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1.
J Hepatol ; 73(4): 896-905, 2020 10.
Article in English | MEDLINE | ID: mdl-32376414

ABSTRACT

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty acid ß-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in patients with NASH; however, the effect of ACC inhibition on liver fibrosis has not been reported. METHODS: A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, diethylnitrosamine. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. RESULTS: Inhibition of ACC reduced the activation of TGF-ß-stimulated HSCs, as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which inhibition of de novo lipogenesis blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require de novo lipogenesis for activation. Consistent with this direct antifibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat choline-deficient, high-fat diet model and in response to chronic diethylnitrosamine-induced liver injury (in the absence of hepatic lipid accumulation). CONCLUSIONS: In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the profibrogenic activity of HSCs. Thus, small molecule inhibitors of ACC may lessen fibrosis by reducing lipotoxicity in hepatocytes and by preventing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH. LAY SUMMARY: Hepatic fibrosis is the most important predictor of liver-related outcomes in patients with non-alcoholic steatohepatitis (NASH). Small molecule inhibitors of acetyl-CoA carboxylase (ACC) reduce hepatic fat content and markers of liver injury in patients with NASH. Herein, we report that inhibition of ACC and de novo lipogenesis also directly suppress the activation of hepatic stellate cells - the primary cell responsible for generating fibrotic scar in the liver - and thus fibrosis. These data provide further evidence for the use of ACC inhibitors to treat patients with NASH and advanced fibrosis.


Subject(s)
Acetyl-CoA Carboxylase/antagonists & inhibitors , Hepatic Stellate Cells/metabolism , Lipogenesis/drug effects , Liver Cirrhosis/metabolism , Liver/pathology , Non-alcoholic Fatty Liver Disease/metabolism , Animals , Biomarkers/metabolism , Cell Line , Diet, High-Fat/adverse effects , Disease Models, Animal , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/pathology , Humans , Liver/metabolism , Liver Cirrhosis/etiology , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred C57BL , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/drug therapy , Rats , Rats, Wistar
2.
Clin Pharmacokinet ; 59(9): 1109-1117, 2020 09.
Article in English | MEDLINE | ID: mdl-32333325

ABSTRACT

BACKGROUND: Selonsertib is a first-in-class inhibitor of apoptosis signal-regulating kinase 1 (ASK1) with therapeutic potential for fibrotic diseases. This phase I study evaluated the safety, tolerability, pharmacokinetics (PK), and food effect of selonsertib in healthy subjects. METHODS: This was a double-blinded, randomized, placebo-controlled dose-escalation study. Healthy subjects received 1, 3, 10, 30, or 100 mg of selonsertib or placebo as single or multiple doses once daily for 14 days in the fasted state, or 30 mg or placebo single dose in the fed state. Blood and urine (single-dose cohorts only) samples for selonsertib PK were collected and safety was assessed throughout the study. Ex vivo pharmacodynamic (PD) assessment was performed in blood from a separate cohort of healthy donors using an auranofin-stimulated C-X-C motif chemokine ligand 1 (CXCL1) assay. RESULTS: Overall, 107 subjects (83 active, 24 placebo) were enrolled and randomized to 11 cohorts. Selonsertib was generally well tolerated; adverse events were generally mild to moderate. Selonsertib was rapidly absorbed with dose-proportional PK of both parent and inactive metabolite GS-607509. There was no food effect on selonsertib PK. Renal excretion was a minor pathway of selonsertib elimination. Selonsertib half maximal effective concentration (EC50) in human whole blood was determined to be 56 ng/mL. CONCLUSIONS: Selonsertib exhibited a favorable PK profile amenable to once-daily dosing without regard to food. PD data suggest pharmacologically relevant exposures were achieved in the dose range evaluated. Study results support further clinical development of selonsertib.


Subject(s)
Benzamides/pharmacokinetics , Imidazoles/pharmacokinetics , MAP Kinase Kinase Kinase 5 , Pyridines/pharmacokinetics , Area Under Curve , Benzamides/administration & dosage , Dose-Response Relationship, Drug , Double-Blind Method , Food-Drug Interactions , Healthy Volunteers , Humans , Imidazoles/administration & dosage , MAP Kinase Kinase Kinase 5/antagonists & inhibitors , Pyridines/administration & dosage
3.
Cancer Immunol Immunother ; 57(8): 1263-70, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18236040

