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The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not recapitulate human physiology at the tissue and organ levels. Here, we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production, and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection, but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential., One-sentence editorial summary: A microfluidic bronchial-airway-on-a-chip lined by human bronchial-airway epithelium and pulmonary endothelium can be used to rapidly identify antiviral therapeutics and prophylactics with repurposing potential.

Background and objectives Impaired nitric oxide signaling through soluble guanylate cyclase has been implicated in the pathophysiology of diabetic kidney disease. Praliciguat, a soluble guanylate cyclase stimulator that amplifies nitric oxide signaling, inhibited kidney inflammation and fibrosis in animal models. Design, setting, participants, & measurements In a phase 2 trial, 156 adults with type 2 diabetes, eGFR 30–75 ml/min per 1.73 m2, and urine albumin-creatinine ratio 200–5000 mg/g treated with renin-angiotensin system inhibitors were randomly allocated 1:1:1 to placebo, 20 mg praliciguat, or 40 mg praliciguat daily for 12 weeks. The primary efficacy and safety outcomes were change from baseline to weeks 8 and 12 in urine albumin-creatinine ratio and treatment-emergent adverse events, respectively. Other outcomes assessed were 24-hour ambulatory BP and metabolic parameters. Results Of 156 participants randomized, 140 (90%) completed the study. The primary efficacy analysis demonstrated a mean change from baseline in urine albumin-creatinine ratio of −28% (90% confidence interval, −36 to −18) in the pooled praliciguat group and −15% (−28 to 0.4) in the placebo group (difference −15%; −31 to 4; P=0.17). Between-group decreases from baseline to week 12 for praliciguat versus placebo were seen in mean 24-hour systolic BP (−4 mm Hg; −8 to −1), hemoglobin A1c (−0.3%; −0.5 to −0.03), and serum cholesterol (−10 mg/dl; −19 to −1). The incidence of treatment-emergent adverse events was similar in the pooled praliciguat and placebo groups (42% and 44%, respectively). Serious adverse events, events leading to study drug discontinuation, and events potentially related to BP lowering were reported at higher frequency in the 40-mg group but were similar in 20-mg and placebo groups. Conclusions Praliciguat treatment for 12 weeks did not significantly reduce albuminuria compared with placebo in the primary efficacy analysis. Nonetheless, the observed changes in urine albumin-creatinine ratio, BP, and metabolic variables may support further investigation of praliciguat in diabetic kidney disease. Clinical Trial registry name and registration number: A Study to Evaluate the Soluble Guanylate Cyclase (sGC) Stimulator IW-1973 in Diabetic Nephropathy/Diabetic Kidney Disease as Measured by Albuminuria, NCT03217591

The current coronavirus disease 2019 (COVID-19) pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III). These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique sequence motif (sense strand, 5'-C; antisense strand, 3'-GGG) that mediates end-to-end dimer self-assembly. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory small interfering RNAs (siRNAs), their activity is independent of Toll-like receptor (TLR) 7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with immunostimulant poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad-spectrum inhibition of infections by many respiratory viruses with pandemic potential, including severe acute respiratory syndrome coronavirus (SARS-CoV)-2, SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus (HCoV)-NL63, and influenza A virus in cell lines, human lung chips that mimic organ-level lung pathophysiology, and a mouse SARS-CoV-2 infection model. These short double-stranded RNAs (dsRNAs) can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics.

The ongoing COVID-19 pandemic has claimed more than 6 million lives and continues to test the world economy and healthcare systems. To combat this pandemic, the biological research community has shifted efforts to the development of medical countermeasures, including vaccines and therapeutics. However, to date, the only small molecules approved for the treatment of COVID-19 in the United States are the nucleoside analogue Remdesivir and the protease inhibitor Paxlovid, though multiple compounds have received Emergency Use Authorization and many more are currently being tested in human efficacy trials. One such compound, Apilimod, is being considered as a COVID-19 therapeutic in a Phase II efficacy trial. However, at the time of writing, there are no published efficacy data in human trials or animal COVID-19 models. Here we show that, while Apilimod and other PIKfyve inhibitors have potent antiviral activity in various cell lines against multiple human coronaviruses, these compounds worsen disease in a COVID-19 murine model when given prophylactically or therapeutically.

