Identification of a Human Whole Blood-Based Endothelial Cell Impedance Assay for Assessing Clinical Transient Receptor Potential Vanilloid 4 Target Engagement Ex Vivo.

Transient receptor potential vanilloid 4 (TRPV4) channels expressed on pulmonary endothelial cells are activated by elevated pulmonary vascular pressure, resulting in endothelial shape change, pulmonary barrier disruption, and edema. As such, TRPV4 blocker GSK2798745 was recently investigated in phase I/IIa trials to reduce pulmonary edema caused by heart failure (HF). In the absence of a suitable TRPV4 target engagement biomarker, we hypothesized that an ex vivo assay could be used to predict pharmacological activity at the intended site of action (endothelial cells) of subjects. In this assay, the ability of GSK2798745 to block TRPV4 agonist GSK1016790-induced impendence reduction in human umbilical vein endothelial cells (HUVECs) in the presence of human whole blood was assessed. Blood from healthy volunteers drawn 1-12 hours after single or repeated dose of GSK2798745 (5 mg) inhibited GSK1016790-induced impedance reduction by ≥85%. Similarly, blood samples from 16 subjects with HF dosed with GSK2798745 (2.4 mg) inhibited GSK1016790-induced HUVEC impedance reduction by ≥58% 1-24 hours after single dosing and ≥78% 1-24 hours after 7 days of repeated dosing. No inhibition was detected using blood from placebo subjects. Using matched GSK2798745 plasma levels, a pharmacokinetic/pharmacodynamic (PK/PD) relationship was calculated as 2.9 nM IC50, consistent with the 6.5 nM IC50 of GSK2798745 obtained from a rat in vivo PK/PD model of pulmonary edema after correcting for rat-to-human differences. These results indicate that circulating levels of GSK2798745 in the recently completed phase I/IIa trials were sufficient to block TRPV4 in lung vascular endothelial cells to a large extent, supporting this dosing regimen for assessing efficacy in HF. SIGNIFICANCE STATEMENT: In the absence of a suitable target engagement biomarker, we developed an ex vivo assay to predict the pharmacological activity of the transient receptor potential vanilloid 4 (TRPV4) blocker GSK2798745 in healthy volunteers and subjects with heart failure (HF) from phase I/IIa trials. The potency values from the ex vivo assay were consistent with those predicted from a rat in vivo pharmacokinetic/pharmacodynamic model of pulmonary edema, strongly suggesting that circulating levels of GSK2798745 were sufficient to robustly block TRPV4, supporting use of GSK2798745 for assessing efficacy in HF.

Risk factors for disease progression in idiopathic pulmonary fibrosis.

In this retrospective study of a randomised trial of simtuzumab in idiopathic pulmonary fibrosis (IPF), prodromal decline in forced vital capacity (FVC) was significantly associated with increased risk of mortality, respiratory and all-cause hospitalisations, and categorical disease progression. Predictive modelling of progression-free survival event risk was used to assess the effect of population enrichment for patients at risk of rapid progression of IPF; C-index values were 0.64 (death), 0.69 (disease progression), and 0.72 (adjudicated respiratory hospitalisation) and 0.76 (all-cause hospitalisation). Predictive modelling may be a useful tool for improving efficiency of clinical trials with categorical end points.

Quality home visits: Activities to promote meaningful interactions.

Examination of and support for specific practices that promote high-quality home visiting are essential as family support programs continue to expand across the country. The current study used direct observation of 91 home visits across 41 home visitors to examine relations among interaction partners, content of the interactions, the home-visitors' activities, and quality of home-visitors' practices and family-members' engagement within programs funded by the Maternal, Infant, and Early Childhood Home Visiting program. More time spent in triadic interactions focused on child-related content, as measured by the Home Visit Rating Scale-Revised, was related to higher quality of family engagement in home visits, as measured with the Home Visit Observation Rating Scales. Time spent in adult-focused interactions and administrative tasks, however, was related to lower quality of home-visiting practices and family engagement. Implications for research and practice are discussed.

