RESUMO
The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.
Assuntos
Pesquisa Biomédica/normas , Avaliação Pré-Clínica de Medicamentos/normas , Laboratórios/normas , Animais , Ensaios Clínicos Fase I como Assunto , Drogas em Investigação/química , Drogas em Investigação/farmacocinética , Humanos , Testes de Mutagenicidade , Farmacologia Clínica/normasRESUMO
The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.
Assuntos
Humanos , Animais , Pesquisa Biomédica/normas , Avaliação Pré-Clínica de Medicamentos/normas , Laboratórios/normas , Ensaios Clínicos Fase I como Assunto , Drogas em Investigação/química , Drogas em Investigação/farmacocinética , Testes de Mutagenicidade , Farmacologia Clínica/normasRESUMO
Evidences suggest protein kinase C epsilon (PKCε) activation is involved in both inflammatory and neuropathic pains. We have previously shown that tetracyclic triterpene euphol produces antinociception in different models of persistent pain, an action associated with its anti-inflammatory properties. Among these properties are the cannabinoid system activation and different PKC isozymes modulation. Herein, we sought to explore the potential role of PKCε modulation on euphol antinociceptive effect, in inflammatory and neuropathic pain models, in rodents. Also, we investigated further mechanisms associated with euphol effects. Oral treatment with euphol (30 mg/kg) prevented the putative effect of PGE2-induced acute and persistent mechanical hypersensitivity in mice and rats, respectively. In the PGE2-induced acute mechanical hypersensitivity euphol promoted an inhibitory effect similar to a PKCε inhibitor peptide. Likewise, in rats it prevented the mechanical hypersensitivity induced by a PKCε activator. Conversely, euphol effectiveness was not observed in a cAMP/PKA-induced mechanical hypersensitivity in mice. Single (1h prior) or repeated (twice daily during 3 or 13 days) treatments with euphol ameliorated painful peripheral neuropathy induced by paclitaxel and also the mechanical hypersensitivity induced by B16F10 melanoma cells injection, in mice. Additionally, in both inflammatory and neuropathic pain models, euphol consistently prevented PKCε up-regulation, as well as, inhibited the up-regulation of PKCε-activated intracellular pathways; namely nuclear factor-κB (NF-κB), cyclic AMP response element binding protein (CREB) and cyclo-oxygenase-2 (COX-2). The present results suggest the antinociceptive effect on persistent pain caused by euphol is likely dependent on the inhibition of pro-inflammatory mediators modulated by PKCε.
Assuntos
Analgésicos/administração & dosagem , Lanosterol/análogos & derivados , Dor/metabolismo , Dor/prevenção & controle , Proteína Quinase C-épsilon/metabolismo , Animais , Anti-Inflamatórios/administração & dosagem , Dinoprostona/administração & dosagem , Mediadores da Inflamação/metabolismo , Lanosterol/administração & dosagem , Masculino , Camundongos , Dor/induzido quimicamente , Limiar da Dor/efeitos dos fármacos , Proteína Quinase C-épsilon/administração & dosagem , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Infraorbital nerve constriction (CION) causes hypersensitivity to facial mechanical, heat and cold stimulation in rats and mice and is a reliable model to study trigeminal neuropathic pain. In this model there is evidence that mechanisms operated by kinin B1 and B2 receptors contribute to heat hyperalgesia in both rats and mice. Herein we further explored this issue and assessed the role of kinin receptors in mechanical hyperalgesia after CION. Swiss and C57Bl/6 mice that underwent CION or sham surgery or dynorphin A (1-17) administration were repeatedly submitted to application of either heat stimuli to the snout or mechanical stimuli to the forehead. Treatment of the animals on the fifth day after CION surgery with DALBK (B1 receptor antagonist) or HOE-140 (B2 receptor antagonist), both at 0.01-1µmol/kg (i.p.), effectively reduced CION-induced mechanical hyperalgesia. Knockout mice for kinin B1, B2 or B1/B2 receptors did not develop heat or mechanical hyperalgesia in response to CION. Subarachnoid dynorphin A (1-17) delivery (15nmol/5µL) also resulted in orofacial heat hyperalgesia, which was attenuated by post-treatment with DALBK (1 and 3µmol/kg, i.p.), but was not affected by HOE-140. Additionally, treatment with an anti-dynorphin A antiserum (200µg/5µL, s.a.) reduced CION-induced heat hyperalgesia for up to 2h. These results suggest that both kinin B1 and B2 receptors are relevant in orofacial sensory nociceptive changes induced by CION. Furthermore, they also indicate that dynorphin A could stimulate kinin receptors and this effect seems to contribute to the maintenance of trigeminal neuropathic pain.