SummaryImportanceDrug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit.ObjectivesTo test if different statins differ in their ability to exert protective effects based on molecular computational predictions and electronic medical record analysis.Main Outcomes and MeasuresA Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2, with a total of 2,436 drugs investigated. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus.ResultsSimvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells.Conclusions and RelevanceDifferent statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

IntroductionThe use and misuse of opioids by active service members has been examined in several studies, but little is known about their spouses’ opioid use. This study estimates the number of military spouses who received high-risk or long-term opioid prescriptions between 2010 and 2014, and addresses how the Military Health System can help prevent risky prescribing in order to improve military force readiness.Materials and MethodsThis study used data from the Millennium Cohort Family Study, a nationwide survey of 9,872 spouses of service members with 2 to 5 years of military service, augmented with information from the military’s Pharmacy Data Transaction Service about prescriptions for controlled drugs dispensed to these service members’ spouses. Our objectives were to estimate the prevalence of opioid prescribing indicative of long-term use (≥60 day supply or at least one extended-release opioid prescription in any 3-month period) and, separately, high-risk use (daily dosage of ≥90 morphine mg equivalent or total dosage of ≥8,190 morphine mg equivalent, or prescriptions from more than three pharmacies, or concurrent prescriptions). For each of these dependent variables, we conducted bivariate analyses and multiple logistic regression models using information about spouses’ physical health, sociodemographic characteristics, substance use behaviors, perceived social support, and stresses associated with military stress, among others. Informed consent, including consent to link survey responses to medical and personnel records, was obtained from all participants. The Naval Health Research Center’s Institutional Review Board and the Office of Management and Budget approved the study.ResultsSpouses were predominantly female (86%), had not served in the military themselves (79%), and were spouses of enlisted (91%) active duty (86%) service members. Almost half (47.6%) of spouses obtained at least one opioid prescription during the 2-year observation window, and 8.5% had received opioid prescriptions that posed risk to their health. About 7% met the criteria for receipt of high-risk opioid prescriptions, 3% obtained opioids from three or more pharmacies during a 3-month period, and 4% of spouses who received any opioids received both long-term and high-risk prescriptions. Adverse childhood experiences, physical pain, and lack of social support were associated with increased odds of obtaining high-risk opioid prescriptions.ConclusionsApproximately 48% of military spouses had used Military Health System insurance to fill at least one opioid prescription during the 2-year observation period. The Department of Defense has taken measures to minimize high-risk opioid prescribing, including passing prescribing guidelines in 2017, establishing the controlled drug management analysis reporting tool, establishing a pain management education and training program, and more. These efforts should continue to expand as reducing the numbers of service members and spouses at risk for adverse events may be effective in reducing opioid misuse and improve the overall health and safety of military spouses and thus, the readiness of the U.S. Armed Forces.

The rising threat of pandemic viruses, such as SARS-CoV-2, requires development of new preclinical discovery platforms that can more rapidly identify therapeutics that are activein vitroand also translatein vivo. Here we show that human organ-on-a-chip (Organ Chip) microfluidic culture devices lined by highly differentiated human primary lung airway epithelium and endothelium can be used to model virus entry, replication, strain-dependent virulence, host cytokine production, and recruitment of circulating immune cells in response to infection by respiratory viruses with great pandemic potential. We provide a first demonstration of drug repurposing by using oseltamivir in influenza A virus-infected organ chip cultures and show that co-administration of the approved anticoagulant drug, nafamostat, can double oseltamivir’s therapeutic time window. With the emergence of the COVID-19 pandemic, the Airway Chips were used to assess the inhibitory activities of approved drugs that showed inhibition in traditional cell culture assays only to find that most failed when tested in the Organ Chip platform. When administered in human Airway Chips under flow at a clinically relevant dose, one drug – amodiaquine - significantly inhibited infection by a pseudotyped SARS-CoV-2 virus. Proof of concept was provided by showing that amodiaquine and its active metabolite (desethylamodiaquine) also significantly reduce viral load in both direct infection and animal-to-animal transmission models of native SARS-CoV-2 infection in hamsters. These data highlight the value of Organ Chip technology as a more stringent and physiologically relevant platform for drug repurposing, and suggest that amodiaquine should be considered for future clinical testing.

