RESUMEN
BACKGROUND AND PURPOSE: While investigating the effects of systemic urotensin II (U-II), a potent vasoactive peptide acting at the UT receptor, we observed ear pinna flushing after systemic administration to conscious rats. In the present study, U-II-induced ear flushing was quantified in terms of ear pinna temperature change and potential mechanisms were explored. EXPERIMENTAL APPROACH: U-II-induced ear flushing was quantified by measuring lateral ear pinna temperature changes and compared to that of calcitonin gene-related peptide (CGRP), a known cutaneous vasodilator. Further, the effects of a variety of pharmacological agents on U-II-induced ear flushing were explored. KEY RESULTS: Subcutaneous injection of U-II (9 microg kg(-1))produced localized ear pinna flushing with an onset of approximately 15 min, a duration of approximately 30 min and a maximal temperature change of 9 degrees C. In contrast, CGRP caused cutaneous flushing within multiple cutaneous beds including the ear pinna with a shorter onset and greater duration than U-II. A potent UT receptor antagonist, urantide, blocked U-II-induced ear flushing but did not affect CGRP-induced ear flushing. Pretreatment with indomethacin or L-Nomega-nitroarginine methylester (L-NAME) abolished U-II-induced ear flushing. Mecamylamine or propranolol did not affect this response to U-II. Direct intracerebroventricular injection studies suggested that the ear flushing response to U-II was not mediated directly by the CNS. CONCLUSION AND IMPLICATIONS: Our results suggest that U-II-induced ear flushing and temperature increase is mediated by peripheral activation of the UT receptor and involves prostaglandin- and nitric oxide-mediated vasodilation of small capillary beds in the rat ear pinna.
Asunto(s)
Oído Externo/irrigación sanguínea , Rubor/inducido químicamente , Urotensinas/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Temperatura Corporal/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/farmacología , Inhibidores Enzimáticos/farmacología , Indometacina/farmacología , Inyecciones Subcutáneas , Masculino , Mecamilamina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Antagonistas Nicotínicos/farmacología , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Propranolol/farmacología , Ratas , Ratas Sprague-Dawley , Flujo Sanguíneo Regional/efectos de los fármacos , Urotensinas/administración & dosificación , Urotensinas/antagonistas & inhibidores , Vasodilatadores/farmacologíaRESUMEN
B lymphocyte stimulator (BLyS; also known as TNFSF20, BAFF, TALL-1, zTNF4, and THANK), a tumor necrosis factor ligand family member, has recently been identified as a factor that promotes expansion and differentiation of the B cell population, leading to increases in serum immunoglobulin levels. Here, pharmacokinetic parameters for BLyS administered i.v. and s.c. to mice are described, and the effects of different dosing regimens on serum and salivary immunoglobulin levels as well as splenic cell populations are reported. The pharmacokinetics of BLyS following i.v. injection are monophasic with a half-life of 160 min, a clearance of 0.22 ml/min-kg, and a volume of distribution of 53 ml/kg. Systemic administration of BLyS to mice resulted in increased serum IgG, IgA, IgM, and IgE and salivary IgA as well as splenic B cell population expansion and differentiation. The i.v. and s.c. routes of administration were pharmacologically equivalent, even though s.c. bioavailability of BLyS is only 25%. BLyS (s.c.) dramatically elevated serum IgG and IgA levels, and the duration of the responses after cessation of treatment (t(1/2) = 4.4 and 1.3 days, respectively) are similar to the half-lives of endogenous IgG and IgA in mice. The IgM response is more modest than that of IgG and IgA but lasts longer (t(1/2) = 7.0 days) than the half-life of endogenous IgM. A linear pharmacodynamic response was identified between days of dosing x log(dose), and increases in serum IgG, IgA, and IgM indicating that the response is more sensitive to the duration of dosing than to the cumulative dose. The implications of these findings for therapeutic administration of BLyS are discussed.
