RESUMO
Neuroactive steroids reduce mortality, decrease edema, and improve functional outcomes in preclinical and clinical traumatic brain injury (TBI) studies. In this study, we tested the efficacy of two related novel neuroactive steroids, NTS-104 and NTS-105, in a rat model of TBI. NTS-104 is a water-soluble prodrug of NTS-105, a partial progesterone receptor agonist. To investigate the effects of NTS-104 on TBI recovery, adult male Sprague Dawley rats received moderate parasagittal fluid-percussion injury or sham surgery and were treated with vehicle or NTS-104 (10 âmg/kg, intramuscularly) at 4, 10, 24, and 48 âh post-TBI. The therapeutic time window was also assessed using the neuroactive steroid NTS-105 (3 âmg/kg, intramuscularly). Edema in the parietal cortex and hippocampus, measured at 3 days post-injury (DPI), was reduced by NTS-104 and NTS-105. NTS-105 was effective in reducing edema when given at 4, 10, or 24 âh post-injury. Sensorimotor deficits in the cylinder test at 3 DPI were ameliorated by NTS-104 and NTS-105 treatment. Cognitive recovery, assessed with cue and contextual fear conditioning and retention of the water maze task assessed subacutely 1-3 weeks post-injury, also improved with NTS-104 treatment. Cortical and hippocampal atrophy at 22 DPI did not improve, indicating that NTS-104/NTS-105 may promote post-TBI cognitive recovery by controlling edema and other processes. These results demonstrate that NTS-104/NTS-105 is a promising therapeutic approach to improve motor and cognitive recovery after moderate TBI.
RESUMO
Traumatic brain injury (TBI) is a major cause of hospitalization and death. To mitigate these human costs, the search for effective drugs to treat TBI continues. In the current study, we evaluated the efficacy of the novel neurosteroid, NTS-105, to reduce post-traumatic pathobiology in an in vitro model of moderate TBI that utilizes an organotypic hippocampal slice culture. NTS-105 inhibited activation of the androgen receptor and the mineralocorticoid receptor, partially activated the progesterone B receptor and was not active at the glucocorticoid receptor. Treatment with NTS-105 starting one hour after injury decreased post-traumatic cell death in a dose-dependent manner, with 10 nM NTS-105 being most effective. Post-traumatic administration of 10 nM NTS-105 also prevented deficits in long-term potentiation (LTP) without adversely affecting neuronal activity in naïve cultures. We propose that the high potency pleiotropic action of NTS-105 beneficial effects at multiple receptors (e.g. androgen, mineralocorticoid and progesterone) provides significant mechanistic advantages over native neurosteroids such as progesterone, which lacked clinical success for the treatment of TBI. Our results suggest that this pleiotropic pharmacology may be a promising strategy for the effective treatment of TBI, and future studies should test its efficacy in pre-clinical animal models of TBI.
Assuntos
Lesões Encefálicas Traumáticas , Potenciação de Longa Duração , Animais , Humanos , Progesterona/farmacologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Neurônios/metabolismo , Morte Celular , Hipocampo/metabolismo , Modelos Animais de DoençasRESUMO
Traumatic brain injury (TBI) triggers wide-ranging pathology that impacts multiple biochemical and physiological systems, both inside and outside the brain. Functional recovery in patients is impeded by early onset brain edema, acute and chronic inflammation, delayed cell death, and neurovascular disruption. Drug treatments that target these deficits are under active development, but it seems likely that fully effective therapy may require interruption of the multiplicity of TBI-induced pathological processes either by a cocktail of drug treatments or a single pleiotropic drug. The complex and highly interconnected biochemical network embodied by the neurosteroid system offers multiple options for the research and development of pleiotropic drug treatments that may provide benefit for those who have suffered a TBI. This narrative review examines the neurosteroids and their signaling systems and proposes directions for their utility in the next stage of TBI drug research and development.
Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Neuroesteroides , Humanos , Neuroesteroides/metabolismo , Neuroesteroides/farmacologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas/patologiaRESUMO
BACKGROUND: Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke. METHODS: The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly. RESULTS: Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1ß, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1. CONCLUSIONS: NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Fármacos Neuroprotetores , Neuroesteroides , Pró-Fármacos , Acidente Vascular Cerebral , Animais , Ratos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Molécula 1 de Adesão Intercelular/uso terapêutico , Galectina 1/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Interleucina-6 , Proteínas Proto-Oncogênicas c-akt , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Modelos Animais de Doenças , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
Multiple sclerosis (MS) is a chronic, inflammatory demyelinating disease of the CNS. In addition to motor, sensory and visual deficits, MS is also characterized by hippocampal demyelination and memory impairment. We recently demonstrated that a recombinant human-derived monoclonal IgM antibody, which is designated rHIgM22 and currently in clinical development for people with MS, accelerates remyelination of the corpus callosum in the brains of cuprizone-treated mice. Here, we investigated the effects of rHIgM22 in the hippocampus and on hippocampal-dependent learning and memory in the same mouse model of cuprizone-induced demyelination and spontaneous remyelination. The degree of hippocampal myelination of cuprizone-fed mice treated with a single dose of rHIgM22 (10â¯mg/kg of body weight) was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. Four or five mice per treatment group were examined for each time point. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated hippocampus. Using two additional cohorts of mice and eight animals per treatment group for each cohort, we also demonstrate that the enhancing effects of rHIgM22 on hippocampal remyelination were accompanied by improved performance in the Morris water maze and amelioration of the memory deficits induced by cuprizone. These results further confirm the remyelination-promoting capabilities of rHIgM22 and support additional investigation of its therapeutic potential in MS.
Assuntos
Anticorpos/farmacologia , Cuprizona/farmacologia , Bainha de Mielina/efeitos dos fármacos , Remielinização/efeitos dos fármacos , Animais , Corpo Caloso/efeitos dos fármacos , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Oligodendroglia/efeitos dos fármacosRESUMO
GGF2 is a recombinant human neuregulin-1ß in development for chronic heart failure. Phase 1 clinical trials of GGF2 were put on hold when transient elevations in serum aminotransferases and total bilirubin were observed in 2 of 43 subjects who received single doses of GGF2 at 1.5 or 0.378 mg/kg. However, aminotransferase elevations were modest and not typical of liver injury sufficient to result in elevated serum bilirubin. Cynomolgus monkeys administered a single 15 mg/kg dose of GGF2 had similar transient elevations in serum aminotransferases and bilirubin as well as transient elevations in serum bile acids. However, no hepatocellular necrosis was observed in liver biopsies obtained during peak elevations. When sandwich-cultured human hepatocytes were treated with GGF2 for up to 72 h at concentrations approximately 0.8-fold average plasma Cmax for the 0.378 mg/kg dose, no cytotoxicity was observed. Gene expression profiling identified approximately 50% reductions in mRNAs coding for bilirubin transporters and bile acid conjugating enzymes, as well as changes in expression of additional genes mimicking the interleukin-6-mediated acute phase response. Similar gene expression changes were observed in GGF2-treated HepG2 cells and primary monkey hepatocytes. Additional studies conducted in sandwich-cultured human hepatocytes revealed a transient and GGF2 concentration-dependent decrease in hepatocyte bile acid content and biliary clearance of taurocholate without affecting biliary taurocholate efflux. Taken together, these data suggest that GGF2 does not cause significant hepatocellular death, but transiently modifies hepatic handling of bilirubin and bile acids, effects that may account for the elevations in serum bilirubin observed in the clinical trial subjects.
Assuntos
Ácidos e Sais Biliares/sangue , Ductos Biliares/efeitos dos fármacos , Bilirrubina/sangue , Hepatócitos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Neuregulina-1/efeitos adversos , Animais , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Ensaios Clínicos Fase I como Assunto , Citocromo P-450 CYP3A/genética , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Fígado/metabolismo , Fígado/patologia , Macaca fascicularis , Masculino , Cultura Primária de Células , Toxicogenética , Transcriptoma/efeitos dos fármacosRESUMO
Neuregulin-1ß is a member of the neuregulin family of growth factors and is critically important for normal development and functioning of the heart and brain. A recombinant version of neuregulin-1ß, cimaglermin alfa (also known as glial growth factor 2 or GGF2) is being investigated as a possible therapy for heart failure. Previous studies suggest that neuregulin-1ß stimulation of skeletal muscle increases glucose uptake and, specifically, sufficient doses of cimaglermin alfa acutely produce hypoglycemia in pigs. Since acute hypoglycemia could be a safety concern, blood glucose changes in the above pig study were further investigated. In addition, basal glucose and glucose disposal were investigated in mice. Finally, as part of standard clinical chemistry profiling in a single ascending-dose human safety study, blood glucose levels were evaluated in patients with heart failure after cimaglermin alfa treatment. A single intravenous injection of cimaglermin alfa at doses of 0.8mg/kg and 2.6mg/kg in mice resulted in a transient reduction of blood glucose concentrations of approximately 20% and 34%, respectively, at 2h after the treatment compared to pre-treatment levels. Similar results were observed in diabetic mice. Treatment with cimaglermin alfa also increased blood glucose disposal following oral challenge in mice. However, no significant alterations in blood glucose concentrations were found in human heart failure patients at 0.5 and 2h after treatment with cimaglermin alfa over an equivalent human dose range, based on body surface area. Taken together, these data indicate strong species differences in blood glucose handling after cimaglermin alfa treatment, and particularly do not indicate that this phenomenon should affect human subjects.
