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1.
Cell Rep Methods ; 3(1): 100388, 2023 01 23.
Article in English | MEDLINE | ID: mdl-36814840

ABSTRACT

CD4+ T cells are critical to the immune system and perform multiple functions; therefore, their identification and characterization are crucial to better understanding the immune system in both health and disease states. However, current methods rarely preserve their ex vivo phenotype, thus limiting our understanding of their in vivo functions. Here we introduce a flexible, rapid, and robust platform for ex vivo CD4+ T cell identification. By combining MHCII allele purification, allele-independent peptide loading, and multiplexed flow cytometry technologies, we can enable high-throughput personalized CD4+ T cell identification, immunophenotyping, and sorting. Using this platform in combination with single-cell sorting and multimodal analyses, we identified and characterized antigen-specific CD4+ T cells relevant to COVID-19 and cancer neoantigen immunotherapy. Overall, our platform can be used to detect and characterize CD4+ T cells across multiple diseases, with potential to guide CD4+ T cell epitope design for any disease-specific immunization strategy.


Subject(s)
CD4-Positive T-Lymphocytes , COVID-19 , Humans , Epitopes, T-Lymphocyte/genetics , Flow Cytometry , Cell Separation
2.
Cancer Cell ; 40(9): 1010-1026.e11, 2022 09 12.
Article in English | MEDLINE | ID: mdl-36027916

ABSTRACT

Neoantigens arising from mutations in tumor DNA provide targets for immune-based therapy. Here, we report the clinical and immune data from a Phase Ib clinical trial of a personalized neoantigen-vaccine NEO-PV-01 in combination with pemetrexed, carboplatin, and pembrolizumab as first-line therapy for advanced non-squamous non-small cell lung cancer (NSCLC). This analysis of 38 patients treated with the regimen demonstrated no treatment-related serious adverse events. Multiple parameters including baseline tumor immune infiltration and on-treatment circulating tumor DNA levels were highly correlated with clinical response. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination. Epitope spread to non-vaccinating neoantigens, including responses to KRAS G12C and G12V mutations, were detected post-vaccination. Neoantigen-specific CD4+ T cells generated post-vaccination revealed effector and cytotoxic phenotypes with increased CD4+ T cell infiltration in the post-vaccine tumor biopsy. Collectively, these data support the safety and immunogenicity of this regimen in advanced non-squamous NSCLC.


Subject(s)
Cancer Vaccines , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Antigens, Neoplasm , CD8-Positive T-Lymphocytes , Cancer Vaccines/adverse effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Humans , Immunotherapy , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics
3.
Nature ; 595(7868): 572-577, 2021 07.
Article in English | MEDLINE | ID: mdl-34044428

ABSTRACT

BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-191. Here we extend a previous phase-I/II trial report2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ+ or IL-2+ CD8+ and CD4+ T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8+ T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8+ T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Adolescent , Adult , BNT162 Vaccine , CD8-Positive T-Lymphocytes/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/adverse effects , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunoglobulin G/immunology , Immunologic Memory , Interferon-gamma/immunology , Interleukin-2/immunology , Male , Middle Aged , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells/immunology , Young Adult
4.
Cell ; 183(2): 347-362.e24, 2020 10 15.
Article in English | MEDLINE | ID: mdl-33064988

ABSTRACT

Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).


Subject(s)
Cancer Vaccines/immunology , Immunotherapy/methods , Precision Medicine/methods , Aged , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/immunology , Female , Humans , Kaplan-Meier Estimate , Male , Melanoma/drug therapy , Melanoma/immunology , Middle Aged , Mutation , Nivolumab/therapeutic use , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/immunology
5.
Bioorg Med Chem ; 26(10): 2842-2849, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29111369

ABSTRACT

Neoantigens arise from somatic mutations that differ from wild-type antigens and are specific to each individual patient, which provide tumor specific targets for developing personalized cancer vaccines. Decades of work has increasingly shown the potential of targeting neoantigens to generate effective clinical responses. Current clinical trials using neoantigen targeting cancer vaccines, including in combination with checkpoint blockade monoclonal antibodies, have demonstrated potent T-cell responses against those neoantigens accompanied by antitumor effects in patients. Personalized neoantigen vaccines represent a potential new class of cancer immunotherapy.


