Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro.

Aspergillosis is difficult to treat and carries a high mortality rate in immunocompromised patients. Neutrophils play a critical role in control of infection but may be diminished in number and function during immunosuppressive therapies. Here, we measure the effect of three bifunctional small molecules that target Aspergillus fumigatus and prime neutrophils to generate a more effective response against the pathogen. The molecules combine two moieties joined by a chemical linker: a targeting moiety (TM) that binds to the surface of the microbial target, and an effector moiety (EM) that interacts with chemoattractant receptors on human neutrophils. We report that the bifunctional compounds enhance the interactions between primary human neutrophils and A. fumigatus in vitro, using three microfluidic assay platforms. The bifunctional compounds significantly enhance the recruitment of neutrophils, increase hyphae killing by neutrophils in a uniform concentration of drug, and decrease hyphal tip growth velocity in the presence of neutrophils compared to the antifungal targeting moiety alone. We validated that the bifunctional compounds are also effective in vivo, using a zebrafish infection model with neutrophils expressing the appropriate EM receptor. We measured significantly increased phagocytosis of A. fumigatus conidia by neutrophils expressing the EM receptor in the presence of the compounds compared to receptor-negative cells. Finally, we demonstrate that treatment with our lead compound significantly improved the antifungal activity of neutrophils from immunosuppressed patients ex vivo. This type of bifunctional compounds strategy may be utilized to redirect the immune system to destroy fungal, bacterial, and viral pathogens.

Causes of Emergency Department Overcrowding and Blockage of Access to Critical Services in Beijing: A 2-Year Study.

BACKGROUND: Emergency department (ED) overcrowding is a serious issue worldwide. OBJECTIVES: This study was done to evaluate the degree of overcrowding in local "teaching hospitals" in Beijing, and to ascertain the apparent root causes for the pervasive degree of overcrowding in these EDs. METHODS: This is a multicenter cross-sectional study. The studied population included all ED patients from 18 metropolitan teaching hospital EDs in Beijing for calendar years 2013 and 2014. Patient characteristics, and the primary reasons that these patients sought care in these EDs, are described. RESULTS: The total numbers of annual emergency visits were 1,554,387 and 1,615,571 in 2013 and 2014, respectively. High acuity cases accounted for 4.6% and 5.5% of the total annual emergency visits in 2013 and 2014, respectively. The percentage of patients placed into "Observation" beds, which were created to accommodate patients deemed to have problems too complex to be treated in an inpatient bed, or to accommodate patients simply needing chronic care, was 11.9% and 13.1% in 2013 and 2014, respectively. The ED-boarded patients accounted for 2.71% and 2.6% of the total annual emergency visits in 2013 and 2014, respectively. The average waiting time to admit the ED-boarded patients was 37.1 h and 36.2 h in 2013 and 2014, respectively. Respiratory symptoms were the most common presenting complaints, and an upper respiratory infection was the most common ED diagnosis. Patients who had pneumonia or various manifestations of end-stage diseases, such as advanced dementia or multiple organ dysfunction, were the most common characteristics of patients who had stays in "Observation" units. CONCLUSIONS: One principal reason for ED crowding in Beijing lies in the large numbers of patients who persist in the expectation of receiving ongoing care in the ED for minor illnesses. However, as is true in many nations, one of the other most important root causes of ED crowding is "access block," the inability to promptly move patients deemed by emergency physicians to need inpatient care to an inpatient bed for that care. However, in our system, another challenge, not widely described as a contributor to crowding in other nations, is that doctors assigned to inpatient services have been empowered to refuse to admit patients perceived to have overly "complex" needs. Further, patients with multisystem illnesses or end-stage status, who need ongoing chronic care to manage activities of daily living, have begun to populate Beijing EDs in increasing numbers. This is an issue with various root causes.

Did you hear the one about the policeman, the doctor and the pharmacist at 30000 feet?