Assuntos
Glicemia/metabolismo , Insuficiência Cardíaca/sangue , Neuregulina-1/farmacologia , Adolescente , Adulto , Idoso , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Relação Dose-Resposta a Droga , Feminino , Humanos , Insulina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Modelos Animais , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Especificidade da Espécie , Suínos , Adulto JovemRESUMO
Failure of oligodendrocyte precursor cells (OPCs) to differentiate and remyelinate axons is thought to be a major cause of the limited ability of the central nervous system to repair plaques of immune-mediated demyelination in multiple sclerosis (MS). Current therapies for MS aim to lessen the immune response in order to reduce the frequency and severity of attacks, but these existing therapies do not target remyelination or stimulate repair of the damaged tissue. Thus, the promotion of OPC differentiation and remyelination is potentially an important therapeutic goal. Previous studies have shown that a recombinant human-derived monoclonal IgM antibody, designated rHIgM22, promotes remyelination, particularly of the spinal cord in rodent models of demyelination. Here, we examined the effects of rHIgM22 in remyelination in the brain using the mouse model of cuprizone-induced demyelination, which is characterized by spontaneous remyelination. The myelination state of the corpus callosum of cuprizone-fed mice treated with rHIgM22 was examined immediately after the end of the cuprizone diet as well as at different time points during the recovery period with regular food, and compared with that of cuprizone-fed animals treated with either vehicle or human IgM isotype control antibody. Mice fed only regular food were used as controls. We demonstrate that treatment with rHIgM22 accelerated remyelination of the demyelinated corpus callosum. The remyelination-enhancing effects of rHIgM22 were found across different, anatomically distinct regions of the corpus callosum, and followed a spatiotemporal pattern that was similar to that of the spontaneous remyelination process. These enhancing effects were also accompanied by increased differentiation of OPCs into mature oligodendrocytes. Our data indicate strong remyelination-promoting capabilities of rHIgM22 and further support its therapeutic potential in MS.
Assuntos
Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Imunoglobulina M/uso terapêutico , Inibidores da Monoaminoxidase/toxicidade , Remielinização/efeitos dos fármacos , Análise de Variância , Animais , Diferenciação Celular/efeitos dos fármacos , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Doenças Desmielinizantes/tratamento farmacológico , Modelos Animais de Doenças , Imunoglobulina M/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Básica da Mielina/imunologia , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Fator de Transcrição 2 de Oligodendrócitos/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/metabolismo , Nervo Óptico/patologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de TempoRESUMO
BACKGROUND: 4-Aminopyridine (4-AP) is a Food and Drug Administration-approved drug to improve motor function in people with multiple sclerosis. Preliminary results suggest the drug may act on intact neural circuits and not just on demyelinated ones. OBJECTIVE: To determine if 4-AP at clinically relevant levels alters the excitability of intact motor circuits. METHODS: In anesthetized rats, electrodes were placed over motor cortex and the dorsal cervical spinal cord for electrical stimulation, and electromyogram electrodes were inserted into biceps muscle to measure responses. The motor responses to brain and spinal cord stimulation were measured before and for 5 hours after 4-AP administration both in uninjured rats and rats with a cut lesion of the pyramidal tract. Blood was collected at the same time as electrophysiology to determine drug plasma concentration with a goal of 20 to 100 ng/mL. RESULTS: We first determined that a bolus infusion of 0.32 mg/kg 4-AP was optimal: it produced on average 61.5 ± 1.8 ng/mL over the 5 hours after infusion. This dose of 4-AP increased responses to spinal cord stimulation by 1.3-fold in uninjured rats and 3-fold in rats with pyramidal tract lesion. Responses to cortical stimulation also increased by 2-fold in uninjured rats and up to 4-fold in the injured. CONCLUSION: Clinically relevant levels of 4-AP strongly augment physiological responses in intact circuits, an effect that was more robust after partial injury, demonstrating its broad potential in treating central nervous system injuries.
