RESUMO
Achieving durable protective immunity following vaccination is dependent on many factors, including vaccine composition and antigen dose, and it has been investigated for various types of vaccines. Aim of the present study was to investigate the overall immune response elicited by two different booster doses in CD-1 mice, by exploiting the largely used 13-valent pneumococcal conjugate vaccine Prevnar 13® (PCV13). Immunization was performed by two primary doses of PCV13 two weeks apart, and a full or fractional (1/5) booster dose on week 10. Serotype-specific antibody titer, avidity, and opsonophagocytic activity were evaluated one week later, and compared to cell-mediated immunity (CMI) responses determined as the frequency of cytokines producing splenocytes by in vitro recall with the antigens (carrier protein and polysaccharides). Data showed that regardless of the booster dose, a comparable humoral response was produced, characterized by similar amounts of serotype-specific antibodies, with analog avidity and opsonophagocytic properties. On the other hand, when CMI was evaluated, the presence of CRM197-specific IL-5 and IL-2 producing cells was evident in splenocytes from mice immunized with the full dose, while in those immunized with the fractional booster dose, IFN-γ producing cells responsive to both protein and polysaccharide antigens were significantly increased, whereas the number of IL-5 and IL-2 positive cells remained unaffected. Overall the present findings show that PCV13 humoral response in mice is associated to a Th2 predominant response at the full booster dose, while the fractional one favors a mixed Th1/Th2 response, suggesting an important role of CMI besides measurement of functional protective antibodies, as an additional and important key information in vaccine development.
RESUMO
Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1ß enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.
Assuntos
Sinalização do Cálcio/fisiologia , Citocinas/metabolismo , Prostaglandinas/metabolismo , Receptor B2 da Bradicinina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Bradicinina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Humanos , Mediadores da Inflamação/metabolismo , Receptor B2 da Bradicinina/agonistas , Epitélio Pigmentado da Retina/efeitos dos fármacosRESUMO
Dexketoprofen and tramadol, alone or in combination, were evaluated after oral or intra-articular administration on knee osteoarthritis nociception induced by intra-articular (i.ar.) monosodium iodoacetate (MIA, 1 mg/25 µl) in the rat right knee while the left knee received saline (25 µl). Seven days after MIA treatment, dexketoprofen, tramadol, their combination or the vehicle were administered. Nociception was evaluated as alteration in hind limb weight distribution with Incapacitance tester at different time-points after drug administration. Oral dexketoprofen (0.1-1 mg/kg) or tramadol (0.5-5 mg/kg) induced maximal antinociception at 1 and 5 mg/kg, respectively. Their combination dose-dependently increased the intensity and duration of antinociception, that was additive and lasted up to 3 days. Also the intra-articular administration of dexketoprofen or tramadol (10-100 µg/25 µl) inhibited MIA-induced nociception, and the combination of the lower doses (10 µg/25 µl) produced a long lasting more than additive antinociceptive effect indicating a synergistic interaction between the two drugs. This effect was significantly reduced by naloxone (10 µg/25 µl, i.ar.) co-administered with both compounds. The intra-articular administration of both drugs at 10 µg/25 µl in the contralateral control knee joint provoked a marked synergistic antinociceptive effect indicating significant systemic diffusion through synovial membrane. The oral or intra-articular combination of dexketoprofen and tramadol produced additive or synergistic antinociceptive effects, respectively, in the model of MIA-induced osteoarthritis in rats, that might allow to obtain therapeutic advantages with lower side effects.
