Analytical method for simultaneously measuring ex vivo drug receptor occupancy and dissociation rate: Application to (R)-dimethindene occupancy of central histamine H1 receptors

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k₄). [Here, we reference k₄ as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan ()]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [³H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k₄ for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60?mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k₄ rate determined by using our ex vivo method was equivalent to k₄determined by in vitro competition kinetics (dissociation half-life t_1/2 ~ 30?min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.     

Assessment of sigma-1 receptor occupancy in mice with non-radiolabelled FTC-146 as a tracer

Abstract

The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) is advantageous in in-vivo receptor occupancy assays at pre-clinical drug developmental stages. Relatively, its application is effective in terms of high throughput, data reproducibility, sensitivity, and sample processing. In this perspective, we have evaluated the use of FTC-146 as a non-radiolabelled tracer to determine the sigma-1 receptor occupancy of test drugs in mice brain. Further, the brain and plasma exposures of test drug were determined at their corresponding occupancies. In this occupancy method, the optimized tracer treatment (sacrification) time after intravenous administration was 30?min. The tracer dose was 3?µg/kg and specific brain regions of interest were frontal cortex, pons and midbrain. Mice were pretreated orally with SA4503, fluspidine, haloperidol, and donepezil followed by tracer treatment. Among the test drugs, SA4503 was used as positive control group at its highest test dose (7?mg/kg, intraperitoneal). There was a dose-dependent decrease in brain regional FTC-146 binding in pretreated mice. From the occupancy curves of SA4503, fluspidine, haloperidol, and donepezil the effective dose (ED₅₀) value ranges are 0.74–1.45, 0.09–0.11, 0.11–0.12, and 0.07–0.09?mg/kg, respectively. Their corresponding brain effective concentration (EC₅₀) values are 74.3–132.5, 3.4–3.7, 122.5–139.5, and 8.8–11.0?ng/g and plasma EC₅₀ values are 34.3–53.7, 0.08–0.10, 7.8–9.5, and 0.6–0.7?ng/mL. Brain regional distribution and binding inhibition upon pretreatment were comparable with data reported with labeled [¹⁸F]FTC-146. Drug exposures were simultaneously determined and correlated with sigma-1 occupancy from the same experiment. Wide category drugs can be assayed for sigma-1 receptor engagement and their correlation with exposures aid in clinical development.     

Block the light and sleep well: Evening blue light filtration as a part of cognitive behavioral therapy for insomnia

ABSTRACT

The objective of the present study was to assess the effect of combining CBT-I with wearing blue-light blocking glasses 90 min prior to bedtime on subjective and objective sleep parameters and daily symptoms (anxiety, depression, hyperarousal). Thirty patients (mean age 48.1 ± 16.13 years, range 21–71, 15 men/15 women) completed a CBT-I group therapy program, with groups randomly assigned to either “active” (blue-light filtering glasses) condition or “placebo” (glasses without filtering properties) condition. Patients were continually monitored by wristwatch actigraphy, kept their sleep diaries and completed a standard questionnaire battery at admission and after the end of the program. Statistical analyses showed a greater reduction of BAI score in “active” (4.33 ± 4.58) versus “placebo” (?0.92 ± 3.68) groups of patients [F = 6.389, p = .019, Cohen’s d = 1.26] and significant prolongation of subjective total sleep time in “active” (?36.88 ± 48.68 min.) versus “placebo” (7.04 ± 47.50 min.) [F = 8.56, p < .01, d = 0.91] group. When pre- and post-treatment results were compared in both groups separately, using paired-samples t-tests, significant differences were observed also in the active group for BDI-II score (t = 3.66, p = .003, Cohen’s d = 0.95) and HAS score (t = 2.90, p = .012, Cohen’s d = 0.75). No significant differences were found in the placebo group. In active group, there was also a significant reduction of subjective sleep latency (t = 2.65, p = .021, d = 0.73) and an increase of subjective total sleep time (t = ?2.73, p = .018, d = ?0.76) without change in objective sleep duration which was significantly shortened in the placebo group. We provide further evidence that blocking short-wavelength light in the evening hours may be beneficial for patients suffering from insomnia.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

