Model-Based Interspecies Scaling for Predicting Human Pharmacokinetics of CB 4332, a Complement Factor I Protein.

Interspecies scaling of the pharmacokinetics (PK) of CB 4332, a 150 kDa recombinant complement factor I protein, was performed using traditional and model-based approaches to inform first-in-human dose selection. Plasma concentration versus time data from four preclinical PK studies of single intravenous and subcutaneous (SC) CB 4332 dosing in mice, rats and nonhuman primates (NHPs) were modeled simultaneously using naive pooling including allometric scaling. The human-equivalent dose was calculated using the preclinical no observed adverse effect level (NOAEL) as part of the dose-by-factor approach. Pharmacokinetic modeling of CB 4332 revealed species-specific differences in the elimination, which was accounted for by including an additional rat-specific clearance. Signs of anti-drug antibodies (ADA) formation in all rats and some NHPs were observed. Consequently, an additional ADA-induced clearance parameter was estimated including the time of onset. The traditional dose-by-factor approach calculated a maximum recommended starting SC dose of 0.9 mg/kg once weekly, which was predicted it to result in a trough steady-state concentration lower than the determined efficacy target range for CB 4332 in humans. Model simulations predicted the efficacy target range to be reached using 5 mg/kg once weekly SC dosing.

Model-Informed Support of Dose Selection for Prophylactic Treatment with Dalcinonacog Alfa in Adult and Paediatric Hemophilia B Patients.

INTRODUCTION: Dalcinonacog alfa (DalcA), a novel subcutaneously administered recombinant human factor IX (FIX) variant is being developed for adult and paediatric patients with hemophilia B (HB). DalcA has been shown to raise FIX to clinically meaningful levels in adults with HB. This work aimed to support dosing regimen selection in adults and perform first-in-paediatric dose extrapolations using a model-based pharmacokinetic (PK) approach. METHODS: A population PK model was built using adult data from two clinical trials (NCT03186677, NCT03995784). With allometry in the model, clinical trial simulations were performed to study alternative dosing regimens in adults and children. Steady-state trough levels and the time-to-reach target were derived to inform dose selection. RESULTS: Almost 90% of the adults were predicted to achieve desirable FIX levels, i.e. 10% FIX activity, following daily 100 IU/kg dosing, with 90% of the subjects reaching target within 1.6-7.1 days. No every-other-day regimen met the target. A dose of 125 IU/kg resulted in adequate FIX levels down to 6 years, whereas a 150 IU/kg dose was needed below 6 down to 2 years of age. For subjects down to 6 years that did not reach target with 125 IU/kg, a dose escalation to 150 IU/kg was appropriate. The children below 6 to 2 years were shown to need a dose escalation to 200 IU/kg if 150 IU/kg given daily was insufficient. CONCLUSION: This study supported the adult dose selection for DalcA in the presence of sparse data and enabled first-in-paediatric dose selection to achieve FIX levels that reduce risk of spontaneous bleeds.

Novel Oxygen Carrier Slows Infarct Growth in Large Vessel Occlusion Dog Model Based on Magnetic Resonance Imaging Analysis.

BACKGROUND: Tissue hypoxia plays a critical role in the events leading to cell death in ischemic stroke. Despite promising results in preclinical and small clinical pilot studies, inhaled oxygen supplementation has not translated to improved outcomes in large clinical trials. Moreover, clinical observations suggest that indiscriminate oxygen supplementation can adversely affect outcome, highlighting the need to develop novel approaches to selectively deliver oxygen to affected regions. This study tested the hypothesis that intravenous delivery of a novel oxygen carrier (Omniox-Ischemic Stroke [OMX-IS]), which selectively releases oxygen into severely ischemic tissue, could delay infarct progression in an established canine thromboembolic large vessel occlusion stroke model that replicates key dynamics of human infarct evolution. METHODS: After endovascular placement of an autologous clot into the middle cerebral artery, animals received OMX-IS treatment or placebo 45 to 60 minutes after stroke onset. Perfusion-weighted magnetic resonance imaging was performed to define infarct progression dynamics to stratify animals into fast versus slow stroke evolvers. Serial diffusion-weighted magnetic resonance imaging was performed for up to 5 hours to quantify infarct evolution. Histology was performed postmortem to confirm final infarct size. RESULTS: In fast evolvers, OMX-IS therapy substantially slowed infarct progression (by ≈1 hour, P<0.0001) and reduced the final normalized infarct volume as compared to controls (0.99 versus 0.88, control versus OMX-IS drug, P<0.0001). Among slow evolvers, OMX-IS treatment delayed infarct progression by approximately 45 minutes; however, this did not reach statistical significance (P=0.09). The final normalized infarct volume also did not show a significant difference (0.93 versus 0.95, OMX-IS drug versus control, P=0.34). Postmortem histologically determined infarct volumes showed excellent concordance with the magnetic resonance imaging defined ischemic lesion volume (bias: 1.33% [95% CI, -15% to 18%). CONCLUSIONS: Intravenous delivery of a novel oxygen carrier is a promising approach to delay infarct progression after ischemic stroke, especially in treating patients with large vessel occlusion stroke who cannot undergo definitive reperfusion therapy within a timely fashion.

