Perturbed liver gene zonation in a mouse model of non-alcoholic steatohepatitis.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.

Glutaminase 2 knockdown reduces hyperammonemia and associated lethality of urea cycle disorder mouse model.

Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otcspf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otcspf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otcspf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle.

Single-cell RNA transcriptome landscape of hepatocytes and non-parenchymal cells in healthy and NAFLD mouse liver.

Nonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers.

Discovery and validation of biomarkers to aid the development of safe and effective pain therapeutics: challenges and opportunities.

Pain medication plays an important role in the treatment of acute and chronic pain conditions, but some drugs, opioids in particular, have been overprescribed or prescribed without adequate safeguards, leading to an alarming rise in medication-related overdose deaths. The NIH Helping to End Addiction Long-term (HEAL) Initiative is a trans-agency effort to provide scientific solutions to stem the opioid crisis. One component of the initiative is to support biomarker discovery and rigorous validation in collaboration with industry leaders to accelerate high-quality clinical research into neurotherapeutics and pain. The use of objective biomarkers and clinical trial end points throughout the drug discovery and development process is crucial to help define pathophysiological subsets of pain, evaluate target engagement of new drugs and predict the analgesic efficacy of new drugs. In 2018, the NIH-led Discovery and Validation of Biomarkers to Develop Non-Addictive Therapeutics for Pain workshop convened scientific leaders from academia, industry, government and patient advocacy groups to discuss progress, challenges, gaps and ideas to facilitate the development of biomarkers and end points for pain. The outcomes of this workshop are outlined in this Consensus Statement.

Large-scale transcriptomic analysis reveals that pridopidine reverses aberrant gene expression and activates neuroprotective pathways in the YAC128 HD mouse.

BACKGROUND: Huntington Disease (HD) is an incurable autosomal dominant neurodegenerative disorder driven by an expansion repeat giving rise to the mutant huntingtin protein (mHtt), which is known to disrupt a multitude of transcriptional pathways. Pridopidine, a small molecule in development for treatment of HD, has been shown to improve motor symptoms in HD patients. In HD animal models, pridopidine exerts neuroprotective effects and improves behavioral and motor functions. Pridopidine binds primarily to the sigma-1 receptor, (IC50 ~ 100 nM), which mediates its neuroprotective properties, such as rescue of spine density and aberrant calcium signaling in HD neuronal cultures. Pridopidine enhances brain-derived neurotrophic factor (BDNF) secretion, which is blocked by putative sigma-1 receptor antagonist NE-100, and was shown to upregulate transcription of genes in the BDNF, glucocorticoid receptor (GR), and dopamine D1 receptor (D1R) pathways in the rat striatum. The impact of different doses of pridopidine on gene expression and transcript splicing in HD across relevant brain regions was explored, utilizing the YAC128 HD mouse model, which carries the entire human mHtt gene containing 128 CAG repeats. METHODS: RNAseq was analyzed from striatum, cortex, and hippocampus of wild-type and YAC128 mice treated with vehicle, 10 mg/kg or 30 mg/kg pridopidine from the presymptomatic stage (1.5 months of age) until 11.5 months of age in which mice exhibit progressive disease phenotypes. RESULTS: The most pronounced transcriptional effect of pridopidine at both doses was observed in the striatum with minimal effects in other regions. In addition, for the first time pridopidine was found to have a dose-dependent impact on alternative exon and junction usage, a regulatory mechanism known to be impaired in HD. In the striatum of YAC128 HD mice, pridopidine treatment initiation prior to symptomatic manifestation rescues the impaired expression of the BDNF, GR, D1R and cAMP pathways. CONCLUSIONS: Pridopidine has broad effects on restoring transcriptomic disturbances in the striatum, particularly involving synaptic transmission and activating neuroprotective pathways that are disturbed in HD. Benefits of treatment initiation at early disease stages track with trends observed in the clinic.

Expression profiling of human immune cell subsets identifies miRNA-mRNA regulatory relationships correlated with cell type specific expression.