A definite measure of occupancy exposures,seeking with non-radiolabeled in vivo 5-HT2A receptor occupancy and in vitro free fractions

Abstract

Unbound drug concentration in the brain would be the true exposure responsible for specific target occupancy. Drug exposures from preclinical are total concentrations of those over/underestimate the clinical dose projection. With the application of mass spectrometry, the current work proposes a definite measure of test drug exposures at serotonin-2A occupancy. The 5-HT_2A occupancy of antagonist in the rat brain has determined with non-radiolabeled tracer MDL-100,907 at an optimized dose (3?µg/kg) and treatment time (30?min). Equilibrium dialysis method determines the in vitro free fraction of the test antagonist in untreated rat brain homogenates and plasma. Drug-free fractions derived the unbound concentration (EC₅₀) in plasma and brain at test doses. The corresponding binding affinities (Ki) correlated with the unbound concentrations. Except for quetiapine, the ED₅₀ values in the dose-occupancy curves of antagonists are close and ranged from 1 to 3?mg/kg. The test drug quetiapine, eplivanserin, and clozapine showed high free fractions in plasma, but for ketanserin and olanzapine, the brain free fraction was higher. The correlation between the unbound EC₅₀ of the antagonists and corresponding Ki values was good (r²=0.828). The improved EC₅₀ accuracy with unbound concentrations was 10–250 folds in plasma and 10–170 folds in the brain. Further, the free fractions (f_{u, plasma}/f_{u, brain}) of test drugs had shown a correlation of ～83% with brain permeability (C_{total brain}/C_{total plasma}), a limiting factor. Thus, correlating the occupancy with unbound exposure and pharmacology would result in an accurate measurement of drug potency and optimizes in selecting the clinical dose.     

Involvement of NMDA receptor in low-frequency magnetic field-induced anxiety in mice

It had been reported that exposure to extremely low-frequency magnetic field (ELFMF) induces anxiety in human and rodents. Anxiety mediates via the activation of N-methyl-d-aspartate (NMDA) receptor, whereas activation of γ-aminobutyric acid (GABA) receptor attenuates the same. Hence, the present study was carried out to understand the contribution of NMDA and/or GABA receptors modulation in ELFMF-induced anxiety for which Swiss albino mice were exposed to ELFMF (50?Hz, 10?G) by subjecting them to Helmholtz coils. The exposure was for 8?h/day for 7, 30, 60, 90 and 120 days. Anxiety level was assessed in elevated plus maze, open field test and social interaction test, on 7th, 30th, 60th, 90th and 120th exposure day, respectively. Moreover, the role of GABA and glutamate in ELFMF-induced anxiety was assessed by treating mice with muscimol [0.25?mg/kg intraperitoneally (i.p.)], bicuculline (1.0?mg/kg i.p.), NMDA (15?mg/kg i.p.) and MK-801 (0.03?mg/kg i.p.), as a GABA_A and NMDA receptor agonist and antagonist, respectively. Glutamate receptor agonist exacerbated while inhibitor attenuated the ELFMF-induced anxiety. In addition, levels of GABA and glutamate were determined in regions of the brain viz, cortex, striatum, hippocampus and hypothalamus. Experiments demonstrated significant elevation of GABA and glutamate levels in the hippocampus and hypothalamus. However, GABA receptor modulators did not produce significant effect on ELFMF-induced anxiety and elevated levels of GABA at tested dose. Together, these findings suggest that ELFMF significantly induced anxiety behavior, and indicated the involvement of NMDA receptor in its effect.     

Zopiclone versus placebo for short-term treatment of insomnia in patients with advanced cancer: study protocol for a double-blind,randomized, placebo-controlled,clinical multicenter trial

Background

Despite the high prevalence of insomnia in patients with advanced cancer, there are no randomized controlled trials on pharmacological interventions for insomnia in this group of patients. A variety of pharmacological agents is recommended to manage sleep disturbance for insomnia in the general population, but their efficacy and safety in adults with advanced cancer are not established. Thus, there is a need to evaluate the effectiveness of medications for insomnia in order to improve the evidence in patients with advanced cancer. One of the most used sleep medications at present in patients with cancer is zopiclone.