Assuntos
Ácido Iodoacético/efeitos adversos , Cetoprofeno/análogos & derivados , Osteoartrite/induzido quimicamente , Osteoartrite/tratamento farmacológico , Tramadol/administração & dosagem , Tramadol/farmacologia , Trometamina/administração & dosagem , Trometamina/farmacologia , Administração Oral , Analgésicos/administração & dosagem , Analgésicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Cetoprofeno/administração & dosagem , Cetoprofeno/farmacologia , Articulação do Joelho/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
Obesity and adipose tissue contribute to local and systemic inflammation. However the role of the inflammatory mediator bradykinin (BK) in this context is not known. We therefore evaluated the effect of BK on adipokines secretion in human preadipocytes during the course of differentiation and characterized the receptors involved. Results obtained from antibody array and ELISA experiments showed that several adipokines are released by human preadipocytes under basal conditions while BK specifically stimulated the production of interleukin(IL)-6 and IL-8. The effect of BK diminished with the progression of differentiation, being almost inactive on adipocytes. In preadipocytes, BK also induced a rapid and transient [Ca²âº](i) mobilization, a rapid and sustained increase in ERK1/2 activation and enhanced forskolin-stimulated cAMP accumulation. BK was without effect on cell proliferation and viability as assessed by bromodeoxyuridine incorporation, WST-1 conversion, or lactate dehydrogenase leakage and was without effect on adipogenesis as measured by triglyceride accumulation, GPDH activity and leptin release. The B1 receptor agonist, Lys-[des-Arg9]-BK, displayed poor activity or was without effect while overall BK effects were prevented by the selective B2 receptor antagonist, fasitibant chloride, but not by the B1 selective antagonist, Lys-[Leu8][des-Arg9]-BK. Immunoblot analysis and immunofluorescence studies showed that the kinin B2 receptor was essentially expressed at the beginning of the differentiation program. In conclusion, human preadipocytes expressed kinin B2 receptors linked to multiple signaling pathways, IL-6 and IL-8 production, and BK proinflammatory response in adipose tissue could be prevented by fasitibant chloride.
Assuntos
Adipócitos/metabolismo , Adipocinas/metabolismo , Bradicinina/metabolismo , Citocinas/metabolismo , Receptor B2 da Bradicinina/metabolismo , Células-Tronco/metabolismo , Adipócitos/citologia , Bradicinina/farmacologia , Cálcio/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , AMP Cíclico/biossíntese , Glicerolfosfato Desidrogenase/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leptina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais , Células-Tronco/citologia , Triglicerídeos/metabolismoRESUMO
A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R'). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.
Assuntos
Antagonistas de Receptor B2 da Bradicinina , Glicina/análogos & derivados , Glicina/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Bradicinina/metabolismo , Broncoconstrição/efeitos dos fármacos , Células CHO , Cricetinae , Glicina/síntese química , Cobaias , Humanos , Hipotensão/tratamento farmacológico , Receptor B2 da Bradicinina/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/síntese químicaRESUMO
BACKGROUND AND PURPOSE: Bradykinin, through the kinin B2 receptor, is involved in inflammatory processes related to arthropathies. B2 receptor antagonists inhibited carrageenan-induced arthritis in rats in synergy with anti-inflammatory steroids. The mechanism(s) underlying this drug interaction was investigated. EXPERIMENTAL APPROACH: Drugs inhibiting inflammatory mediators released by carrageenan were injected, alone or in combination, into the knee joint of pentobarbital anaesthetized rats 30 min before intra-articular administration of carrageenan. Their effects on the carrageenan-induced inflammatory responses (joint pain, oedema and neutrophil recruitment) and release of inflammatory mediators (prostaglandins, IL-1ß, IL-6 and the chemokine GRO/CINC-1), were assessed after 6 h. KEY RESULTS: The combination of fasitibant chloride (MEN16132) and dexamethasone was more effective than each drug administered alone in inhibiting knee joint inflammation and release of inflammatory mediators. Fasitibant chloride, MK571, atenolol, des-Arg9-[Leu8]-bradykinin (B2 receptor, leukotriene, catecholamine and B1 receptor antagonists, respectively) and dexketoprofen (COX inhibitor), reduced joint pain and, except for the latter, also diminished joint oedema. A combination of drugs inhibiting joint pain (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, dexketoprofen, MK571 and atenolol) and oedema (fasitibant chloride, des-Arg9-[Leu8]-bradykinin, MK571 and atenolol) abolished the respective inflammatory response, producing inhibition comparable with that achieved with the combination of fasitibant chloride and dexamethasone. MK571 alone was able to block neutrophil recruitment. CONCLUSIONS AND IMPLICATIONS: Bradykinin-mediated inflammatory responses to intra-articular carrageenan were not controlled by steroids, which were not capable of preventing bradykinin effects either by direct activation of the B2 receptor, or through the indirect effects mediated by release of eicosanoids and cytokines.