In vivo functions of p75NTR: challenges and opportunities for an emerging therapeutic target.

The p75 neurotrophin receptor (p75NTR) functions at the molecular nexus of cell death, survival, and differentiation. In addition to its contribution to neurodegenerative diseases and nervous system injuries, recent studies have revealed unanticipated roles of p75NTR in liver repair, fibrinolysis, lung fibrosis, muscle regeneration, and metabolism. Linking these various p75NTR functions more precisely to specific mechanisms marks p75NTR as an emerging candidate for therapeutic intervention in a wide range of disorders. Indeed, small molecule inhibitors of p75NTR binding to neurotrophins have shown efficacy in models of Alzheimer's disease (AD) and neurodegeneration. Here, we outline recent advances in understanding p75NTR pleiotropic functions in vivo, and propose an integrated view of p75NTR and its challenges and opportunities as a pharmacological target.

Correction: Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

[This corrects the article DOI: 10.1371/journal.pbio.2005924.].

Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX-based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX-based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV-treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.

p75 Neurotrophin Receptor Regulates Energy Balance in Obesity.

Obesity and metabolic syndrome reflect the dysregulation of molecular pathways that control energy homeostasis. Here, we show that the p75 neurotrophin receptor (p75(NTR)) controls energy expenditure in obese mice on a high-fat diet (HFD). Despite no changes in food intake, p75(NTR)-null mice were protected from HFD-induced obesity and remained lean as a result of increased energy expenditure without developing insulin resistance or liver steatosis. p75(NTR) directly interacts with the catalytic subunit of protein kinase A (PKA) and regulates cAMP signaling in adipocytes, leading to decreased lipolysis and thermogenesis. Adipocyte-specific depletion of p75(NTR) or transplantation of p75(NTR)-null white adipose tissue (WAT) into wild-type mice fed a HFD protected against weight gain and insulin resistance. Our results reveal that signaling from p75(NTR) to cAMP/PKA regulates energy balance and suggest that non-CNS neurotrophin receptor signaling could be a target for treating obesity and the metabolic syndrome.

Hypoxia Inducible Factor-1α in Astrocytes and/or Myeloid Cells Is Not Required for the Development of Autoimmune Demyelinating Disease

Hypoxia-like tissue alterations, characterized by the upregulation of hypoxia-inducible factor-1α (HIF-1α), have been described in the normal appearing white matter and pre-demyelinating lesions of multiple sclerosis (MS) patients. As HIF-1α regulates the transcription of a wide set of genes involved in neuroprotection and neuroinflammation, HIF-1α expression may contribute to the pathogenesis of inflammatory demyelination. To test this hypothesis, we analyzed the effect of cell-specific genetic ablation or overexpression of HIF-1α on the onset and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. HIF-1α was mainly expressed in astrocytes and microglia/macrophages in the mouse spinal cord at the peak of EAE. However, genetic ablation of HIF-1α in astrocytes and/or myeloid cells did not ameliorate clinical symptoms. Furthermore, conditional knock-out of Von Hippel Lindau, a negative regulator of HIF-1α stabilization, failed to exacerbate the clinical course of EAE. In accordance with clinical symptoms, genetic ablation or overexpression of HIF-1α did not change the extent of spinal cord inflammation and demyelination. Overall, our data indicate that despite dramatic upregulation of HIF-1α in astrocytes and myeloid cells in EAE, HIF-1α expression in these two cell types is not required for the development of inflammatory demyelination. Despite numerous reports indicating HIF-1α expression in glia, neurons, and inflammatory cells in the CNS of MS patients, the cell-specific contribution of HIF-1α to disease pathogenesis remains unclear. Here we show that although HIF-1α is dramatically upregulated in astrocytes and myeloid cells in EAE, cell-specific depletion of HIF-1α in these two cell types surprisingly does not affect the development of neuroinflammatory disease. Together with two recently published studies showing a role for oligodendrocyte-specific HIF-1α in myelination and T-cell-specific HIF-1α in EAE, our results demonstrate a tightly regulated cellular specificity for HIF-1α contribution in nervous system pathogenesis.

Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-ß signaling and astrocyte functions.

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-ß signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

Suppression of oxidative stress by ß-hydroxybutyrate, an endogenous histone deacetylase inhibitor.