Methods

This is a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. A total of 100 patients with metastatic cancer who report insomnia will be randomly allocated to zopiclone or placebo. The treatment duration with zopiclone/placebo is 6 consecutive nights. The primary endpoint is patient-reported sleep quality during the final study night (night 6) assessed on a numerical rating scale of 0–10, where 0?=?Best sleep and 10?=?Worst possible sleep. Secondary endpoints include the mean patient-reported total sleep time and sleep onset latency during the final study night (night 6).

Discussion

Results from this study on treatment of insomnia in advanced cancer will contribute to clinical decision-making and improve the treatment of sleep disturbance in this patient cohort.

Trial registration

ClinicalTrials.gov, NCT02807922. Registered on 21 June 2016.

     

Oximes short-acting CB1 receptor agonists

New oximes short-acting CB1 agonists were explored by the introduction of an internal oxime and polar groups at the C3 alkyl tail of Δ⁸-THC. The scope of the research was to drastically alter two important physicochemical properties hydrophobicity (log P) and topological surface area (tPSA) of the compound, which play a critical role in tissue distribution and sequestration (depot effect). Key synthesized analogs demonstrated sub-nanomolar affinity for CB1, marked reduction in hydrophobicity (ClogP～2.5–3.5 vs 9.09 of Δ⁸-THC-DMH), and found to function as either agonists (trans-oximes) or neutral antagonists (cis-oximes) in a cAMP functional assay. All oxime analogs showed comparable affinity at the CB2 receptor, but surprisingly they were found to function as inverse agonists for CB2. In behavioral studies (i.e. analgesia, hypothermia) trans-oxime 8a exhibited a predictable fast onset (～20?min) and short duration of pharmacological action (～180?min), in contrast to the very prolonged duration of Δ⁸-THC-DMH (>24?h), thus limiting the potential for severe psychotropic side-effects associated with persistent activation of the CB1 receptor. We have conducted 100?ns molecular dynamic (MD) simulations of CB1 complexes with AM11542 (CB1 agonist) and both trans-8a and cis-8b isomeric oximes. These studies revealed that the C3 alkyl tail of cis-8b orientated within the CB1 binding pocket in a manner that triggered a conformational change that stabilized the CB1 receptor at its inactive-state (antagonistic functional effect). In contrast, the trans-8a isomer’s conformation was coincided with that of the AM11542 CB1 agonist-bound structure, stabilizing the CB1 receptor at the active-state (agonistic functional effect). We have selected oxime trans-8a based on its potency for CB1, and favorable pharmacodynamic profile, such as fast onset and predictable duration of pharmacological action, for evaluation in pre-clinical models of anorexia nervosa.     