Subject(s)
Cancer Vaccines/therapeutic use , Immunotherapy/methods , Neoplasms/prevention & control , Peptides/therapeutic use , Precision Medicine/methods , Amino Acid Sequence , Animals , Antigens, Neoplasm/administration & dosage , Antigens, Neoplasm/immunology , Antigens, Neoplasm/therapeutic use , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , Humans , Neoplasms/immunology , Peptides/administration & dosage , Peptides/immunology , T-Lymphocytes/immunology
6.
Life Sci ; 109(1): 20-9, 2014 Jul 25.
Article in English | MEDLINE | ID: mdl-24931905

ABSTRACT

AIMS: The 28 amino acid hormone ghrelin, the natural ligand for the growth hormone secretagogue, or ghrelin receptor (GHR), has diverse physiological functions, including a possible role as a gastrointestinal prokinetic. The synthetic ghrelin mimetic RM-131 is in Phase II clinical trials for treatment of diabetic gastroparesis and other gastrointestinal (GI) disorders. We aimed to determine the relative potency of RM-131, when compared to other GI ghrelin mimetics, to predict efficacy and determine the role of RM-131 in models of inflammatory bowel disease. MAIN METHODS: We evaluated and compared ghrelin, RM-131 and other synthetic ghrelin mimetics for their prokinetic potency in models of gastrointestinal disorders in the rat and we evaluated the endocrine (rats and dogs) and anti-inflammatory effects (mice) of the ghrelin mimetic RM-131. KEY FINDINGS: The pentapeptide RM-131 increased gastric emptying in rodent models of ileus. RM-131 is about 100-fold more potent than human ghrelin and is 600 to 1800-fold more potent, when compared to several investigational ghrelin mimetics tested in clinical trials. RM-131 has anti-inflammatory effects and significantly increases survival and reduces macroscopic markers of tissue damage in a TNBS model of inflammatory bowel disease. RM-131 treatment shows a transient increase in growth hormone levels in Beagle dogs and rats, returning to baseline upon chronic treatment. Significant effects on glucose and insulin are not observed in chronic studies. SIGNIFICANCE: RM-131's potency, efficacy and endocrine profile, are promising attributes for the treatment of diverse functional gastrointestinal disorders in humans.


Subject(s)
Gastrointestinal Agents/chemistry , Gastrointestinal Agents/therapeutic use , Gastrointestinal Diseases/drug therapy , Ghrelin/chemistry , Ghrelin/therapeutic use , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/therapeutic use , Diabetes Complications/drug therapy , Dogs , Gastric Emptying/drug effects , Gastrointestinal Motility/drug effects , Gastroparesis/drug therapy , Growth Hormone/metabolism , Humans , Ileus/drug therapy , Inflammatory Bowel Diseases/drug therapy , Male , Mice , Rats , Rats, Sprague-Dawley
7.
Arthritis Rheumatol ; 66(5): 1247-55, 2014 May.
Article in English | MEDLINE | ID: mdl-24470361

ABSTRACT

OBJECTIVE: Acute articular injuries lead to an increased risk of progressive joint damage and osteoarthritis (OA), and no therapies are currently available to repair or protect the injured joint tissue. Intraarticular delivery of therapeutic proteins has been limited by their rapid clearance from the joint space and lack of retention within cartilage. The aim of this study was to test whether targeted delivery to cartilage by fusion with a heparin-binding domain would be sufficient to prolong the in vivo function of the insulin-like growth factor 1 (IGF-1). METHODS: We produced a humanized and optimized recombinant HB-IGF-1 fusion protein. By injecting HB-IGF-1, IGF-1, or saline alone into the knee joints of adult Lewis rats, we tested whether fusion with a heparin-binding domain 1) altered the kinetics of retention in joint tissues, 2) prolonged functional stimulation as measured by radiolabel incorporation, and 3) enhanced efficacy in a rat model of surgically induced OA, using weekly injections. RESULTS: Fusion of heparin-binding domain with IGF-1 prolonged retention in articular and meniscal cartilage from <1 day to 8 days after injection. Unmodified IGF-1 had no functional effect 2 days after injection, whereas HB-IGF-1 stimulated meniscal cartilage at least 4 days after injection. HB-IGF-1, but not IGF-1, significantly slowed cartilage damage in a rat model of OA. CONCLUSION: Heparin-binding domain fusions can transform rapidly cleared proteins into potential intraarticular therapies by targeting them to cartilage.