Concentrations of acetyl-coenzyme A and nicotinamide adenine dinucleotide (NAD(+)) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-ß-hydroxybutyrate (ßOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous ßOHB, or fasting or calorie restriction, two conditions associated with increased ßOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by ßOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with ßOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with ßOHB conferred substantial protection against oxidative stress.

Fibrinogen-induced perivascular microglial clustering is required for the development of axonal damage in neuroinflammation.

Blood-brain barrier disruption, microglial activation and neurodegeneration are hallmarks of multiple sclerosis. However, the initial triggers that activate innate immune responses and their role in axonal damage remain unknown. Here we show that the blood protein fibrinogen induces rapid microglial responses toward the vasculature and is required for axonal damage in neuroinflammation. Using in vivo two-photon microscopy, we demonstrate that microglia form perivascular clusters before myelin loss or paralysis onset and that, of the plasma proteins, fibrinogen specifically induces rapid and sustained microglial responses in vivo. Fibrinogen leakage correlates with areas of axonal damage and induces reactive oxygen species release in microglia. Blocking fibrin formation with anticoagulant treatment or genetically eliminating the fibrinogen binding motif recognized by the microglial integrin receptor CD11b/CD18 inhibits perivascular microglial clustering and axonal damage. Thus, early and progressive perivascular microglial clustering triggered by fibrinogen leakage upon blood-brain barrier disruption contributes to axonal damage in neuroinflammatory disease.

p75 neurotrophin receptor regulates glucose homeostasis and insulin sensitivity.

Insulin resistance is a key factor in the etiology of type 2 diabetes. Insulin-stimulated glucose uptake is mediated by the glucose transporter 4 (GLUT4), which is expressed mainly in skeletal muscle and adipose tissue. Insulin-stimulated translocation of GLUT4 from its intracellular compartment to the plasma membrane is regulated by small guanosine triphosphate hydrolases (GTPases) and is essential for the maintenance of normal glucose homeostasis. Here we show that the p75 neurotrophin receptor (p75(NTR)) is a regulator of glucose uptake and insulin resistance. p75(NTR) knockout mice show increased insulin sensitivity on normal chow diet, independent of changes in body weight. Euglycemic-hyperinsulinemic clamp studies demonstrate that deletion of the p75(NTR) gene increases the insulin-stimulated glucose disposal rate and suppression of hepatic glucose production. Genetic depletion or shRNA knockdown of p75(NTR) in adipocytes or myoblasts increases insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, overexpression of p75(NTR) in adipocytes decreases insulin-stimulated glucose transport. In adipocytes, p75(NTR) forms a complex with the Rab5 family GTPases Rab5 and Rab31 that regulate GLUT4 trafficking. Rab5 and Rab31 directly interact with p75(NTR) primarily via helix 4 of the p75(NTR) death domain. Adipocytes from p75(NTR) knockout mice show increased Rab5 and decreased Rab31 activities, and dominant negative Rab5 rescues the increase in glucose uptake seen in p75(NTR) knockout adipocytes. Our results identify p75(NTR) as a unique player in glucose metabolism and suggest that signaling from p75(NTR) to Rab5 family GTPases may represent a unique therapeutic target for insulin resistance and diabetes.

p75 (NTR) and hypoxia: a breath of fresh air in neurotrophin receptor signaling.

Comment on: Le Moan N, et al. Mol Cell 2011; 44:476-90.

Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1α.

Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75(NTR)) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75(NTR) directly interacts with the evolutionarily conserved zinc finger domains of the E3 RING ubiquitin ligase Siah2 (seven in absentia homolog 2), which regulates HIF-1α degradation. p75(NTR) stabilizes Siah2 by decreasing its auto-ubiquitination. Genetic loss of p75(NTR) dramatically decreases Siah2 abundance, HIF-1α stabilization, and induction of HIF-1α target genes in hypoxia. p75(NTR-/-) mice show reduced HIF-1α stabilization, vascular endothelial growth factor (VEGF) expression, and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75(NTR) constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.

Hepatic stellate cells and astrocytes: Stars of scar formation and tissue repair.

Scar formation inhibits tissue repair and regeneration in the liver and central nervous system. Activation of hepatic stellate cells (HSCs) after liver injury or of astrocytes after nervous system damage is considered to drive scar formation. HSCs are the fibrotic cells of the liver, as they undergo activation and acquire fibrogenic properties after liver injury. HSC activation has been compared to reactive gliosis of astrocytes, which acquire a reactive phenotype and contribute to scar formation after nervous system injury, much like HSCs after liver injury. It is intriguing that a wide range of neuroglia-related molecules are expressed by HSCs. We identified an unexpected role for the p75 neurotrophin receptor in regulating HSC activation and liver repair. Here we discuss the molecular mechanisms that regulate HSC activation and reactive gliosis and their contributions to scar formation and tissue repair. Juxtaposing key mechanistic and functional similarities in HSC and astrocyte activation might provide novel insight into liver regeneration and nervous system repair.

