RESUMEN
OBJECTIVE: The aim of the present study was to investigate the intra-articular duration of Durolane™ in a rabbit model to allow comparison between Durolane™ residence time and data reported for other hyaluronic acid products as well as native hyaluronic acid. DESIGN: (14)C-labeled Durolane™ was manufactured by labeling the cross-linker used for stabilization. A single injection of approximately 0.3 mL (14)C-labeled Durolane™ was administered intra-articularly in both knee joints of male New Zealand White rabbits. At days 1, 2, 3, 7, 28, 60, 96, and 120 after injection, the knee joints of 4 animals were collected, and the radioactivity of the remaining gel was measured. The obtained data were fitted by exponential models to calculate the half-life of the gel. Two additional rabbits were used for histology of the joint 127 days after the injection. RESULTS: The elimination of (14)C-labeled Durolane™ followed first-order kinetics with an apparent half-life of approximately 32 days. Histology showed no morphological changes in the knee joints. CONCLUSIONS: This study shows that Durolane™ has a half-life of 32 days in the rabbit knee joint, which is much longer compared to literature data on hyaluronic acid and other modified hyaluronic acid products.
RESUMEN
Active efflux transporters in the blood-brain barrier lower the brain concentrations of many drug molecules and endogenous substances and thus affect their central action. The objective of this investigation was to study the dynamics of the entire inhibition process of the efflux transporter P-glycoprotein (P-gp), using positron emission tomography (PET). The P-gp marker [(11)C]verapamil was administered to anesthetized rats as an i.v. bolus dose followed by graded infusions via a computerized pump system to obtain a steady-state concentration of [(11)C]verapamil in brain. The P-gp modulator cyclosporin A (CsA) (3, 10 and 25 mg/kg) was administered as a short bolus injection 30 min after the start of the [(11)C]verapamil infusion. The CsA pharmacokinetics was studied in whole blood in a parallel group of rats. The CsA blood concentrations were used as input to model P-gp inhibition. The inhibition of P-gp was observed as a rapid increase in brain concentrations of [(11)C]verapamil, with a maximum after 5, 7.5 and 17.5 min for the respective doses. The respective increases in maximal [(11)C]verapamil concentrations were 1.5, 2.5 and 4 times the baseline concentration. A model in which CsA inhibited P-gp by decreasing the transport of [(11)C]verapamil out from the brain resulted in the best fit. Our data suggest that it is not the CsA concentration in blood, but rather the CsA concentration in an effect compartment, probably the endothelial cells of the blood-brain barrier that is responsible for the inhibition of P-gp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/diagnóstico por imagen , Ciclosporina/farmacología , Inmunosupresores/farmacología , Algoritmos , Animales , Biotransformación , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacocinética , Radioisótopos de Carbono , Ciclosporina/sangre , Inmunosupresores/sangre , Marcaje Isotópico , Masculino , Modelos Estadísticos , Tomografía de Emisión de Positrones , Ratas , Ratas Sprague-Dawley , Verapamilo/administración & dosificación , Verapamilo/farmacocinéticaRESUMEN
The general anaesthetic ketamine affects the central cholinergic system in several manners, but its effect on spinal acetylcholine release, which may be an important transmitter in spinal antinociception, is unknown. This study aimed to investigate the effect of ketamine on spinal acetylcholine release. Microdialysis probes were placed intraspinally in male rats, and acetylcholine was quantified with HPLC. Anaesthesia was switched from isoflurane (1.3%) to ketamine (150 mg/kg h), which resulted in a 500% increased acetylcholine release. The increase was attenuated during nicotinic receptor blockade (50 microM mecamylamine). The nicotinic receptor agonist epibatidine (175 microM) produced a ten-fold higher relative increase of acetylcholine release during isoflurane anaesthesia compared to ketamine anaesthesia (270% to 27%). Intraspinal administration of ketamine and norketamine both increased the acetylcholine release in high concentrations (100 microM to 10 mM). The results indicate that spinal nicotinic receptors are important for the ketamine-induced acetylcholine release, and that the effect is partly mediated at the spinal level.