Assuntos
Bradicinina/metabolismo , Dinorfinas/metabolismo , Dor Facial/metabolismo , Neuralgia/metabolismo , Animais , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Modelos Animais de Doenças , Dinorfinas/farmacologia , Temperatura Alta , Hiperalgesia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurotransmissores/farmacologia , Medição da Dor , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , TatoRESUMO
BACKGROUND AND PURPOSE: Phosphoinositide 3-kinase-γ (PI3Kγ) is implicated in many pathophysiological conditions, and recent evidence has suggested its involvement in colitis. In the present study, we investigated the effects of AS605240, a relatively selective PI3Kγ inhibitor, in experimental colitis and its underlying mechanisms. EXPERIMENTAL APPROACH: Acute colitis was induced in mice by treatment with trinitrobenzene sulphonic acid (TNBS), and the effect of AS605240 on colonic injury was assessed. Pro-inflammatory mediators and cytokines were measured by immunohistochemistry, elisa, real time-polymerase chain reaction and flow cytometry. KEY RESULTS: Oral administration of AS605240 significantly attenuated TNBS-induced acute colitis and diminished the expression of matrix metalloproteinase-9 and vascular endothelial growth factor. The colonic levels and expression of IL-1ß, CXCL-1/KC, MIP-2 and TNF-α were also reduced following therapeutic treatment with AS605240. Moreover, AS605240 reduced MIP-2 levels in a culture of neutrophils stimulated with lipopolysaccharide. The mechanisms underlying these actions of AS605240 are related to nuclear factor-κ (NF-κB) inhibition. Importantly, the PI3Kγ inhibitor also up-regulated IL-10, CD25 and FoxP3 expression. In addition, a significant increase in CD25 and FoxP3 expression was found in isolated lamina propria CD4+ T cells of AS605240-treated mice. The effect of AS605240 on Treg induction was further confirmed by showing that concomitant in vivo blockade of IL-10R significantly attenuated its therapeutic activity. CONCLUSIONS AND IMPLICATIONS: These results suggest that AS605240 protects mice against TNBS-induced colitis by inhibiting multiple inflammatory components through the NF-κB pathway while simultaneously inducing an increase in the functional activity of CD4+CD25+ Treg. Thus, AS605240 may offer a promising new therapeutic strategy for the treatment of inflammatory bowel diseases.
Assuntos
Antígenos CD4/metabolismo , Colite/tratamento farmacológico , Fatores de Transcrição Forkhead/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Quinoxalinas/uso terapêutico , Linfócitos T Reguladores/metabolismo , Tiazolidinedionas/uso terapêutico , Ácido Trinitrobenzenossulfônico , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Colite/induzido quimicamente , Colite/imunologia , Colo/efeitos dos fármacos , Colo/imunologia , Colo/patologia , Citocinas/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , NF-kappa B/metabolismo , Infiltração de Neutrófilos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Quinoxalinas/farmacologia , Tiazolidinedionas/farmacologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
BACKGROUND AND PURPOSE: The effects of systemic treatment with indomethacin-loaded nanocapsules (IndOH-NC) were compared with those of free indomethacin (IndOH) in rat models of acute and chronic oedema. EXPERIMENTAL APPROACH: The following models of inflammation were employed: carrageenan-induced acute oedema (measured between 30 min and 4 h), sub-chronic oedema induced by complete Freund's adjuvant (CFA) (determined between 2 h and 72 h), and CFA-induced arthritis (oedema measured between 14 and 21 days). KEY RESULTS: IndOH or IndOH-NC produced equal inhibition of carrageenan-elicited oedema. However, IndOH-NC was more effective in both the sub-chronic (33 +/- 4% inhibition) and the arthritis (35 +/- 2% inhibition) model of oedema evoked by CFA, when compared with IndOH (21 +/- 2% and 14 +/- 3% inhibition respectively) (P < 0.01). In the CFA arthritis model, treatment with IndOH-NC markedly inhibited the serum levels of the pro-inflammatory cytokines tumour necrosis factor alpha and IL-6 (by 83 +/- 8% and 84 +/- 11% respectively), while the levels of the anti-inflammatory cytokine IL-10 were significantly increased (196 +/- 55%). The indices of gastrointestinal damage in IndOH-NC-treated animals were significantly less that those after IndOH treatment (58 +/- 16%, 72 +/- 6% and 69 +/- 2%, for duodenum, jejunum and ileum respectively). CONCLUSIONS AND IMPLICATIONS: IndOH-NC produced an increased anti-inflammatory efficacy in long-term models of inflammation, allied to an improved gastrointestinal safety. This formulation might represent a promising alternative for treating chronic inflammatory diseases, with reduced undesirable effects.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Indometacina/uso terapêutico , Inflamação/tratamento farmacológico , Nanocápsulas/uso terapêutico , Animais , Anti-Inflamatórios/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Indometacina/efeitos adversos , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND AND PURPOSE: Kinins are implicated in many pathophysiological conditions, and recent evidence has suggested their involvement in colitis. This study assessed the role of the kinin B1 receptors in a mouse model of colitis. EXPERIMENTAL APPROACH: Colitis was induced in mice by 2,4,6-trinitrobenzene sulphonic acid (TNBS), and tissue damage and myeloperoxidase activity were assessed. B1 receptor induction was analysed by organ bath studies, binding assay and reverse transcription PCR. KEY RESULTS: TNBS-induced colitis was associated with tissue damage, neutrophil infiltration and time-dependent increase of colon B1 receptor-mediated contraction, with the maximal response observed at 72 h. The upregulation of the B1 receptor at this time point was also confirmed by means of binding studies. B1 receptor mRNA levels were elevated as early as 6 h after colitis induction and remained high for up to 48 h. TNBS-evoked tissue damage and neutrophil influx were reduced by the selective B1 receptor antagonist SSR240612, and in B1 receptor knockout mice. In vivo treatment with inhibitors of protein synthesis, nuclear factor-kappaB activation, inducible nitric oxide synthase (iNOS) or tumour necrosis factor alpha (TNFalpha) significantly reduced B1 receptor agonist-induced contraction. Similar results were observed in iNOS and TNF receptor 1-knockout mice. CONCLUSIONS AND IMPLICATIONS: These results provide convincing evidence on the role of B1 receptors in the pathogenesis of colitis. Therefore, the blockade of kinin B1 receptors might represent a new therapeutic option for treating inflammatory bowel diseases.