Asunto(s)
Inmunidad Celular/efectos de los fármacos , Proteínas de la Membrana/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Factor Activador de Células B , Semivida , Humanos , Inmunoglobulinas/metabolismo , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Tamaño de los Órganos/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Saliva/inmunología , Bazo/citología , Bazo/efectos de los fármacosRESUMEN
The potential acute toxicity of a ribozyme (ANGIOZYME) targeting the flt-1 vascular endothelial growth factor (VEGF) receptor mRNA was evaluated in cynomolgus monkeys following i.v. infusion or s.c. injection. ANGIOZYME was administered as a 4-hour i.v. infusion at doses of 10, 30, or 100 mg/kg or a s.c. bolus at 100 mg/kg. End points included blood pressure, electrocardiogram (ECG), clinical chemistry, hematology, complement factors, coagulation parameters, and ribozyme plasma concentrations. ANGIOZYME was well tolerated, with no drug-associated morbidity or mortality. There was no clear evidence of ANGIOZYME-related adverse effects in this study. Slight increases in spleen weight and lymphoid hyperplasia were observed in several animals. However, these changes were not dose dependent. Steady-state concentrations of ANGIOZYME were achieved during the 4-hour infusion of 10, 30, or 100 mg/kg. Dose-dependent elimination of ANGIOZYME was observed, with faster clearance at the two highest doses. ANGIOZYME was slowly absorbed after s.c. administration, resulting in steady-state concentrations for the 9-hour sampling period. Monkeys in this toxicology study received significant plasma ANGIOZYME exposure by both the s.c. and i.v. routes.
Asunto(s)
Marcación de Gen , ARN Catalítico/farmacocinética , ARN Catalítico/toxicidad , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/sangre , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/toxicidad , Animales , Análisis Químico de la Sangre , Factores de Coagulación Sanguínea/análisis , Cromatografía Líquida de Alta Presión , Proteínas del Sistema Complemento/análisis , Esquema de Medicación , Femenino , Infusiones Intravenosas , Inyecciones Intravenosas , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , ARN Catalítico/administración & dosificación , ARN Catalítico/sangre , Receptores de Factores de Crecimiento Endotelial VascularRESUMEN
In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.
Asunto(s)
Artritis/enzimología , Artritis/terapia , Marcación de Gen , ARN Catalítico/uso terapéutico , Animales , Artritis/genética , Artritis/metabolismo , Cartílago Articular/enzimología , Cartílago Articular/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/enzimología , Marcación de Gen/métodos , Humanos , Hidrólisis , Inyecciones Intraarticulares , Masculino , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Metaloproteinasa 3 de la Matriz/fisiología , Inhibidores de la Metaloproteinasa de la Matriz , Técnicas de Cultivo de Órganos , ARN Catalítico/administración & dosificación , ARN Catalítico/metabolismo , Conejos , Reproducibilidad de los Resultados , Especificidad por Sustrato , Membrana Sinovial/enzimología , Membrana Sinovial/metabolismoRESUMEN
Chemically stabilized hammerhead ribozymes are nuclease-resistant, RNA-based oligonucleotides that selectively bind and cleave specific target RNAs. Due to their potential for specifically inhibiting gene expression, ribozymes are being investigated for therapeutic applications as well as for the elucidation of gene function. In particular, we have investigated ribozymes that target the mRNA of the vascular endothelial growth factor (VEGF) receptors because VEGF signaling is an important mediator of tumor angiogenesis and metastasis. Here we report pharmacodynamic studies testing anti-Flt-1 (VEGFR-1) and anti-KDR (VEGFR-2) ribozymes in animal models of solid tumor growth and metastasis. Ribozymes targeting either Flt-1 or KDR significantly inhibited primary tumor growth in a highly metastatic variant of Lewis lung carcinoma. However, only treatment with the anti-Flt-1 ribozyme resulted in a statistically significant and dose-dependent inhibition of lung metastasis in this model. The anti-Flt-1 ribozyme was then tested in a xenograft model of human metastatic colorectal cancer in which significant inhibition of liver metastasis was observed. Taken together, these data represent the first demonstration that synthetic ribozymes targeting VEGF receptor mRNA reduced the growth and metastasis of solid tumors in vivo.
