What evidence do we need for biomarker qualification?

Biomarkers can facilitate all aspects of the drug development process. However, biomarker qualification-the use of a biomarker that is accepted by the U.S. Food and Drug Administration-needs a clear, predictable process. We describe a multistakeholder effort including government, industry, and academia that proposes a framework for defining the amount of evidence needed for biomarker qualification. This framework is intended for broad applications across multiple biomarker categories and uses.

Advancing Value Assessment in the United States: A Multistakeholder Perspective.

Rising costs without perceived proportional improvements in quality and outcomes have motivated fundamental shifts in health care delivery and payment to achieve better value. Aligned with these efforts, several value assessment frameworks have been introduced recently to help providers, patients, and payers better understand the potential value of drugs and other interventions and make informed decisions about their use. Given their early stage of development, it is imperative to evaluate these efforts on an ongoing basis to identify how best to support and improve them moving forward. This article provides a multistakeholder perspective on the key limitations and opportunities posed by the current value assessment frameworks and areas of and actions for improvement. In particular, we outline 10 fundamental guiding principles and associated strategies that should be considered in subsequent iterations of the existing frameworks or by emerging initiatives in the future. Although value assessment frameworks may not be able to meet all the needs and preferences of stakeholders, we contend that there are common elements and potential next steps that can be supported to advance value assessment in the United States.

Combinatorial pharmacogenomic guidance for psychiatric medications reduces overall pharmacy costs in a 1 year prospective evaluation.

OBJECTIVES: The objective of this project was to determine pharmacy cost savings and improvement in adherence based on a combinatorial pharmacogenomic test (CPGx ) in patients who had switched or added a new psychiatric medication after having failed monotherapy for their psychiatric disorder. RESEARCH DESIGN AND METHODS: The prospective project compared 1 year pharmacy claims between a GeneSight CPGx guided cohort and a propensity-matched control group. Patients were project eligible if they augmented or switched to a different antidepressant or antipsychotic medication within the previous 90 days. Following the medication switch or augmentation, pharmacogenomic (PGx) testing was offered to each patient's treating clinician. Pharmacy claims were extracted from the Medco pharmacy claims database for each patient (n = 2168) for 1 year following testing and compared to a 5-to-1 propensity-matched treatment as usual (TAU), standard of care control group (n = 10,880). MAIN OUTCOME MEASURES: Total pharmacy spend per member per year; adherence. RESULTS: Patients who received PGx testing saved $1035.60 in total medication costs (both CNS and non-CNS medications) over 1 year compared to the non-tested standard of care cohort (p = 0.007). PGx testing improved adherence compared to standard of care (ΔPDCCPGx = 0.11 vs ΔPDCTAU = -0.01; p < 0.0001). Pharmacy cost savings averaged $2774.53 for patients who were changed to a CPGx congruent medication regimen, compared to those who were not (p < 0.0001). CONCLUSIONS: PGx testing provides significant 'real world' cost savings, while simultaneously improving adherence in a difficult to treat psychiatric population. Limitations of this study include the lack of therapeutic efficacy follow-up data and possible confounding due to matching only on demographic and psychiatric variables.

Monitoring phosphoproteomic response to targeted kinase inhibitors using reverse-phase protein microarrays.

Phosphoproteomic networks mediated by protein kinases are the key drivers of proliferative and survival signals underlying human cancers, and as such a number of kinases have been the subject of intensive drug discovery efforts. A key question that must be answered during clinical development is whether a kinase inhibitor is effectively inhibiting its appropriate target kinase and pathway in the tumor. Reverse-phase protein arrays (RPMAs) offer the ability to analyze behavior of entire signaling networks in response to drug treatment and thus have promise as a technology for monitoring cellular response to kinase inhibitors. We have shown that it is possible to use RPMAs to detect phosphorylation changes in key multiple signaling pathway proteins in response to targeted inhibitors of EGFR, MEK, and PI3 kinase.

Inhibition of transforming growth factor beta receptor I kinase blocks hepatocellular carcinoma growth through neo-angiogenesis regulation.

UNLABELLED: Curative therapies for patients with hepatocellular carcinoma (HCC) are mainly invasive, and with the exception of sorafenib, no medical treatments are available for advanced or metastatic stages of HCC. We investigated the antitumoral effect of blocking the transforming growth factor beta (TGF-beta) signaling pathway in HCC with LY2109761, a kinase inhibitor of TGF-beta receptor I kinase. The antitumor activity of LY2109761 was associated with inhibition of molecular pathways involved in neo-angiogenesis and tumor growth of HCC. This anti-angiogenic effect is more effective than that of bevacizumab, which specifically targets vascular endothelial growth factor (VEGF). We found that the paracrine cross-talk between HCC and endothelial cells is blocked by LY210976, inhibiting blood vessel formation. This effect was mediated by SMAD2/3 and affected the secretion of VEGF. Finally, LY2109761 does not show significant effects on physiological angiogenetic development. CONCLUSION: These data support the rationale for targeting TGF-beta signaling in patients with HCC.

Corequirement of PICK1 binding and PKC phosphorylation for stable surface expression of the metabotropic glutamate receptor mGluR7.

The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding, thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo.

mGluR7 undergoes rapid internalization in response to activation by the allosteric agonist AMN082.