Objectives This research looked at whether notifying prescribers and pharmacies about patients who use multiple providers to obtain opioids reduces their prescribing activity (including use of multiple providers, numbers of opioid prescriptions, or amounts of opioids obtained). Design Nevada's prescription drug monitoring program (PDMP) identified patients using multiple providers to obtain opioids and assigned them alternately to an intervention or control group. Controlled substance prescription histories were sent only to intervention patients' providers. Subsequent opioid purchases by patients in both groups were compared. Setting All pharmacies and dispensers in the state are required to report every prescription for Schedule II-IV opioids dispensed to the PDMP. Subjects All patients receiving opioids from more than four different Nevada prescribers and more than four pharmacies during the previous six months assigned to the intervention (N = 436) or control group (N = 441). Methods We used ordinary least squares regression to estimate notification effects on each outcome, accounting for preexisting differences among groups. Results Providers receiving unsolicited notices were 13% less likely to continue prescribing to patients than providers not notified. Eighty-four percent of the intervention patients' prescribers discontinued prescribing to them after assignment, compared with 80.5% of the control group's prescribers-who were not notified. Because patients in both groups found other prescribers to replace discontinued prescribers, notification had at most a small effect on patients' use of multiple providers, numbers of opioid prescriptions, or amounts of opioids purchased. Conclusions Requiring prescribers to solicit patients' prescription histories is likely to be a more effective use of PDMP resources than proactive notification.

Highlights • Military spouses were more likely to have risky opioid Rx if their spouse did. • High-risk opioid therapy was linked to pain, disability, smoking, and ACES • Reducing risky opioid Rx for service members may reduce similar risky Rx for spouses., Background Estimates suggest approximately 2.4% of service members, and 15% of service members who have engaged in recent combat, report misusing pain relievers in the past year. This study explores the extent to which military spouses’ obtainment of opioids is associated with their service member partners’ obtainment of opioid prescriptions, in addition to other factors such as service member health, state prescribing patterns, and sociodemographic characteristics. Methods Data were drawn from the Millennium Cohort Family Study, a large, longitudinal survey of married spouses of service members from all service branches, and archival data analyzed from 2018 to 2020. The dependent variables were spouse long-term opioid therapy and spouse opioid prescriptions that pose a high risk of adverse outcomes. Results Seven percent of spouse and service member dyads met the criteria for high-risk opioid use, generally because they had purchased a prescription for a ≥90 Morphine Milligram Equivalents daily dose (76.7% for spouses, 72.8% for service members). Strong associations were found between spouse and service member opioid therapies (OR = 5.53 for long-term; OR = 2.20 for high-risk). Conclusions Findings suggest that reducing the number of long-term and high-risk opioid prescriptions to service members may subsequently reduce the number of similar prescriptions obtained by their spouses. Reducing the number of service members and spouses at risk for adverse events may prove to be effective in stemming the opioid epidemic and improve the overall health and safety of military spouses and thus, the readiness of the U.S. Armed Forces.

Aims/hypothesis Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, amplifies nitric oxide signalling. This exploratory trial investigated the safety, tolerability, pharmacokinetic profile and pharmacodynamic effects of praliciguat in individuals with type 2 diabetes and hypertension. Methods This Phase IIA, double-blind, placebo-controlled trial investigated praliciguat in 26 participants with type 2 diabetes and hypertension on stable glucose- and BP-lowering therapies. Participants were randomly allocated in a 3:5:5 ratio to three groups: placebo (n = 6), praliciguat 40 mg once daily for days 1–14 (n = 10), or praliciguat 20 mg twice daily for days 1–7 then 40 mg once daily for days 8–14 (n = 10). Assessments were made in clinic and included treatment-emergent adverse events, pharmacokinetics, metabolic variables, 24 h BP and heart rate, platelet function, reactive hyperaemia index (RHI) and plasma biomarkers. Participants, the sponsor, the investigator and clinic study staff (except designated pharmacy personnel) were blinded to group assignment. Results Participants treated for 14 days with praliciguat had least-square mean change-from-baseline differences vs placebo (95% CI) of −0.7 (−1.8, 0.4) mmol/l for fasting plasma glucose, −0.7 (−1.1, −0.2) mmol/l for total cholesterol, −0.5 (−1.0, −0.1) mmol/l for LDL-cholesterol, −23 (−56, 9) for HOMA-IR in those not being treated with insulin, and −5 (−10, 1) mmHg and 3 (−1, 6) beats/min for average 24 h mean arterial pressure and heart rate, respectively. Apart from one serious adverse event (SAE; upper gastrointestinal haemorrhage), praliciguat was well tolerated. Praliciguat did not affect platelet function or RHI. Among exploratory biomarkers, plasma levels of asymmetric dimethylarginine decreased in praliciguat vs placebo recipients. Conclusions/interpretation In participants with type 2 diabetes and hypertension on standard therapies, over 14 days praliciguat was well tolerated, except for a single SAE, and showed positive trends in metabolic and BP variables. These results support further clinical investigation of praliciguat. Trial registration ClinicalTrials.gov NCT03091920. Funding This trial was funded by Cyclerion Therapeutics.