Low-density lipoprotein receptor-related protein 1 is an essential receptor for myelin phagocytosis.

Multiple sclerosis (MS) is an autoimmune disease in which myelin is progressively degraded. Because degraded myelin may both initiate and accelerate disease progression, clearing degraded myelin from extracellular spaces may be critical. In this study, we prepared myelin vesicles (MV) from rat brains as a model of degraded myelin. Murine embryonic fibroblasts (MEFs) rapidly internalized MVs, which accumulated in lysosomes only when these cells expressed low-density lipoprotein receptor-related protein (LRP1). Receptor-associated protein (RAP), which binds LRP1 and inhibits interaction with other ligands, blocked MV uptake by LRP1-expressing MEFs. As a complementary approach, we prepared primary cultures of rat astrocytes, microglia and oligodendrocytes. All three cell types expressed LRP1 and mediated MV uptake, which was inhibited by RAP. LRP1 gene-silencing in oligodendrocytes also blocked MV uptake. Myelin basic protein (MBP), which was expressed as a recombinant protein, bound directly to LRP1. MBP-specific antibody inhibited MV uptake by oligodendrocytes. In experimental autoimmune encephalomyelitis in mice, LRP1 protein expression was substantially increased in the cerebellum and spinal cord. LRP1 colocalized with multiple CNS cell types. These studies establish LRP1 as a major receptor for phagocytosis of degraded myelin, which may function alone or in concert with co-receptors previously implicated in myelin phagocytosis.

Protein-thiol oxidation, from single proteins to proteome-wide analyses.

Protein-thiol oxidation subserves multiple biological functions, from enzymatic catalysis to protein oxidative folding, protein trafficking, reactive oxygen (ROS) and nitrogen (RNS) species sensing and signaling and, more generally, protein redox regulation. Protein-thiol oxidation may also constitute a sequel of ROS and RNS toxicity. Accurate and robust methods aimed at monitoring the in vivo redox state of cysteine residues are thus warranted. To this aim, we have developed biochemical approaches that rely on trapping cysteine residues in their in vivo redox state using acidic conditions, followed by the differential labeling of reduced versus oxidized cysteine residues by thiol-specific reagents. These methods have been instrumental in the discovery of eukaryotic peroxide receptors and new ROS-scavenging enzymes and in identifying the repertoire of cytoplasmic oxidized protein thiols. Proteome-wide approaches also contributed to establish the functions of the thioredoxin and glutathione pathways in eukaryotic cytoplasmic thiol-redox control.

The system biology of thiol redox system in Escherichia coli and yeast: differential functions in oxidative stress, iron metabolism and DNA synthesis.

By its ability to engage in a variety of redox reactions and coordinating metals, cysteine serves as a key residue in mediating enzymatic catalysis, protein oxidative folding and trafficking, and redox signaling. The thiol redox system, which consists of the glutathione and thioredoxin pathways, uses the cysteine residue to catalyze thiol-disulfide exchange reactions, thereby controlling the redox state of cytoplasmic cysteine residues and regulating the biological functions it subserves. Here, we consider the thiol redox systems of Escherichia coli and Saccharomyces cerevisiae, emphasizing the role of genetic approaches in the understanding of the cellular functions of these systems. We show that although prokaryotic and eukaryotic systems have a similar architecture, they profoundly differ in their overall cellular functions.

The Saccharomyces cerevisiae proteome of oxidized protein thiols: contrasted functions for the thioredoxin and glutathione pathways.

Protein thiol oxidation subserves important biological functions and constitutes a sequel of reactive oxygen species toxicity. We developed two distinct thiol-labeling approaches to identify oxidized cytoplasmic protein thiols in Saccharomyces cerevisiae. Inone approach, we used N-(6-(biotinamido)hexyl)-3'-(2'-pyridyldithio)-propionamide to purify oxidized protein thiols, and in the other, we used N-[(14)C]ethylmaleimide to quantify this oxidation. Both approaches showed a large number of the same proteins with oxidized thiols ( approximately 200), 64 of which were identified by mass spectrometry. We show that, irrespective of its mechanism, protein thiol oxidation is dependent upon molecular O(2). We also show that H(2)O(2) does not cause de novo protein thiol oxidation, but rather increases the oxidation state of a select group of proteins. Furthermore, our study reveals contrasted differences in the oxidized proteome of cells upon inactivation of the thioredoxin or GSH pathway suggestive of very distinct thiol redox control functions, assigning an exclusive role for thioredoxin in H(2)O(2) metabolism and the presumed thiol redox buffer function for GSH. Taken together, these results suggest the high selectivity of cytoplasmic protein thiol oxidation.