The G-protein coupled receptor (GPCR) metabotropic glutamate receptor 7 (mGluR7) is widely expressed throughout the nervous system and is implicated in diverse physiological processes ranging from synaptic plasticity to neuroprotection. To date, unequivocally assigning specific functions to mGluR7 has been hampered by a lack of specific pharmacological tools, however, an mGluR7 specific allosteric agonist, AMN082, was recently discovered. Accumulating evidence indicates that in addition to G-protein activation, GPCRs trigger critical intracellular signalling cascades during agonist-induced internalization. Thus, to determine if AMN082 will be useful for evaluating signalling events related to mGluR7 internalization as well as receptor activation we have examined whether AMN082 induces mGluR7 endocytosis. Using an immunofluorescence assay we demonstrate that AMN082 induces robust internalization of mGluR7 overexpressed in dissociated hippocampal neurons. AMN082-induced mGluR7 internalization was resistant to inhibition by a competitive antagonist consistent with the distinct binding site of the allosteric agonist from the glutamate-binding pocket utilized by conventional orthosteric ligands. Finally, as an independent assay of receptor internalization we overexpressed N-terminal pHluorin-tagged mGluR7 in neurons, allowing live imaging of surface receptors in real time. AMN082 treatment produced a rapid loss of surface mGluR7 as indicated by decreased fluorescence confirming the ability of allosteric receptor activation to trigger mGluR7 endocytosis. Thus, AMN082 will be effective for investigating physiological processes related to both mGluR7 activation and internalization such as control of bidirectional plasticity at mossy fiber-st. lucidum interneuron synapses.

Constitutive endocytosis of the metabotropic glutamate receptor mGluR7 is clathrin-independent.

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are widely expressed throughout the brain and are involved in synaptic development, transmission, and plasticity. The endocytosis of several members of the GPCR superfamily of receptors, such as beta-adrenergic receptors, has been studied extensively. In contrast, the mechanisms regulating mGluR endocytosis and intracellular trafficking remain poorly defined. We describe here for the first time a distinct endocytic and intracellular sorting pathway utilized by mGluR7. We show that mGluR7 constitutively internalizes via a non-clathrin mediated pathway in heterologous cells and in neurons. Unlike clathrin-mediated NMDAR endocytosis, mGluR7 traffics via an Arf6-positive endosomal pathway, similar to other well-characterized proteins such as major histocompatibility complex class I (MHC I) and the GPI-anchored protein CD59. Thus constitutive endocytosis of mGluR7 in neurons is not regulated by clathrin-dependent mechanisms, and this clathrin-independent pathway ultimately determines the amount of receptor present on the plasma membrane available to bind and respond to glutamate.

mGluR7 is a metaplastic switch controlling bidirectional plasticity of feedforward inhibition.

Plasticity of feedforward inhibition in the hippocampal mossy fiber (MF) pathway can dramatically influence dentate gyrus-CA3 dialog. Interestingly, MF inputs to CA3 stratum lucidum interneurons (SLINs) undergo long-term depression (LTD) following high-frequency stimulation (HFS), in contrast to MF-pyramid (PYR) synapses, where long-term potentiation (LTP) occurs. Furthermore, activity-induced potentiation of MF-SLIN transmission has not previously been observed. Here we report that metabotropic glutamate receptor subtype 7 (mGluR7) is a metaplastic switch at MF-SLIN synapses, whose activation and surface expression governs the direction of plasticity. In naive slices, mGluR7 activation during HFS generates MF-SLIN LTD, depressing presynaptic release through a PKC-dependent mechanism. Following agonist exposure, mGluR7 undergoes internalization, unmasking the ability of MF-SLIN synapses to undergo presynaptic potentiation in response to the same HFS that induces LTD in naive slices. Thus, selective mGluR7 targeting to MF terminals contacting SLINs and not PYRs provides cell target-specific plasticity and bidirectional control of feedforward inhibition.

Subunit-specific regulation of NMDA receptor endocytosis.

At excitatory synapses, both NMDA and AMPA receptors are localized to the postsynaptic density (PSD). However, unlike AMPA receptors, synaptic NMDA receptors are stable components of the PSD. Even so, surface-expressed NMDA receptors undergo endocytosis, which is more robust early in development and declines during synaptic development. We investigated the subunit-specific contributions to NMDA receptor endocytosis, specifically defining the endocytic motifs and endocytic pathways preferred by the NR2A and NR2B subunits. We find that NR2A and NR2B have distinct endocytic motifs encoded in their distal C termini and that these interact with clathrin adaptor complexes with differing affinities. We also find that NR2A and NR2B sort into different intracellular pathways after endocytosis, with NR2B preferentially trafficking through recycling endosomes. In mature cultures, we find that NR2B undergoes more robust endocytosis than NR2A, consistent with previous studies showing that NR2A is more highly expressed at stable synaptic sites. Our findings demonstrate fundamental differences between NR2A and NR2B that help clarify developmental changes in NMDA receptor trafficking and surface expression.

Differential binding of the AP-2 adaptor complex and PSD-95 to the C-terminus of the NMDA receptor subunit NR2B regulates surface expression.

NMDA receptor expression on the plasma membrane and at synaptic sites is tightly regulated. We have recently shown that the NMDA receptor subunit NR2B has an endocytic motif contained within its C-terminus. We now identify this motif as a consensus tyrosine-based motif (YEKL) and demonstrate that this sequence binds directly to the medium chain of the AP-2 adaptor, a protein complex that links internalized proteins to clathrin. Although the AP-2 binding site on NR2B is adjacent to the PSD-95 binding site, it is distinct, as mutation of tyrosine 1472 of the endocytic motif disrupts AP-2 binding but not binding to PSD-95. Internalization assays reveal that like PSD-95, both SAP97 and PSD-93 inhibit NR2B-mediated endocytosis. Furthermore, we find that co-expression of a PSD-95 mutant that is unable to cluster NMDA receptors also inhibits NR2B-mediated endocytosis. Together, these data demonstrate that AP-2 and PSD-95 bind to unique sites on the C-terminus of NR2B and have antagonistic functional consequences that are independent of the ability of the PSD-95 to cluster receptors on the plasma membrane.