Assuntos
Colite/fisiopatologia , Receptor B1 da Bradicinina/fisiologia , Animais , Colite/induzido quimicamente , Colite/genética , Colo/patologia , Técnicas In Vitro , Indicadores e Reagentes , Calidina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Óxido Nítrico Sintase Tipo II/biossíntese , Peroxidase/metabolismo , Receptor B1 da Bradicinina/biossíntese , Receptor B1 da Bradicinina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Trinitrobenzenossulfônico , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/fisiologiaRESUMO
BACKGROUND AND AIMS: It has recently been described that bradykinin B(2) receptors are expressed in the human gallbladder and that their activation induces a powerful contraction, especially in acute cholecystitis tissues. Here the role of the B(1) receptor in the contractility of control and inflamed human gallbladder was investigated. METHODS: Strips of human gallbladder from either acute gallstone cholecystitis or elective gastro-entero-pancreatic surgery (control) were assessed in vitro and processed for reverse transcription-PCR analysis. Cumulative concentration-response curves with the selective B(1) receptor agonist, Lys-Des-Arg(9)-bradykinin, cholecystokinin and carbachol were performed in control and cholecystitis specimens. RESULTS: Lys-Des-Arg(9)-bradykinin concentration-dependently contracted strips of control gallbladders and its motor effect was higher in inflamed gallbladders. Lys-Des-Arg(9)-bradykinin-induced contraction was not altered by pretreatment with the selective bradykinin B(2) receptor antagonist, HOE140 (1 microM), the NK(1) (SR140333), NK(2) (SR48968) and NK(3) (SR142801) tachykinin receptor antagonists (all 1 microM), the muscarinic acetylcholine receptor antagonist, atropine (1 microM), and the cyclo-oxygenase inhibitor, indomethacin (5 microM). In contrast, the Lys-Des-Arg(9)-bradykinin-induced motor response was significantly reduced by the selective B(1) receptor antagonist, R-715. Finally, quantitative real-time PCR analysis indicated that B(1) receptor mRNA levels were significantly higher in cholecystitis smooth muscle specimens, when compared with that observed in control tissues. CONCLUSIONS: Bradykinin B(1) receptor has an important role as a spasmogen of human gallbladder, and selective antagonists of the B(1) receptor may represent a valid therapeutic option to control pain in patients with acute cholecystitis.
Assuntos
Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Colecistite/tratamento farmacológico , Vesícula Biliar/efeitos dos fármacos , Adulto , Idoso , Atropina/farmacologia , Colecistite/metabolismo , Inibidores de Ciclo-Oxigenase/farmacologia , Feminino , Vesícula Biliar/fisiologia , Humanos , Indometacina/farmacologia , Masculino , Pessoa de Meia-Idade , Antagonistas Muscarínicos/farmacologia , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The kinin system can contribute distinctly to the sensory changes associated with different models of nerve injury-induced neuropathic pain. This study examines the roles of kinin B(1) and B(2) receptor-operated mechanisms in alterations in nociceptive responses of rats submitted to unilateral L5/L6 spinal nerve ligation (SNL) injury. Behavioural responses to ipsilateral hind paw stimulation with acetone (evaporation-evoked cooling), radiant heat (Hargreaves method) or von Frey hairs revealed that SNL rats developed long-lasting cold allodynia (from Days 3 to 40 post-surgery, peak on Day 6), heat hyperalgesia (stable peak from Days 9 to 36) and tactile allodynia (stable peak from Days 3 to 51). SNL rats manifested nocifensive responses to intraplantar injections on Day 12 of the selective B(1) receptor agonist des-Arg(9)-bradykinin (DABK) and augmented responses to the selective B(2) receptor agonist bradykinin (BK; each at 0.01-1nmol/paw). Systemic treatment of SNL rats with des-Arg(9)-Leu(8)-BK or HOE 140 (peptidic B(1) and B(2) receptor antagonists, respectively; 0.1-1mumol/kg, i.p.) selectively blocked responses triggered by DABK and BK (1nmol/paw) and alleviated partially and transiently established cold allodynia, heat hyperalgesia and (to a lesser extent) tactile allodynia. Western blot analysis revealed enhanced expression of kinin B(1) and B(2) receptor protein in ipsilateral L4-L6 spinal nerve and hind paw skin samples collected on Day 12 after SNL surgery. These results indicate that peripheral pronociceptive kinin B(1) and B(2) receptor-operated mechanisms contribute significantly to the maintenance of hind paw cold and mechanical allodynia and heat hyperalgesia induced by L5/L6 SNL in rats.