Blood consists of different cell populations with distinct functions and correspondingly, distinct gene expression profiles. In this study, global miRNA expression profiling was performed across a panel of nine human immune cell subsets (neutrophils, eosinophils, monocytes, B cells, NK cells, CD4 T cells, CD8 T cells, mDCs and pDCs) to identify cell-type specific miRNAs. mRNA expression profiling was performed on the same samples to determine if miRNAs specific to certain cell types down-regulated expression levels of their target genes. Six cell-type specific miRNAs (miR-143; neutrophil specific, miR-125; T cells and neutrophil specific, miR-500; monocyte and pDC specific, miR-150; lymphoid cell specific, miR-652 and miR-223; both myeloid cell specific) were negatively correlated with expression of their predicted target genes. These results were further validated using an independent cohort where similar immune cell subsets were isolated and profiled for both miRNA and mRNA expression. miRNAs which negatively correlated with target gene expression in both cohorts were identified as candidates for miRNA/mRNA regulatory pairs and were used to construct a cell-type specific regulatory network. miRNA/mRNA pairs formed two distinct clusters in the network corresponding to myeloid (nine miRNAs) and lymphoid lineages (two miRNAs). Several myeloid specific miRNAs targeted common genes including ABL2, EIF4A2, EPC1 and INO80D; these common targets were enriched for genes involved in the regulation of gene expression (p<9.0E-7). Those miRNA might therefore have significant further effect on gene expression by repressing the expression of genes involved in transcriptional regulation. The miRNA and mRNA expression profiles reported in this study form a comprehensive transcriptome database of various human blood cells and serve as a valuable resource for elucidating the role of miRNA mediated regulation in the establishment of immune cell identity.

The multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities.

Exosomes are bioactive vesicles derived from the cell's endosomal membrane system and secreted into surrounding body fluids. Exosomes contain cell and cell-state specific cargos of protein, mRNA and miRNA. Exosome formation, cargo content, and delivery to surrounding cells is of immense biological interest considering the role that exosomes are believed to play in various pathological conditions. They aid antigen presentation by immune cells and can exhibit either anti-inflammatory or pro-inflammatory properties depending on the parent antigen-presenting cell's conditioning. Viruses can hijack a host cell's exosomal machinery to evade host defense systems aiding in the trans-infection of viruses. Tumor derived exosomes may help establish an oncogenic niche systemically via delivery of protein, mRNA, and miRNA that can aid angiogenesis, cell proliferation, and cell survival. Exosomes have also been implicated in the spread of neurodegenerative diseases. Studies have shown that exosomes are selectively taken up by cells distal from their release. They can reprogram the recipient cells due to their active molecular cargo. Cell-lineage and state-specific exosomes imply that they may therefore harbor body fluid-based biomarkers of unparalleled accuracy, particularly for tissues that are difficult or impossible to access. Exosome-specific membrane proteins provide markers enabling exosome identity and selection, while cell type and cell condition-specific protein, mRNA and miRNA cargo provide a rich potential source of biomarkers. This review serves to provide an overview of the current state of the science in the burgeoning field of exosome biology.

Approaches and limitations of phosphatidylinositol-3-kinase pathway activation status as a predictive biomarker in the clinical development of targeted therapy.

The central role played by the class I(A) phosphatidylinositol-3-kinase (PI3K) signaling node in human cancer is highlighted in the multiple mechanisms by which these signals become dysregulated. Many studies suggest that constitutive PI3K activation in human cancer contributes to drug resistance, including targeted agents and standard cytotoxic therapy. The combination of activation mechanisms and the multiple downstream cascades that emanate from the PI3K node contributes to the difficulty in measuring PI3K activation as a biomarker. Although many agents suppress the pathway in models, the challenge remains to translate this biology into a patient selection strategy (i.e., identify patients with "PI3K activated" tumors) and subsequently link this biomarker definition to drug responses in patients. The various genetic and epigenetic lesions resulting in pathway activation necessitate combined approaches using genetic, genomic, and protein biomarkers to accurately characterize "PI3K activated" tumors. Such a combined approach to pathway status can be assessed using a statistical stratification of patients in a randomized trial into "pathway on" and "pathway off" subsets to compare the treatment effect in each arm. Instead of considering individual biomarkers for their predictive ability, this strategy proposes the use of a collection of biomarkers to identify a specific "pathway on" patient population predicted to have clinical benefit from a pathway inhibitor. Here, we review the current understanding of the mechanisms of PI3K activation in breast cancer and discuss a pathway-based approach using PI3K as a predictive biomarker in clinical development, which is currently in use in a global phase 3 setting.

Voluntary exploratory data submissions to the US FDA and the EMA: experience and impact.

Heterogeneity in the underlying mechanisms of disease processes and inter-patient variability in drug responses are major challenges in drug development. To address these challenges, biomarker strategies based on a range of platforms, such as microarray gene-expression technologies, are increasingly being applied to elucidate these sources of variability and thereby potentially increase drug development success rates. With the aim of enhancing understanding of the regulatory significance of such biomarker data by regulators and sponsors, the US Food and Drug Administration initiated a programme in 2004 to allow sponsors to submit exploratory genomic data voluntarily, without immediate regulatory impact. In this article, a selection of case studies from the first 5 years of this programme - which is now known as the voluntary exploratory data submission programme, and also involves collaboration with the European Medicines Agency - are discussed, and general lessons are highlighted.