Subject(s)
Cartilage, Articular/drug effects , Insulin-Like Growth Factor I/pharmacology , Insulin-Like Growth Factor I/therapeutic use , Osteoarthritis/drug therapy , Recombinant Fusion Proteins/pharmacology , Recombinant Fusion Proteins/therapeutic use , Animals , Blood Glucose/metabolism , Disease Models, Animal , Drug Delivery Systems , Heparin/metabolism , Injections, Intra-Articular , Insulin-Like Growth Factor I/metabolism , Male , Osteoarthritis/metabolism , Protein Binding , Rats , Rats, Inbred Lew , Recombinant Fusion Proteins/administration & dosage , Treatment Outcome
8.
Int J Cardiol ; 168(3): 2369-74, 2013 Oct 03.
Article in English | MEDLINE | ID: mdl-23465234

ABSTRACT

Chronic heart failure (CHF) remains one of the most challenging diseases in terms of numbers and disease management, particularly so, if the CHF patient develops cardiac cachexia. Ghrelin and its analogs have been suggested to improve body weight and cardiac function in heart failure models and exploratory human clinical studies. However, most ghrelin compounds are peptides and need to be injected several times per day, which affects the quality of life of patients. Here, we compared two application routes, three times daily subcutaneous (sc) injections to continuous infusion using osmotic mini-pumps in a rat model of CHF. Moreover, the effects were also compared to three times daily sc injections of growth hormone (GH). Rats were treated for 28 d. The results show that treatment with 50 or 100 nmol/kg/d BIM-28131 (RM-131) potently induces body weight gain, fat and lean mass compared to placebo. The gain of lean mass was equal to the gain of lean mass in the 2mg/kg/d GH group and superior to 250 µg/kg/d GH. Both GH and BIM-28131 increased levels of insulin-like growth factor-1 to a similar extent. Little effect was seen on cardiac function; only cardiac output was improved by either high dose BIM-28131 or GH. Overall the effects of BIM-28131 were similar in both application routes.


Subject(s)
Body Composition/drug effects , Body Weight/drug effects , Ghrelin/analogs & derivatives , Heart Failure/physiopathology , Myocardial Infarction/physiopathology , Oligopeptides/administration & dosage , Animals , Disease Models, Animal , Infusion Pumps , Injections, Subcutaneous , Male , Rats , Rats, Sprague-Dawley
9.
J Cachexia Sarcopenia Muscle ; 4(1): 63-9, 2013 Mar.
Article in English | MEDLINE | ID: mdl-22987245

ABSTRACT

BACKGROUND: In chronic heart failure (CHF), cachexia is a hallmark of the terminal stage of this disease and is associated with a severely reduced quality of life and poor prognosis. Therapeutic options are currently not available. Ghrelin and its analogues BIM-28125 and BIM-28131 (now known as RM-131) have been shown to increase weight in a rat model of CHF. It has been further demonstrated that the expression of myostatin, a negative regulator of skeletal muscle mass, is increased in CHF. The aim of the study was to investigate the influence of ghrelin or its analogues on myostatin in CHF. METHODS: In an animal model of CHF, Sprague-Dawley rats received either ghrelin or two ghrelin analogues BIM-28125 and BIM-28131 in two different concentrations (50 and 500 nmol/kg/day) compared to placebo. The compounds were delivered using osmotic mini pumps. The expression of myostatin was analyzed in skeletal muscle by RT-PCR and Western blot, and muscle mass of gastrocnemius muscle was measured. The plasma levels of tumor necrosis factor alpha (TNF-α) were measured. RESULTS: The relative weight of the gastrocnemius muscle of the sham-operated group was significantly increased compared to placebo-treated CHF rats. The application of ghrelin analogue BIM-28125 and BIM-28131 in their higher concentrations led to a significant reduction in myostatin mRNA expression in comparison to placebo. Myostatin protein expression was significantly reduced in both concentrations of ghrelin and BIM-28131 and in the lower concentration of BIM-28125. The increase of TNF-α plasma concentration in the CHF-animals could be abolished by all used substances. CONCLUSIONS: In an animal model of CHF, the expression of myostatin is significantly reduced in the skeletal muscle after application of ghrelin and most concentrations of its analogues BIM-28125 and BIM-28131 possibly due to anti-inflammatory effects.