ABSTRACT

Promising anti-tumor responses have been observed in the clinic using monoclonal antibodies (mAbs) that block immune checkpoints. One concern with these therapeutic agents remains the potential induction of immune breakthrough events (IBEs) resulting from the disruption of T cell homeostasis or the breaking of tolerance to self antigens. As an approach to maintaining anti-tumor responses but decreasing the likelihood of these events, the local expression of a mAb in combination with a GM-CSF-secreting cancer immunotherapy was evaluated. Using anti-cytotoxic T lymphocyte antigen (CTLA)-4 as a model antibody to test this hypothesis, tumor cell lines were generated that expressed the full-length mAb in addition to GM-CSF. Evaluation of these cell lines in two therapeutic tumor models revealed that local, cell-mediated delivery of anti-CTLA-4 from a GM-CSF-secreting tumor cell immunotherapy activated potent anti-tumor responses and prolonged overall survival at significantly lower serum mAb levels in the host. Furthermore, lowering the systemic exposure of the host to the immune modulatory mAb correlated with reduced evidence of systemic autoimmunity. This approach has broad utility for the delivery of mAbs or proteins locally from cellular immunotherapies to minimize IBEs while retaining the potent therapeutic effects of such combination treatments.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Antigens, CD/immunology , Autoimmunity/immunology , Immunotherapy , Neoplasms, Experimental/immunology , Neoplasms, Experimental/therapy , Animals , Antibodies, Monoclonal/genetics , CTLA-4 Antigen , Cell Line, Tumor , Cloning, Molecular , Disease Models, Animal , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Hybridomas/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasm Transplantation , Neoplasms, Experimental/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
4.
Mol Ther ; 15(6): 1153-9, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17375065

ABSTRACT

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.


Subject(s)
Antibodies, Monoclonal/genetics , Dependovirus/genetics , Genetic Vectors/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/metabolism , Binding Sites , Blotting, Western , CHO Cells , Cell Line , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Drug Delivery Systems/methods , Enzyme-Linked Immunosorbent Assay , Female , Furin/metabolism , Gene Expression/drug effects , Genetic Vectors/administration & dosage , Humans , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mice, Nude , Molecular Sequence Data , Sirolimus/pharmacology
5.
J Biol Chem ; 280(48): 40364-74, 2005 Dec 02.
Article in English | MEDLINE | ID: mdl-16183991

ABSTRACT

Accumulating evidence suggests that neurodegeneration induced by pathogenic proteins depends on contributions from surrounding glia. Here we demonstrate that NF-kappaB signaling in microglia is critically involved in neuronal death induced by amyloid-beta (Abeta) peptides, which are widely presumed to cause Alzheimer disease. Constitutive inhibition of NF-kappaB signaling in microglia by expression of the nondegradable IkappaBalpha superrepressor blocked neurotoxicity, indicating a pivotal role for microglial NF-kappaB signaling in mediating Abeta toxicity. Stimulation of microglia with Abeta increased acetylation of RelA/p65 at lysine 310, which regulates the NF-kappaB pathway. Overexpression of SIRT1 deacetylase and the addition of the SIRT1 agonist resveratrol markedly reduced NF-kappaB signaling stimulated by Abeta and had strong neuroprotective effects. Our results support a glial loop hypothesis by demonstrating a critical role for microglial NF-kappaB signaling in Abeta-dependent neurodegeneration. They also implicate SIRT1 in this pathway and highlight the therapeutic potential of resveratrol and other sirtuin-activating compounds in Alzheimer disease.


Subject(s)
Amyloid beta-Peptides/chemistry , Microglia/metabolism , NF-kappa B/metabolism , Sirtuins/physiology , Alzheimer Disease/metabolism , Animals , Bromodeoxyuridine/pharmacology , Cells, Cultured , Genes, Reporter , Genetic Vectors , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry , Lentivirus/genetics , Lysine/chemistry , Microscopy, Fluorescence , Models, Biological , Models, Genetic , Neurons/metabolism , Rats , Rats, Sprague-Dawley , Resveratrol , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Sirtuin 1 , Sirtuins/metabolism , Stilbenes/pharmacology
6.
Nat Biotechnol ; 23(5): 584-90, 2005 May.
Article in English | MEDLINE | ID: mdl-15834403

ABSTRACT

Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 microg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.