Analytical validation of a highly sensitive microparticle-based immunoassay for the quantitation of IL-13 in human serum using the Erenna immunoassay system.

IL-13 is a Th2 cytokine that has been shown to be an important mediator of airway inflammation contributing to asthma lesions. Given its proposed role in asthma, measurements of this cytokine in serum may provide insights into disease mechanisms, progression and pharmacodynamic effects of IL-13 targeted therapeutics. However, current commercially available ELISA immunoassays are frequently unable to detect baseline concentrations of IL-13 in serum from healthy individuals, which are below the limit of detection. Here we describe the use of the novel microparticle-based Erenna IL-13 human immunoassay (Singulex, Inc.), which utilizes proprietary antibodies and single molecule counting technology, to quantify IL-13 from 100 microL of serum from apparently healthy subjects and clinically defined symptomatic and asymptomatic asthma subjects. The lower limit of quantification of the Erenna assay was validated at 0.07 pg/mL and the assay detected baseline concentrations of IL-13 in 98% of serum samples tested. The calibration curve showed good precision over the entire linear range of 0.07-50 pg/mL, with inter-assay imprecision <10% CV except at the lowest concentration tested (<15%). The intra- and inter-assay imprecision of spiked serum samples containing three different IL-13 concentrations (2, 8, and 25 pg/mL) ranged from 2.2-2.4% and 6.1-6.8%, respectively. Using the Erenna IL-13 assay, we observe that serum IL-13 concentrations range from <0.07-1.02 pg/mL in apparently healthy subjects (N=60) with similar ranges in asymptomatic (0.07-0.66 pg/mL, N=26) and symptomatic (<0.07-1.26 pg/mL, N=96) asthma subjects. The Erenna immunoassay improved sensitivity by over two full logs compared to previous ELISA methods, while using smaller sample volumes. In addition, the Erenna assay reliably measured IL-13 in endogenous and spiked human serum samples that were not quantifiable using other methods. Taken together, these results show that this novel assay offers a significant improvement over previous methods for high-sensitive quantitative measurement of IL-13 in human serum samples obtained from both apparently healthy and asthmatic subjects, and can be used in future clinical studies to accurately measure concentrations of this cytokine prior to and following drug therapy in human serum.

Begacestat (GSI-953): a novel, selective thiophene sulfonamide inhibitor of amyloid precursor protein gamma-secretase for the treatment of Alzheimer's disease.

The presenilin containing gamma-secretase complex is responsible for the regulated intramembraneous proteolysis of the amyloid precursor protein (APP), the Notch receptor, and a multitude of other substrates. gamma-Secretase catalyzes the final step in the generation of Abeta(40) and Abeta(42) peptides from APP. Amyloid beta-peptides (Abeta peptides) aggregate to form neurotoxic oligomers, senile plaques, and congophilic angiopathy, some of the cardinal pathologies associated with Alzheimer's disease. Although inhibition of this protease acting on APP may result in potentially therapeutic reductions of neurotoxic Abeta peptides, nonselective inhibition of the enzyme may cause severe adverse events as a result of impaired Notch receptor processing. Here, we report the preclinical pharmacological profile of GSI-953 (begacestat), a novel thiophene sulfonamide gamma-secretase inhibitor (GSI) that selectively inhibits cleavage of APP over Notch. This GSI inhibits Abeta production with low nanomolar potency in cellular and cell-free assays of gamma-secretase function, and displaces a tritiated analog of GSI-953 from enriched gamma-secretase enzyme complexes with similar potency. Cellular assays of Notch cleavage reveal that this compound is approximately 16-fold selective for the inhibition of APP cleavage. In the human APP-overexpressing Tg2576 transgenic mouse, treatment with this orally active compound results in a robust reduction in brain, plasma, and cerebral spinal fluid Abeta levels, and a reversal of contextual fear-conditioning deficits that are correlated with Abeta load. In healthy human volunteers, oral administration of a single dose of GSI-953 produces dose-dependent changes in plasma Abeta levels, confirming pharmacodynamic activity of GSI-953 in humans.

Safety, pharmacokinetics, and pharmacodynamics of single doses of LXR-623, a novel liver X-receptor agonist, in healthy participants.