Assuntos
Neuralgia/fisiopatologia , Receptor B1 da Bradicinina/fisiologia , Receptor B2 da Bradicinina/fisiologia , Nervos Espinhais/fisiologia , Animais , Comportamento Animal , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina , Antagonistas de Receptor B2 da Bradicinina , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Lateralidade Funcional , Hiperalgesia/fisiopatologia , Ligadura , Masculino , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Wistar , Receptor B1 da Bradicinina/agonistas , Receptor B2 da Bradicinina/agonistas , Nervos Espinhais/efeitos dos fármacos , Nervos Espinhais/lesões , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: alpha-Humulene and trans-caryophyllene are sesquiterpene compounds identified in the essential oil of Cordia verbenacea which display topical and systemic anti-inflammatory effects in different experimental models. However, the molecular mechanisms through which they exert their anti-inflammatory activity still remain unclear. Here, we evaluate the effects of alpha-humulene and trans-caryophyllene on the acute inflammatory responses elicited by LPS. EXPERIMENTAL APPROACH: The biological activities of alpha-humulene and trans-caryophyllene were investigated in a model of acute inflammation in rat paw, induced by LPS and characterized by paw oedema, neutrophil recruitment, cytokine production, activation of MAP kinases and NF-kappaB and up-regulated expression of kinin B(1) receptors. KEY RESULTS: Treatment with either alpha-humulene or trans-caryophyllene effectively reduced neutrophil migration and activation of NF-kappaB induced by LPS in the rat paw. However, only alpha-humulene significantly reduced the increase in TNF-alpha and IL-1beta levels, paw oedema and the up-regulation of B(1) receptors following treatment with LPS. Both compounds failed to interfere with the activation of the MAP kinases, ERK, p38 and JNK. CONCLUSIONS AND IMPLICATIONS: Both alpha-humulene and trans-caryophyllene inhibit the LPS-induced NF-kappaB activation and neutrophil migration, although only alpha-humulene had the ability to prevent the production of pro-inflammatory cytokines TNF-alpha and IL-1beta and the in vivo up-regulation of kinin B(1) receptors. These data provide additional molecular and functional insights into the beneficial effects of the sesquiterpenes alpha-humulene and trans-caryophyllene isolated from the essential oil of Cordia verbenacea as agents for the management of inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides , Cordia/química , Edema/induzido quimicamente , Edema/prevenção & controle , Lipopolissacarídeos , Óleos Voláteis/farmacologia , Animais , Western Blotting , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Edema/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Pé/patologia , Interleucina-1beta/biossíntese , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Peroxidase/metabolismo , Sesquiterpenos Policíclicos , Ratos , Ratos Wistar , Receptor B1 da Bradicinina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sesquiterpenos/farmacologia , Fator de Necrose Tumoral alfa/biossíntese , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Receptor subtypes involved in PGE(2)-induced nociception are still controversial. The present study investigated the prostanoid E receptor (EP) subtypes and the protein kinase (PK) pathways involved in the nociception induced by PGE(2) injection in the mouse paw. EXPERIMENTAL APPROACH: Paw-licking and mechanical allodynia were measured in vivo and protein kinase activation ex vivo by Western blots of extracts of paw skin. KEY RESULTS: Intraplantar (i.pl.) injection of PGE(2) into the mouse paw caused nociceptive behaviour of short duration with mean ED(50) of 1.43 nmol. PGE(2) produced a longer-lasting mechanical allodynia, with an ED(50) of 0.05 nmol. Intraplantar injection of antagonists at EP(3) or EP(4), but not at EP(1) or EP(2) receptors inhibited PGE(2)-induced paw-licking. Paw-licking caused by PGE(2) was blocked by an inhibitor of PKA but only partially decreased by inhibition of the extracellular-regulated kinase (ERK). Selective inhibitors of PKC, c-Jun N-terminal kinase (JNK) or p38, all failed to affect PGE(2)-induced paw-licking. An EP(3) antagonist inhibited PGE(2)-induced mechanical allodynia. However, inhibitors of PKA, PKC or ERK, but not p38 or JNK, also partially inhibited PGE(2)-induced mechanical allodynia. Western blot analyses confirmed that i.pl. injection of PGE(2) activated PKA, PKCalpha, and mitogen activated kinases (MAPKs) in the paw. Co-treatment with EP(3) or EP(4) receptor antagonists reduced PGE(2)-induced PKA and ERK, but not PKCalpha activation. CONCLUSIONS AND IMPLICATIONS: The present results indicate that the nociceptive behaviour and mechanical allodynia caused by i.pl. PGE(2) are mediated through activation of distinct EP receptors and PK-dependent mechanisms.