Sibutramine Produces Dose-Related Weight Loss

BRAY, GEORGE A., GEORGE L. BLACKBURN, JAMES M. FERGUSON, FRANK L. GREENWAY, ADESH K. JAIN, CARL M. MENDEL, JOSEPH MEN-DELS, DONNA H. RYAN, SHERWYN L. SCHWARTZ, MONTE L. SCHEINBAUM, AND TIMOTHY B. SEATON. Sibutramine produces dose-related weight loss. Obes Res. Objective Sibutramine is a weight control drug that inhibits the reuptake of both serotonin and norepinephrine. In animals, it reduces food intake and increases thermogenesis and preliminary data in human beings showed weight loss. This paper reports a 24-week dose-ranging study to determine the effect of sibutramine on body weight of patients with obesity. Research Methods and Procedures Seven clinical centers screened 1463 patients with obesity and randomized 1047 to 24 weeks of treatment with 1 of 6 doses of sibutramine (1, 5, 10, 15, 20, or 30 mg) or placebo once daily. Six hundred eighty-three patients completed the study. A two-week placebo run-in period was used to initiate a standardized program of diet, physical activity, and lifestyle changes. Results Weight loss was dose-related and statistically significant vs. placebo (p <0.05) across all time-points for a 5 mglday to 30 mglday dosage of sibutramine. At week 24, percent weight loss from baseline for completers was: placebo, 1.2%; 1 mg, 2.7%; 5 mg, 3.9%; 10 mg, 6.1%; 15 mg, 7.4%; 20 mg, 8.8%; and 30 mg, 9.4%. Weight loss achieved at week 4 was predictive of weight loss achieved at week 24. Patients losing weight demonstrated an increase in serum high density lipoprotein cholesterol and reductions in serum triglycerides, total cholesterol, low density lipoprotein cholesterol, and uric acid. Small mean increases in blood pressure and pulse rate (with considerable individual variability) were observed in patients treated with sibutramine. The most frequent adverse events were dry mouth, anorexia, and insomnia. Discussion Sibutramine administered once daily for 24 weeks in the weight loss phase of treatment for uncomplicated obesity produced dose-related weight loss and was well tolerated. Improvements in serum lipids and uric acid accompany sibutramine-induced weight loss. Most of the adverse events observed on sibutramine are related to its pharmacology, including small mean increases in blood pressure and heart rate.     

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs)

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX₁R/OX₂R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.     

Circadian Phase,Sleepiness, and Light Exposure Assessment in Night Workers With and Without Shift Work Disorder

Most night workers are unable to adjust their circadian rhythms to the atypical hours of sleep and wake. Between 10% and 30% of shiftworkers report symptoms of excessive sleepiness and/or insomnia consistent with a diagnosis of shift work disorder (SWD). Difficulties in attaining appropriate shifts in circadian phase, in response to night work, may explain why some individuals develop SWD. In the present study, it was hypothesized that disturbances of sleep and wakefulness in shiftworkers are related to the degree of mismatch between their endogenous circadian rhythms and the night-work schedule of sleep during the day and wake activities at night. Five asymptomatic night workers (ANWs) (3 females; [mean?±?SD] age: 39.2?±?12.5 yrs; mean yrs on shift?=?9.3) and five night workers meeting diagnostic criteria (International Classification of Sleep Disorders [ICSD]-2) for SWD (3 females; age: 35.6?±?8.6 yrs; mean years on shift?=?8.4) participated. All participants were admitted to the sleep center at 16:00?h, where they stayed in a dim light (<10 lux) private room for the study period of 25 consecutive hours. Saliva samples for melatonin assessment were collected at 30-min intervals. Circadian phase was determined from circadian rhythms of salivary melatonin onset (dim light melatonin onset, DLMO) calculated for each individual melatonin profile. Objective sleepiness was assessed using the multiple sleep latency test (MSLT; 13 trials, 2-h intervals starting at 17:00?h). A Mann-Whitney U test was used for evaluation of differences between groups. The DLMO in ANW group was 04:42?±?3.25?h, whereas in the SWD group it was 20:42?±?2.21?h (z = 2.4; p?<?.05). Sleep did not differ between groups, except the SWD group showed an earlier bedtime on off days from work relative to that in ANW group. The MSLT corresponding to night work time (01:00–09:00?h) was significantly shorter (3.6?±?.90?min: [M?±?SEM]) in the SWD group compared with that in ANW group (6.8?±?.93?min). DLMO was significantly correlated with insomnia severity (r = ?.68; p < .03), indicating that the workers with more severe insomnia symptoms had an earlier timing of DLMO. Finally, SWD subjects were exposed to more morning light (between 05:00 and 11:00?h) as than ANW ones (798 vs. 180 lux [M?±?SD], respectively z?=??1.7; p?<?.05). These data provide evidence of an internal physiological delay of the circadian pacemaker in asymptomatic night-shift workers. In contrast, individuals with SWD maintain a circadian phase position similar to day workers, leading to a mismatch/conflict between their endogenous rhythms and their sleep-wake schedule. (Author correspondence: vgumeny1@hfhs.org)     

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10⁻⁶M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.     

