A randomized, double-blind trial investigating the efficacy of caspofungin versus amphotericin B deoxycholate in the treatment of invasive candidiasis in neonates and infants younger than 3 months of age.

OBJECTIVES: Investigate the efficacy of caspofungin in participants <3 months of age with invasive Candida infection (ICI). METHODS: This multicentre, randomized, double-blind, comparator-controlled, Phase 2 study (protocol MK0991-064; NCT01945281) enrolled participants <3 months of age with culture-confirmed ICI within 96 h of study entry. Participants were randomly assigned 2:1 to once-daily intravenous 2 mg/kg caspofungin or intravenous 1 mg/kg amphotericin B deoxycholate (dAMB). The primary endpoint was fungal-free survival (FFS) 2 weeks after treatment in the full-analysis-set (FAS) population, defined as participants with culture-confirmed ICI who received ≥1 dose of therapy. Planned enrolment was 90 participants. RESULTS: Fifty-one participants were enrolled; 49 received treatment (caspofungin, n=33; dAMB, n=16); 2 additional participants did not have confirmed infections at study entry. The study was terminated after ∼ 3.5 years because of low enrolment. Forty-seven participants were included in the FAS population (caspofungin, n=31; dAMB, n=16). FFS rate at 2 weeks after treatment was 71.0% (22/31) in the caspofungin arm and 68.8% (11/16) in the dAMB arm [difference, stratified by weight, - 0.9% (95% CI, - 24.3%-27.7%)]. Adverse events developed in 84.8% (28/33) of participants in the caspofungin arm and 100% (16/16) in the dAMB arm. CONCLUSIONS: Among neonates and infants with confirmed ICI, FFS at 2 weeks was similar in the caspofungin and dAMB treatment arms. A smaller proportion of participants who received caspofungin experienced adverse events.

Efficacy and safety of ruzasvir 60 mg and uprifosbuvir 450 mg for 12 weeks in adults with chronic hepatitis C virus genotype 1, 2, 3, 4 or 6 infection.

In clinical trials, the three-drug regimen of ruzasvir (RZR) 60 mg, uprifosbuvir (UPR) 450 mg and grazoprevir 100 mg, with or without ribavirin, has demonstrated promising efficacy and excellent tolerability across a wide range of hepatitis C virus (HCV)-infected individuals. The present study assessed the efficacy and safety of the two-drug combination of RZR 60 mg plus UPR 450 mg administered for 12 weeks in participants with HCV genotype (GT) 1-6 infection. In this open-label clinical trial, treatment-naive or -experienced and cirrhotic or noncirrhotic participants with chronic HCV GT1-6 infection received RZR 60 mg plus UPR 450 mg orally once daily for 12 weeks (NCT02759315/protocol PN035). The primary efficacy endpoint was sustained virologic response at 12 weeks after the end of therapy (SVR12). One hundred and sixty participants were enrolled. SVR12 rates were 96% (52 of 54) in participants with GT1a infection; 100% (15 of 15) in those with GT1b infection; 97% (28 of 29) in those with GT2 infection; 77% (30 of 39) in those with GT3 infection; 90% (18 of 20) in those with GT4 infection; and 67% (2 of 3) in those with GT6 infection. Drug-related adverse events (AEs) reported by >5% of participants were fatigue (n = 10, 6.3%) and diarrhoea (n = 9, 5.6%). Five participants reported a total of 11 serious AEs, none considered drug-related. One participant experienced on-treatment alanine aminotransferase/aspartate aminotransferase elevations that resolved without intervention. Data from the present study indicate that the combination of RZR 60 mg plus UPR 450 mg once daily for 12 weeks was well tolerated overall but was effective only for certain genotypes.

Inhibition of cyclophilins alters lipid trafficking and blocks hepatitis C virus secretion.