Assuntos
Dinoprostona/administração & dosagem , Nociceptores/efeitos dos fármacos , Proteínas Quinases/metabolismo , Receptores de Prostaglandina E/fisiologia , Acrilamidas/administração & dosagem , Animais , Carragenina/administração & dosagem , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Naftalenos/administração & dosagem , Nociceptores/fisiologia , Medição da Dor , Prostaglandinas E Sintéticas/administração & dosagem , Proteína Quinase C/metabolismo , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E/classificação , Estresse MecânicoRESUMO
Changes in responsiveness of the vas deferens and urinary bladder to bradykinin (BK) receptor agonists (Tyr8-BK and des-Arg9-BK), substance P (SP), and endothelin-1 (ET-1) were assessed 8 weeks after streptozotocin (STZ)-induced diabetes. Preparations from control or STZ-treated (60 mg/kg i.p.) male rats were tested for contractile and neurogenic twitch potentiating (TP, in VD only) effects of all four agonists (1 nM to 0.3 or 3 microM). In diabetic VD, contractile effects of Tyr8-BK, des-Arg9-BK, and SP were enhanced, but ET-1 effects were unchanged. In contrast, TP by des-Arg9-BK was unaffected, that by Tyr8-BK was decreased, and those by SP and ET-1 were increased. In diabetic UB, only contractions to des-Arg9-BK and SP were enhanced. Following insulin replacement (human, 1-3 U/day s.c.), starting 1 week after STZ, TP induced by Tyr8-BK and des-Arg9-BK in VD were further inhibited, but all other changes in both preparations were reversed at least partially. Insulin treatment of nondiabetic rats, however, also affected VD (but not UB) responsiveness, such that contractions to Tyr8-BK and TP by ET-1 were increased, but TP by Tyr8-BK was decreased. Thus, STZ-induced type I diabetes causes important alterations in responsiveness of non-vascular smooth muscle tissues of the rat to BK, SP, and ET-1. Long term insulin replacement, at doses normalising glycaemia, effectively reversed most changes in VD or UB responsiveness, but it is unclear if this is truly due to blocking of STZ-induced changes, since the treatment also affected responsiveness of nondiabetic tissues.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Insulina/farmacologia , Peptídeos/farmacologia , Bexiga Urinária/fisiologia , Ducto Deferente/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Endotelina-1/farmacologia , Endotelina-1/fisiologia , Técnicas In Vitro , Masculino , Peptídeos/fisiologia , Ratos , Ratos Wistar , Receptores da Bradicinina/agonistas , Receptores da Bradicinina/fisiologia , Substância P/farmacologia , Substância P/fisiologia , Bexiga Urinária/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
OBJECTIVE: This study evaluates the action of celecoxib and rofecoxib, two selective cyclooxygenase-2 (COX-2) inhibitors in two acute models of inflammation, carrageenan (Cg)-induced rat pleurisy, and paw oedema formation. MATERIAL: Male Wistar rats (N = 4-10 per group) were used. A fixed volume of PBS or carrageenan was injected into the pleural cavity or into the paw. Furthermore, the myeloperoxidase (MPO) activity and the levels of nitrite/nitrate (NOx), interleukin-1beta (IL-1beta), tumor necrosis factor-a (TNF-a) and PGE2 were also assessed in the paw tissue or in pleural exudate. RESULTS: Dexamethasone (DEX, 0.5 mg kg(-1), s.c., -4 h) and indomethacin (INDO, 3 mg kg(-1), p.o., -1 h) suppressed Cg-induced pleural exudate accumulation by 84 and 77% and inflammatory cell influx by 66 and 47%, respectively. In contrast, celecoxib (CLX, 10 mg kg(-1), p.o., -1 h) or rofecoxib (RFX, 10 mg kg(-1) , p.o., -1 h) only reduced the Cg-induced pleural exudate volume by 44 and 40%, respectively, but had no significant effect over inflammatory cell influx. At the same doses used for pleurisy, DEX, INDO, CLX, RFX and SC-560 (a selective COX-1 inhibitor, 40 mg kg(-1), p.o., -1 h), inhibited the Cg-induced paw oedema by 49, 31, 21, 21 and 17%. DEX, INDO or SC-560 reduced the level of MPO by 71, 78 and 59%, while CLX or RFX produced a small, but significant increase (28 or 16%) in MPO activity. In the rat model of pleurisy, PGE2 levels in cell-free exudates were significantly attenuated by 91, 89, 57 and 65% in animals treated with DEX, INDO, CLX or RFX. In contrast, INDO reduced significantly the whole bloodTXB, synthesis (59%) while DEX and INDO reduced the pleural content of NOx significantly. Treatment of animals with CLX or RFX did not alter the content of pro-inflammatory cytokines IL-1beta or TNF-alpha in the pleural exudate, but CLX reduced IL-1beta levels in the rat paw tissue and RFX increased TNF-alpha in this tissue. CONCLUSION: Together these results provide consistent evidence