Liver X-receptor (LXR) agonists have been postulated to enhance reverse cholesterol transport (RCT), a process believed to shuttle cholesterol from the periphery back to the liver. Enhancing RCT via the upregulation of cholesterol transporters such as the adenosine triphosphate-binding cassettes ABCA1 and ABCG1 could therefore inhibit the progression of atherosclerosis. LXR-623 is a synthetic ligand for LXRs alpha and beta that has shown promise in animal models of atherosclerosis. The authors present results from a single ascending-dose study of the safety, pharmacokinetics, and pharmacodynamics of LXR-623 in healthy participants. LXR-623 was absorbed rapidly with peak concentrations (C(max)) achieved at approximately 2 hours. The C(max) and area under the concentration-time curve increased in a dose-proportional manner. The mean terminal disposition half-life was between 41 and 43 hours independently of dose. LXR activation resulted in a dose-dependent increase in ABCA1 and ABCG1 expression. The effect of LXR-623 concentration on ABCA1 and ABCG1 expression was further characterized via a population pharmacokinetic-pharmacodynamic analysis, yielding EC(50) estimates of 526 ng/mL and 729 ng/mL, respectively. Central nervous system-related adverse events were observed at the 2 top doses tested. The pharmacodynamic effects described here are the first demonstration of "target engagement" by an LXR agonist in humans.

Measurement of myostatin concentrations in human serum: Circulating concentrations in young and older men and effects of testosterone administration.

UNLABELLED: Methodological problems, including binding of myostatin to plasma proteins and cross-reactivity of assay reagents with other proteins, have confounded myostatin measurements. Here we describe development of an accurate assay for measuring myostatin concentrations in humans. Monoclonal antibodies that bind to distinct regions of myostatin served as capture and detector antibodies in a sandwich ELISA that used acid treatment to dissociate myostatin from binding proteins. Serum from myostatin-deficient Belgian Blue cattle was used as matrix and recombinant human myostatin as standard. The quantitative range was 0.15-37.50 ng/mL. Intra- and inter-assay CVs in low, mid, and high range were 4.1%, 4.7%, and 7.2%, and 3.9%, 1.6%, and 5.2%, respectively. Myostatin protein was undetectable in sera of Belgian Blue cattle and myostatin knockout mice. Recovery in spiked sera approximated 100%. ActRIIB-Fc or anti-myostatin antibody MYO-029 had no effect on myostatin measurements when assayed at pH 2.5. Myostatin levels were higher in young than older men (mean+/-S.E.M. 8.0+/-0.3 ng/mL vs. 7.0+/-0.4 ng/mL, P=0.03). In men treated with graded doses of testosterone, myostatin levels were significantly higher on day 56 than baseline in both young and older men; changes in myostatin levels were significantly correlated with changes in total and free testosterone in young men. Myostatin levels were not significantly associated with lean body mass in either young or older men. CONCLUSION: Myostatin ELISA has the characteristics of a valid assay: nearly 100% recovery, excellent precision, accuracy, and sufficient sensitivity to enable measurement of myostatin concentrations in men and women.

Comparison of the pharmacokinetics of venlafaxine extended release and desvenlafaxine in extensive and poor cytochrome P450 2D6 metabolizers.

BACKGROUND: The goal of this study was to evaluate the impact of cytochrome P450 2D6 extensive metabolizer (EM) or poor metabolizer (PM) status on the pharmacokinetics of single doses of venlafaxine extended release (ER) and desvenlafaxine (administered as desvenlafaxine succinate) in healthy adults. METHODS: In an open-label, crossover study, 14 healthy volunteers (aged 18-55 years; 7 EMs and 7 PMs) received, in randomized sequence, single doses of venlafaxine ER 75 mg/d or desvenlafaxine 100 mg/d. Cytochrome P450 2D6 genotyping was performed, and plasma drug levels were measured. The arithmetic means and standard deviation (SD) for area under the plasma concentration-versus-time curve (AUC) and peak plasma concentration (Cmax) were calculated. Comparisons of AUC and Cmax between cytochrome P450 2D6 EMs and PMs were calculated using a Wilcoxon exact test. RESULTS: After administration of venlafaxine ER, mean Cmax and AUC of venlafaxine were significantly greater in PMs compared with EMs, whereas mean Cmax and AUC of its metabolite, desvenlafaxine, were significantly lower for PMs than for EMs (P = 0.001, all comparisons). In contrast, mean Cmax and AUC of desvenlafaxine after administration of desvenlafaxine were comparable between EMs and PMs. CONCLUSIONS: Cytochrome P450 2D6 genetic polymorphisms had no discernible impact on exposure to desvenlafaxine after desvenlafaxine administration; in contrast, compared with an EM phenotype, a PM phenotype had a significant effect on venlafaxine and desvenlafaxine plasma concentrations after venlafaxine ER administration. This reduced pharmacokinetic variability of desvenlafaxine may translate into better uniformity of response for patients receiving desvenlafaxine versus venlafaxine, but additional studies are required to test this hypothesis.