Host cyclophilin (cyp) inhibitors, such as NIM811, efficiently inhibit replication of hepatitis C virus (HCV) and have shown significant promise in recent clinical trials for the treatment of chronic HCV. It is therefore important to fully understand the mechanism of action of these therapeutic agents. Data obtained from comprehensive systems biology approaches have led to the hypothesis that the antiviral activity of cyclophilin inhibitors is mediated through impairing the cellular machinery on which HCV relies to traffic cofactors necessary for formation of the replication complex. Indeed, our results demonstrate when cyclophilins are inhibited by NIM811, lipid and protein trafficking within the VLDL pathway is impaired. Following treatment of replicon or HCV infected cells with NIM811, intracellular lipid droplets (LD) more than double in size and decrease in number. Changes in the LDs in response to cyclophilin inhibition are dependent upon expression of viral proteins. Additionally, in cells treated with NIM811, apoB accumulates in a crescent or ring shaped structure surrounding the enlarged LDs and is no longer secreted. Silencing of cypA or cyp40 using siRNA had a similar effect on LD size and apoB localization as compound treatment, suggesting these cyclophilins may play an important role in lipid and apoB trafficking. Interestingly, the decrease in apoB secretion correlates with a decrease in release of viral particles in HCV infected cells. Altogether, these results add a new level of complexity to the mechanism of action of cyclophilin inhibition, and suggest the role for cyclophilins in the virus life cycle extends beyond replication to virus release.

Mechanism of resistance of hepatitis C virus replicons to structurally distinct cyclophilin inhibitors.

The current standard of care for hepatitis C virus (HCV) infection, pegylated alpha interferon in combination with ribavirin, has a limited response rate and adverse side effects. Drugs targeting viral proteins are in clinical development, but they suffer from the development of high viral resistance. The inhibition of cellular proteins that are essential for viral amplification is thought to have a higher barrier to the emergence of resistance. Three cyclophilin inhibitors, the cyclosporine analogs DEBIO-025, SCY635, and NIM811, have shown promising results for the treatment of HCV infection in early clinical trials. In this study, we investigated the frequency and mechanism of resistance to cyclosporine (CsA), NIM811, and a structurally unrelated cyclophilin inhibitor, SFA-1, in replicon-containing Huh7 cells. Cross-resistance between all clones was observed. NIM811-resistant clones were selected only after obtaining initial resistance to either CsA or SFA-1. The time required to select resistance against cyclophilin inhibitors was significantly longer than that required for resistance selection against viral protein inhibitors, and the achievable resistance level was substantially lower. Resistance to cyclophilin inhibitors was mediated by amino acid substitutions in NS3, NS5A, and NS5B, with NS5A mutations conferring the majority of resistance. Mutation D320E in NS5A mediated most of the resistance conferred by NS5A. Taken together, the results indicate that there is a very low frequency and level of resistance to cyclophilin-binding drugs mediated by amino acid substitutions in three viral proteins. The interaction of cyclophilin with NS5A seems to be the most critical, since the NS5A mutations have the largest impact on resistance.

Multiple cyclophilins involved in different cellular pathways mediate HCV replication.

Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.

Epstein-Barr virus latent membrane protein 2A exploits Notch1 to alter B-cell identity in vivo.

Expression of latent membrane protein 2 (LMP2A) during B-cell development leads to global alterations in gene transcription similar to those seen in Hodgkin Reed-Sternberg cells of Hodgkin lymphoma (HL). Along with the consistent detection of LMP2A in Epstein-Barr virus-associated HL, this implicates a role for LMP2A in the pathogenesis of HL. We have shown that LMP2A constitutively activates the Notch1 pathway to autoregulate the LMP2A promoter. To determine whether constitutive activation of the Notch pathway is important for LMP2A-mediated alterations in B-cell development in vivo, TgE-LMP2A-transgenic mice were intercrossed with mice expressing loxP-flanked Notch1 genes and Cre recombinase. B cells from TgE Notch1(lox/lox)-CD19(+/Cre) mice have an increase in immunoglobulin M and CD43 and a decrease in CD5 expression in the bone marrow compared with TgE Notch1(lox/lox) mice, indicating the LMP2A signal for developmental aberrations is impaired in the absence of Notch1. Real-time reverse-transcribed polymerase chain reaction analysis reveals that LMP2A requires the Notch1 pathway to alter levels of B cell-specific transcription factors, E2A and EBF. Interestingly, Notch1 appears to be important for LMP2A-mediated survival in low interleukin-7. We propose that LMP2A and the Notch1 pathway may cooperate to induce the alterations in B-cell identity seen in Hodgkin Reed-Sternberg cells.