A medida que los programas de apoyo familiar continúan expandiéndose a lo largo del país, se hace esencial examinar y apoyar prácticas específicas que promueven la alta calidad de las visitas a casa. El presente estudio usó observaciones directas de 91 visitas a casa llevadas a cabo por 41 visitadores con el fin de examinar las relaciones entre la participación de todas las partes involucradas, el contenido de las interacciones, las actividades de los visitadores a casa, y la calidad tanto de las prácticas de los visitadores como de la participación de los miembros de la familia dentro de los programas subvencionados por MIECHV. Más tiempo empleado en interacciones tríadicas enfocadas en contenidos relacionados con el niño, tal como se midió por medio de las Escalas Revisadas de Evaluación de Visitas a Casa (HVOF-R; McBride y Peterson, 1996), estuvo relacionado con más alta calidad de la participación de la familia en las visitas a casa, tal como se midió por medio de las Escalas de Evaluación de la Observación de Visitas a Casa (HOVRS; Roggman et al., 2014). El tiempo empleado en interacciones enfocadas en los adultos y tareas administrativas, sin embargo, estuvo relacionado con más baja calidad de las prácticas de visita a casa y participación de la familia. Se discuten las implicaciones para la investigación y la práctica.

L'examen et le soutien de pratiques spécifiques qui promeuvent la visite à domicile de qualité sont essentiels alors que les programmes de soutien à famille continuent de se développer aux Etats-Unis. Cette étude a utilisé une observation directe de 91 visites à domicile effectuées par 41 visiteurs afin d'examiner les relations entre les partenaires d'interaction, le contenu des interactions, les activités des visiteurs ou visiteuses à domicile, et la qualité des pratiques des visiteurs ou visiteuses à domicile ainsi que l'engagement des membres de la famille au sein des programmes américains de visite à domicile subventionnés par le programme américain Maternal, Infant, and Early Childhood Home Visiting. Plus de temps passé dans les interactions triadiques mettant l'accent sur le contenu lié à l'enfant tel qu'il est mesuré par la version révisée de l'Echelle d'Evaluation de la Visite à Domicile (HVOF-R; McBride & Peterson, 1996) a été lié à une plus grande qualité de l'engagement de la famille durant les visites à domicile telles qu'elles ont été mesurées au moyen des Echelles d'Evaluation de l'Observation de la Visite à Domicile (HOVRS; Roggman et al., 2014). Le temps passé en interactions avec les adultes et en tâches administratives étaient lié à des pratiques de visite à domicile et à un engagement familial de moindre qualité. Les implications pour les recherches et la pratique sont discutées.

Engaging, recruiting, and retaining black men who have sex with men in research studies: don't underestimate the importance of staffing--lessons learned from HPTN 061, the BROTHERS study.

CONTEXT: HIV/AIDS in the United States continues to primarily impact men who have sex with men (MSM), with disproportionately high rates among black MSM. OBJECTIVE: The purpose of this study was to identify factors that may influence engagement and retention of black MSM in HIV research. DESIGN AND PARTICIPANTS: This was a qualitative evaluation of study implementation within a multisite, prospective, observational study (HIV Prevention Trials Network 061, BROTHERS) that enrolled 1553 black MSM in 6 cities throughout the United States. Data collection for this evaluation included a written, structured survey collected from each of the sites describing site characteristics including staff and organizational structure, reviews of site standard operating procedures, and work plans; semistructured key informant interviews were conducted with site coordinators to characterize staffing, site-level factors facilitating or impeding effective community engagement, study recruitment, and retention. Data from completed surveys and site standard operating procedures were collated, and notes from key informant interviews were thematically coded for content by 2 independent reviewers. RESULTS: Several key themes emerged from the data, including the importance of inclusion of members of the community being studied as staff, institutional hiring practices that support inclusive staffing, cultivating a supportive working environment for study implementation, and ongoing relationships between research institutions and community. CONCLUSIONS: This study underscores the importance of staffing in implementing research with black MSM. Investigators should consider how staffing and organizational structures affect implementation during study design and when preparing to initiate study activities. Ongoing monitoring of community engagement can inform and improve methods for engagement and ensure cultural relevance while removing barriers for participation.

NADPH oxidase-dependent and -independent mechanisms of reported inhibitors of reactive oxygen generation.

NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CßII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

Sociodemographic and risk behavior characteristics associated with unprotected sex with women among black men who have sex with men and women in New York City.

The objectives of this cross-sectional study were to compare sociodemographic and risk behavior characteristics between black men who have sex with both men and women (MSMW) and those who have sex with men only (MSMO) and assess factors associated with having any unprotected vaginal and/or anal intercourse (UVAI) with women in the last 3 months. Data from 326 black men who reported recent unprotected anal intercourse with a man in an HIV behavioral intervention study in New York City were analyzed. Baseline characteristics were compared between MSMW and MSMO, and factors associated with having any UVAI in the past 3 months with women among MSMW were evaluated. In total, 26.8% reported having sex with both men and women in the last 3 months. MSMW were less likely to be HIV infected, use amyl nitrates, and have unprotected receptive anal sex with most recent male partner. MSMW were more likely to be over 40 years old and use heroin. A total of 55.6% of MSMW reported having UVAI with women in the last 3 months. Compared to MSMW having only protected sex, MSMW having any UVAI with women were less likely to be HIV infected and to disclose having sex with men to female partners; they were more likely to have greater than four male sex partners in the last 3 months. In conclusion, HIV prevention interventions among black MSMW should directly address the risk of HIV transmission to both their female and male partners. Disclosure of bisexuality to female partners may be an important component of future prevention efforts.

