RESUMO
The recently described 'gasomediator' hydrogen sulfide (H2S) has been involved in pain mechanisms, but its effect on pruritus, a sensory modality that similarly to pain acts as a protective mechanism, is poorly known and controversial. The effects of the slow-releasing (GYY4137) and spontaneous H2S donors (Na2S and Lawesson's reagent, LR) were evaluated in histamine and compound 48/80 (C48/80)-dependent dorsal skin pruritus and inflammation in male BALB/c mice. Animals were intradermally (i.d.) injected with C48/80 (3µg/site) or histamine (1µmol/site) alone or co-injected with Na2S, LR or GYY4137 (within the 0.3-100nmol range). The involvement of endogenous H2S and KATP channel-dependent mechanism were also evaluated. Pruritus was assessed by the number of scratching bouts, whilst skin inflammation was evaluated by the extravascular accumulation of intravenously injected 125I-albumin (plasma extravasation) and myeloperoxidase (MPO) activity (neutrophil recruitment). Histamine or C48/80 significantly evoked itching behavior paralleled by plasma extravasation and increased MPO activity. Na2S and LR significantly ameliorated histamine or C48/80-induced pruritus and inflammation, although these effects were less pronounced or absent with GYY4137. Inhibition of endogenous H2S synthesis increased both Tyrode and C48/80-induced responses in the skin, whereas the blockade of KATP channels by glibenclamide did not. H2S-releasing donors significantly attenuate C48/80-induced mast cell degranulation either in vivo or in vitro. We provide first evidences that H2S donors confer protective effect against histamine-mediated acute pruritus and cutaneous inflammation. These effects can be mediated, at least in part, by stabilizing mast cells, known to contain multiple mediators and to be primary initiators of allergic processes, thus making of H2S donors a potential alternative/complementary therapy for treating inflammatory allergic skin diseases and related pruritus.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Inflamação/tratamento farmacológico , Mastócitos/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Prurido/tratamento farmacológico , Pele/efeitos dos fármacos , Animais , Glibureto/farmacologia , Histamina/metabolismo , Inflamação/metabolismo , Canais KATP/metabolismo , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Prurido/metabolismo , Pele/metabolismoRESUMO
The purpose of this study was to evaluate iffreezing-thawing and cooling processes affect thestructural properties and biological activity ofcommercial equine chorionic gonadotropin (eCG). First,the structure profile of diluted eCG underwent none,one or three cycles of freezing-thawing was analysed byreverse phase high-performance liquid chromatography(RP-HPLC). In a second experiment, groups ofprepuberal rats were treated with sterile water forinjection USP or eCG that underwent none, one or threecycles of freezing-thawing to assess the increase ofovarian weigh. Finally, groups of prepubertal gilts weretreated with diluted eCG immediately afterreconstitution (T1), after refrigeration for six months(T2) and after freezing and subsequently thawing for one(T3) or three (T4) cycles. The control group (T5) receivedsterile water for injection USP without eCG. Ovulationwas induced with human chorionic gonadotropin (hCG),administered 72 h after the eCG. Gilts were slaughteredfive days after the hCG injection and ovaries wererecovered and analysed for the presence of corporalutea. Data were analysed by ANOVA and Fishersexact tests. In the analyses by RP-HPLC, the retentiontimes of cold stressed eCG were similar to unstressedcontrol. The mean ovarian weight of rats treated with coldstressed and unstressed eCG was statistically higher thanwater control (P < 0.05). Lastly, significantly more giltsovulated in groups T1, T2, T3 and T4 than in thecontrol T5 (P < 0.05). It was concluded that freezingthawing,as well as cooling over a period of up to sixmonths, did not significantly change the structuralproperties or biological activity of eCG.