indicating that the selective COX-2 inhibitors CLX and RFX, in contrast to DEX, INDO or SC-560, despite reducing greatly the Cg-induced pleural exudation, PGE2 content and paw oedema have only partial acute anti-inflammatory properties in two different rat acute models of inflammation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Inflamação/tratamento farmacológico , Isoenzimas/metabolismo , Lactonas/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Sulfonamidas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Carragenina , Celecoxib , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Dexametasona/farmacologia , Dinoprostona/metabolismo , Edema/induzido quimicamente , Edema/patologia , Indometacina/farmacologia , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucina-1/metabolismo , Masculino , Óxido Nítrico/metabolismo , Peroxidase/metabolismo , Pleurisia/induzido quimicamente , Pleurisia/tratamento farmacológico , Pleurisia/patologia , Pirazóis , Ratos , Ratos Wistar , Sulfonas , Tromboxano B2/sangue , Fator de Necrose Tumoral alfa/metabolismoRESUMO
This study investigates the antinociception caused by i.p. and p.o. administration of ether fraction and the triterpene identified as urs-12-ene-3beta-16beta-diol, known as Brein, isolated from Protium kleinii in several models of nociception in mice. The systemic administration of ether fraction (0.3 to 10 mg/kg, i.p. or 3 to 60 mg/kg, p.o.) caused a dose-related antinociception when assessed against acetic acid-induced writhing, with mean ID50 values of 1.2 and 16.4 mg/kg, respectively. The ether fraction (5 to 60 mg/kg, i.p. or 30 to 300 mg/kg, p.o.) also produced dose-related inhibition of both phases of formalin induced licking. The mean ID50s values for the early phase were > 60.0 and 62.1 mg/kg, while for the late phase they were 15.4 and 60.0 mg/kg, respectively, given by i.p. and p.o. routes. The ether fraction (3 to 30 mg/kg, i.p. or 10 to 100 mg/kg, p.o.) produced significant inhibition of the neurogenic nociception caused by topical injection of capsaicin, with mean ID50 values of 6.2 and 16.0 mg/kg, respectively. Given orally (1 to 30 mg/kg) the ether fraction produced graded and pronounced inhibition of glutamate-induced hyperalgesia in mice with a mean ID50 value of 15.2 mg/kg. In contrast, the ether fraction failed to produce antinociception when assessed in the thermal model of pain, the tail flick and hot plate tests. The antinociception caused by the ether fraction, in contrast to that of morphine, was not reversed by naloxone when assessed in the formalin-induced licking. The ether fraction did not affect motor coordination or the core body temperature in mices. The triterpene Brein isolated from P. kleinii, given by i.p. route (10 to 100 mg/kg) produced dose-related inhibition of both phases of formalin induced-licking, with mean ID50s values of 15.3 and 20.6 for the early and the late phases, respectively. These data show that the active principle(s) present in the ether fraction from the resin of P. kleinii elicited pronounced antinociception when assessed by i.p. or p.o routes, against both inflammatory and neurogenic nociception. Such effects seem, at least in part, to be related to the presence of the triterpene Brein in the extract. The mechanisms responsible for the antinociceptive action are at this moment not completely understood, but the involvement of the opioid pathway seems unlikely.
Assuntos
Analgésicos/farmacologia , Dor/tratamento farmacológico , Triterpenos/farmacologia , Analgésicos/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Camundongos , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Resinas Vegetais/farmacologia , Resinas Vegetais/uso terapêutico , Triterpenos/uso terapêuticoRESUMO
We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 æM and Emax of 100 percent (N = 10) or 20 æM and Emax of 92 percent (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 + or - 7.0, 43 + or - 3.9 and 78 + or - 5.6 percent) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 æM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 æM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 + or - 12 percent. Glibenclamide (1 or 3 æM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 æM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 æM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 æM, while methylene blue (10 or 30 æM) or ODQ (1 æM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 æM), a VIP receptor antagonist, significantly inhibited (37 + or - 7 percent) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 æM), a CGRP antagonist, only slightly enhanced the action of gentisic acid.