Sexual partner characteristics, serodiscordant/serostatus unknown unprotected anal intercourse and disclosure among human immunodeficiency virus-infected and uninfected black men who have sex with men in New York City.

OBJECTIVES: Black men who have sex with men (MSM) are disproportionately infected with human immunodeficiency virus (HIV) in the United States. This study describes sexual partner characteristics and disclosure of HIV serostatus and evaluates factors associated with sexual risk behaviors during last sex among black MSM. DESIGN AND METHODS: Between 2008 and 2009, 328 black MSM who reported recent unprotected anal intercourse were enrolled in an HIV behavioral intervention study in New York City. Factors associated with serodiscordant/serostatus unknown UAI (defined as having UAI with a partner of different or unknown HIV serostatus) with a male partner during last sex were assessed using logistic regression. RESULTS: A total of 205 HIV-infected and 123 uninfected men were enrolled in this study. Almost all men (91.6%) reported having a black male partner during last sex. About half (47.3%) of men used alcohol and 38.7% used other substances before or during last sex. About two-thirds (68.8%) of participants disclosed their HIV status to their last sex partner, while 57.2% of partners disclosed. In multivariate analysis, meeting a partner on the internet or chat line was associated with serodiscordant/serostatus unknown UAI during last sex among HIV-infected men. The only factor associated with serodiscordant/serostatus unknown UAI during last sex among HIV-uninfected men was the partner being a non-main partner. CONCLUSIONS: A significant proportion of black MSM in this study did not disclose their HIV status. Our data highlight the need for more data on dyadic variables and sexual risk behaviors among black MSM, as well as interventions to encourage communication between partners.

Pharmacological inhibition of C-C chemokine receptor 2 decreases macrophage infiltration in the aortic root of the human C-C chemokine receptor 2/apolipoprotein E-/- mouse: magnetic resonance imaging assessment.

UNLABELLED: Purpose- This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. METHODS AND RESULTS: Apolipoprotein E(-/-) mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05). CONCLUSIONS: An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

CCR2 receptor antagonists: optimization of biaryl sulfonamides to increase activity in whole blood.

A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.

Discovery of potent, selective sulfonylfuran urea endothelial lipase inhibitors.

Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and atherogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease. Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selective EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and modifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irreversible inhibitors.

Discovery of GSK2798745: A Clinical Candidate for Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4).

GSK2798745, a clinical candidate, was identified as an inhibitor of the transient receptor potential vanilloid 4 (TRPV4) ion channel for the treatment of pulmonary edema associated with congestive heart failure. We discuss the lead optimization of this novel spirocarbamate series and specifically focus on our strategies and solutions for achieving desirable potency, rat pharmacokinetics, and physicochemical properties. We highlight the use of conformational bias to deliver potency and optimization of volume of distribution and unbound clearance to enable desirable in vivo mean residence times.

Aminomethylpiperazines as selective urotensin antagonists.

Aminomethylpiperazines, reported previously as being kappa-opioid receptor agonists, were identified as lead compounds in the development of selective urotensin receptor antagonists. Optimized substitution of the piperazine moiety has provided high affinity urotensin receptor antagonists with greater than 100-fold selectivity over the kappa-opioid receptor. Select compounds were found to inhibit urotensin-induced vasoconstriction in isolated rat aortic rings consistent with the hypothesis that an urotensin antagonist may be useful for the treatment of hypertension.

Potent and selective small-molecule human urotensin-II antagonists with improved pharmacokinetic profiles.

Lead compound 1 was successfully redesigned to provide compounds with improved pharmacokinetic profiles for this series of human urotensin-II antagonists. Replacement of the 2-pyrrolidinylmethyl-3-phenyl-piperidine core of 1 with a substituted N-methyl-2-(1-pyrrolidinyl)ethanamine core as in compound 7 resulted in compounds with improved oral bioavailability in rats. The relationship between stereochemistry and selectivity for hUT over the kappa-opioid receptor was also explored.

