Closing in on the target: sustained virologic response in hepatitis C virus genotype 1 infection response-guided therapy.

Retrospective analyses of the boceprevir and telaprevir phase 3 trial data demonstrate the clinical relevance of detected but not quantifiable hepatitis C virus (HCV) genotype 1 RNA during treatment. These analyses illustrate the importance of using precise and standard terminology in reporting low-level HCV RNA results for consistent data collection across clinical trials, and to ensure optimal virologic response-guided treatment decision making in clinical practice. In the context of currently available quantitative HCV RNA assays, we clarify that unquantifiable HCV RNA should be classified as target detected or target not detected, as both have been shown to reflect clinically different qualitative HCV RNA levels during treatment. Additionally, use of terms such as "undetectable" or "below limit of detection" should be avoided as such terms are imprecise, not consistently defined, and often misinterpreted.

Recommendations for standardized nomenclature and definitions of viral response in trials of hepatitis C virus investigational agents.

Outdated virological response terms used at key trial timepoints in clinical trials with first-generation direct-acting antivirals plus pegylated interferon and ribavirin have failed to keep pace with hepatitis C virus (HCV) drug development. A more intuitive and flexible nomenclature capable of adapting to continuing advances in HCV drug development is needed. Assistance in standardization of the field was provided by members of the Hepatitis C Virus Drug Development Advisory Group, a project of the Forum for Collaborative HIV Research with participation from the American Association for the Study of Liver Diseases, European Association for the of the liver, and the Infectious Diseases Society of America. Our proposed descriptive, virological response nomenclature for key decision points in trials (with and without lead-in treatment) is based on an assay-specified lower limit of quantitation cutoff. This allows responses to be categorized as either quantifiable or unquantifiable HCV RNA, with unquantifiable responses further divided based on whether target HCV RNA was detected or not detected. The unified reporting recommendations will facilitate interpretation of results across clinical trials and validation of new response-guided timepoints. As time-critical treatment parameters are shortened in HCV trials, the proposed nomenclature will greatly simplify and facilitate future adaptations of virological response terms. Our proposed nomenclature will also be helpful in developing treatment guidelines for use in clinical practice.

Novel clinical trial designs for the development of new antiretroviral agents.

The resounding success of combination antiretroviral efficacy for both treatment-naïve and treatment-experienced patients - with 70-90% viral suppression rates in recent studies - has made registration trials for new agents challenging. With the inevitable specter of drug resistance, new agents must have a pathway to approval. The Forum for Collaborative HIV Research obtained input from concerned stakeholders including industry, clinical sciences, community advocacy, and regulatory sciences (Food and Drug Administration and European Medicines Agency) to discuss how safety and efficacy of new agents could be demonstrated. Recognizing the shortfalls of superiority or noninferiority trials in this environment, a new trial design for treatment-experienced patients, minimizing the risk for drug resistance but allowing full assessment of safety, was proposed. The antiviral efficacy of an active investigational drug would be assessed by comparison to placebo as an add-on to a failing regimen in a short, 10-14-day study followed by institution of an optimized background regimen (OBR) in both arms with investigational drug given to all patients. The follow-on stage would assess dose response, safety, durability of initial response, and development of resistance. Additionally, a second safety trial could be conducted comparing patients randomized to the investigational agent with a new OBR to those on a new OBR and placebo. Finally, approval decisions could consider other long-term safety endpoints. Exposing treatment-naïve patients to investigational agents remains a controversial issue; stakeholders have different interpretations of risk-benefit for trials in this population that necessitate careful consideration before initiating trials in them.

Vascular endothelial growth factor enhances migration of astroglial cells in subventricular zone neurosphere cultures.