Assuntos
4-Aminopiridina/farmacologia , Fármacos do Sistema Nervoso Central/farmacologia , Medula Cervical/efeitos dos fármacos , Córtex Motor/efeitos dos fármacos , Tratos Piramidais/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , 4-Aminopiridina/sangue , 4-Aminopiridina/farmacocinética , Animais , Fármacos do Sistema Nervoso Central/sangue , Fármacos do Sistema Nervoso Central/farmacocinética , Medula Cervical/lesões , Medula Cervical/fisiologia , Medula Cervical/fisiopatologia , Avaliação Pré-Clínica de Medicamentos , Estimulação Elétrica , Eletromiografia , Potencial Evocado Motor/efeitos dos fármacos , Potencial Evocado Motor/fisiologia , Feminino , Membro Anterior/efeitos dos fármacos , Membro Anterior/fisiologia , Membro Anterior/fisiopatologia , Microeletrodos , Córtex Motor/fisiologia , Córtex Motor/fisiopatologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , Músculo Esquelético/fisiopatologia , Tratos Piramidais/lesões , Tratos Piramidais/fisiologia , Tratos Piramidais/fisiopatologia , Distribuição Aleatória , Ratos Sprague-Dawley , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Neuregulins are important growth factors involved in cardiac development and response to stress. Certain isoforms and fragments of neuregulin have been found to be cardioprotective. The effects of a full-length neuregulin-1ß isoform, glial growth factor 2 (GGF2; USAN/INN; also called cimaglermin) were investigated in vitro. Various dosing regimens were then evaluated for their effects on left ventricular (LV) function in rats with surgically-induced myocardial infarction. In vitro, GGF2 bound with high affinity to erythroblastic leukemia viral oncogene (ErbB) 4 receptors, potently promoted Akt phosphorylation, as well as reduced cell death following doxorubicin exposure in HL1 cells. Daily GGF2 treatment beginning 7-14 days after left anterior descending coronary artery ligation produced improvements in LV ejection fraction and other measures of LV function and morphology. The improvements in LV function (e.g. 10% point increase in absolute LV ejection fraction) with GGF2 were dose-dependent. LV performance was substantially improved when GGF2 treatment was delivered infrequently, despite a serum half-life of less than 2h and could be maintained for more than 10 months with treatment once weekly or once every 2 weeks. These studies confirm previous findings that GGF2 may improve contractile performance in the failing rat heart and that infrequent exposure to GGF2 may improve LV function and impact remodeling in the failing myocardium. GGF2 is now being developed for the treatment of heart failure in humans.
Assuntos
Ventrículos do Coração/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Neuregulina-1/farmacologia , Disfunção Ventricular/tratamento farmacológico , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Citoproteção/efeitos dos fármacos , Doxorrubicina/efeitos adversos , Esquema de Medicação , Insuficiência Cardíaca/complicações , Humanos , Camundongos , Infarto do Miocárdio/complicações , Neuregulina-1/administração & dosagem , Neuregulina-1/química , Neuregulina-1/metabolismo , Ratos , Receptor ErbB-4/metabolismoRESUMO
BACKGROUND: Peripheral nerve injury (PNI) can result in neurodegenerative changes leading to motor, sensory and autonomic dysfunction. Injury to the rat sciatic nerve is used to model pathophysiologic processes following PNI and assess the efficacy of therapeutic interventions. Frequently, temporal changes in the sciatic functional index (SFI), a measure of sensorimotor integration are measured in rats to assess functional recovery following sciatic nerve injury. However, multiple rat strains and behavioral endpoints have been employed to investigate pathophysiology of PNI and impact of therapeutic intervention on recovery, raising the possibility that rat strain may influence the outcome of such studies. NEW METHOD: The temporal course of recovery from sham, sciatic nerve crush or transection injury was assessed using SFI determined by two methods (footprint and DigiGait), and proprioceptive hind limb placement (a measure of proprioceptive integrity) of the sciatic nerve innervation, in male Sprague Dawley, Lewis, Fischer, Wistar and Long Evans rats. RESULTS: The SFI profile, as assessed by both inked footprint analysis and DigiGait, following sciatic nerve injury was remarkably conserved across strains. Dramatic strain-related differences were observed in the latency to place the crush- or transection-injured hind limb following proprioceptive hind limb stimulation. COMPARISON WITH EXISTING METHOD: The novelty of this study is the parallel comparison of multiple strains using existing and novel tests. CONCLUSION: These results suggest that some sensorimotor function tests may be sensitive to the choice of strain, as evidenced by the differences between SFI and proprioceptive function outcomes.