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/prevenção & controle , Antagonistas de Receptor B2 da Bradicinina , Dexametasona/uso terapêutico , Ornitina/análogos & derivados , Sulfonamidas/uso terapêutico , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/fisiopatologia , Citocinas/metabolismo , Sinergismo Farmacológico , Quimioterapia Combinada , Edema/prevenção & controle , Mediadores da Inflamação/metabolismo , Articulações/efeitos dos fármacos , Articulações/imunologia , Articulações/patologia , Antagonistas de Leucotrienos/uso terapêutico , Masculino , Infiltração de Neutrófilos/efeitos dos fármacos , Ornitina/uso terapêutico , Propionatos/uso terapêutico , Quinolinas/uso terapêutico , Ratos , Ratos Wistar , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismoRESUMO
Dry and persistent cough is one of the commonest side effects experienced by patients treated with angiotensin-converting enzyme (ACE) inhibitors for the therapy of hypertension and congestive heart failure. The present study investigated the effect of zofenopril and ramipril on cough induced by citric acid in guinea pig and the involvement of bradykinin (BK) and prostaglandin E2 (PGE2) in mediating the responses of these drugs. Zofenopril (10 mg/kg) or ramipril (3-10 mg/kg), which is threefold more potent than zofenopril, on a mg basis, in lowering blood pressure, was orally administered daily in drinking water for 2 weeks. At the end of this period, aerosol of citric acid solution (0.1 M) was performed and the number of cough counted for 10 min. The role of the kinin B(2) receptor was also investigated. BK and PGE2 levels in the bronchoalveolar lavage (BAL) fluid were measured after repeated oral treatment with zofenopril or ramipril (10 mg/kg). Ramipril (3-10 mg/kg) increased citric acid-induced cough by 40% and 60%, respectively, as compared to the vehicle control group (15.0 ± 1.8), while zofenopril (10 mg/kg) was without effect. The enhancement of citric acid-induced cough caused by ramipril (10 mg/kg) was reduced by the kinin B(2) receptor antagonist MEN16132 (0.25 mg/kg ip). BK and PGE2 levels in the BAL fluid were increased, in comparison to the control group, after ramipril treatment, while they were unchanged after zofenopril administration. Zofenopril, contrary to ramipril, did not affect either citric acid-induced cough in the guinea pigs or BK and PGE2 production in the airways.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/fisiologia , Captopril/análogos & derivados , Tosse/fisiopatologia , Dinoprostona/farmacologia , Ramipril/farmacologia , Animais , Antagonistas de Receptor B2 da Bradicinina , Líquido da Lavagem Broncoalveolar/química , Captopril/farmacologia , Ácido Cítrico , Tosse/induzido quimicamente , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Cobaias , Masculino , Ornitina/análogos & derivados , Ornitina/farmacologia , Sulfonamidas/farmacologiaRESUMO
The present study was designed to investigate the role of bradykinin (BK) in the knee joint osteoarthritis induced by intra-articular (i.ar.) administration of monosodium iodoacetate (MIA) in the rat, and to determine the efficacy of the kinin B(2) receptor antagonists, 4-(S)-amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl} piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132) and icatibant, in reducing pain. Rats received MIA (1 mg/25 microl i.ar.) in the right knee. MEN16132, icatibant (1, 3, and 10 microg/25 microl i.ar.), or saline were administered 7 days after MIA treatment, and their antinociceptive effect was observed for 2 weeks. MEN16132 induced a marked and sustained reduction of incapacitation produced by MIA, approximately 56% inhibition of pain at 3 microg/knee. MEN16132 analgesia was more potent and longer lasting, up to 10 days, than icatibant. MEN16132 (3 microg/knee), at different time points from MIA treatment in separate groups of animals, produced comparable maximal antinociceptive effects, whereas the pain response induced by MIA was unaffected if MEN16132 (10 microg/knee) was administered in the contralateral knee. Indomethacin at high doses (100-625 microg/knee) inhibited by approximately 40% but with a short duration the MIA-induced pain. MIA treatment produced a significant increase of BK and prostaglandin E(2) (PGE(2)) metabolite levels in synovial fluid up to 21 days, and PGE(2) metabolite levels were reduced almost to basal values by MEN16132. In conclusion the potent and long-lasting analgesic effect of MEN16132 in MIA-induced osteoarthritis indicates an important role for BK in osteoarthritic pain, and suggests that MEN16132 can be a candidate for the treatment of this chronic disease.