Vascular endothelial growth factor (VEGF) is an endothelial and neuronal survival factor and a mitogen for endothelial cells and astrocytes in both explant and in vivo injury models. In the CNS, interplay between the vasculature and neural stem progenitor (NSP) cells is required for the maintenance of angiogenic/neurogenic coordination in the germinal niche in the subventricular zone (SVZ) of the lateral ventricle. Using an in vitro SVZ neurosphere (NS) model, this study aimed to understand the direct effects of VEGF and its receptor signaling on neonatal NSP cell growth and migration. Our data indicate that VEGF administration, compared with untreated or brain-derived neurotrophic factor-treated NS, significantly increased growth and migratory capacity of glial fibrillary acidic protein (GFAP)(+) and nestin(+) NSP cells and in secondary cultures induced a stellate astrocyte morphology. Blockade of both VEGF, which is normally expressed in some NS cells, and its flt-1 receptor signaling by neutralizing antibodies caused morphological changes specifically in GFAP(+) cells and disrupted sphere formation and outward migration. These cells did not appear as conventional polygonal astrocytes; their process growth was severely restricted, and overall migration was reduced by up to 76% of control cultures. Blockade of VEGF's flk-1 receptor reduced VEGF expression and caused a lesser, though significant, decrease (29%) in NSP (GFAP(+)) cell migration. The results show that both VEGF and, in particular, flt-1 receptor signaling are critical to the proper configuration of the NS and its subsequent development. VEGF is also an important growth and migratory factor particularly for GFAP(+) cells developing in SVZ-derived NS in culture.

Roles of the endogenous VEGF receptors flt-1 and flk-1 in astroglial and vascular remodeling after brain injury.

Following trauma to the brain significant changes occur in both the astroglial and vascular components of the neuropil. Angiogenesis is required to re-establish metabolic support and astrocyte activation encompasses several functions including scar formation and the production of growth factors. VEGF has seminal involvement in the process of brain repair and is upregulated during many pathological events. VEGF signaling is regulated mainly through its two primary receptors: flk-1 (KDR/VEGF-R2) is expressed on vascular endothelium and some neurons and flt-1 (VEGF-R1) in the CNS, is expressed predominantly by activated astrocytes. Using an injury model of chronic minipump infusion of neutralizing antibodies (NA) to block VEGF receptor signaling, this study takes advantage of these differences in VEGF receptor distribution in order to understand the role the cytokine plays after brain injury. Infusion of NA to flk-1 caused a significant decrease in vascular proliferation and increased endothelial cell degeneration compared to control IgG infusions but had no effect on astrogliosis. By contrast infusion of NA to flt-1 significantly decreased astroglial mitogenicity and scar formation and caused some increase in endothelial degeneration. Neutralization of the flt-1 receptor function, but not flk-1, caused significant reduction in the astroglial expression of the growth factors, CNTF and FGF by 7days. These data suggest that after CNS injury, endogenous VEGF upregulation (by astrocytes) induces angiogenesis and, by autocrine signaling, increases both astrocyte proliferation and facilitates expression of growth factors. It is likely that VEGF plays an important role in aspects of astroglial scar formation.

Astrocyte growth effects of vascular endothelial growth factor (VEGF) application to perinatal neocortical explants: receptor mediation and signal transduction pathways.

The non-angiogenic role of vascular endothelial growth factor (VEGF), and its receptors flt-1 and flk-1, together with downstream signaling pathways were examined in fetal and postnatal rat cerebral cortical organotypic explants. VEGF application in both paradigms caused a significant increase in astroglial proliferation and a dose-dependent increase in GFAP and nestin immunoreactivity. The VEGF receptor flt-1 was observed on most, though not all astrocytes, while flk-1 receptor immunoexpression was absent. Treatment with antisense oligonucleotides (AS-ODNs) to flt-1 resulted in a dramatic decrease in GFAP and nestin immunoreactivity, which further confirmed the role of flt-1 in mediating VEGF's gliotrophic effects, while AS-ODNs to flk-1 had no effect. VEGF-induced gliotrophic effects were found to be mediated by the MAPK/ERK and PI-3 kinase signaling pathways, since the both the MEK1 inhibitor, PD98059 and the PI-3 kinase inhibitor, Wortmannin abolished VEGF-induced astrocytic GFAP(+) expression. Although high dose VEGF application resulted in strong upregulation of both GFAP and nestin immunoreactivity in astrocytes, overlap of the two proteins was not observed in all cells, suggesting that some of the nestin(+) cells might be neural progenitors. Exposure to VEGF resulted in upregulation of both VEGF and bFGF mRNA at the one-day time point, and bFGF protein by 3 days; VEGF activated astrocytes expressed bFGF to a much greater degree than those in untreated explants. The increased expression of bFGF induced by VEGF, may serve in the proliferation of multipotential neural stem/progenitor cells in vitro. VEGF, an established angiogenic factor, appears to play a significant role in the growth and differentiation of astrocytes in the CNS.