10.
Diabetes ; 62(2): 490-7, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23048186

ABSTRACT

The melanocortin-4 receptor (MC4R) is well recognized as an important mediator of body weight homeostasis. Activation of MC4R causes dramatic weight loss in rodent models, and mutations in human are associated with obesity. This makes MC4R a logical target for pharmacological therapy for the treatment of obesity. However, previous studies in rodents and humans have observed a broad array of side effects caused by acute treatment with MC4R agonists, including increased heart rate and blood pressure. We demonstrate that treatment with a highly-selective novel MC4R agonist (BIM-22493 or RM-493) resulted in transient decreases in food intake (35%), with persistent weight loss over 8 weeks of treatment (13.5%) in a diet-induced obese nonhuman primate model. Consistent with weight loss, these animals significantly decreased adiposity and improved glucose tolerance. Importantly, we observed no increases in blood pressure or heart rate with BIM-22493 treatment. In contrast, treatment with LY2112688, an MC4R agonist previously shown to increase blood pressure and heart rate in humans, caused increases in blood pressure and heart rate, while modestly decreasing food intake. These studies demonstrate that distinct melanocortin peptide drugs can have widely different efficacies and side effects.


Subject(s)
Anti-Obesity Agents/therapeutic use , Cardiovascular System/drug effects , Diet, High-Fat/adverse effects , Insulin Resistance , Obesity/drug therapy , Peptides/therapeutic use , Receptor, Melanocortin, Type 4/agonists , Weight Loss/drug effects , alpha-MSH/analogs & derivatives , Adiposity/drug effects , Animals , Blood Pressure/drug effects , Chronic Disease , Eating/drug effects , Glucose Intolerance/drug therapy , Heart Rate/drug effects , Macaca mulatta , Male , Obesity/etiology , alpha-MSH/therapeutic use
11.
PLoS One ; 6(11): e26865, 2011.
Article in English | MEDLINE | ID: mdl-22102869

ABSTRACT

UNLABELLED: Cardiac cachexia is a serious complication of chronic heart failure with a prevalence of 10-16% and poor prognosis. There are no current therapy options for cardiac cachexia. Ghrelin is the natural ligand for the GHS-1a-receptor and a potential target for conditions associated with cachexia. Ghrelin has been shown to increase weight in several species. The GHS-1a-receptor is not only found in the brain, but also in other tissues, including the myocardium. Human clinical trials with native ghrelin in cardiac cachexia demonstrated increases in appetite, weight and cardiac output. METHODS: Human ghrelin or one of two analogues BIM-28125 and BIM-28131 (also known as RM-131) were tested at 50 nmole/kg/d and 500 nmole/kg/d versus placebo in a rat model of heart failure (myocardial infarction). Animals (SD-rats, approx. 225 g at surgery) received diuretics from day 14 and compounds from day 28 for 4 weeks using osmotic pumps. Weight was monitored and body composition analysed (NMR-scanning). Cardiac function was assessed by echocardiography and hemodynamics. RESULTS: Animals with MI gained less weight compared to sham rats until start of the therapy (311 g vs 324 g, p = 0.0129). Animals treated with BIM-28131 at 50 nmole/kg/d or all compounds at 500 nmole/kg/d displayed stronger weight gain compared to placebo and sham (all p<0.001). Before treatment, body composition was similar in all groups (average: 36 g fat, 248 g lean). Placebo-treated rats gained no fat, but only lean mass. The active compounds induced both fat and lean mass gain, but to a different extent. The fat-to-muscle-ratio of tissue gain was 0.9±0.07 for BIM-28131 at 50 nmole/kg/d, whereas at 500 nmole/kg/d it was 0.76±0.07 for BIM-28131, 0.68±0.12 for BIM-28125, and 0.48±0.05 for ghrelin. MuRF-1 and MAFbx were differentially regulated by treatment. CONCLUSION: Ghrelin is a very promising treatment option for cardiac cachexia, with the analogue BIM-28131 (RM-131) being the most effective compound.