EBV LMP2A provides a surrogate pre-B cell receptor signal through constitutive activation of the ERK/MAPK pathway.

Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) provides developmental and survival signals that mimic those of a B-cell receptor (BCR). Expression of LMP2A during B-cell development results in the ability of B cells to exit the bone marrow in the absence of a BCR and persist in the periphery, where they would normally undergo apoptosis. This study extends the current knowledge of LMP2A function by examining the growth properties of bone marrow B cells from TgE LMP2A mice. Despite the lack of pre-BCR expression, bone marrow B cells from TgE LMP2A mice proliferate and survive in low concentrations of interleukin 7, similar to wild-type cells. Constitutive phosphorylation of ERK/MAPK and PI3K/Akt in TgE LMP2A bone marrow B cells is also reminiscent of signalling through the pre-BCR, altogether demonstrating that LMP2A provides a pre-BCR-like signal to developing B cells.

An auto-regulatory loop for EBV LMP2A involves activation of Notch.

LMP2A is consistently detected in Hodgkin's lymphoma, nasopharyngeal carcinoma and has also been detected in Burkitt's lymphoma. Interestingly, LMP2A is detected in the absence of the transcriptional activator EBNA2, suggesting that an alternative mechanism is responsible for LMP2A expression. The intracellular domain of Notch (Notch-IC) and EBNA2 are functional homologs and recent microarray analysis indicates that LMP2A may constitutively activate the Notch pathway in vivo. Coupled with evidence that Notch-IC can bind to and activate the LMP2A promoter, we hypothesized that expression of LMP2A results in the constitutive activation of the Notch pathway to auto-regulate its promoter. Our data indicate that LMP2A constitutively activates the Notch pathway in B cells and epithelial cells. Expression of LMP2A alone is sufficient to activate its own expression and the amino-terminal signaling domain is required as LMP2B is unable to activate the LMP2A promoter. In addition, point mutations in tyrosines 31, 101 and 112 each results in a significant decrease in LMP2A promoter activation. Deletion of the RBP-Jkappa consensus sequences results in a significant decrease in promoter activity. The observation that LMP2A activates its own promoter suggests that LMP2A exploits the Notch pathway in order to control its own expression and may explain EBNA2-independent expression of LMP2A in EBV-associated malignancies.

Cholinergic and peptidergic regulation of phenylethanolamine N-methyltransferase gene expression.

The splanchnic nerve, innervating the adrenal medulla, releases a variety of neurotransmitters that stimulate genes involved in catecholamine biosynthesis. In particular, cholinergic agonists have been shown to induce phenylethanolamine N-methyltransferase (PNMT) gene expression through activation of both nicotinic and muscarinic receptors in vivo and in vitro. By contrast, the role of peptidergic neurotransmitters in adrenal medullary PNMT gene expression remains unclear. Using transient transfection assays, we demonstrate that rat PNMT promoter-luciferase reporter gene constructs are markedly activated by 10 nM PACAP when expressed in PC12 cells. PACAP appears to mediate its effects primarily through PAC1 receptors and, subsequently, cAMP-protein kinase A (PKA) and extracellular Ca(2+) signaling mechanisms. Activation of these signal transduction pathways markedly increases nuclear levels of the immediately early gene transcription factor Egr-1 and the developmental factor AP2. A slight decrease in Sp1 expression may also occur, whereas MAZ and glucocorticoid receptor expression remains unaltered. Although PACAP stimulates rapid changes in transcription factor expression and PNMT promoter activity, its effects are long lasting. PNMT promoter induction continues to rise and is sustained for > or=48 hours. By contrast, while muscarine, nicotine, or carbachol (100 micro M) also evoke rapid increases in rat PNMT promoter activity, peak activity is observed at 6 hours, followed by a decline and restoration to basal levels by 24 hours. Cholinergic activation of the PNMT promoter also seems to involve the cAMP-PKA signaling mechanism. However, the magnitude of stimulation and antagonist blockade with H-89 or the polypeptide inhibitor PKI suggests that the extent of activation is much less than that with PACAP.