PURPOSE: This study estimates the prevalence in US counties of opioid patients who use large numbers of prescribers, the amounts of opioids they obtain, and the extent to which their prevalence is predicted by ecological attributes of counties, including general medical exposure to opioids. METHODS: Finite mixture models were used to estimate the size of an outlier subpopulation of patients with suspiciously large numbers of prescribers (probable doctor shoppers), using a sample of 146 million opioid prescriptions dispensed during 2008. Ordinary least squares regression models of county-level shopper rates included independent variables measuring ecological attributes of counties, including rates of patients prescribed opioids, socioeconomic characteristics of the resident population, supply of physicians, and measures of healthcare service utilization. RESULTS: The prevalence of shoppers varied widely by county, with rates ranging between 0.6 and 2.5 per 1000 residents. Shopper prevalence was strongly correlated with opioid prescribing for the general population, accounting for 30% of observed county variation in shopper prevalence, after adjusting for physician supply, emergency department visits, in-patient hospital days, poverty rates, percent of county residents living in urban areas, and racial/ethnic composition of resident populations. Approximately 30% of shoppers obtained prescriptions in multiple states. CONCLUSIONS: The correlation between prevalence of doctor shoppers and opioid patients in a county could indicate either that easy access to legitimate medical treatment raises the risk of abuse or that drug abusers take advantage of greater opportunities in places where access is easy. Approaches to preventing excessive use of different prescribers are discussed. Copyright © 2014 John Wiley & Sons, Ltd. Language: en

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with

Pepducins are a class of lipidated peptide G-protein coupled receptor (GPCR) allosteric modulators that exhibit unique pharmacological properties. Because of their rational design, pepducins can potentially be used to target all members of this gene family. In addition to enormous therapeutic potential, pepducins are proving to be valuable tools to elucidate the molecular mechanisms involved in GPCR signaling. In this review, we focus on the characterization of pepducin biology and discuss their utility as novel therapeutics.

At the 2nd Pepducin Science Symposium held in Cambridge, Massachusetts, on November 4-5, 2010, investigators working in G protein-coupled receptor (GPCR) research convened to discuss progress since last year's inaugural conference. This year's symposium focused on increasing knowledge of the structure and function of this ubiquitous superfamily of membrane receptors and their potential modulation for disease treatment. Presentations also focused on how GPCR mechanisms might be exploited to treat diseases with pepducins, novel synthetic lipopeptide pharmacophores that modulate heptahelical GPCR activity. While the multiple roles of GPCRs in physiological and pathophysiological processes offer significant opportunities for novel drug development, the global nature of their activity challenges drug-specific and validated target identification. This year's conference highlighted advances in understanding of GPCR agonist and antagonist ligand-binding motifs, their ligand-independent functions, structure-activity relationships (SARs), and evolving unique methods to probe GPCR structure and function. Study results summarized at the meeting also provided evidence for evolving views of how signaling mechanisms work through these receptors.

The cannabinoid CB 1 G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB 1 receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB 1 receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB 1 receptor ligands.

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341–mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.

It is well established that melanocortins are peptides that have potent anti-inflammatory activity. Recent research has focused on understanding which of the known melanocortin receptors mediates the anti-inflammatory actions of the melanocortins. The aim of this study was to assess the anti-inflammatory activity of a synthetic MC-1R agonist. BMS-470539 is a potent, selective, full agonist of human and murine MC-1R with EC50 values in a cAMP accumulation assay of 16.8 and 11.6 nM, respectively. BMS-470539 dose-dependently inhibited TNF-α-induced activation of a NF-κB transcriptional reporter in human melanoma cells, which endogenously express MC-1R. In vivo studies with BMS-470539 demonstrated that subcutaneous administration of BMS-470539 resulted in a dose-dependent inhibition of LPS-induced TNF-α production in BALB/c mice. In this model, the compound had an ED50 of approximately 10 μmol/kg and a pharmacodynamic half-life of ∼8 h. Pharmacokinetic analysis of the compound indicated that the compound had a t1/2 of 1.7 h. In a model of lung inflammation, administration of 15 μmol/kg BMS-470539 resulted in a 45% reduction in LPS-induced leukocyte infiltration (an infiltrate comprised primarily of neutrophils). The compound was also effective in a model of delayed-type hypersensitivity, reducing paw swelling by 59%, comparable with that seen with 5 mg/kg dexamethasone. These studies demonstrate that a selective small molecule agonist of the melanocortin-1 receptor is a potent anti-inflammatory agent in vivo and provides compelling evidence for the involvement of this receptor in the modulation of inflammation.