Asunto(s)
Acetilcolina/metabolismo , Anestésicos Disociativos/farmacología , Ketamina/análogos & derivados , Mecamilamina/farmacología , Receptores Nicotínicos/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Anestésicos por Inhalación/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Isoflurano/farmacología , Ketamina/farmacología , Masculino , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Factores de TiempoRESUMEN
It has been shown that analgesics such as morphine, lidocaine and clonidine increase the release of spinal acetylcholine. Acetylcholine may therefore play an important role in the regulation of spinal pain threshold. Since behavioral as well as in vitro studies have shown a clear involvement of GABA (gamma-amino butyric acid) receptors in the regulation of spinal nociceptive mechanisms, the present study focused on the role of GABA receptors for spinal acetylcholine release control. GABA receptor agonists and antagonists were infused via a spinal microdialysis probe and acetylcholine release was measured. The GABA(A) receptor agonist muscimol decreased acetylcholine release and the antagonist bicuculline increased acetylcholine release. The GABA(B) receptor agonist baclofen decreased acetylcholine release whereas the antagonist saclofen did not change acetylcholine release. The results suggest that both GABA receptor subtypes have an inhibitory role on spinal dorsal horn acetylcholine release and that the GABA(A) receptors are tonically regulating acetylcholine release.
Asunto(s)
Acetilcolina/metabolismo , Células del Asta Posterior/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Animales , Baclofeno/análogos & derivados , Baclofeno/farmacología , Bicuculina/farmacología , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Agonistas de Receptores GABA-B , Antagonistas de Receptores de GABA-B , Masculino , Microdiálisis , Muscimol/farmacología , Células del Asta Posterior/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
UNLABELLED: Detection of epidermal growth factor receptor (EGFR) overexpression in many carcinomas provides important diagnostic information, which can influence patient management. The use of PET may enable such detection in vivo by a noninvasive procedure with high sensitivity. The aim of this study was to develop a method for preparation of a positron-emitting tracer based on a natural ligand to EGFR, the recombinant human epidermal growth factor (hEGF), and to perform a preclinical evaluation of the tracer. METHODS: DOTA-hEGF (DOTA is 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) was prepared by coupling of a N-sulfosuccinimide ester of DOTA to hEGF. The conjugate was labeled with a generator-produced positron-emitting nuclide, (68)Ga (half-life = 68 min), using microwave heating. Binding specificity, affinity, internalization, and retention of (68)Ga-DOTA-hEGF was studied in 2 EGFR-expressing cell lines, U343 glioma cells and A431 cervical carcinoma cells. Biodistribution and microPET visualization studies were performed in BALB/c nu/nu mice bearing A431 carcinoma xenografts. RESULTS: A 1-min-long microwave-assisted labeling provided radioactivity incorporation of 77% +/- 4%. Both cell lines demonstrated receptor-specific uptake of the conjugate, rapid internalization of the tracer, and good retention of radioactivity. Binding to both cell lines occurred with high affinity, approximately 2 nmol/L. The biodistribution study demonstrated accumulation of radioactivity in xenografts and in EGFR-expressing organs. The microPET imaging study enabled visualization of tumors and demonstrated quick--within 5 min--localization of radioactivity in tumors. CONCLUSION: (68)Ga-DOTA-hEGF has potential for imaging EGFR overexpression in tumors.