Subject(s)
Disease Models, Animal , Ghrelin/analogs & derivatives , Ghrelin/pharmacology , Heart Failure/metabolism , Muscle Proteins/metabolism , SKP Cullin F-Box Protein Ligases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Blotting, Western , Body Weight/drug effects , Echocardiography , Heart Failure/drug therapy , Hemodynamics , Humans , Injections, Subcutaneous , Male , Rats , Rats, Sprague-Dawley , Tripartite Motif Proteins , Weight Gain/drug effects
12.
J Endocrinol ; 207(2): 177-83, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20696697

ABSTRACT

Human melanocortin 4 receptor (hMC4R) mutations with in vitro functional effects are responsible for 0.5-2.5% of severe obesity. Designing ligands that are able to counteract this in vitro-associated molecular defect is crucial to develop specific anti-obesity drugs in these genetically associated cases. We analyzed the in vitro effect of two novel melanocortin agonists, IRC-022493 and IRC-022511, on typical hMC4R mutations chosen based on the nature of their functional alterations, i.e. intracytoplasmic retention and/or reduced basal activity and/or reduced α-MSH potency. We assessed the in vitro ability of IRC-022493 and IRC-022511 to bind and activate hMC4R mutants. These mutations were found earlier in 11 obese French patients (median age (range) was 17.6 years (5.7-48.0) and body mass index (BMI)-Z-score 4.2 s.d. (1.5-5.5). The MC4R agonists were responsible for a significant activation of mutated hMC4R depending on the functional characteristics of the mutations. Both agonists were able to activate mutated hMC4R with decreased α-MSH potency, associated with or without decreased basal activity, to the same extent than α-MSH in wild-type MC4R. This result suggests that those mutations would be the best targets for the MC4R agonists among MC4R mutation-bearing obese patients. No specific clinical phenotype was associated with the differential response to pharmacological agonists. We identified two novel melanocortin agonists that were able in vitro to efficiently activate mutated hMC4R with impaired endogenous agonist functional response. These results stimulate interest in the development of these drugs for hMC4R mutations-associated obesity.


Subject(s)
Melanocortins/pharmacology , Obesity/genetics , Receptor, Melanocortin, Type 4/agonists , Receptor, Melanocortin, Type 4/genetics , Adolescent , Adult , Amino Acid Sequence , Animals , CHO Cells , Child , Child, Preschool , Cloning, Molecular , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Drug Design , Humans , Middle Aged , Molecular Sequence Data , Mutation , Obesity/drug therapy , Obesity/metabolism , Protein Binding , Receptor, Melanocortin, Type 4/metabolism , Young Adult
13.
Endocrinology ; 151(6): 2474-82, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20382695