Assuntos
Traumatismos dos Nervos Periféricos/fisiopatologia , Ratos/fisiologia , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/lesões , Animais , Marcha/fisiologia , Membro Posterior/fisiopatologia , Masculino , Atividade Motora/fisiologia , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Tamanho do Órgão , Traumatismos dos Nervos Periféricos/patologia , Propriocepção/fisiologia , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologia , Especificidade da EspécieRESUMO
Magnesium (Mg2+) homeostasis is impaired following spinal cord injury (SCI) and the loss of extracellular Mg2+ contributes to secondary injury by various mechanisms, including glutamate neurotoxicity. The neuroprotective effects of high dose Mg2+ supplementation have been reported in many animal models. Recent studies found that lower Mg2+ doses also improved neurologic outcomes when Mg2+ was formulated with polyethylene glycol (PEG), suggesting that a PEG/ Mg2+ formulation might increase Mg2+ delivery to the injured spinal cord, compared with that of MgSO4 alone. Here, we assessed spinal extracellular Mg2+ and glutamate levels following SCI in rats using microdialysis. Basal levels of extracellular Mg2+ (â¼0.5 mM) were significantly reduced to 0.15 mM in the core and 0.12 mM in the rostral peri-lesion area after SCI. A single intravenous infusion of saline or of MgSO4 at 192 µmoL/kg did not significantly change extracellular Mg2+ concentrations. However, a single infusion of AC105 (a MgCl2 in PEG) at an equimolar Mg2+ dose significantly increased the Mg2+ concentration to 0.3 mM (core area) and 0.25 mM (rostral peri-lesion area). Moreover, multiple AC105 treatments completely restored the depleted extracellular Mg2+ concentrations after SCI to levels in the uninjured spinal cord. Repeated MgSO4 infusions slightly increased the Mg2+ concentrations while saline infusion had no effect. In addition, AC105 treatment significantly reduced extracellular glutamate levels in the lesion center after SCI. These results indicate that intravenous infusion of PEG-formulated Mg2+ normalized the Mg2+ homeostasis following SCI and reduced potentially neurotoxic glutamate levels, consistent with a neuroprotective mechanism of blocking excitotoxicity.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Líquido Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Sulfato de Magnésio/administração & dosagem , Polietilenoglicóis/administração & dosagem , Traumatismos da Medula Espinal/metabolismo , Animais , Agonistas de Aminoácidos Excitatórios , Líquido Extracelular/efeitos dos fármacos , Feminino , Infusões Intravenosas , Sulfato de Magnésio/metabolismo , Microdiálise/métodos , Polietilenoglicóis/metabolismo , Ratos , Ratos Long-Evans , Traumatismos da Medula Espinal/tratamento farmacológico , Vértebras TorácicasRESUMO
Cimaglermin (neuregulin 1ß3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb- and hindlimb-placing scores (proprioceptive behavioral tests), body-swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin-mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose-frequency study used the dose-ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose- and frequency-dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth-associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Infarto da Artéria Cerebral Média/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neuregulina-1/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Análise de Variância , Animais , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/etiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Neuregulin-1ß (NRG-1ß) is a growth factor critical for cardiac development and repair with therapeutic potential for heart failure. We previously showed that the glial growth factor 2 (GGF2) isoform of NRG-1ß improves cardiac function in rodents after myocardial infarction (MI), but its efficacy in a large animal model of cardiac injury has not been examined. We therefore sought to examine the effects of GGF2 on ventricular remodeling, cardiac function, and global transcription in post-MI swine, as well as potential mechanisms for anti-remodeling effects. METHODS AND RESULTS: MI was induced in anesthetized swine (n=23) by intracoronary balloon occlusion. At 1 week post-MI, survivors (n=13) received GGF2 treatment (intravenous, biweekly for 4 weeks; n=8) or were untreated (n=5). At 5 weeks post-MI, fractional shortening was higher (32.8% versus 25.3%, P=0.019), and left ventricular (LV) end-diastolic dimension lower (4.5 versus 5.3 cm, P=0.003) in GGF2-treated animals. Treatment altered expression of 528 genes, as measured by microarrays, including collagens, basal lamina components, and matricellular proteins. GGF2-treated pigs exhibited improvements in LV cardiomyocyte mitochondria and intercalated disk structures and showed less fibrosis, altered matrix structure, and fewer myofibroblasts (myoFbs), based on trichrome staining, electron microscopy, and immunostaining. In vitro experiments with isolated murine and rat cardiac fibroblasts demonstrate that NRG-1ß reduces myoFbs, and suppresses TGFß-induced phospho-SMAD3 as well as αSMA expression. CONCLUSIONS: These results suggest that GGF2/NRG-1ß prevents adverse remodeling after injury in part via anti-fibrotic effects in the heart.