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Antagonistas de Receptor B2 da Bradicinina , Ornitina/análogos & derivados , Osteoartrite/tratamento farmacológico , Dor/tratamento farmacológico , Sulfonamidas/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/uso terapêutico , Dinoprostona/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Membro Posterior/fisiopatologia , Injeções Intra-Articulares , Iodoacetatos , Masculino , Ornitina/administração & dosagem , Ornitina/uso terapêutico , Osteoartrite/induzido quimicamente , Osteoartrite/metabolismo , Osteoartrite/fisiopatologia , Dor/metabolismo , Dor/fisiopatologia , Ratos , Ratos Wistar , Sulfonamidas/administração & dosagem , Líquido Sinovial/metabolismoRESUMO
We have investigated the effect of intravaginal application of capsaicin on micturition reflex in female rats. Urinary bladder contractility was measured by transurethral pressure recording at isovolumetric and subthreshold conditions in anaesthetized rats. The intravaginal application of capsaicin (15 microg/50 microl rat) induced reproducible bladder phasic contractions, without desensitization upon repeated applications, that were blocked by intravenous atropine (1 mg/kg) or hexamethonium (5 mg/kg) and prevented by removal of paracervical ganglia or systemic capsaicin pretreatment (125 mg/kg, s.c.). The inhibition of sympathetic transmission by guanethidine (30 mg/kg, s.c.) produced significant increase of the bladder reflex contractions activated by intravaginal capsaicin. Intravenous administration of the TRPV1 antagonist, capsazepine (3 mg/kg), significantly reduced the excitatory reflex response to capsaicin. Intravaginal administration of capsaicin (15 microg/50 microl), during distension-induced reflex bladder contractions, produced a transient block of reflexes, unaffected by guanethidine pretreatment. In conclusion, the stimulation of capsaicin-sensitive sensory nerve endings in the rat cervix-vagina induced a dual excitatory or inhibitory bladder response in anaesthetized female rats depending on the degree of bladder distension.
Assuntos
Capsaicina/farmacologia , Colo do Útero/inervação , Fibras Nervosas/efeitos dos fármacos , Reflexo/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Vagina/inervação , Animais , Atropina/farmacologia , Capsaicina/administração & dosagem , Capsaicina/análogos & derivados , Feminino , Ganglionectomia , Guanetidina/farmacologia , Hexametônio/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/inervação , Músculo Liso/fisiologia , Fibras Nervosas/fisiologia , Ratos , Ratos Wistar , Reflexo/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPV/antagonistas & inibidores , Bexiga Urinária/inervação , Bexiga Urinária/fisiologia , Micção/efeitos dos fármacosRESUMO
Kinins have been suggested to be involved in human airway diseases such as asthma and rhinitis. MEN16132 is a non-peptide kinin B(2) receptor antagonist able to inhibit the responses produced by intravenous bradykinin into the airways, as bronchoconstriction and microvascular leakage; we tested the effect of MEN16132 on endogenously generated bradykinin through the dextran sulfate-induced contact activation of kinin-kallikrein cascade in guinea-pigs. After dextran sulfate administration (1.5 mg/kg i.v.), the pulmonary insufflation pressure was monitored and the microvascular leakage of upper and lower airways was assessed using Evans blue as tracer of plasma protein extravasation. Our results demonstrated that topical MEN16132 strongly inhibited the dextran sulfate-induced bronchoconstriction (0.3 mM solution aerosol for 5 min) and plasma protein extravasation in both lower airways (3-10 microM solution aerosol for 5 min) and nasal mucosa (0.3 nmol/nostril); Icatibant, the peptide antagonist of kinin B(2) receptor, exerted a 3-30-fold less potent inhibitory effect than MEN16132. We conclude that local application of MEN16132 into the airways abolishes the responses produced by the endogenous generation of bradykinin and it can be useful as new pharmacological tool to check the role of kinins in human diseases.