Asunto(s)
Carcinoma de Células Escamosas/diagnóstico por imagen , Carcinoma de Células Escamosas/metabolismo , Factor de Crecimiento Epidérmico/análogos & derivados , Receptores ErbB/metabolismo , Glioma/diagnóstico por imagen , Glioma/metabolismo , Compuestos Organometálicos/farmacocinética , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Factor de Crecimiento Epidérmico/farmacocinética , Estudios de Factibilidad , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Marcaje Isotópico/métodos , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Especificidad de Órganos , Cintigrafía , Radiofármacos/farmacocinética , Distribución TisularRESUMEN
Stimulation of spinal serotonin (5-HT) receptors results in analgesia and release of acetylcholine. We investigated the involvement of 5-HT1, 5-HT2, and 5-HT3 receptor subtypes in the regulation of spinal acetylcholine release. A spinal microdialysis probe was placed dorsally at about the C5 level in anaesthetized rats. The selective serotonin reuptake inhibitor citalopram was found to increase acetylcholine release when infused via the microdialysis probe. Several doses of the 5-HT receptor agonists 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT, 5-HT1A), 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP93129, 5-HT1B), alpha-methyl-5-hydroxytryptamine maleate (m5-HT, 5-HT2), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 5-HT2C), and 1-(m-chlorophenyl)-biguanide (5-HT3) were subsequently infused via the microdialysis probe. Only 8-OH-DPAT, CP93129, and m5-HT increased acetylcholine release dose dependently. The 5-HT1A receptor selective antagonist (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide hydrochloride and the 5-HT2A receptor selective antagonist ketanserin tartrate inhibited the 8-OH-DPAT and the m5-HT induced acetylcholine release. The results suggest that 5-HT1A and the 5-HT2A receptors are involved in the regulation of acetylcholine release in the spinal cord.
Asunto(s)
Acetilcolina/metabolismo , Receptores de Serotonina/metabolismo , Serotonina/análogos & derivados , Médula Espinal/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Anfetaminas/farmacología , Animales , Biguanidas/farmacología , Citalopram/farmacología , Relación Dosis-Respuesta a Droga , Ketanserina/farmacología , Masculino , Microdiálisis , Piperazinas/farmacología , Piridinas/farmacología , Pirroles/farmacología , Ratas , Ratas Sprague-Dawley , Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Médula Espinal/efectos de los fármacos , Factores de TiempoRESUMEN
The serotonin transporter radioligand [11C]-3-amino-4-(2-dimethylaminomethylphenylsulfanyl)-benzonitrile, or [11C]DASB, was examined in order to assess its potential for measuring fluctuations in endogenous serotonin concentrations with positron emission tomography. Binding characteristics of [11C]DASB and the propensity for serotonin to displace the tracer were explored in rat brain homogenates. Experiments showed that serotonin displaced [11C]DASB in vitro. Ex vivo experiments performed after tranylcypromine injection (3 or 15 mg/kg) showed a dose-dependent trend in radioactivity uptake and suggested that serotonin may compete with [11C]DASB for transporter binding.
Asunto(s)
Compuestos de Anilina/farmacocinética , Compuestos de Anilina/uso terapéutico , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Tomografía de Emisión de Positrones/métodos , Serotonina/metabolismo , Sulfuros/farmacocinética , Sulfuros/uso terapéutico , Animales , Estudios de Factibilidad , Masculino , Tasa de Depuración Metabólica , Neurotransmisores/metabolismo , Especificidad de Órganos , Unión Proteica , Radiofármacos/farmacocinética , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Aspirin and paracetamol have been shown to suppress non-inflammatory pain conditions like thermal, visceral and mechanical pain in mice and rats. The non-inflammatory antinociception appears to be mediated by central receptor mechanisms, such as the cholinergic system. In this study, we tested the hypothesis that the non-inflammatory antinociception of aspirin and paracetamol could be mediated by an increase of intraspinal acetylcholine release. Microdialysis probes were placed intraspinally in anesthetized rats for acetylcholine sampling. Subcutaneously administered aspirin 100 and 300 mg/kg increased, while paracetamol 300 mg/kg decreased intraspinal acetylcholine release. Intraspinal drug administration did not affect acetylcholine release. Our results suggest that an increased intraspinal acetylcholine release could be involved in part of the non-inflammatory pain suppression by aspirin, but not by paracetamol.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Sistema Nervioso Parasimpático/fisiología , Médula Espinal/fisiología , Acetilcolina/farmacología , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Aspirina/administración & dosificación , Temperatura Corporal/efectos de los fármacos , Inyecciones Subcutáneas , Masculino , Microdiálisis , Dolor/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
Epibatidine has been used in a wide dose range and was found to produce both nociceptive and antinociceptive effects. The different effects were partly explained by an action on multiple nicotinic receptor systems. The present study investigated the possibility that part of the action of intraspinally or subcutaneously administered (+/-) epibatidine, is mediated through an action on muscarinic receptors. Radioligand receptor assays were performed using homogenates of rat spinal cord and muscarinic M1-M5 receptors expressed in Sf9 cells. The intraspinal acetylcholine releasing effect of intraspinally and subcutaneously administered (+/-) epibatidine was studied with and without with atropine pretreatment. (+/-) Epibatidine has affinity for muscarinic receptors both in spinal cord tissue and expressed in Sf9 cells. The intraspinal administration of 160 microM (+/-) epibatidine produced an increase in acetylcholine release that was reduced by pretreatment with 100 microM atropine. Subcutaneous administration of 30 microg/kg (+/-) epibatidine produced an increase in intraspinal acetylcholine release that was not inhibited by 5 mg/kg subcutaneous atropine pretreatment. We conclude that (+/-) epibatidine, in microM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase acetylcholine release. Epibatidine induced spinal acetylcholine release observed after subcutaneous administration appears not to be mediated via muscarinic receptor. The dual action on both nicotinic receptors and muscarinic receptors may explain the potent analgesic effect observed after epibatidine administration.