Assuntos
Insuficiência Cardíaca/tratamento farmacológico , Miocárdio/patologia , Neuregulina-1/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Actinas/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fosforilação , Ratos Sprague-Dawley , Proteína Smad3/metabolismo , Suínos , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacos , Remodelação Ventricular/genéticaRESUMO
BACKGROUND AND PURPOSE: Stroke survivors often have permanent deficits that are only partially addressed by physical therapy. This study evaluated the effects of dalfampridine, a potassium channel blocker, on persistent sensorimotor deficits in rats with treatment initiated 4 or 8 weeks after stroke. METHODS: Rats underwent permanent middle cerebral artery occlusion. Sensorimotor function was measured using limb-placing and body-swing symmetry tests, which normally show a partial recovery from initial deficits that plateaus ≈4 weeks after permanent middle cerebral artery occlusion. Dalfampridine was administered starting at 4 or 8 weeks after permanent middle cerebral artery occlusion in 2 blinded, vehicle-controlled studies. Plasma samples were collected and brain tissue was processed for histologic assessment. RESULTS: Dalfampridine treatment (0.5-2.0 mg/kg) improved forelimb- and hindlimb-placing responses and body-swing symmetry in a reversible and dose-dependent manner. Plasma dalfampridine concentrations correlated with dose. Brain infarct volumes showed no differences between treatment groups. CONCLUSIONS: Dalfampridine improves sensorimotor function in the rat permanent middle cerebral artery occlusion model. Dalfampridine extended-release tablets (prolonged release fampridine outside the United States) are used to improve walking in patients with multiple sclerosis, and these preclinical data provide a strong rationale for examining the potential of dalfampridine to treat chronic stable deficits in stroke patients. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01605825.
Assuntos
4-Aminopiridina/uso terapêutico , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média/tratamento farmacológico , Bloqueadores dos Canais de Potássio/farmacologia , Desempenho Psicomotor/efeitos dos fármacos , 4-Aminopiridina/administração & dosagem , Animais , Estudos Cross-Over , Relação Dose-Resposta a Droga , Infarto da Artéria Cerebral Média/sangue , Infarto da Artéria Cerebral Média/fisiopatologia , Masculino , Bloqueadores dos Canais de Potássio/administração & dosagem , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
AIMS: Recombinant Neuregulin (NRG)-1ß has multiple beneficial effects on cardiac myocytes in culture, and has potential as a clinical therapy for heart failure (HF). A number of factors may influence the effect of NRG-1ß on cardiac function via ErbB receptor coupling and expression. We examined the effect of the NRG-1ß isoform, glial growth factor 2 (GGF2), in rats with myocardial infarction (MI) and determined the impact of high-fat diet as well as chronicity of disease on GGF2 induced improvement in left ventricular systolic function. Potential mechanisms for GGF2 effects on the remote myocardium were explored using microarray and proteomic analysis. METHODS AND RESULTS: Rats with MI were randomized to receive vehicle, 0.625 mg/kg, or 3.25 mg/kg GGF2 in the presence and absence of high-fat feeding beginning at day 7 post-MI and continuing for 4 weeks. Residual left ventricular (LV) function was improved in both of the GGF2 treatment groups compared with the vehicle treated MI group at 4 weeks of treatment as assessed by echocardiography. High-fat diet did not prevent the effects of high dose GGF2. In experiments where treatment was delayed until 8 weeks after MI, high but not low dose GGF2 treatment was associated with improved systolic function. mRNA and protein expression analysis of remote left ventricular tissue revealed a number of changes in myocardial gene and protein expression altered by MI that were normalized by GGF2 treatment, many of which are involved in energy production. CONCLUSIONS: This study demonstrates that in rats with MI induced systolic dysfunction, GGF2 treatment improves cardiac function. There are differences in sensitivity of the myocardium to GGF2 effects when administered early vs. late post-MI that may be important to consider in the development of GGF2 in humans.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Neuregulina-1/farmacologia , Neuregulina-1/uso terapêutico , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Dieta Hiperlipídica , Eletrocardiografia , Fibrose , Glucose/metabolismo , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Humanos , Injeções Intravenosas , Masculino , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/metabolismo , Miocárdio/patologia , Neuregulina-1/administração & dosagem , Neuregulina-1/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Tomografia por Emissão de Pósitrons , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptores Proteína Tirosina Quinases/metabolismo , Sobrevivência de Tecidos/efeitos dos fármacos , UltrassonografiaRESUMO