The melanocortin-1 receptor (MC-1R) is a G-protein-coupled receptor involved in inflammation and skin pigmentation. Compound 2 is the first highly potent and selective MC-1R small-molecule agonist reported. Compound 2 showed efficacy in an acute model of inflammation, which has demonstrated the role of MC-1R in modulation of inflammation.

Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.

BackgroundAbuse of prescription opioid analgesics is a serious threat to public health, resulting in rising numbers of overdose deaths and admissions to emergency departments and treatment facilities. Absent adequate patient information systems, "doctor shopping" patients can obtain multiple opioid prescriptions for nonmedical use from different unknowing physicians. Our study estimates the prevalence of doctor shopping in the US and the amounts and types of opioids involved.Methods and findingsThe sample included records for 146.1 million opioid prescriptions dispensed during 2008 by 76% of US retail pharmacies. Prescriptions were linked to unique patients and weighted to estimate all prescriptions and patients in the nation. Finite mixture models were used to estimate different latent patient populations having different patterns of using prescribers. On average, patients in the extreme outlying population (0.7% of purchasers), presumed to be doctor shoppers, obtained 32 opioid prescriptions from 10 different prescribers. They bought 1.9% of all opioid prescriptions, constituting 4% of weighed amounts dispensed.ConclusionsOur data did not provide information to make a clinical diagnosis of individuals. Very few of these patients can be classified with certainty as diverting drugs for nonmedical purposes. However, even patients with legitimate medical need for opioids who use large numbers of prescribers may signal dangerously uncoordinated care. To close the information gap that makes doctor shopping and uncoordinated care possible, states have created prescription drug monitoring programs to collect records of scheduled drugs dispensed, but the majority of physicians do not access this information. To facilitate use by busy practitioners, most monitoring programs should improve access and response time, scan prescription data to flag suspicious purchasing patterns and alert physicians and pharmacists. Physicians could also prevent doctor shopping by adopting procedures to screen new patients for their risk of abuse and to monitor patients' adherence to prescribed treatments.

Ibipinabant (IBI), a potent cannabinoid-1 receptor (CB1R) antagonist, previously in development for the treatment of obesity, causes skeletal and cardiac myopathy in beagle dogs. This toxicity was characterized by increases in muscle-derived enzyme activity in serum and microscopic striated muscle degeneration and accumulation of lipid droplets in myofibers. Additional changes in serum chemistry included decreases in glucose and increases in non-esterified fatty acids and cholesterol, and metabolic acidosis, consistent with disturbances in lipid and carbohydrate metabolism. No evidence of CB1R expression was detected in dog striated muscle as assessed by polymerase chain reaction, immunohistochemistry, Western blot analysis, and competitive radioligand binding. Investigative studies utilized metabonomic technology and demonstrated changes in several intermediates and metabolites of fatty acid metabolism including plasma acylcarnitines and urinary ethylmalonate, methylsuccinate, adipate, suberate, hexanoylglycine, sarcosine, dimethylglycine, isovalerylglycine, and 2-hydroxyglutarate. These results indicated that the toxic effect of IBI on striated muscle in beagle dogs is consistent with an inhibition of the mitochondrial flavin-containing enzymes including dimethyl glycine, sarcosine, isovaleryl-CoA, 2-hydroxyglutarate, and multiple acyl-CoA (short, medium, long, and very long chain) dehydrogenases. All of these enzymes converge at the level of electron transfer flavoprotein (ETF) and ETF oxidoreductase. Urinary ethylmalonate was shown to be a biomarker of IBI-induced striated muscle toxicity in dogs and could provide the ability to monitor potential IBI-induced toxic myopathy in humans. We propose that IBI-induced toxic myopathy in beagle dogs is not caused by direct antagonism of CB1R and could represent a model of ethylmalonic-adipic aciduria in humans.