Asunto(s)
Acetilcolina/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Receptores Muscarínicos/metabolismo , Médula Espinal/metabolismo , Acetilcolina/análisis , Analgésicos no Narcóticos/farmacología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Inyecciones Espinales , Inyecciones Subcutáneas , Masculino , Microdiálisis , Agonistas Muscarínicos/farmacología , Piridinas/administración & dosificación , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/genética , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Médula Espinal/efectos de los fármacos , Transfección , TritioRESUMEN
Cholinergic agonists produce spinal antinociception via mechanisms involving an increased release of intraspinal acetylcholine. The cholinergic receptor system interacts with several other receptor types, such as alpha2-adrenergic receptors. To fully understand these interactions, the effects of various receptor ligands on the cholinergic system must be investigated in detail. This study was initiated to investigate the effects of the alpha2-adrenergic receptor agonists clonidine and rilmenidine and the alpha2-adrenergic receptor antagonists yohimbine and efaroxan on spinal cholinergic receptors in the rat. Spinal microdialysis was used to measure in vivo changes of acetylcholine after administration of the ligands, with or without nicotinic receptor blockade. In addition, in vitro binding properties of the ligands on muscarinic and nicotinic receptors were investigated. It was found that clonidine and rilmenidine increased, while yohimbine decreased spinal acetylcholine release. Efaroxan affected acetylcholine release differently depending on concentration. Nicotinic receptor blockade attenuated the effect of all ligands. All ligands showed poor binding affinity for muscarinic receptors. On the other hand, all ligands possessed affinity for nicotinic receptors. Clonidine and yohimbine binding was best fit to a one site binding curve and rilmenidine and efaroxan to a two site binding curve. The present study demonstrates that the tested alpha2-adrenergic receptor ligands affect intraspinal acetylcholine release in the rat evoked by nicotinic receptor mechanisms in vivo, and that they possess binding affinity to nicotinic receptors in vitro. The binding of alpha2-adrenergic receptor ligands to nicotinic receptors might affect the intraspinal release of acetylcholine.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas de Receptores Adrenérgicos alfa 2 , Receptores Colinérgicos/metabolismo , Médula Espinal/efectos de los fármacos , Acetilcolina/biosíntesis , Animales , Benzofuranos/farmacología , Unión Competitiva , Clonidina/farmacología , Relación Dosis-Respuesta a Droga , Imidazoles/farmacología , Técnicas In Vitro , Inyecciones Espinales , Ligandos , Masculino , Microdiálisis , Oxazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/metabolismo , Rilmenidina , Médula Espinal/metabolismo , Factores de Tiempo , Yohimbina/farmacologíaRESUMEN
Activation of cholinergic receptors in the spinal cord increases the intraspinal release of acetylcholine (ACh) and produces potent analgesia. The mechanisms that regulate the release of spinal ACh are not fully known. In the present study, we investigated the role of nicotinic ACh receptors in the regulation of intraspinal ACh release. Using an in vivo intraspinal microdialysis technique, nicotine was administered alone and in combination with the nicotinic antagonists mecamylamine (50 microM), dihydro-beta-erythroidine (DbetaE) (500 microM) and methyllycaconitine (MLA) (40 nM). Administration of nicotine (1 microM-1 mM) produced a dose dependent increase of intraspinal ACh release, while 10 mM nicotine resulted in dramatic increase in ACh release followed by a decrease to baseline. Administration of mecamylamine or DbetaE also induced an increased ACh release while MLA caused a decreased release. Mecamylamine and DbetaE, but not MLA pretreatment attenuated the stimulatory effect of 100 microM nicotine on intraspinal ACh release. It is suggested that spinal ACh release is regulated by different nicotinic ACh receptors. These receptors may tonically regulate spinal ACh release either directly or indirectly via inhibitory interneurones. Some of these receptors may be desensitised by high nicotine concentrations leading to a reduction of ACh release.