BACKGROUND: Dalfampridine (4-aminopyridine; 4-AP) is a potassium channel blocker that has been available in the United States as a treatment to improve walking in patients with multiple sclerosis. 4-AP is well-characterized in vitro with regard to inhibition of neuronal potassium channels, but the potential contribution of its metabolites to clinical activity has not been determined. This study evaluated the concentration-response of 4-AP and its two primary metabolites, 3-hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate, for inhibition of the potassium channels Kv 1.1, Kv 1.2, and Kv 1.4, which are considered candidates for mediating effects of 4-AP on action potential conduction because of their presence in axonal membranes. METHODS: Stable transfection of cDNA for Kv 1.1, Kv 1.2, and Kv 1.4 was performed into HEK293 cells, and colonies of cells containing each channel were selected and maintained under appropriate cell culture conditions. Electrophysiological measurements were performed using a patch-clamp technique in at least three cells for each concentration (50, 500, 5000, and 50,000 µM) of 4-AP and the two metabolites, with each cell acting as its own control. Concentration-response curves were constructed for 4-AP and each metabolite. Data were analyzed using nonlinear least-squares fit, and concentrations inhibiting the channels by 50% (IC50) were estimated. RESULTS: 4-AP induced similar concentration-dependent inhibition profiles of all three potassium channels, resulting in a narrow range of IC50 values across channels (242 µM to 399 µM). Across the three channels, the IC50 values of 3-hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate were 1-2 orders of magnitude higher (less potent) than those of 4-AP. CONCLUSIONS: 3-Hydroxy-4-aminopyridine and 3-hydroxy-4-aminopyridine sulfate demonstrated low in vitro potency for Kv 1.1, Kv 1.2, and Kv 1.4 inhibition, suggesting that these metabolites are unlikely to contribute to the positive pharmacodynamic effects of 4-AP. A limitation of this study is that while the metabolites were substantially less active at these representative potassium channels in vitro, the untested possibility exists that they may be active at one or more of the many other channel types that occur in vivo.
RESUMO
BACKGROUND: Dalfampridine (4-aminopyridine; 4-AP) is a potassium channel blocker available in the United States to improve walking in patients with multiple sclerosis as demonstrated by an increase in walking speed. Its pharmacokinetics have been evaluated in human studies but its metabolites are not well characterized. This study characterizes the metabolic profile of dalfampridine in two animal species that were used to support nonclinical toxicology evaluation. METHODS: Metabolic profiling of single oral (14)C-4-AP doses was performed in 12 adult male Sprague-Dawley rats. Similarly, metabolic profiling was performed in beagle dogs in two studies that administered (14)C-4-AP by gastric intubation; the first study included six animals (three males, three females), and the second study included two animals (one male, one female). Blood and urine samples were evaluated using high performance liquid chromatography, thin layer chromatography, and radioanalysis (liquid scintillation counting), with further identification of components by gas chromatography/mass spectrometry. RESULTS: Five radioactive components, M1-M5, were detected in rat plasma, although most of the radioactivity corresponded with unchanged 4-AP. Based on Rf values, M1 and M2 coseparated with reference standards of 3-hydroxy-4-AP and 4-AP, respectively. Additionally, components M1, M2, and M3 coseparated with the same components isolated from the urine of a dog dosed with (14)C-4-AP and identified as 3-hydroxy-4-AP, 4-AP, and 3-hydroxy-4-AP sulfate, respectively; M4 and M5 could not be identified because of low concentrations. In dogs, most of the radioactivity was excreted within the first 24 hours as unchanged compound. CONCLUSIONS: Following oral dosing, 4-AP was rapidly absorbed in rats and dogs, with rapid excretion and almost complete urinary recovery in dogs. The primary metabolites in both animal models were 3-hydroxy-4-AP and 3-hydroxy-4-AP sulfate. Systemic clearance not accounted for by renal excretion of 4-AP may occur by liver metabolism by hydroxylation of 4-AP to 3-hydroxy-4-AP followed by sulfate conjugation to 3-hydroxy-4-AP sulfate.