Assuntos
Antagonistas de Receptor B2 da Bradicinina , Bradicinina/efeitos dos fármacos , Broncoconstrição/efeitos dos fármacos , Ornitina/análogos & derivados , Sulfonamidas/farmacologia , Administração Intranasal , Aerossóis , Animais , Proteínas Sanguíneas/metabolismo , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Bradicinina/metabolismo , Bradicinina/farmacologia , Sulfato de Dextrana , Relação Dose-Resposta a Droga , Cobaias , Sistema Calicreína-Cinina/efeitos dos fármacos , Masculino , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Ornitina/administração & dosagem , Ornitina/farmacologia , Sulfonamidas/administração & dosagemRESUMO
Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.
Assuntos
Antagonistas de Receptor B2 da Bradicinina , Broncodilatadores/síntese química , Ornitina/análogos & derivados , Sulfonamidas/síntese química , Animais , Pressão Sanguínea/efeitos dos fármacos , Broncoconstrição/efeitos dos fármacos , Broncodilatadores/química , Broncodilatadores/farmacologia , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Cobaias , Humanos , Íleo/efeitos dos fármacos , Íleo/fisiologia , Técnicas In Vitro , Fosfatos de Inositol/biossíntese , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ornitina/síntese química , Ornitina/química , Ornitina/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2Assuntos
Membrana Celular/efeitos dos fármacos
, Dipeptídeos/farmacologia
, Receptores da Neurocinina-2/antagonistas & inibidores
, Tiofenos/farmacologia
, Idoso
, Animais
, Ligação Competitiva/efeitos dos fármacos
, Broncoconstrição/efeitos dos fármacos
, Células CHO
, Membrana Celular/metabolismo
, Colo/efeitos dos fármacos
, Colo/fisiologia
, Cricetinae
, Cricetulus
, Dipeptídeos/química
, Dipeptídeos/metabolismo
, Relação Dose-Resposta a Droga
, Feminino
, Cobaias
, Humanos
, Técnicas In Vitro
, Masculino
, Camundongos
, Pessoa de Meia-Idade
, Contração Muscular/efeitos dos fármacos
, Neurocinina A/análogos & derivados
, Neurocinina A/farmacologia
, Fragmentos de Peptídeos/farmacologia
, Coelhos
, Ensaio Radioligante
, Ratos
, Ratos Wistar
, Receptores da Neurocinina-2/agonistas
, Receptores da Neurocinina-2/metabolismo
, Suínos
, Tiofenos/química
, Tiofenos/metabolismo
, Vasoconstrição/efeitos dos fármacos
RESUMO
We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.
Assuntos
Antagonistas de Receptor B2 da Bradicinina , Quinolinas/síntese química , Sarcosina/análogos & derivados , Sulfonamidas/síntese química , Animais , Bradicinina/metabolismo , Broncoconstritores/síntese química , Broncoconstritores/química , Broncoconstritores/farmacologia , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , Desenho de Fármacos , Cobaias , Humanos , Hipotensão/induzido quimicamente , Fosfatos de Inositol/biossíntese , Modelos Moleculares , Estrutura Molecular , Piperazina , Piperazinas/química , Quinolinas/química , Quinolinas/farmacologia , Ensaio Radioligante , Receptor B2 da Bradicinina/metabolismo , Sarcosina/síntese química , Sarcosina/química , Sarcosina/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
We have tested the activity of 4-(S)-amino-5-(4-[4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl] piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132), a novel nonpeptide kinin B(2) receptor antagonist, on bradykinin (BK)-induced inflammatory responses, bronchoconstriction, and hypotension in guinea pigs. After i.v. (1-10 nmol/kg i.v.), intratracheal (i.t.) (10-100 nmol/kg i.t.), or aerosol (0.01-0.1 mM/5 min) administration, MEN16132 inhibited in a dose-dependent manner the bronchoconstriction induced by BK (10 nmol/kg i.v.). MEN16132 was more potent and possessed a longer duration of action as compared with the peptide B(2) receptor antagonist icatibant (HOE140; H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH trifluoroacetate). After i.v. administration, its inhibitory effect on bronchoconstriction lasted more than 8 h at 30 nmol/kg. When administered by i.v. or i.t. routes, the dose completely inhibiting bronchoconstriction also partially reduced the hypotensive response to BK, whereas after aerosol administration, the