Randomized controlled trials of the D1/D5 antagonist ecopipam for weight loss in obese subjects

Objective: To evaluate the efficacy and safety of the selective dopamine D1/D5 antagonist ecopipam for the treatment of obesity. Research Methods and Procedures: Four randomized, double‐blind, multicenter trials compared ecopipam (n = 1667) and placebo (n = 1118) in obese subjects including type 2 diabetic subjects. Subjects received oral ecopipam 10, 30, or 100 mg daily for 12 weeks (Phase 2) or 50 or 100 mg daily for 52 weeks (Phase 3) combined with a weight loss program. Primary efficacy variables were the proportion of subjects with ≥5% weight loss from baseline at 12 weeks (Phase 2) or the distribution of percentage weight loss from baseline at 52 weeks (Phase 3). Results: In the Phase 2 study, 26% of subjects administered ecopipam 100 mg vs. 6% of placebo subjects achieved ≥5% weight loss after 12 weeks (p < 0.01). In the Phase 3 studies, ecopipam 100 mg produced a 3.1% to 4.3% greater weight loss than placebo at 52 weeks. More subjects administered ecopipam vs. placebo achieved a 5% to 10% or >10% weight loss in two non‐diabetic phase 3 trials. Ecopipam‐treated subjects also maintained more weight loss compared with placebo subjects at 52 weeks. Phase 3 studies were discontinued because of unexpected psychiatric adverse events (ecopipam 31% vs. placebo 15%), including depression, anxiety, and suicidal ideation. Discussion: Ecopipam was effective for achieving and maintaining weight loss in obese subjects, including type 2 diabetic subjects; however, the adverse effects on mood observed in the Phase 3 studies exclude its projected use in weight management.     

The D2 dopamine receptor occupancy of risperidone and its relationship to extrapyramidal symptoms: A pet study

Risperidone is a recently introduced neuroleptic distinguished by a decreased incidence of extrapyramidal side effects (EPS). The mechanism of its low EPS is unclear. Since it has been shown that EPS is related to the level of D₂ receptor occupancy, we studied nine patients receiving 2–6 mg/day of risperidone using [¹¹C]-raclopride PET scans in order to determine the in vivo D₂ receptor binding characteristics of risperidone. The mean level of receptor occupancy was 66% at 2 mg; 73% at 4 mg; and 79% at 6 mg. Three patients, those with the highest receptor occupancies, exhibited mild EPS, though none required anitparkinsonian medications. Our results suggest that at doses of 4–6 mg the in vivo D₂ receptor occupancy of risperidone is similar to that of typical neuroleptics and higher than that of clozapine. This would suggest that the EPS benefits of risperidone cannot be explained by a low D₂ binding but may be related to its high 5-HT₂ affinity. However, the emergence of EPS at higher levels of D₂ receptor occupancy, in this study and in previous clinical trials, would suggest that risperidone's high 5-HT₂ affinity provides only a relative protection from EPS. and once the D₂ occupancy exceeds a certain threshold this ‘relative’ 5-HT₂-mediated protection from EPS may be lost.     

Organization of the human interleukin-1 receptor antagonist gene IL1HY1

 The interleukin (IL)-1 family of proteins plays an important role in inflammatory and defense mechanisms. The recently characterized IL1HY1 cDNA encodes a new member of the IL-1 receptor antagonist family (IL-1ra). In this report, we describe the complete nucleotide sequence of the human IL1HY1 gene. We sequenced approximately 7600 nucleotides and found four coding exons ranging in size from 55 to 2288 nucleotides. The 5′ untranslated region is formed by one of two alternatively used exons and one invariably present exon which also contains the region encoding the first nine amino acids of the protein. IL1HY1 and IL-1ra intron positions are well conserved within the protein-coding region, providing evidence that these genes arose from a duplication of a primordial IL-1 receptor antagonist gene. Received: 15 October 1999 / Revised: 30 December 1999     

A constitutively active form of neurokinin 1 receptor and neurokinin 1 receptor-mediated apoptosis in glioblastomas