RESUMO
Rivaroxaban, an oral, direct factor Xa inhibitor, has been approved in several countries for thromboprophylaxis after elective hip or knee arthroplasty based on favorable benefit-risk profile and improved efficacy compared to enoxaparin in reducing the composite of symptomatic and asymptomatic deep vein thrombosis, nonfatal pulmonary embolism, and all-cause mortality. Given the potential therapeutic utility of factor Xa inhibition in arterial thrombosis, we evaluated the antithrombotic activity of rivaroxaban in a model of arterial thrombosis in anesthetized rats in which thrombotic occlusion was induced by electrolytic injury of the carotid artery. Rivaroxaban, 0.3, 1 or 3âmg/kg, enoxaparin, 10âmg/kg, or vehicle were infused intravenously to anesthetized rats and time to occlusion as well as coagulation parameters monitored following carotid electrolytic injury. Although the lowest dose of rivaroxaban (0.3âmg/kg) did not prolong occlusion time compared to vehicle, rivaroxaban at 1 or 3âmg/kg prevented occlusion in all vessels during the 30-min observation period (median occlusion time >30âmin), which was greater than that following a single dose of enoxaparin infused at a dose of 10âmg/kg (median time to occlusion = 21.6âmin). Rivaroxaban was also effective following oral dosing at 3âmg/kg. Rivaroxaban's antithrombotic activity was paralleled by dose-dependent increases in prothrombin time (PT) and activated clotting time (ACT) without significant changes in activated partial thromboplastin time. Rivaroxaban also markedly increased Russell's viper venom time (RVVT) and decreased thrombin-antithrombin complex concentrations at all doses. These findings support the potential utility of rivaroxaban in arterial thrombotic disorders such as acute coronary syndrome, stroke and peripheral arterial disease.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Lesões das Artérias Carótidas/sangue , Oclusão Coronária/sangue , Trombose Coronária/sangue , Inibidores do Fator Xa , Morfolinas/administração & dosagem , Tiofenos/administração & dosagem , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Anticoagulantes/uso terapêutico , Lesões das Artérias Carótidas/induzido quimicamente , Oclusão Coronária/induzido quimicamente , Trombose Coronária/induzido quimicamente , Modelos Animais de Doenças , Eletrólitos/efeitos adversos , Enoxaparina/administração & dosagem , Enoxaparina/uso terapêutico , Fator Xa/metabolismo , Fibrinolíticos/administração & dosagem , Fibrinolíticos/uso terapêutico , Injeções Intravenosas , Masculino , Morfolinas/uso terapêutico , Tempo de Tromboplastina Parcial , Tempo de Protrombina , Ratos , Ratos Sprague-Dawley , Rivaroxabana , Tiofenos/uso terapêuticoRESUMO
Neuregulins are a family of growth factors essential for normal cardiac and nervous system development. The EGF-like domain of neuregulins contains the active site which binds and activates signaling cascades through ErbB receptors. A neuregulin-1 gene EGF-like fragment demonstrated neuroprotection in the transient middle cerebral artery occlusion (MCAO) stroke model and drastically reduced infarct volume (Xu et al., 2004). Here we use a permanent MCAO rat model to initially compare two products of the neuregulin-1 gene and also assess levels of recovery with acute versus delayed time to treatment. In the initial study full-length glial growth factor 2 (GGF2) and an EGF-like domain fragment were compared with acute intravenous delivery. In a second study GGF2 only was delivered starting at 24h, 3 days or 7 days after permanent ischemia was induced. In both studies daily intravenous administration continued for 10 days. Recovery of neurological function was assessed using limb placing and body swing tests. GGF2 had similar functional improvements compared to the EGF-like domain fragment at equimolar doses, and a higher dose of GGF2 demonstrated more robust functional improvements compared to a lower dose. GGF2 improved sensorimotor recovery with all treatment paradigms, even enhancing recovery of function with a delay of 7 days to treatment. Histological assessments did not show any associated reduction in infarct volume at either 48 h or 21 days post-ischemic event. Neurorestorative effects of this kind are of great potential clinical importance, given the difficulty of delivering neuroprotective therapies within a short time after an ischemic event in human patients. If confirmed by additional work including additional data on mechanism(s) of improved outcome with verification in other stroke models, one can make a compelling case to bring GGF2 to clinical trials as a neurorestorative approach to improving outcome following stroke injury.