Asunto(s)
Antineoplásicos/uso terapéutico , Metástasis de la Neoplasia/prevención & control , ARN Catalítico/uso terapéutico , ARN Mensajero/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Animales , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Carcinoma Pulmonar de Lewis/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Trasplante de Neoplasias , ARN Catalítico/genética , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
A significant amount of research has been devoted to the chemical stabilization of synthetic ribozymes, in part, so that applications to systemic disease can be explored. A nuclease-stabilized synthetic hammerhead ribozyme, ANGIOZYME, has been developed which targets the mRNA encoding a vascular endothelial growth factor receptor, Flt-1. Because the stimulation of this receptor may contribute to tumor neovascularization and subsequent tumor growth and metastasis, we have explored the systemic use of ANGIOZYME to down regulate this receptor in a syngeneic model of metastatic cancer. We describe here the application of pharmacokinetic analysis to the selection of a dosing regimen for pharmacodynamic screening in this murine cancer model. These studies demonstrate that the appropriate application of pharmacokinetic analysis is necessary for the optimization of systemic pharmacodynamic studies using synthetic ribozymes.
Asunto(s)
ARN Catalítico/farmacología , ARN Catalítico/farmacocinética , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/farmacocinética , Inhibidores de la Angiogénesis/farmacología , Animales , Secuencia de Bases , Proteínas de la Matriz Extracelular/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Cadenas Pesadas de Miosina , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Miosina Tipo IIB no Muscular , Estabilidad del ARN , ARN Catalítico/administración & dosificación , ARN Catalítico/genética , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
Vascular endothelial growth factor (VEGF) is a growth factor that contributes to the angiogenesis of developing tumors. To interfere with the action of VEGF, a nuclease-stabilized ribozyme, ANGIOZYME, has been developed against VEGF receptor subtype Flt-1 mRNA. To determine which routes of administration would be useful for systemic delivery of this ribozyme, a dose of 30 mg/kg [32P]ANGIOZYME was administered as an i.v., i.p., or s.c. bolus. Concentrations of ANGIOZYME in plasma, femur, kidney, liver, and lung were examined. ANGIOZYME was well absorbed after i.p. (90%) or s.c. administration (77%), with peak plasma concentrations occurring 30 minutes after dosing. Total body clearance after a single dose of 30 mg/kg ANGIOZYME was 20 ml/min/kg, and the elimination half-life was 33 minutes. The apparent volume of distribution at steady-state ranged from 0.5 to 1.3 L/kg. ANGIOZYME was detected in the four tissues examined through the 3 hour sampling period after i.v. or i.p. administration. After s.c. administration, ANGIOZYME was detected in femur, kidney, and lung but not in the liver. The highest concentrations of ANGIOZYME were found in kidney and femur with all three routes. Because of the rapid and extensive absorption after extravascular injections, either i.p. or s.c. administration could be considered for use in pharmacodynamic studies examining the effects of ANGIOZYME or other ribozymes with similar chemical modifications.
Asunto(s)
Neovascularización Patológica , ARN Catalítico/farmacocinética , Animales , Femenino , Semivida , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/prevención & control , ARN Catalítico/farmacología , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento Endotelial Vascular , Distribución TisularRESUMEN
Vascular endothelial growth factor (VEGF) and its receptors Flt-1 and KDR play important roles in physiological and pathological angiogenesis. Ribozymes that target the VEGF receptor mRNAs were developed and their biological activities in cell culture and an animal model were assessed. Ribozymes targeting Flt-1 or KDR mRNA sites reduced VEGF-induced proliferation of cultured human vascular endothelial cells and specifically lowered the level of Flt-1 or KDR mRNA present in the cells. Anti- Flt-1 and KDR ribozymes also exhibited anti-angiogenic activity in a rat corneal pocket assay of VEGF-induced angiogenesis. This report illustrates the anti-angiogenic potential of these ribozymes as well as their value in studying VEGF receptor function in normal and pathophysiologic states.