ABSTRACT

Taspoglutide is a novel analog of human glucagon-like peptide-1 [hGLP-1(7-36)NH2] in clinical development for the treatment of type 2 diabetes. Taspoglutide contains alpha-aminoisobutyric acid substitutions replacing Ala(8) and Gly(35) of hGLP-1(7-36)NH2. The binding affinity [radioligand binding assay using [(125)I]hGLP-1(7-36)NH2], potency (cAMP production in CHO cells stably overexpressing hGLP-1 receptor), and in vitro plasma stability of taspoglutide compared with hGLP-1(7-36)NH2 have been evaluated. Effects on basal and glucose-stimulated insulin secretion were determined in vitro in INS-1E cells and in vivo in normal rats. Taspoglutide has comparable affinity (affinity constant 1.1 +/- 0.2 nm) to the natural ligand (affinity constant 1.5 +/- 0.3 nm) for the hGLP-1 receptor and exhibits comparable potency in stimulating cAMP production (EC(50) Taspo 0.06 nm and EC(50) hGLP-1(7-36)NH2 0.08 nm). Taspoglutide exerts insulinotropic action in vitro and in vivo and retains the glucoincretin property of hGLP-1(7-36)NH2. Stimulation of insulin secretion is concentration dependent and evident in the presence of high-glucose concentrations (16.7 mm) with a taspoglutide concentration as low as 0.001 nm. Taspoglutide is fully resistant to dipeptidyl peptidase-4 cleavage (during 1 h incubation at room temperature with purified enzyme) and has an extended in vitro plasma half-life relative to hGLP-1(7-36)NH2 (9.8 h vs. 50 min). In vitro, taspoglutide does not inhibit dipeptidyl peptidase-4 activity. This study provides the biochemical and pharmacological basis for the sustained plasma drug levels and prolonged therapeutic activity seen in early clinical trials of taspoglutide. Excellent stability and potency with substantial glucoincretin effects position taspoglutide as a promising new agent for treatment of type 2 diabetes.


Subject(s)
Glucagon-Like Peptide 1/analogs & derivatives , Peptides/pharmacology , Animals , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Dipeptidyl Peptidase 4/metabolism , Drug Stability , Enzyme-Linked Immunosorbent Assay , Glucagon-Like Peptide-1 Receptor , Humans , Insulin/metabolism , Peptides/blood , Peptides/chemistry , Peptides/pharmacokinetics , Protein Binding , Rats , Rats, Sprague-Dawley , Receptors, Glucagon/metabolism
14.
Peptides ; 30(10): 1892-900, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19646498

ABSTRACT

Melanocortin receptor agonists act in the brain to regulate food intake and body weight and, independently of these actions, affect insulin sensitivity. These experiments investigated the function of novel non-selective melanocortin receptor agonists (BIM-22493, BIM-22511) that cross the blood-brain barrier when administered peripherally. Treatment of diet induced obese C57BL/6J (B6) mice with melanocortin agonists administered peripherally improved obesity, hyperinsulinemia (approximately 50%) and fatty liver disease. Specificity of function was determined using B6 melanocortin-3 and melanocortin-4 receptor knockout mice (MC3RKO, MC4RKO). Chow fed MC4RKO but not MC3RKO used for these tests exhibited obesity, hyperinsulinemia and severe hepatosteatosis associated with increased expression of insulin-stimulated genes involved in lipogenesis. Reduced food intake associated with acute BIM-22493 treatment, and weight loss associated with 14 days of treatment with BIM-22511, required functional MC4R but not MC3R. However, while 14 days of treatment with BIM-22511 did not affect body weight and even increased cumulative food intake in MC4RKO, a significant reduction (approximately 50%) in fasting insulin was still observed. Despite lowering insulin, chronic treatment with BIM-22511 did not improve hepatosteatosis in MC4RKO, and did not affect hepatic lipogenic gene expression. Together, these results demonstrate that peripherally administered melanocortin receptor agonists regulate body weight, liver metabolism and glucose homeostasis through independent pathways. MC4R are necessary for melanocortin agonist-induced weight loss and improvements in liver metabolism, but are not required for improvements in hyperinsulinemia. Agonists with activity at MC4R improve glucose homeostasis at least partially by causing weight loss, however other melanocortin receptors may have potential for treating aberrations in glucose homeostasis associated with obesity.