Discovery and implementation of transcriptional biomarkers of synthetic LXR agonists in peripheral blood cells.

BACKGROUND: LXRs (Liver X Receptor alpha and beta) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds. OBJECTIVE: Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators. METHODS: Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRalpha, LXRbeta, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated ex vivo with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623. RESULTS: A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRalpha and LXRbeta, and all cell types significantly increased ABCA1 and ABCG1 expression upon ex vivo LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription. CONCLUSION: Peripheral blood cells express LXRalpha and LXRbeta, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.

An assessment of drug-drug interactions: the effect of desvenlafaxine and duloxetine on the pharmacokinetics of the CYP2D6 probe desipramine in healthy subjects.

A number of antidepressants inhibit the activity of the cytochrome P450 2D6 enzyme system, which can lead to drug-drug interactions. Based on its metabolic profile, desvenlafaxine, administered as desvenlafaxine succinate, a new serotonin-norepinephrine reuptake inhibitor, is not expected to have an impact on activity of CYP2D6. This single-center, randomized, open-label, four-period, crossover study was undertaken to evaluate the effect of multiple doses of desvenlafaxine (100 mg/day, twice the recommended therapeutic dose for major depressive disorder in the United States) and duloxetine (30 mg b.i.d.) on the pharmacokinetics (PK) of a single dose of desipramine (50 mg). A single dose of desipramine was given first to assess its PK. Desvenlafaxine or duloxetine was then administered, in a crossover design, so that steady-state levels were achieved; a single dose of desipramine was then coadministered. The geometric least-square mean ratios (coadministration versus desipramine alone) for area under the plasma concentration versus time curve (AUC) and peak plasma concentrations (C(max)) of desipramine and 2-hydroxydesipramine were compared using analysis of variance. Relative to desipramine alone, increases in AUC and C(max) of desipramine associated with duloxetine administration (122 and 63%, respectively) were significantly greater than those associated with desvenlafaxine (22 and 19%, respectively; P < 0.001). Duloxetine coadministered with desipramine was also associated with a decrease in 2-hydroxydesipramine C(max) that was significant compared with the small increase seen with desvenlafaxine and desipramine (-24 versus 9%; P < 0.001); the difference between changes in 2-hydroxydesipramine AUC did not reach statistical significance (P = 0.054). Overall, desvenlafaxine had a minimal impact on the PK of desipramine compared with duloxetine, suggesting a lower risk for CYP2D6-mediated drug interactions.

Use of ex vivo systems for biomarker discovery.

Patients exhibit a range of responses to drug treatment owing to individual genetic variation and biology. A deeper understanding of the human genome, enabled by increasingly powerful technologies to measure both its genes and gene products, has unleashed the concept of tailoring therapy to the individual patient upon pharmaceutical and clinical sciences. The successful application of personalized medicine depends upon the discovery and development of biomarkers. Biomarkers that either indicate pharmacodynamic effects or constitute predictive measures of individual patient responses can support dose selection and/or help determine therapeutic options. The development of biomarkers for clinical testing and validation can be facilitated by the use of ex vivo systems utilizing clinically relevant human tissues for the discovery of biomarkers of drug activity before first in human (FIH) studies. In this review we discuss the uses of ex vivo systems for both disease tissues and surrogate normal tissues to provide mechanistic insights into drug action and for the purpose of identifying candidate biomarkers.

Opportunities posed by novel patient selection biomarker approaches in oncology drug development: going beyond the cytotoxics.

An area of unmet medical need in clinical oncology has been optimizing patient selection for a given therapeutic with the goal of getting the right drug to the right patient. Recent studies have developed preclinical approaches to identifying molecular 'signatures of resistance' for cytotoxic therapies and prospective validation of this strategy is ongoing in the clinic. New challenges in this setting include identifying approaches to patient selection for cytostatic compounds such as signaling pathway inhibitors and stem cell targets. Here, we discuss the biomarker methodologies developed using traditional cytotoxic drugs and how these approaches can be adapted to identify biomarkers of patient selection for novel signaling inhibitors and other novel targets. It has become increasingly clear that such biomarker discovery and validation needs to begin early and continue throughout the drug development process.

Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray.

Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene() RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2() enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene() blood samples also advocate a short, fixed room temperature storage time for all PAXgene() blood samples collected for the purposes of global transcriptional profiling in clinical studies.