic fatty liver of nutritional origin. They reveal novel aspects of the disease and allow identification of candidate genes that may underlie the initiation of hepatic steatosis and progression to non-alcoholic steatohepatitis. CONCLUSIONS: HCD-fed laboratory animals provide a model of early non-alcoholic fatty liver disease resembling the disease in humans. The genome wide gene profiling of the liver reveals the complexity of the disease, unravels novel aspects of HCD-induced hepatic steatosis, and helps elucidate its nature and mechanisms.

Bortezomib induces caspase-dependent apoptosis in Hodgkin lymphoma cell lines and is associated with reduced c-FLIP expression: a gene expression profiling study with implications for potential combination therapies.

The Hodgkin cells and Reed-Sternberg cells (HRS) of classical Hodgkin lymphoma (CHL) are derived from germinal center B cells. The pathogenesis of CHL is unclear but constitutive activation of NFkappaB may contribute. Proteasome inhibition aimed at inhibiting NFkappaB has been shown to result in apoptosis in HRS cells. Here we investigated the effects of bortezomib, a proteasome inhibitor, in HRS cells with a combination of functional assays and gene expression profiling (GEP). Exposure of KMH2 and L428 cells to bortezomib resulted in inhibition of proliferation and induction of apoptosis. Gene expression analysis of KMH2 cells by oligonucleotide cDNA microarrays showed that a limited set of genes were differentially expressed involving several key cellular pathways including cell cycle and apoptosis. Among them, the caspase 8 inhibitor cFLIP was down-regulated and confirmed by Q-PCR. Given the evidence that cFLIP in HRS cells contribute to cells' insensitive to death receptor-mediated apoptosis, we combined bortezomib and TRAIL. This combination caused further down-regulation of cFLIP protein and increased apoptosis in CHL cells demonstrated by PARP p85 immunohistochemistry and immunoblotting. Such apoptotic effects were inhibited by caspase inhibitor z-VAD-FMK, confirming the pro-apoptotic effects of bortezomib and TRAIL are caspase-dependent. Bortezomib has no detectable effect on expression of TRAIL receptor DR4/DR5 in these two cell lines. Tissue microarray analysis of primary Hodgkin lymphomas displayed that 82% cases (95/116) expressed cFLIP in Reed-Sternberg cells. The discovery of apoptotic pathways that can be manipulated by proteasome inhibition provides rationale for the combination of bortezomib and agents such as TRAIL in CHL treatment.

Developing and evaluating genomics- or proteomics-based diagnostic tests: statistical perspectives.

The completion of the Human Genome Project and the ongoing sequencing of mouse, rat, and other genomes have led to an explosion of genetics-related technologies that are finding their way into all areas of biological research in both basic sciences and clinical applications. High-throughput genomics and proteomics technology has been quickly adapted to develop tools for clinical and pharmacological applications. Because molecular alterations usually occur much earlier than histological, physiological, and clinical abnormality, researchers hope to extend the applications of genomics and/or proteomics technology to early diagnosis of diseases and clinical outcome prognosis. Recently, some successful attempts in molecular diagnosis or prognosis have been published. However, for such tests to be translated from the bench to the bed, they must meet some rigorous standards. To develop a clinically meaningful genomics-based diagnostic test, we must have good study design, appropriate statistical analyses, and valid assessment of its clinical efficacy. In this chapter, we discuss statistical considerations on the process of developing reliable and useful genomics- or proteomics-based tests.

Increased vulnerability of ApoE4 neurons to HIV proteins and opiates: protection by diosgenin and L-deprenyl.

Human immunodeficiency virus (HIV) infection continues to rise in drug-abusing populations and causes a dementing illness in a subset of individuals. Factors contributing to the development of dementia in this population remain unknown. We found that HIV-infected individuals with the E4 allele of Apolipoprotein E (ApoE) or history of intravenous drug abuse had increased oxidative stress in the CNS. In vitro studies showed that HIV proteins, gp120 and Tat, Tat + morphine but not tumor necrosis factor-alpha (TNF-alpha), caused increased neurotoxicity in human neuronal cultures with ApoE4 allele. Microarray analysis showed a differential alteration of transcripts involved in energy metabolism in cultures of ApoE3 and 4 neurons upon treatment with Tat + morphine. This was confirmed using assays of mitochondrial function and exposure of the neurons to Tat + morphine. Using this in vitro model, we screened a number of novel antioxidants and found that only L-deprenyl and diosgenin protected against the neurotoxicity of Tat + morphine. Furthermore, Tat-induced oxidative stress impaired morphine metabolism which could also be prevented by diosgenin. In conclusion, opiate abusers with HIV infection and the ApoE4 allele may be at increased risk of developing dementia. L-deprenyl and a plant estrogen, diosgenin, may have therapeutic potential in this population.