This is the remarkable story of survival against all the odds. A passenger had a myocardial infarction complicated by a witnessed cardiac arrest while on a commercial flight through some of the most remote airspace on the planet. Immediate cardiopulmonary resuscitation and use of an automatic external defibrillator achieved rapid return of spontaneous circulation. Passengers and crew worked effectively together, under the guidance of a physician, to provide critical care to the patient while the flight diverted so he could be transferred to an emergency hospital in Beijing for eventual thrombolysis and postresuscitation care. He made a rapid and full recovery to be discharged from hospital, neurologically intact, 10 days later.

11ß-HSD1 inhibition reduces atherosclerosis in mice by altering proinflammatory gene expression in the vasculature.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is implicated in the etiology of metabolic syndrome. We previously showed that pharmacological inhibition of 11ß-HSD1 ameliorated multiple facets of metabolic syndrome and attenuated atherosclerosis in ApoE-/- mice. However, the molecular mechanism underlying the atheroprotective effect was not clear. In this study, we tested whether and how 11ß-HSD1 inhibition affects vascular inflammation, a major culprit for atherosclerosis and its associated complications. ApoE-/- mice were treated with an 11ß-HSD1 inhibitor for various periods of time. Plasma lipids and aortic cholesterol accumulation were quantified. Several microarray studies were carried out to examine the effect of 11ß-HSD1 inhibition on gene expression in atherosclerotic tissues. Our data suggest 11ß-HSD1 inhibition can directly modulate atherosclerotic plaques and attenuate atherosclerosis independently of lipid lowering effects. We identified immune response genes as the category of mRNA most significantly suppressed by 11ß-HSD1 inhibition. This anti-inflammatory effect was further confirmed in plaque macrophages and smooth muscle cells procured by laser capture microdissection. These findings in the vascular wall were corroborated by reduction in circulating MCP1 levels after 11ß-HSD1 inhibition. Taken together, our data suggest 11ß-HSD1 inhibition regulates proinflammatory gene expression in atherosclerotic tissues of ApoE-/- mice, and this effect may contribute to the attenuation of atherosclerosis in these animals.

Metabolic syndrome in mice induced by expressing a transcriptional activator in adipose tissue.

Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11ß-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11ß-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.

Travelers' diarrhea in Nepal: an update on the pathogens and antibiotic resistance.

BACKGROUND: Diarrhea is the most common illness among travelers and expatriates in Nepal. Published data on the etiology of travelers' diarrhea (TD) in Nepal are over 13 years old and no prior data exist on antibiotic susceptibility for currently used drugs. We investigated the etiology of diarrhea and antimicrobial susceptibility pattern of bacterial pathogens and compared the results to previous work from the same clinical setting. METHODS: A total of 381 cases and 176 controls were enrolled between March 2001 and 2003 in a case-control study. Enrollees were over age 18 years from high socioeconomic countries visiting or living in Nepal. Stool samples were assessed by microbiologic, molecular identification, and enzyme immunoassay (EIA) methods, and antimicrobial susceptibility was determined by disk diffusion. Risk factors were assessed by questionnaires. RESULTS: At least one enteropathogen was identified in 263 of 381 (69%) cases and 47 of 176 (27%) controls (p ≤ 0.001). Pathogens significantly detected among cases were Campylobacter (17%), enterotoxigenic Escherichia coli (ETEC) (15%), Shigella (13%), and Giardia (11%). Cyclospora was detected only in cases (8%) mainly during monsoon season. Although 71% of Campylobacter isolates were resistant to ciprofloxacin, 80% of bacterial isolates overall were sensitive to either ciprofloxacin or azithromycin while 20% were intermediately sensitive or resistant. No bacterial isolates were resistant to both drugs. CONCLUSIONS: The most common pathogens causing TD in Nepal were Campylobacter, ETEC, and Shigella. Because resistance to fluoroquinolone or azithromycin was similar, one of these drugs could be used as empiric therapy for TD with the other reserved for treatment failures.

CCR5 blockade modulates inflammation and alloimmunity in primates.

Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Delta32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck's compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.

Alpha-aminothiazole-gamma-aminobutanoic amides as potent, small molecule CCR2 receptor antagonists.