Subject(s)
Receptor, Melanocortin, Type 3/agonists , Receptor, Melanocortin, Type 3/genetics , Receptor, Melanocortin, Type 4/agonists , Receptor, Melanocortin, Type 4/genetics , alpha-MSH/analogs & derivatives , Animals , Diet , Eating , Energy Metabolism , Female , Glucose/metabolism , Humans , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Obesity/metabolism , Receptor, Melanocortin, Type 3/metabolism , Receptor, Melanocortin, Type 4/metabolism , Weight Loss , alpha-MSH/pharmacology
17.
Int J Cardiol ; 137(3): 267-75, 2009 Nov 12.
Article in English | MEDLINE | ID: mdl-18723230

ABSTRACT

BACKGROUND: Ghrelin, was observed to have treatment-potential for severe chronic heart failure (CHF) and cardiac cachexia based on anti-cachectic and cardio-protective effects. METHODS: We performed two studies to assess the effects of human ghrelin on food intake, body weight and body composition, as well as heart function in a rat model of CHF. Study-1 (50 or 500 nmole/kg/d ghrelin by pump infusion) was focused on food intake and body composition, study-2 (50 or 100 nmole/kg/d ghrelin by subcutaneous injection (3-times daily) was focused on heart function due to a lack of cardiac effects observed in study-1. In both studies, myocardial infarction was induced by LAD ligation. On day 28 after surgery, rats were randomized and treated with ghrelin or placebo for 4 weeks. Food intake (study-1), body composition (NMR) cardiac function (echocardiography and invasive hemodynamics (study-2 only) were assessed. RESULTS: In study-1, CHF rats treated with high dose ghrelin showed an increase in body weight (+25%, p<0.001), lean mass (+16%, p<0.01) and fat mass (+17%, p=0.001) vs placebo. In study-2, CHF rats treated with both low- and high dose ghrelin showed an increase in body weight (both +18%, p

Subject(s)
Body Composition/drug effects , Eating , Ghrelin/pharmacology , Analysis of Variance , Animals , Body Weight/drug effects , Disease Models, Animal , Echocardiography , Ghrelin/administration & dosage , Heart Failure/drug therapy , Heart Failure/physiopathology , Hemodynamics , Humans , Injections, Subcutaneous , Male , Random Allocation , Rats , Rats, Sprague-Dawley
18.
Cell Metab ; 8(6): 468-81, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19041763

ABSTRACT

Obesity and nutrient homeostasis are linked by mechanisms that are not fully elucidated. Here we describe a secreted protein, adropin, encoded by a gene, Energy Homeostasis Associated (Enho), expressed in liver and brain. Liver Enho expression is regulated by nutrition: lean C57BL/6J mice fed high-fat diet (HFD) exhibited a rapid increase, while fasting reduced expression compared to controls. However, liver Enho expression declines with diet-induced obesity (DIO) associated with 3 months of HFD or with genetically induced obesity, suggesting an association with metabolic disorders in the obese state. In DIO mice, transgenic overexpression or systemic adropin treatment attenuated hepatosteatosis and insulin resistance independently of effects on adiposity or food intake. Adropin regulated expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma, a major regulator of lipogenesis. Adropin may therefore be a factor governing glucose and lipid homeostasis, which protects against hepatosteatosis and hyperinsulinemia associated with obesity.


Subject(s)
Blood Proteins/physiology , Energy Metabolism , Lipid Metabolism , Proteins/physiology , Adipose Tissue, Brown/metabolism , Adipose Tissue, White/metabolism , Amino Acid Sequence , Animals , Base Sequence , Benzoates/chemistry , Benzoates/metabolism , Benzylamines/chemistry , Benzylamines/metabolism , Blood Proteins/genetics , Blood Proteins/metabolism , Cells, Cultured , DNA-Binding Proteins/agonists , DNA-Binding Proteins/metabolism , Fasting , Fatty Liver/metabolism , Female , Humans , Intercellular Signaling Peptides and Proteins , Leptin/metabolism , Liver X Receptors , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Obesity/genetics , Obesity/metabolism , Orphan Nuclear Receptors , Peptides , Proteins/genetics , Proteins/metabolism , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism
19.
Endocr Relat Cancer ; 15(2): 583-96, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18509006