Development of potent and selective small-molecule human Urotensin-II antagonists.

This work describes the development of potent and selective human Urotensin-II receptor antagonists starting from lead compound 1, (3,4-dichlorophenyl)methyl{2-oxo-2-[3-phenyl-2-(1-pyrrolidinylmethyl)-1-piperidinyl]ethyl}amine. Several problems relating to oral bioavailability, cytochrome P450 inhibition, and off-target activity at the kappa opioid receptor and cardiac sodium channel were addressed during lead development. hUT binding affinity relative to compound 1 was improved by more than 40-fold in some analogs, and a structural modification was identified which significantly attenuated both off-target activities.

Development of dihydropyridone indazole amides as selective Rho-kinase inhibitors.

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

Discovery of aminofurazan-azabenzimidazoles as inhibitors of Rho-kinase with high kinase selectivity and antihypertensive activity.

The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

Discovery of GSK2193874: An Orally Active, Potent, and Selective Blocker of Transient Receptor Potential Vanilloid 4.

Transient Receptor Potential Vanilloid 4 (TRPV4) is a member of the Transient Receptor Potential (TRP) superfamily of cation channels. TRPV4 is expressed in the vascular endothelium in the lung and regulates the integrity of the alveolar septal barrier. Increased pulmonary vascular pressure evokes TRPV4-dependent pulmonary edema, and therefore, inhibition of TRPV4 represents a novel approach for the treatment of pulmonary edema associated with conditions such as congestive heart failure. Herein we report the discovery of an orally active, potent, and selective TRPV4 blocker, 3-(1,4'-bipiperidin-1'-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide (GSK2193874, 28) after addressing an unexpected off-target cardiovascular liability observed from in vivo studies. GSK2193874 is a selective tool for elucidating TRPV4 biology both in vitro and in vivo.

A randomized trial of a behavioral intervention for black MSM: the DiSH study.

OBJECTIVE: To test a new behavioral intervention for black MSM in reducing sexual risk and increasing social support and intentions to use condoms. DESIGN: A single-site, unblinded randomized trial in New York City with 3-month follow-up. METHODS: Participants (n = 283) reporting at least two sexual partners and unprotected anal intercourse with a man in the past 3 months were enrolled and randomized to a social-cognitive theory-based intervention or control comparison. Men in the intervention group participated in five 2-h group sessions focused on creating a group environment with sexual risk-reduction information and exercises woven into joint meal preparation and sharing activities, while exploring self-efficacy perceptions and outcome expectancies. Intervention (n = 142) and control (n = 141) groups received standard HIV counseling and testing at baseline. RESULTS: No significant differences were found between study arms at 3 months in number of male partners, number of unprotected anal intercourse partners, proportion reporting unprotected sex, number of acts protected by condoms, self-efficacy, condom attitudes, condom intentions, social isolation and psychological distress. In both arms combined, declines from baseline to 3 months were observed in sexual risk behaviors, social isolation and psychological distress, whereas self-efficacy, condom attitudes and condom intentions improved. CONCLUSION: As the HIV epidemic continues to have a dramatic impact on black MSM in the USA, the urgency to design innovative interventions continues.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

Engaging members of African American and Latino communities in preventive HIV vaccine trials.

BACKGROUND: African Americans (AAs) and Latinos in United States bear a disproportionate burden of HIV infection, yet remain underrepresented in HIV vaccine trials. The success in engaging and enrolling AAs and Latinos in phase 1 and phase 2 vaccine trials at 2 research sites in New York City is described. METHODS: A retrospective analysis of 1683 HIV-uninfected individuals who completed > or = 1 stage of the screening process from 2002 to 2006. Data on sociodemographic, behavioral characteristics, medical eligibility, and enrollment in National Institutes of Health-sponsored vaccine trials were collected. RESULTS: 7.5% of screening participants completed enrollment; 33% were AAs, 24% Latinos. The proportion of enrollees did not differ significantly by race/ethnicity. Low-risk vs. high-risk AAs (49% vs. 23%, P = 0.006) and high-risk vs. low-risk Latinos (31% vs. 13%, P = 0.006) were more likely to enroll. Among them, loss to follow-up was the most common reason for not completing screening. In multivariate analysis, older participants, high-risk men, and high-risk women were more likely to complete enrollment. CONCLUSIONS: Once potential minority participants are identified and engaged in the screening process, it is possible to enroll them at rates comparable to white participants. Experience at these sites suggests that the challenge in achieving high rates of minority participation is in increasing the initial pool of candidates prescreening for HIV vaccine studies.