Estimates of geographic variation among states and counties in the prevalence of opioid prescribing are developed using data from a large (135 million) representative national sample of opi- oid prescriptions dispensed during 2008 by 37,000 retail pharmacies. Statistical analyses are used to estimate the extent to which county variation is explained by characteristics of resident populations, their healthcare utilization, proxy measures of morbidity, availability of healthcare resources, and prescription monitoring laws. Geographic variation in prevalence of prescribed opioids is large, greater than the variation observed for other healthcare services. Counties having the highest pre- scribing rates for opioids were disproportionately located in Appalachia and in southern and western states. The number of available physicians was by far the strongest predictor of amounts prescribed, but only one-third of county variation is explained by the combination of all measured factors. Wide variation in prescribing opioids reflects weak consensus regarding the appropriate use of opioids for treating pain, especially chronic noncancer pain. Patients' demands for treatment have increased, more potent opioids have become available, an epidemic of abuse has emerged, and calls for in- creased government regulation are growing. Greater guidance, education, and training in opioid pre- scribing are needed for clinicians to support appropriate prescribing practices. Perspective: Wide geographic variation that does not reflect differences in the prevalence of in- juries, surgeries, or conditions requiring analgesics raises questions about opioid prescribing prac- tices. Low prescription rates may indicate undertreatment, while high rates may indicate overprescribing and insufficient attention to risks of misuse.

We have developed a solid phase synthesis route to 1,5-substituted pyrazole-4-carboxamides with three diversity points aimed at the discovery of new compounds as potential G-Protein coupled receptor (GPCR) ligands. The new chemistry involves acylation of a resin bound secondary amine with a β-ketoester via transamidation, conversion of the resulting β-ketoamide to the corresponding vinylogous amide, pyrazole formation upon reaction with a aryl hydrzine, and cleavage of the product from the resin. Using the reported methodology, we describe the syntheses of multiple arrays of pyrazoles that were used collectively to construct a library of more than 1000 analogues. Several members of this library displayed submicromolar antagonist activities at the cannabinoid subtype 1 (CB-1) receptor.

Although the intent of the Sentencing Reform Act of 1984 ("SRA," hereafter) was to eliminate disparities in sentences imposed by federal judges, differences in imprisonment sentences received by black, white, and Hispanic offenders grew more pronounced after full implementation of the guidelines. That is, imprison ment sentences meted out to blacks grew increasingly harsher, on average, relative to whites and Hispanics, during the first year and a half following full imple mentation of the guidelines (that is, after the U.S. Supreme Court's decision in Mistretta). At the request of the U.S. Department of Justice's Bureau of Justice Statistics, we undertook an analysis of sentencing decisions in all federal district courts between 1986

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.

Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease. CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affinity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radioligand was used to conduct ex vivo receptor occupancy studies.

Structure-activity relationships for a series of pyrazine carboxamide CB1 antagonists are reported. Pharmaceutical properties of the series are improved via inclusion of hydroxyl-containing sidechains. This structural modification sufficiently improved ADME properties of an orally inactive series such that food intake reduction was achieved in rat feeding models. Compound 35 elicits a 46% reduction in food intake in ad libidum fed rats 4-h post-dose.

We have previously shown that tumour necrosis factor-alpha (TNF-alpha) upregulates human melanoma cell integrin expression, migration and invasion in vitro. The aim of this study was to investigate the effect of the non-steroidal anti-inflammatory agent sodium salicylate on TNF-alpha-induced activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and upregulation of intercellular adhesion molecule-1 (ICAM-1), and TNF-alpha-stimulated cell migration and invasion through fibronectin. HBL human melanoma cells were pre-incubated with sodium salicylate prior to stimulation with TNF-alpha for 24 h. NF-kappaB activation was measured using an assay that detects changes in the expression of a luciferase reporter gene under the direct control of NF-kappaB transcriptional activity. The effect of sodium salicylate and TNF-alpha on HBL cell invasion over 20 h and migration over 24 h was studied using fibronectin invasion and 'scratch wound' migration models in vitro, as described previously. Sodium salicylate inhibited TNF-alpha-stimulated NF-kappaB activation in melanoma cells in a concentration-dependent manner, and this was achieved with pre-incubation times as short as 15 min. TNF-alpha-stimulated ICAM-1 expression in HBL cells was also downregulated by sodium salicylate, although in a manner inversely related to the concentration of this agent. In functional assays, TNF-alpha stimulated migration and invasion, and sodium salicylate significantly reduced the extent of melanoma invasion and migration in both the presence and absence of TNF-alpha. In conclusion, sodium salicylate effectively inhibited TNF-alpha-induced upregulation of NF-kappaB, ICAM-1 expression, in-vitro migration and invasion in human melanoma cells, indicating that non-steroidal anti-inflammatory drugs may be a useful therapeutic approach to oppose inflammation-induced melanoma invasion and metastasis in vivo.