Activation of receptor-mediated angiogenesis and signaling pathways after VEGF administration in fetal rat CNS explants.

SUMMARY: The angiogenic role of vascular endothelial growth factor (VEGF) receptors, flk-1 and flt-1, and their downstream signaling pathways, MAPK/ERK and PI-3 kinase, were examined in a fetal rat cortical explant model after exposure to exogenous VEGF. Treatment with VEGF resulted in substantial neovascularization characterized by increased vascular flk-1 receptor expression, whereas flt-1 receptor protein expression was absent. The specific role of flk-1 receptors in the angiogenic process was confirmed by the addition of antisense oligonucleotides (AS-ODNs) to flk-1, which blocked angiogenesis, whereas AS-ODNs to flt-1 had no effect. These results were further supported by the finding that specific chemical inhibition of flk-1 receptors caused disruption of the angiogenic response, whereas inhibition of the flt-1 receptors had no effect. Application of either MAPK/ERK or PI-3 kinase pathway inhibitors disrupted VEGF-induced angiogenesis, thereby indicating that both signaling pathways mediate this process. Thus VEGF binding to the endothelial flk-1 receptor activates the MAPK/ERK and PI-3 kinase pathways, resulting in neoangiogenic events. Of interest is the fact that although VEGF is regarded as a vascular permeability factor, its application to nascent cortical tissue caused an increase in a key physiologic protein of the blood-brain barrier function, glucose transporter-1, suggesting that the cytokine may have a role in blood-brain barrier development.

Neurotrophic effects of vascular endothelial growth factor on organotypic cortical explants and primary cortical neurons.

Vascular endothelial growth factor (VEGF) is well known to play an important regulatory role in vascular growth and development. Because gene knock-outs of VEGF and its receptors flk-1 and flt-1 result in early embryonic lethality, determining roles for VEGF in CNS development has been particularly difficult. Recent studies have shown that VEGF is upregulated after various injuries to the adult brain and that the cytokine affords protection to cultured neurons affected by oxidative or excitotoxic stress. The present study demonstrates, for the first time, that VEGF is directly neurotrophic to CNS neurons in culture. We applied VEGF to normoxic fetal organotypic cortical explants as a model of CNS neuropil, in addition to primary cortical neurons, to assess direct growth effects absent vascular or astroglial activity. We found that VEGF provided a significant dose-responsive increase in the neuronal microtubule markers TUJ1 and MAP-2, as well as mRNA for MAP-2 and flk-1. Antisense oligodeoxynucleotides to flk-1, but not flt-1, inhibited neuritic outgrowth, whereas inhibitors of the signaling pathways MEK1 and P13-AKT both abrogated VEGF-induced growth. VEGF applied to primary cortical neurons produced significant increases in neuronal cell body diameter and the number of emerging neurites mediated by flk-1. Possibly, VEGF achieves its effects by acting on the neuronal microtubular content, which is involved with growth, stability and maturation. Several studies have now shown that VEGF is neurotrophic and neuroprotective independent of a vascular component; we suggest that VEGF plays seminal pleiotrophic roles in CNS development and repair.