Previous studies have shown that neurokinin 1 receptor (NK1R) occurs naturally in human glioblastomas and its stimulation causes cell proliferation. In the present study we show that stimulation of NK1R in human U373 glioblastoma cells by substance P increases Akt phosphorylation by 2.5-fold, with an EC₅₀ of 57 nM. Blockade of NK1R lowers basal phosphorylation of Akt, indicating the presence of a constitutively active form of NK1R; similar results are seen in U251 MG and DBTRG-05 glioblastoma cells. Linkage of NK1R to Akt implicates NK1R in apoptosis of glioblastoma cells. Indeed, treatment of serum-starved U373 cells with substance P reduces apoptosis by 53 ± 1% ( p  <   0.05), and treatment with NK1R antagonist L-733,060 increases apoptosis by 64 ± 16% ( p  <   0.01). Further, the blockade of NK1R in human glioblastoma cells with L-733,060 causes cleavage of Caspase-3 and proteolysis of poly (ADP-ribose) polymerase. Experiments designed to elucidate the mechanism of NK1R-mediated Akt phosphorylation revealed total involvement of non-receptor tyrosine kinase Src and phosphatidyl-3-kinase, a partial involvement of epidermal growth factor receptor, and no involvement of mitogen-activated protein/extracellular signal-related kinase. Taken together, the results of the present study indicate a key role for NK1R in glioblastoma apoptosis.     

Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons

Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pig ileum, including inhibitory motor neurons and secretomotor neurons. In the present work, endocytosis of NK1 receptors in these enteric neurons was studied following addition of substance P (SP) to isolated ileum. NK1 receptors were localised with antibodies against the C-terminus of this receptor. Some preparations were incubated with SP tagged with the fluorescent label, Cy3.18, so that the fate of SP bound to receptors could be followed. Preparations were analysed by confocal microcopy. In tissue that was incubated at 4° C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37° C in the presence of 10⁻⁷ M SP (plus 3×10⁻⁷M tetrodotoxin to prevent indirect activation via other neurons) the neuronal NK1 receptor was rapidly internalised. After 5 min, NK1 receptor IR was partially internalised, at 20 min NK1 receptor IR was throughout the cytoplasm and in perinuclear aggregates and at 30 min it was again at the cell surface. SP-induced NK1 receptor endocytosis was inhibited by the specific NK1 receptor antagonist, SR140333. Cy3-SP was colocalised with NK1 receptor IR and was internalised with the NK1 receptor. These results show that enteric neurons exhibit authentic NK1 receptors that are rapidly internalised when exposed to their preferred ligand.     

Effect of blue-blocking glasses in major depressive disorder with sleep onset insomnia: A randomized,double-blind,placebo-controlled study

Blue wavelengths form the portion of the visible electromagnetic spectrum that most potently regulates circadian rhythm. We hypothesized that wearing blue-blocking (BB) glasses in the evening may influence circadian rhythm disturbances in patients with major depressive disorder (MDD), resulting in improved sleep and mood. We used a randomized placebo-controlled double-blinded design. Patients with MDD with sleep onset insomnia were randomly assigned to wearing either BB glasses or clear glasses (placebo). Patients were instructed to wear the glasses from 20:00 hours until bedtime for 2 weeks. We assessed sleep state (sleep quality on a visual analog scale, the Morningness–Eveningness Questionnaire [MEQ], and a sleep diary) and depressive symptoms at baseline and after 2 weeks. Data were analyzed with a full analysis set. In total, 20 patients were randomly assigned to the BB and placebo groups (BB group, n = 10; placebo group, n = 10). There were three dropouts (BB group, n = 1; placebo group, n = 2). At baseline, sleep quality, sleep latency (assessed via a sleep diary), and antipsychotics use differed between the groups. To take account of these differences, the baseline sleep state or depressive symptoms and antipsychotics use were used as covariates in the later analysis. The change scores for sleep quality did not show a significant improvement in the BB group compared with the placebo group (mean [standard deviation, SD] scores for BB versus placebo: 36.1 [31.7] versus 16.2 [15.1], p = 0.43), although half of the BB group showed a clear improvement in sleep quality. The change in MEQ scores did not significantly differ between the groups (p = 0.14), although there was a trend of a shift to morning type in the BB group (3.10 [4.95] points) and to evening type in the placebo group (0.50 [3.89] points). There were no statistically significant changes in depressive symptoms in either group. Across both groups, 40% of the participants reported pain or discomfort from wearing the glasses, which were available in only one size. Thus, the failure to find significant differences may have resulted from the glasses used in this study. Glasses fitted to individual patients may improve efficacy and safety. Replication of the study with a larger sample size and size-adjustable glasses is needed.     