A series of racemic and homochiral alpha-aminothiazole-gamma-aminobutyroamides that display high affinities for human and murine CCR2 and functional antagonism by inhibition of monocyte recruitment are described. A representative example is (2S)-2-[2-(acetylamino)-1,3-thiazol-4-yl]-N-[3-methyl-5-(trifluoromethyl)benzyl]-4-(4-phenylpiperidin-1-yl)butanamide, which shows 5 nM affinity for human monocytes and CHO cells expressing the human CCR2b receptor. It also inhibited MCP-1 initiated chemotaxis of human monocytes with an IC50 of 0.69 nM.

Inhibition of human immunodeficiency virus envelope glycoprotein- mediated single cell lysis by low-molecular-weight antagonists of viral entry.

The coexpression of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins and receptors leads to the lysis of single cells by a process that is dependent upon membrane fusion. This cell lysis was inhibited by low-molecular-weight compounds that interfere with receptor binding or with receptor-induced conformational transitions in the envelope glycoproteins. A peptide, T20, potently inhibited cell-cell fusion but had no effect on single cell lysis mediated by the HIV-1 envelope glycoproteins. Thus, critical events in the lysis of single cells by the HIV-1 envelope glycoproteins occur in intracellular compartments accessible only to small inhibitory compounds.

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion.

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.

Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor.

Pre-exposure oral prophylaxis with antiviral drugs is a potential method for preventing transmission of human immunodeficiency virus type 1 (HIV-1). We show that oral delivery of CMPD167, a small molecule that binds to the CCR5 coreceptor, for 10-14 d can protect a substantial proportion of macaques from vaginal infection with a CCR5-using virus (SHIV-162P3). The macaques that became infected despite receiving CMPD167 had reduced plasma viremia levels during the earliest stages of infection.

11beta-HSD1 inhibition ameliorates metabolic syndrome and prevents progression of atherosclerosis in mice.

The enzyme 11beta-hydroxysteroid dehydrogenase (HSD) type 1 converts inactive cortisone into active cortisol in cells, thereby raising the effective glucocorticoid (GC) tone above serum levels. We report that pharmacologic inhibition of 11beta-HSD1 has a therapeutic effect in mouse models of metabolic syndrome. Administration of a selective, potent 11beta-HSD1 inhibitor lowered body weight, insulin, fasting glucose, triglycerides, and cholesterol in diet-induced obese mice and lowered fasting glucose, insulin, glucagon, triglycerides, and free fatty acids, as well as improved glucose tolerance, in a mouse model of type 2 diabetes. Most importantly, inhibition of 11beta-HSD1 slowed plaque progression in a murine model of atherosclerosis, the key clinical sequela of metabolic syndrome. Mice with a targeted deletion of apolipoprotein E exhibited 84% less accumulation of aortic total cholesterol, as well as lower serum cholesterol and triglycerides, when treated with an 11beta-HSD1 inhibitor. These data provide the first evidence that pharmacologic inhibition of intracellular GC activation can effectively treat atherosclerosis, the key clinical consequence of metabolic syndrome, in addition to its salutary effect on multiple aspects of the metabolic syndrome itself.

Potent 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists: effects of fused heterocycles on antiviral activity and pharmacokinetic properties.

A series of 1,3,4-trisubstituted pyrrolidine CCR5 receptor antagonists containing a variety of fused heterocycles at the 4-position of the piperidine side chain has been discovered, which are orally bioavailable with potent anti-HIV activity.

Synthesis and evaluation of CCR5 antagonists containing modified 4-piperidinyl-2-phenyl-1-(phenylsulfonylamino)-butane.

Synthesis of analogs containing more rigid bicyclic piperidine replacements for the 4-benzyloxycarbonyl-(ethyl)amino-piperidine moiety of the CCR5 antagonist structure, 1, is described. Although similar binding affinity to the lead was achieved with some analogs they were overall less potent anti-HIV agents suggesting that other features besides CCR5 binding are required for good anti-viral activity.

Effect of a CCR5 inhibitor on viral loads in macaques dual-infected with R5 and X4 primate immunodeficiency viruses.