Asunto(s)
Endotelio Vascular/fisiología , Proteínas Proto-Oncogénicas/genética , ARN Catalítico/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factores de Crecimiento/genética , Animales , Endotelio Vascular/patología , Regulación de la Expresión Génica/fisiología , Marcación de Gen , Humanos , Masculino , Neovascularización Patológica/genética , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/genética , Ratas , Ratas Sprague-Dawley , Receptores de Factores de Crecimiento Endotelial Vascular , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
Lesions of the subthalamic nucleus block behavioral effects of nigrostriatal dopamine depletion in rats and primates, but the contribution of this region to the molecular effects of dopaminergic lesions is unknown. The effects of subthalamic nucleus lesions alone or in combination with a 6-hydroxydopamine-induced lesion of the substantia nigra were examined in adult rats. Unilateral subthalamic nucleus lesions caused ipsiversive rotation after peripheral administration of apomorphine and a small decrease in glutamic acid decarboxylase (GAD) mRNA in the ipsilateral globus pallidus (external pallidum). Confirming previous results, nigrostriatal dopaminergic lesions caused contraversive rotation after apomorphine injection, and increased enkephalin mRNA in the striatum, GAD mRNA in the globus pallidus, and somatostatin mRNA in the entopeduncular nucleus (internal pallidum) ipsilateral to the lesion. In addition, the lesion decreased substance P mRNA in the ipsilateral striatum compared to the contralateral side, and GAD mRNA in the contralateral entopeduncular nucleus. These effects were abolished in rats with lesions of the subthalamic nucleus and substantia nigra on the same side. Thus, the subthalamic lesions prevented changes in gene expression induced by dopamine depletion, not only in regions receiving a direct input from the subthalamic nucleus (ipsilateral pallidum), but also in regions which do not (striatum and contralateral pallidum). This suggests that polysynaptic pathways regulated by the subthalamic nucleus contribute to the effects of dopaminergic lesions in many regions of the basal ganglia. This pivotal role of the subthalamic nucleus may account for the beneficial effects of subthalamic nucleus lesions on motor symptoms resulting from dopamine depletion.
Asunto(s)
Cuerpo Estriado/metabolismo , Dopamina/fisiología , Regulación de la Expresión Génica , Sustancia Negra/metabolismo , Núcleos Talámicos/fisiología , Animales , Apomorfina/farmacología , Conducta Animal/efectos de los fármacos , Dopamina/deficiencia , Encefalinas/genética , Globo Pálido/metabolismo , Glutamato Descarboxilasa/genética , Masculino , Oxidopamina/farmacología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Somatostatina/genética , Conducta Estereotipada , Sustancia P/genética , Sustancia Negra/efectos de los fármacos , Sustancia Negra/patología , Núcleos Talámicos/patologíaRESUMEN
The effects of dopaminergic stimulation of the subthalamic nucleus (STh) on motor behavior were examined in conscious rats. Unilateral infusion of apomorphine (0.1 to 3.2 micrograms) into the STh induced a dose-dependent increase in abnormal, nondirected orofacial movements without altering turning, sniffing, grooming, or rearing behaviors. Orofacial movements elicited by local infusion of apomorphine (1.0 microgram) into the STh were blocked by peripheral administration of the D1 antagonist, SCH 23390 (0.1 mg/kg, sc), but not by the D2 antagonists haloperidol (1.0 mg/kg, sc) or sulpiride (50 mg/kg, sc). Furthermore, coinfusion of SCH 23390 (1.0 microgram), but not sulpiride (5.0 micrograms), with apomorphine (1.0 microgram) into the STh blocked oral dyskinesia. Oral movements could not be reelicited by an infusion of apomorphine into the STh after a kainic acid lesion of the STh. In addition, infusion of apomorphine (1.0 microgram) into sites proximal to but deliberately outside of the STh failed to elicit nondirected oral movements above baseline levels. The results indicate that stimulation of D1 dopaminergic receptors within the STh induces abnormal orofacial movements. This highlights the importance of the dopaminergic input to the STh in the regulation of motor function and suggests that D1 receptor antagonists could prove useful in the treatment of orofacial dyskinesia in humans.