Assuntos
Animais , Masculino , Feminino , Cobaias , Hidroxibenzoatos/farmacologia , Técnicas In Vitro , Relaxamento Muscular/efeitos dos fármacos , Canais de Potássio/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Traqueia/efeitos dos fármacos , Epitélio/fisiologia , Cobaias , Músculo Liso/efeitos dos fármacos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologiaRESUMO
We describe here the modulation caused by intrapleural (i.pl.) injection of the cytokines TNF-alpha and IL-1beta and their specific antibodies in the early (4 h) and late (48 h) inflammatory responses caused by injection of carrageenan (Cg) into the mouse pleural cavity. The antibodies against TNF-alpha and IL-1beta, when injected 30 min prior to Cg, reduced, in a graded and significant manner, both exudation and cell migration in the early (4 h) phase, while they potentiated or had no effect in the late (48 h) phase of Cg response. The natural IL-1 receptor antagonist IL-1RA, given 30 min prior to Cg, reduced the exudation by about 50% and abolished the total and differential cell migration in the early (4 h) and late (48 h) phases of the Cg responses. The i.pl. injection of TNF-alpha or IL-1beta, 5 min prior to Cg, caused graded increase in the exudation of the early (4 h) and late (48 h) phases of the Cg-induced inflammatory responses. In contrast, these treatments markedly reduced the total and differential cell migration at 4 h, while having little or no effect on the late (48 h) phase of the Cg pleurisy. These findings extend previous results and demonstrate that the pro-inflammatory cytokines TNF-alpha and IL-1beta have a critical role in controlling both cell migration and exudation caused by injection of Cg in the mouse pleural cavity. Together, these findings may be relevant to the understanding of the mechanisms involved in airway inflammatory responses.
Assuntos
Carragenina/administração & dosagem , Interleucina-1/fisiologia , Pleurisia/imunologia , Pleurisia/patologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Movimento Celular/imunologia , Modelos Animais de Doenças , Feminino , Inflamação/imunologia , Inflamação/patologia , Injeções , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/imunologia , Interleucina-1/metabolismo , Masculino , Camundongos , Pleura , Pleurisia/induzido quimicamente , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/administração & dosagem , Fator de Necrose Tumoral alfa/imunologiaRESUMO
This review highlights the current advances in knowledge about the safety, efficacy, quality control, marketing and regulatory aspects of botanical medicines. Phytotherapeutic agents are standardized herbal preparations consisting of complex mixtures of one or more plants which contain as active ingredients plant parts or plant material in the crude or processed state. A marked growth in the worldwide phytotherapeutic market has occurred over the last 15 years. For the European and USA markets alone, this will reach about $7 billion and $5 billion per annum, respectively, in 1999, and has thus attracted the interest of most large pharmaceutical companies. Insufficient data exist for most plants to guarantee their quality, efficacy and safety. The idea that herbal drugs are safe and free from side effects is false. Plants contain hundreds of constituents and some of them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs, digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of phytotherapeutic agents are less frequent compared with synthetic drugs, but well-controlled clinical trials have now confirmed that such effects really exist. Several regulatory models for herbal medicines are currently available including prescription drugs, over-the-counter substances, traditional medicines and dietary supplements. Harmonization and improvement in the processes of regulation is needed, and the general tendency is to perpetuate the German Commission E experience, which combines scientific studies and traditional knowledge (monographs). Finally, the trend in the domestication, production and biotechnological studies and genetic improvement of medicinal plants, instead of the use of plants harvested in the wild, will offer great advantages, since it will be possible to obtain uniform and high quality raw materials which are fundamental to the efficacy and safety of herbal drugs.
Assuntos
Fitoterapia , Ensaios Clínicos Controlados como Assunto , Países Desenvolvidos , Países em Desenvolvimento , Aprovação de Drogas , Guias como Assunto , Legislação de Medicamentos , Controle de QualidadeRESUMO
This review highlights the current advances in knowledge about the safety, efficacy, quality control, marketing and regulatory aspects of botanical medicines. Phytotherapeutic agents are standardized herbal preparations consisting of complex mixtures of one or more plants which contain as active ingredients plant parts or plant material in the crude or processed state. A marked growth in the worldwide phytotherapeutic market has occurred over the last 15 years. For the European and USA markets alone, this will reach about $7 billion and $5 billion per annum, respectively, in 1999, and has thus attracted the interest of most large pharmaceutical companies. Insufficient data exist for most plants to guarantee their quality, efficacy and safety. The idea that herbal drugs are safe and free from side effects is false. Plants contain