In a previous report we demonstrated that merging together key structural elements present in an AT(1) receptor antagonist (1, irbesartan) with key structural elements in a biphenylsulfonamide ET(A) receptor antagonist (2) followed by additional optimization provided compound 3 as a dual-action receptor antagonist (DARA), which potently blocked both AT(1) and ET(A) receptors. Described herein are our efforts directed toward improving both the pharmacokinetic profile as well as the AT(1) and ET(A) receptor potency of 3. Our efforts centered on modifying the 2'-side chain of 3 and examining the isoxazolylsulfonamide moiety in 3. This effort resulted in the discovery of 7 as a highly potent second-generation DARA. Compound 7 also showed substantially improved pharmacokinetic properties compared to 3. In rats, DARA 7 reduced blood pressure elevations caused by intravenous infusion of Ang II or big ET-1 to a greater extent and with longer duration than DARA 3 or AT(1) or ET(A) receptor antagonists alone. Compound 7 clearly demonstrated superiority over irbesartan (an AT(1) receptor antagonist) in the normal SHR model of hypertension in a dose-dependent manner, demonstrating the synergy of AT(1) and ET(A) receptor blockade in a single molecule.

alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

Angiotensin II and endothelin-1 activate their respective AT(1) and ET(A) receptors on vascular smooth muscle cells, producing vasoconstriction, and both peptides are implicated in the pathogenesis of essential hypertension. Angiotensin II potentiates the production of endothelin, and conversely endothelin augments the synthesis of angiotensin II. Both AT(1) and ET(A) receptor antagonists lower blood pressure in hypertensive patients; thus, a combination AT(1)/ET(A) receptor antagonist may have greater efficacy and broader utility compared with each drug alone. By rational drug design a biphenyl ET(A) receptor blocker was modified to acquire AT(1) receptor antagonism. These compounds (C and D) decreased Sar-Ile-Angiotensin II binding to AT(1) receptors and endothelin-1 binding to ET(A) receptors, and compound C inhibited angiotensin II- and endothelin-1-mediated Ca(2+) transients. In rats compounds C and D reduced blood pressure elevations caused by intravenous infusion of angiotensin II or big endothelin-1. Compound C decreased blood pressure in Na(+)-depleted spontaneously hypertensive rats and in rats with mineralocorticoid hypertension. Compound D was more efficacious than AT(1) receptor antagonists at reducing blood pressure in spontaneously hypertensive rats, and its superiority was likely due to its partial blockade of ET(A) receptors. Therefore compounds C and D are novel agents for treating a broad spectrum of patients with essential hypertension and other cardiovascular diseases.

A series of 4'-[(imidazol-1-yl)methyl]biphenylsulfonamides has potent antagonist activity against both angiotensin II AT(1) and endothelin ET(A) receptors. Such dual-acting antagonists could have utility in the treatment of hypertension, heart failure, and other cardiovascular diseases in a broad patient population. Certain compounds in the present series are orally active in a rat model of angiotensin II-mediated hypertension.

The ET(A) receptor antagonist (2) (N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)-[1,1'-biphenyl]-2-sulfonamide, BMS-193884) shares the same biphenyl core as a large number of AT(1) receptor antagonists, including irbesartan (3). Thus, it was hypothesized that merging the structural elements of 2 with those of the biphenyl AT(1) antagonists (e.g., irbesartan) would yield a compound with dual activity for both receptors. This strategy led to the design, synthesis, and discovery of (15) (4'-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-N-(3,4-dimethyl-5-isoxazolyl)-2'-[(3,3-dimethyl-2-oxo-1-pyrrolidinyl)methyl]-[1,1'-biphenyl]-2-sulfonamide, BMS-248360) as a potent and orally active dual antagonist of both AT(1) and ET(A) receptors. Compound 15 represents a new approach to treating hypertension.