Asunto(s)
Apomorfina/administración & dosificación , Dopaminérgicos , Discinesia Inducida por Medicamentos , Enfermedades de la Boca/inducido químicamente , Núcleos Talámicos/fisiología , Animales , Apomorfina/antagonistas & inhibidores , Benzazepinas/administración & dosificación , Benzazepinas/metabolismo , Benzazepinas/farmacología , Dopaminérgicos/administración & dosificación , Discinesia Inducida por Medicamentos/prevención & control , Haloperidol/administración & dosificación , Haloperidol/farmacología , Ácido Kaínico/farmacología , Masculino , Ratas , Ratas Sprague-Dawley , Sulpirida/administración & dosificación , Sulpirida/farmacología , Núcleos Talámicos/efectos de los fármacos , Núcleos Talámicos/patologíaRESUMEN
Kainic acid was microinjected or microdialyzed into the rostral medial aspect of the fastigial nucleus to determine its effect on mean arterial pressure and heart rate. This was carried out in both the awake and the anesthetized (alpha-chloralose) rat. In awake animals, kainic acid elicited an initial phasic pressor response which was followed by a long-term elevation of mean arterial pressure that lasted for the duration of the experiment (2 h). Rats anesthetized with alpha-chloralose exhibited only a tonic depressor response. This converted to a pressor response as the rats began to emerge from anesthesia after 2 h. Both the awake and the anesthetized rats exhibited regular phasic changes in mean arterial pressure that was superimposed on the longer term changes in the mean arterial pressure. Similar results were obtained in both the microinjected and the microdialyzed animals. Thus, stimulation of the intrinsic fastigial neurons by kainic acid evokes an elevation of the mean arterial pressure in the awake rat. This is manifested as a decrease in pressure in the anesthetized animal. Thus, stimulation of the cardiovascular region of the fastigial nucleus can increase or decrease mean arterial pressure. It is possible that the direction of the change in mean arterial pressure is dependent on the level of afferent or intrinsic fastigial neural activity.
Asunto(s)
Sistema Cardiovascular/efectos de los fármacos , Núcleos Cerebelosos/efectos de los fármacos , Ácido Kaínico/administración & dosificación , Animales , Núcleos Cerebelosos/citología , Cloralosa , Estimulación Eléctrica , Masculino , Microdiálisis , Microinyecciones , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Valores de ReferenciaRESUMEN
A method for restraining awake rats using head implant immobilization is described. In order to reduce stress to the individual animal, rats were restrained side by side in pairs during the adaptation and experimental periods. This technique was used in studies of central regulation of cardiovascular function. In particular, microdialysis probes were placed stereotaxically in deep nuclei of the cerebellum while mean arterial pressure and heart rate were monitored in the awake rat. After initial habituation, normal levels of heart rate and blood pressure obtained during restraint indicated that this is essentially a nonstressful procedure. This technique could also be used for a variety of other experimental conditions where head movement is not desirable, such as during oculomotor or vestibular experiments.
Asunto(s)
Encéfalo/fisiología , Inmovilización , Restricción Física/instrumentación , Animales , Presión Sanguínea/fisiología , Núcleos Cerebelosos/fisiología , Diálisis/instrumentación , Frecuencia Cardíaca/fisiología , Microelectrodos , Neurotransmisores/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The object of the present study was to examine the effects of temperature, oxidation, and pH on in vitro relative recovery of catecholamine and indoleamine neurotransmitters and their metabolites using microdialysis probes. Relative recovery of norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), homovanillic acid (HVA), and 5-hydroxytryptamine (5HT) increased with temperature from 0 to 46 degrees C. For each compound, the increase in the amount recovered with increasing temperature was different. The stability of norepinephrine and dopamine was not affected at any temperature using deoxygenated calibration standard solutions containing ascorbic acid but was greatly reduced when exposed to ambient air without antioxidant treatment; catecholamine metabolites and the indole compounds were less affected. No change for in vitro relative recovery was observed by varying the pH of the perfusing solution from 6 to 8. Thus, temperature control in probe calibration as well as analyte stability using antioxidant treatment are important in reducing the error when estimating extracellular concentrations of neurotransmitter and metabolites.