Asunto(s)
Acetilcolina/biosíntesis , Aconitina/análogos & derivados , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/farmacología , Receptores Nicotínicos/metabolismo , Médula Espinal/efectos de los fármacos , Aconitina/farmacología , Animales , Dihidro-beta-Eritroidina/farmacología , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Masculino , Mecamilamina/farmacología , Microdiálisis , Nicotina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , Médula Espinal/metabolismo , Factores de TiempoRESUMEN
Both systemically and intrathecally administered cholinergic agonists produce antinociception while cholinergic antagonists decrease pain threshold. The mechanism and the site of action of these substances are not known. In the present study it was hypothesized that systemically administered muscarinic agonists and antagonists modify nociceptive threshold by affecting intraspinal release of acetylcholine (ACh). Catheters were inserted into the femoral vein in rats maintained on isoflurane anaesthesia for administration of oxotremorine (10-300 microg/kg) and atropine (0.1, 10, 5000 microg/kg). Spinal microdialysis probes were placed intraspinally at approximately the C2-C5 spinal level for sampling of acetylcholine and dialysis delivery of atropine (0.1, 1, 10 nM). Additionally, the tail-flick behaviour was tested on conscious rats injected intraperitoneally with saline, atropine (10, 100 and 5000 microg/kg), or subcutaneously with oxotremorine (30, 100, 300 microg/kg). Subcutaneous administration of oxotremorine (30, 100, 300 microg/kg) significantly increased the tail-flick latency. These doses of oxotremorine dose-dependently increased the intraspinal release of acetylcholine. Intravenously administered atropine, in a dose that produced hyperalgesia (5000 microg/kg) in the tail-flick test, significantly decreased the intraspinal release of acetylcholine. Our results suggest an association between pain threshold and acetylcholine release in spinal cord. It is also suggested that an approximately 30% increase in basal ACh release produces antinociception and that a 30% decrease in basal release produces hyperalgesia.
Asunto(s)
Acetilcolina/metabolismo , Atropina/farmacología , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Oxotremorina/farmacología , Médula Espinal/efectos de los fármacos , Animales , Atropina/administración & dosificación , Relación Dosis-Respuesta a Droga , Hiperalgesia/inducido químicamente , Inyecciones Intravenosas , Inyecciones Subcutáneas , Masculino , Agonistas Muscarínicos/administración & dosificación , Antagonistas Muscarínicos/administración & dosificación , Oxotremorina/administración & dosificación , Dolor/tratamiento farmacológico , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Cola (estructura animal)RESUMEN
The local anesthetic lidocaine suppresses different pain conditions when administered systemically. Part of the antinociceptive effect appears to be mediated via receptor mechanisms. We have previously shown that muscarinic and nicotinic agonists that produce antinociception increase the intraspinal release of acetylcholine. In the present study it was hypothesized that systemically administered lidocaine is acting through the same mechanisms as cholinergic agonists and affects the intraspinal release of acetylcholine. Microdialysis probes were placed in anesthetized rats for sampling of acetylcholine. Ten and 30 mg/kg lidocaine injected intravenously significantly increased the intraspinal release of acetylcholine. The effect of lidocaine could be reduced by pretreatment with intraspinally administered atropine or mecamylamine. Our results suggest that the antinociceptive effect produced by systemically administered lidocaine is mediated through an action on muscarinic and nicotinic receptors.