inhibitory effect was limited to respiratory level. Intranasal (i.n.) administration of MEN16132 (0.01-0.3 nmol/nostril) reduced, in a dose-dependent and long-lasting manner, the nasal mucosa plasma protein extravasation induced by BK (100 nmol/nostril), and it exerted a complete inhibition at about 30-fold lower dose than icatibant. At 1 nmol/nostril, MEN16132 activity was significant for at least 6 h with no systemic effect measured as inhibition of BK-induced hypotension, and at 10 nmol/nostril, the inhibitory effect lasted for more than 15 h with only a weak effect on hypotension. These findings indicate that in vivo MEN16132 is a potent kinin B(2) receptor antagonist with long duration of action, both after i.v. and local administration. A complete and prolonged inhibition of BK-induced bronchoconstriction or nasal inflammation can be achieved with MEN16132 topical administration (aerosol or i.n.) at doses devoid of systemic effects.
Assuntos
Antagonistas de Receptor B2 da Bradicinina , Bradicinina/antagonistas & inibidores , Broncoconstrição/efeitos dos fármacos , Mucosa Nasal/irrigação sanguínea , Ornitina/análogos & derivados , Sulfonamidas/farmacologia , Anestesia , Animais , Pressão Sanguínea/efeitos dos fármacos , Proteínas Sanguíneas/metabolismo , Bradicinina/administração & dosagem , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cobaias , Hipotensão/induzido quimicamente , Hipotensão/tratamento farmacológico , Técnicas In Vitro , Injeções Intravenosas , Masculino , Microcirculação/efeitos dos fármacos , Ornitina/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacosRESUMO
Since its introduction a decade ago, the Caco-2 in vitro model for testing intestinal permeability has found wide application, in particular for screening new molecules in the pharmaceutical industry. An important issue for the validation of the model is to verify integrity and proper functionality of the Caco-2 cells monolayer, to ensure reproducibility and consistency of results. Several of these methods are based on measuring the apparent permeability coefficients (Papp) of well-characterized reference compounds, having known absorption characteristics, and comparing the observed values to those expected. Most of them use fluorescent or radioactive chemicals to test different parameters. The main limitation of such approaches is that each parameter to be tested is associated with a single specific method, so that multiple procedures are needed to adequately characterize the cell monolayer. This work describes the use of a unique LC-MS/MS method to simultaneously determine the Papp of a set of reference substances having well-characterized absorption behavior, i.e., phenylalanine, atenolol, and propranolol. The method is routinely used in our laboratory to check on model reproducibility and proved useful in verifying the consistency of the results derived from the experiments.
Assuntos
Absorção Intestinal , Mucosa Intestinal/metabolismo , Preparações Farmacêuticas/análise , Absorção , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Condutividade Elétrica , Humanos , Espectrometria de Massas , Permeabilidade , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Tachykinins (TKs) are abundantly expressed in the gastrointestinal (GI) tract in intrinsic excitatory motor neurons, interneurons, sensory neurons and extrinsic sensory neurons. Their role in the regulation of enteric secretomotor functions is well established, especially following pathophysiological stimuli. Recent evidence emphasizes the role of TKs in the sensitization of peripheral branches of visceral afferent neurons, implying a role in determining visceral hypersensitivity. Furthermore, the involvement of both CNS and peripheral TK receptors in autonomic reactions to stress, render these receptors an appealing target for the development of drugs aimed at the treatment of irritable bowel syndrome (IBS), a functional GI disorder. The available preclinical evidence indicates that TK receptor antagonists could normalize motor disturbance (diarrhea and constipation) and reduce the painful symptoms that characterize IBS.