Subject(s)
Adenoviridae/genetics , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/therapeutic use , Antibody Formation/genetics , Genetic Therapy/methods , Kidney/metabolism , Neoplasms/therapy , Viral Proteins/metabolism , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/isolation & purification , Cell Line , Gene Transfer Techniques , Humans , Hybridomas , Kidney/immunology , Mice , Neoplasms/genetics , Neoplasms/immunology , Protein Engineering/methods , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/immunology , Viral Proteins/genetics
7.
Hypertension ; 45(5): 867-73, 2005 May.
Article in English | MEDLINE | ID: mdl-15837831

ABSTRACT

We tested the hypothesis that in the stroke-prone spontaneously hypertensive rat (SHRSP), the Cl- component of dietary NaCl dominantly determines its pressor effect (salt-sensitivity). We telemetrically measured systolic aortic blood pressure (SBP) in SHRSP loaded with: nothing (CTL); NaCl alone (NaCl) (44 mmol/100 grams chow); KCl (KCl) alone (44 mmol); NaCl (44 mmol) combined with KHCO3 (77 mmol) (NaCl/KBC) or with KCl (77 mmol) (NaCl/KCl). Across all groups, from age 10 to 15 or 16 weeks, SBP increased linearly (mm Hg/week) (dp/dt, change in SBP as a function of time): CTL, 5.6; NaCl, 9.5; KCl, 8.8; NaCl/KBC, 9.1; and NaCl/KCl, 14.6. Thus, the value of dp/dt in KCl matched that in NaCl. The value of dp/dt in NaCl/KCl exceeded that in NaCl in direct proportion to the greater Cl- load. Across all groups, only Cl- load bore a direct, highly linear relationship with dp/dt. Strokes occurred only, but always with SBP >250 mm Hg, a value observed almost exclusively in NaCl/KCl. Thus, Cl- dominantly determined the pressor effect induced with dietary NaCl, both with NaCl loaded alone and combined with either KCl or KHCO3, and thereby likely determined the occurrence of stroke with NaCl loading. Over the initial 3-day period of NaCl loading and exacerbating hypertension, external balance of Na+ increased similarly among all groups. However, within 24 hours of initiating NaCl loading, urinary creatinine excretion decreased in direct proportion to dp/dt and urinary Cl- excretion. We conclude that in the SHRSP, the Cl- component of a dietary NaCl dominantly determines salt sensitivity and thereby phenotypic expression. We suggest that Cl- might do so by inducing renal vasoconstriction.


Subject(s)
Chlorides , Genetic Predisposition to Disease , Hypertension/chemically induced , Rats, Inbred SHR/genetics , Sodium Chloride , Stroke/genetics , Animals , Bicarbonates/pharmacology , Blood Pressure/drug effects , Body Weight/drug effects , Chlorides/urine , Creatinine/urine , Drug Combinations , Electrolytes/urine , Hypertension/urine , Incidence , Kidney/drug effects , Kidney/physiopathology , Male , Potassium Chloride/pharmacology , Potassium Compounds/pharmacology , Rats , Sodium Chloride/pharmacology , Stroke/epidemiology
8.
J Biol Chem ; 279(7): 5565-72, 2004 Feb 13.
Article in English | MEDLINE | ID: mdl-14612454

ABSTRACT

Alzheimer's disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimer's disease. Activation of cultured murine microglial BV2 cells by freshly sonicated Abeta42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying Abeta-induced microglial activation, we analyzed the expression levels of transcripts isolated from Abeta42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by Abeta42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in Abeta-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by Abeta42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by Abeta-activated inflammatory response.


Subject(s)
Cathepsin B/physiology , Microglia/metabolism , Neurons/cytology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Calcium-Binding Proteins/metabolism , Cathepsin B/metabolism , Cathepsin L , Cathepsins/metabolism , Cell Death , Cell Line , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Cysteine Endopeptidases/metabolism , DNA, Complementary/metabolism , Electron Transport Complex IV/metabolism , Gene Library , Genome , Inflammation , Mice , Microfilament Proteins , Neurons/metabolism , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Peptide Fragments/metabolism , Peptides/chemistry , RNA/metabolism , RNA, Small Interfering/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinase-2/metabolism , Transfection , Up-Regulation
9.
Nat Med ; 9(8): 1062-8, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12858170

ABSTRACT

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.


Subject(s)
Brain Injuries/physiopathology , Brain/pathology , Membrane Transport Proteins , Mitochondrial Proteins , Neurons/metabolism , Neuroprotective Agents/metabolism , Proteins/metabolism , Stroke/physiopathology , Animals , Brain/metabolism , Brain Injuries/pathology , Caspase 3 , Caspases/metabolism , Cell Death/physiology , Cells, Cultured , Humans , Ion Channels , Ischemia/metabolism , Mice , Mice, Transgenic , Mitochondria/metabolism , Neurons/cytology , Oligonucleotide Array Sequence Analysis , Proteins/genetics , Rats , Reactive Oxygen Species/metabolism , Stroke/pathology , Uncoupling Agents/metabolism , Uncoupling Protein 2
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