hundreds of constituents and some of them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs, digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of phytotherapeutic agents are less frequent compared with synthetic drugs, but well-controlled clinical trials have now confirmed that such effects really exist. Several regulatory models for herbal medicines are currently available including prescription drugs, over-the-counter substances, traditional medicines and dietary supplements. Harmonization and improvement in the processes of regulation is needed, and the general tendency is to perpetuate the German Commission E experience, which combines scientific studies and traditional knowledge (monographs). Finally, the trend in the domestication, production and biotechnological studies and genetic improvement of medicinal plants, instead of the use of plants harvested in the wild, will offer great advantages, since it will be possible to obtain uniform and high quality raw materials which are fundamental to the efficacy and safety of herbal drugs
Assuntos
Qualidade de Produtos para o Consumidor , Indústria Farmacêutica , Medicina Herbária , Ensaios Clínicos como Assunto , Países Desenvolvidos , Países em Desenvolvimento , Aprovação de Drogas , Guias como Assunto , Controle de QualidadeRESUMO
Bradykinin (BK) induces increases in cytosolic calcium concentration [Ca++]i in several cell lines. Because the role of BK in the renal system, particularly in mesangial cell (MC), is not clear, we investigated the effects of kinins on [Ca++]i in mouse-immortalized MC. [Ca++]i was evaluated by spectrofluorometry and expressed as a ratio between the obtained and basal [Ca++]i. BK (0.1 microM) induced a non-sustained increase in [Ca++]i (4.70 +/- 0.27; N = 28). A similar effect was observed with the B2 receptor agonist, Tyr8-BK (0.1 microM, 3.34 +/- 0.48; N = 7), while B1 receptor agonists, des-Arg10-Kallidin (Kal) (1 microM, N = 11) and des-Arg9-BK (1 microM, N = 8), exhibited only discrete responses (1.45 +/- 0.08 and 1.12 +/- 0.04, respectively). Cross-desensitization was seen between BK and Tyr8-BK, but not between BK and des-Arg10-Kal. The BK response was decreased (5.09 +/- 0.30, N = 6 to 1.57 +/- 0.12, N = 7, P < 0.001) by the B2 receptor antagonist HOE 140 (0.1 microM, 15 min), while the B1 receptor antagonist des-Arg9-[Leu8]-BK (1 microM, 15 min) had no effect on BK or des-Arg10-Kal actions. Incubation of cells with Escherichia coli lipopolysaccharide (100 microg/ml, 24 h) alone or in association with tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml, N = 6) did not enhance B1 agonist responses. BK was inhibited by repeated cell washouts in zero Ca++ solution (2.04 +/- 0.19, N = 6 P < 0.001), and the residual response was almost abolished by thapsigargin (Thaps) a sarcoplasmic reticulum (SR) calcium-ATPase inhibitor (1 microM) (1.18 +/- 0.08, N = 5 P < 0.001). Additionally, BK was not inhibited by verapamil (50 microM), nifedipine (30 microM), Ni++ (300 microM) or La (10 microM). In conclusion, BK induces [Ca++]i in mouse MC mainly by B2 receptor activation. B1 receptors have a minor role in this phenomenon even in the presence of known B1 receptor synthesis inducers. Finally, BK mobilizes extracellular calcium sources and, to a lesser extent, intracellular Thaps-sensitive calcium stores. The ion channels involved in calcium influx remain to be detected.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Mesângio Glomerular/efeitos dos fármacos , Mesângio Glomerular/metabolismo , Cininas/farmacologia , Animais , Linhagem Celular , Linhagem Celular Transformada , Citosol/efeitos dos fármacos , Mesângio Glomerular/citologia , Camundongos , Receptores da Bradicinina/metabolismoRESUMO
1. Intradermal (i.d.) injection of cytokines, IL-1beta and TNFalpha (5 ng, 60 and 30 min prior) produces a rapid onset up-regulation of des-Arg9-BK-mediated rat paw oedema. Here we analyse the mechanisms involved in des-Arg9-BK-induced oedema in animals pre-treated with IL-1beta or TNFalpha. 2. Co-injection of anti-IL-1beta, anti-TNFalpha and anti-IL-8 (50 ng) significantly inhibited des-Arg9-BK-induced oedema in animals pre-treated with IL-1beta (65, 37 and 42%) or TNFalpha (39, 64, 25%). IL-1 receptor antagonist (IRA, 100 microg) or IL-10 (10 ng) inhibited the oedema caused by des-Arg9-BK, in rats that had received either IL-1beta (67 and 63%) or TNFalpha (46 and 35%). 3. Co-injection of the PKC inhibitors, staurosporine (10 nmol) or RO 318220 (30 nmol) inhibited des-Arg9-BK-induced paw oedema (44 and 42% for IL-1beta and, 53 and 30% for TNFalpha, respectively). Genistein (tyrosine kinase inhibitor, 2.5 mg kg-1, s.c.) or PD 098059 (MAP-kinase inhibitor, 30 nmol) produced marked inhibition of des-Arg9-BK-induced oedema (58 and 39% for IL-1beta and 31 and 35% for TNFalpha respectively). 4. The NF-kappaB inhibitors TLCK (2 mg kg-1, i.p.) and PDCT (100 mg kg-1, i.p.) significantly inhibited the oedema of des-Arg9-BK in IL-1beta (27 and 83%) or TNFalpha (28 and 80%) pre-treated animals. 5. It is concluded that up-regulation of B1 receptors modulated by IL-1beta or TNFalpha involves the release of other cytokines, activation of PKC and tyrosine kinase pathways, co-ordinated with the activation of MAP-kinase and nuclear factor kappaB, reinforcing the view that B1 receptors may exert a pivotal role in modulating chronic inflammatory processes.