ABSTRACT

Dopamine D2 and somatostatin receptors (sstrs) were reported to affect non-functioning pituitary adenoma (NFPA) proliferation in vitro. However, the reported results differ according to the experimental conditions used. We established an experimental protocol allowing reproducible evaluation of NFPA cell proliferation in vitro, to test and compare the antiproliferative effects of dopamine and somatostatin analogs (alone or in combination) with the activity of the dopamine-somatostatin chimeric molecule BIM-23A760. The protocol was utilized by four independent laboratories, studying 38 fibroblast-deprived NFPA cell cultures. Cells were characterized for GH, POMC, sstr1-sstr5, total dopamine D2 receptor (D2R) (in all cases), and D2 receptor long and short isoforms (in 15 out of 38 cases) mRNA expression and for alpha-subunit, LH, and FSH release. D2R, sstr3, and sstr2 mRNAs were consistently observed, with the dominant expression of D2R (2.9+/-2.6 copy/copy beta-glucuronidase; mean+/-s.e.m.), when compared with sstr3 and sstr2 (0.6+/-1.0 and 0.3+/-0.6 respectively). BIM-23A760, a molecule with high affinity for D2R and sstr2, significantly inhibited [3H]thymidine incorporation in 23 out of 38 (60%) NFPA cultures (EC50=1.2 pM and Emax=-33.6+/-3.7%). BIM-23A760 effects were similar to those induced by the selective D2R agonist cabergoline that showed a statistically significant inhibition in 18 out of 27 tumors (compared with a significant inhibition obtained in 17 out of 27 tumors using BIM-23A760, in the same subgroup of adenomas analyzed), while octreotide was effective in 13 out of 27 cases. In conclusion, superimposable data generated in four independent laboratories using a standardized protocol demonstrate that, in vitro, chimeric dopamine/sstr agonists are effective in inhibiting cell proliferation in two-thirds of NFPAs.


Subject(s)
Adenoma/drug therapy , Adenoma/pathology , Dopamine/analogs & derivatives , Pituitary Neoplasms/drug therapy , Pituitary Neoplasms/pathology , Somatostatin/analogs & derivatives , Adult , Aged , Antineoplastic Agents, Hormonal/pharmacology , Cabergoline , Cell Division/drug effects , Dopamine/pharmacology , Dopamine Antagonists/pharmacology , Dose-Response Relationship, Drug , Ergolines/pharmacology , Female , Fibroblasts/cytology , Humans , Male , Middle Aged , Octreotide/pharmacology , RNA, Messenger/metabolism , Receptors, Dopamine D2/genetics , Receptors, Somatostatin/genetics , Somatostatin/pharmacology , Sulpiride/pharmacology , Thymidine/metabolism , Tritium , Tumor Cells, Cultured
20.
Cell Metab ; 7(5): 389-99, 2008 May.
Article in English | MEDLINE | ID: mdl-18460330

ABSTRACT

Current evidence suggests that hypothalamic fatty acid metabolism may play a role in regulating food intake; however, confirmation that it is a physiologically relevant regulatory system of feeding is still incomplete. Here, we use pharmacological and genetic approaches to demonstrate that the physiological orexigenic response to ghrelin involves specific inhibition of fatty acid biosynthesis induced by AMP-activated protein kinase (AMPK) resulting in decreased hypothalamic levels of malonyl-CoA and increased carnitine palmitoyltransferase 1 (CPT1) activity. In addition, we also demonstrate that fasting downregulates fatty acid synthase (FAS) in a region-specific manner and that this effect is mediated by an AMPK and ghrelin-dependent mechanisms. Thus, decreasing AMPK activity in the ventromedial nucleus of the hypothalamus (VMH) is sufficient to inhibit ghrelin's effects on FAS expression and feeding. Overall, our results indicate that modulation of hypothalamic fatty acid metabolism specifically in the VMH in response to ghrelin is a physiological mechanism that controls feeding.


Subject(s)
Fatty Acids/metabolism , Ghrelin/physiology , Hypothalamus/metabolism , AMP-Activated Protein Kinase Kinases , Animals , Blotting, Western , Carnitine O-Palmitoyltransferase/metabolism , Fasting/physiology , Fatty Acid Synthases/antagonists & inhibitors , Fatty Acid Synthases/metabolism , Feeding Behavior , Hypothalamus/pathology , In Situ Hybridization , Leptin/metabolism , Male , Malonyl Coenzyme A/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Obese , Phosphorylation , Protein Kinases/metabolism , Rats , Rats, Sprague-Dawley , fas Receptor
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