Sibutramine plus meal replacement therapy for body weight loss and maintenance in obese patients

Objective: Our objective was to assess the efficacy and safety of sibutramine with a low‐calorie diet (LCD) and commercial meal‐replacement product in achieving weight loss and weight‐loss maintenance in obese patients. Research Methods and Procedures: Eight U.S. centers recruited 148 obese patients for a 3‐month comprehensive weight‐loss therapy (Phase I) comprising daily sibutramine 10 mg + LCD (two Slim‐Fast meal‐replacement shakes, one low‐calorie meal; total kcal/d = 1200–1500). Patients (N = 113) who lost ≥5% of initial body weight during Phase I were randomized for a 9‐month period (Phase II) to daily sibutramine 15 mg + LCD (one meal‐replacement shake; two low‐calorie meals: total kcal/d ～1200–1500) or daily placebo + three low‐calorie meals (total kcal/d ～1200–1500). Both phases included behavior modification. Efficacy was assessed by body weight change during each phase and by the number of patients at endpoint maintaining ≥80% of the weight they had lost by the end of Phase I. Other outcomes included changes in cardiovascular and metabolic risk factors, adverse events, and vital signs. Results: Mean body weight change during Phase I was ?8.3 kg (p < 0.001). Patients randomized to sibutramine in Phase II had an additional ?2.5 kg mean weight loss vs. a 2.8‐kg increase in the placebo group (p < 0.001). More sibutramine patients maintained ≥80% of their Phase I weight loss at the end of Phase II (85.5% vs. placebo 36.7%, p < 0.001). Most adverse events were mild or moderate in severity, and all serious adverse events were unrelated to sibutramine. Discussion: Sibutramine plus LCD with meal replacements and behavior modification is a safe and effective strategy for achieving and sustaining weight loss in obese patients.     

Weight loss produced by tesofensine in patients with Parkinson's or Alzheimer's disease

Objective: Tesofensine (TE) is a norepinephrine, dopamine, and serotonin reuptake inhibitor. We conducted a meta‐analysis of TE's effect on body weight in trials investigating its potential for treatment of Parkinson's or Alzheimer's disease. Methods and Procedures: Four randomized, double‐blind, multicenter trials compared TE (n = 740) and placebo (n = 228), two in each disease. Patients received oral TE or placebo once daily for 14 weeks without any weight loss program. Results were adjusted for baseline values, age, and study. Results: In the placebo group, 14% were obese and 21% were in the TE group. In the total cohort, weight change after 14 weeks was +0.5, ?0.5, ?0.9, ?1.8, ?2.8% in the placebo, 0.125, 0.25, 0.5 and 1.0 mg in the TE groups, respectively (P = 0.015 for dose effect). In the obese subgroup, weight changes were ?0.2, ?1.7, ?1.6, ?1.5, ?3.7%, and 2.1, 8.2, 14.1, 20.9, 32.1% of the obese patients achieved ≥5% weight loss (P < 0.001 for 0.25, 0.5, and 1.0 mg vs. placebo for both end points). Changes in heart rate were ?0.4, 2.1, 4.2, 6.0, and 6.8 bpm after 14 weeks (TE vs. placebo: P < 0.001 from 0.25 mg), but no effect on blood pressure was observed. Discussion: TE produced a placebo‐subtracted weight loss of ～4% for >14 weeks without any diet and lifestyle therapy, which is similar to that of sibutramine, but with no effect on blood pressure. On the basis of these results, TE is now being developed for obesity management.