Phosphorylation of Gi by the phospholipid- and Ca2+-dependent enzyme protein kinase C has been proposed as a mechanism by which phorbol esters and certain agonists either attenuate inhibition of adenylyl cyclase or impair stimulation of phospholipase C (Jakobs, et al., 1985; Orellana et al., 1987; Smith et al., 1987; Pyne et al., 1989). Support for the phosphorylation of Giα is provided by the occurrence of this modification for one or more forms of Giα in solution with partially purified protein kinase C (Katada et al., 1985). Indeed, phosphorylation of the purified subunit(s) can also be accomplished with cAMP dependent and insulin receptor kinases (O’Brien et al., 1987; Krupinski et al., 1988; Watanabe et al., 1988). The occurrence of phosphorylation within the cell itself, however, has not been fully documented, nor have functional correlates of phosphorylation been established. In unstimulated rat hepatocytes incubated with [32P] H3PO4, a 41-kDa phosphoprotein can be immunoprecipitated with a Giα-directed antiserum (Rothenberg and Kahn, 1988; Pyne et al., 1989). Treatment of these cells with phorbol 12-myristate 13- acetate (PMA) results in a modest (i.e., 70%) increase in incorporated phosphate, while treatment with insulin has no effect. Incorporation of radiolabel into proteins having isoelectric or immunologic properties similar to Giα has also been reported for PMA-treated human platelets (Halenda et al., 1986; Crouch and Lapetina, 1988). The identities of these proteins as Giα, however, have not been rigorously established.

1. The specific retrograde axonal transport of free glycine within the identified neurons R3-14 ofAplysia californica was studied. 2. The soma of the R3-14 neurons are located in the parietovisceral ganglion and their axons project down the branchial nerve to end in a large peripheral field. Using a double-chambered apparatus, the peripheral tissue was incubated in medium containing a3H-amino acid for 4–48 hr, while the nerve and ganglion were isolated and perfused with plain or chemically altered medium. The nerve and ganglion were then either rapidly frozen for scintillation counting or fixed for autoradiography. 3. When3H-glycine was used, radioactivity entered the nerve rapidly, reached the ganglion in 3 hr, and was transported largely (>80%) in the free amino acid form [trichloroacetic acid (TCA) soluble]. The right parietovisceral hemiganglion accumulated up to nine times more radioactivity than the left hemiganglion, reflecting the presence of the R3-14 axons and soma. Two phases of radioactivity were observed, a fast component moving at about 3 mm/hr and a slower (but larger) component moving at about 0.4 mm/hr. 4. Light microscope autoradiography on nerves containing3H-glycine revealed that the R3-14 axons accounted for more than 30% of the total label in the nerve but occupied less than 7% of the total cross-sectional area of the axonal core. Electron microscope autoradiography showed a close association of silver grains and dense core vesicles in the R3-14 axons. 5. Retrograde axonal transport of free glycine was inhibited by (in decreasing order of effectiveness) mercuric chloride, vinblastine, colchicine, Nocodazole, and 2,4-dinitrophenol (2,4-DNP). 6. Comparative studies of other amino acids [3H-leucine,3H-serine,3H-glutamic acid,3H-γ-aminobutyric acid (3H-GABA), and3H-alanine] showed that3H-glycine is the only amino acid that is rapidly axonally transported in large quantities within the R3-14 axons. 7. This work demonstrates, for the first time, that a free amino acid, glycine, is transported in the retrograde direction within a select group of axons. The significance of this transport of glycine is discussed in relation to its use as a neural messenger by neurons R3-14.

RECOGNITION of the syndrome caused by myxoma of the left atrium is of the utmost importance now that surgery offers the opportunity for complete cure. In most instances, the disease is confused with more common cardiac disorders. To serve as a reminder, the following case report is added to the literature. Clinical Summary A 59-year-old woman was admitted to the Aliuippa Hospital on July 4, 1957, because of congestive heart failure. She had been well until 10 months previously, when, mowing her lawn, she developed severe pain in the anterior chest and left arm, associated with profuse perspiration and dyspnea. Because of the possibility of a myocardial infarction, she was hospitalized from Aug. 31 to Sept. 26, 1956. Blood pressure was 120/80 mm. Hg, and cardiac examination, including roentgen study, was normal. Electrocardiograms were normal with a Q wave of questionable significance in aVL. Hemoglobin was 9.5 gm.%; white blood-cell

CARDIAC SARCOIDOSIS has been encountered frequently enough to warrant its consideration in the differential diagnosis of heart disease of unknown origin. It is the purpose of this report to describe an autopsied case and to emphasize sarcoidosis as a cause of paroxysmal ventricular tachycardia. Report of a Case A 40-year-old Italian male was first admitted to the Aliquippa Hospital on June 4, 1957, because of severe anterior chest pain radiating down the inner aspect of the left arm. Mild pain had been present for 2 weeks. In 1937, in Italy, the patient was told that he had "suffocating heart disease." Nevertheless, he served in the Italian Army without restriction. During a preemployment examination in 1953, shortly after arriving in this country, again he was told he had heart trouble, but again was permitted to do manual labor. The patient was first examined 6 months prior to admission because of right