Human immunodeficiency virus type 1 (HIV-1) fusion with its target cells is initiated by sequential interactions between its envelope glycoprotein, CD4, and a co-receptor, usually CCR5 or CXCR4. Small molecules that bind to CCR5 and prevent its use by R5 HIV-1 strains are now being developed clinically as antiviral drugs. To test whether a block to CCR5 promotes the replication of viruses that enter cells via CXCR4 and are associated with accelerated disease progression, we administered a small molecule CCR5 inhibitor, CMPD 167, to three macaques dual-infected with both R5 (SIVmac251) and X4 (SHIV-89.6P) viruses. CMPD 167 caused a rapid and substantial (on average, 50-fold) suppression of R5 virus replication in each animal. In two of the animals, but not in the third, a rapid, transient, 8- to 15-fold increase in the amount of plasma X4 virus occurred. In neither animal was the increase in X4 viral load sustained throughout therapy, however. These observations may have relevance for the development of CCR5 inhibitors for treatment of HIV-1 infection of humans.

Syntheses and SAR studies of 4-(heteroarylpiperdin-1-yl-methyl)-pyrrolidin-1-yl-acetic acid antagonists of the human CCR5 chemokine receptor.

Efforts toward the exploration of the title compounds as CCR5 antagonists are disclosed. The basis for such work stems from the fact that cellular proliferation of HIV-1 requires the cooperative assistance of both CCR5 and CD4 receptors. The synthesis and SAR of pyrrolidineacetic acid derivatives as CCR5 antagonists displaying potent binding and antiviral properties in a HeLa cell-based HIV-1 infectivity assay are discussed.

Syntheses and biological evaluation of 5-(piperidin-1-yl)-3-phenyl-pentylsulfones as CCR5 antagonists.

Cellular proliferation of HIV-1 requires the cooperative assistance of both the CCR5 and CD4 receptors. Our medicinal chemistry efforts in this area have resulted in the identification of N-alkyl piperidine sulfones as CCR5 antagonists. These compounds display potent binding and show antiviral properties in HIV-1 spread cell-based assays.

Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 1: Discovery and SAR study of 4-pyrazolylpiperidine side chains.

Replacement of the flexible connecting chains between the piperidine moiety and an aromatic group in previous CCR5 antagonists with heterocycles, such as pyrazole and isoxazole, provided potent CCR5 antagonists with excellent anti-HIV-1 activity in vitro. SAR studies revealed optimal placement of an unsubstituted nitrogen atom in the heterocycle to be meta to the bond connected to the 4-position of piperidine. Truncation of a benzyl group to a phenyl group afforded compounds with dramatically improved oral bioavailability, albeit with reduced activity.

Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 2: Discovery of potent, selective, and orally bioavailable compounds.

Modifications of the alkyl acetic acid portion and the phenyl on pyrrolidine in our lead pyrazole compound 1 afforded the isopropyl compound 9. This compound is a potent CCR5 antagonist showing good in vitro antiviral activity against HIV-1, an excellent selectivity profile, and good oral bioavailability in three animal species. During this investigation, a new method for the preparation of alpha-(pyrrolidin-1-yl)-alpha,alpha-dialkyl acetic acid from a pyrrolidine and alpha-bromo-alpha,alpha-dialkyl acetic acid using silver triflate was discovered. This allowed us to prepare compounds such as 24 and 25 for the first time. A novel Pd-mediated N-dealkylation of alpha-(pyrrolidin-1-yl)acetic acid was also uncovered.

Antagonists of human CCR5 receptor containing 4-(pyrazolyl)piperidine side chains. Part 3: SAR studies on the benzylpyrazole segment.

Extensive SAR studies in our benzylpyrazole series of CCR5 antagonists have shown that both lipophilic and hydrophilic substituents on the phenyl of the benzyl group increase antiviral potency. However, improvements in pharmacokinetic profiles were generally only observed with more lipophilic substitutions. 4-Biphenyl (51) performed the best in this regard. Highly lipophilic substituents impart undesirable ion channel activity to these CCR5 antagonists. Alkoxy substituents provide a good balance of antiviral activity, pharmacokinetic parameters, and selectivity. Compounds 42b and 42d, containing a 3,4-dimethoxy substituent, are considered the most promising improvements over parent compounds 9. They demonstrate improved antiviral activity while retaining good pharmacokinetic profile and selectivity.