Assuntos
Animais , Cavalos/anatomia & histologia , Cavalos/fisiologia , Fenômenos Químicos , Gonadotropina Coriônica/efeitos adversos , Transtornos de Estresse por Calor/veterinária , Bioensaio , EletrocardiografiaRESUMO
The purpose of this study was to evaluate iffreezing-thawing and cooling processes affect thestructural properties and biological activity ofcommercial equine chorionic gonadotropin (eCG). First,the structure profile of diluted eCG underwent none,one or three cycles of freezing-thawing was analysed byreverse phase high-performance liquid chromatography(RP-HPLC). In a second experiment, groups ofprepuberal rats were treated with sterile water forinjection USP or eCG that underwent none, one or threecycles of freezing-thawing to assess the increase ofovarian weigh. Finally, groups of prepubertal gilts weretreated with diluted eCG immediately afterreconstitution (T1), after refrigeration for six months(T2) and after freezing and subsequently thawing for one(T3) or three (T4) cycles. The control group (T5) receivedsterile water for injection USP without eCG. Ovulationwas induced with human chorionic gonadotropin (hCG),administered 72 h after the eCG. Gilts were slaughteredfive days after the hCG injection and ovaries wererecovered and analysed for the presence of corporalutea. Data were analysed by ANOVA and Fishersexact tests. In the analyses by RP-HPLC, the retentiontimes of cold stressed eCG were similar to unstressedcontrol. The mean ovarian weight of rats treated with coldstressed and unstressed eCG was statistically higher thanwater control (P < 0.05). Lastly, significantly more giltsovulated in groups T1, T2, T3 and T4 than in thecontrol T5 (P < 0.05). It was concluded that freezingthawing,as well as cooling over a period of up to sixmonths, did not significantly change the structuralproperties or biological activity of eCG.(AU)
Assuntos
Animais , Cavalos/anatomia & histologia , Cavalos/fisiologia , Fenômenos Químicos , Transtornos de Estresse por Calor/veterinária , Gonadotropina Coriônica/efeitos adversos , Bioensaio , EletrocardiografiaRESUMO
The present work describes reversed-phase high performance liquid chromatographic methodologies (RP-HPLC) for the qualitative and quantitative analysis of the human glycoprotein hormones thyrotropin (hTSH), follitropin (hFSH), choriogonadotropin (hCG) and lutropin (hLH) in the presence of a large excess (up to 250:1) of human serum albumin (HSA). Chromatographic profiles with a good separation between the hormone and HSA were obtained by using a C4 column and specific gradient elution conditions for each hormone. Parameters such as resolution factor, tailing factor and relative retention time, were determined, and are useful for the evaluation of the quality of the separation obtained between the active pharmaceutical ingredient and the excipient present in the final formulation. The potential of each method for quantification of both HSA and the hormone was also demonstrated. Besides furnishing chromatographic quantifications that can substitute for in vivo bioassays and animal use, the chromatograms also provide a direct panorama of the quality and heterogeneity of the protein of interest.
Assuntos
Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Hormônios Peptídicos/análise , Albumina Sérica/química , Gonadotropina Coriônica/análise , Cromatografia Líquida de Alta Pressão/normas , Cromatografia de Fase Reversa/normas , Hormônio Foliculoestimulante Humano/análise , Humanos , Hormônio Luteinizante/análise , Ligação Proteica , Padrões de Referência , Tireotropina/análiseRESUMO
Reversed-phase high-performance liquid chromatography (RP-HPLC) was compared with the classical Steelman-Pohley bioassay (BA), based on animal use, for the determination of human follicle-stimulating hormone (hFSH) biological activity. A linear relationship (BA(IU)=0.9925 RP-HPLC(IU)-1.3165) with a highly significant correlation (r=0.9371; p<0.0001; n=24) was found for these two methods for six hFSH preparations of different origins. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for six other hFSH preparations was -1.4%, with a 95% confidence interval of -9.3 to +6.6%. The precision of these parameters was 1.63% and 2.82%, respectively. These results demonstrate that RP-HPLC is a viable physical-chemical alternative to the use of an in vivo bioassay for hFSH potency determination, applicable also to hFSH Standards containing large amounts of human serum albumin.
Assuntos
Hormônio Foliculoestimulante Humano/análise , Hormônio Foliculoestimulante Humano/farmacologia , Tecnologia Farmacêutica , Algoritmos , Alternativas aos Testes com Animais , Animais , Bioensaio , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Tamanho do Órgão/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Projetos Piloto , Controle de Qualidade , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/análise , Proteínas Recombinantes/farmacologia , Reprodutibilidade dos Testes , Albumina SéricaRESUMO
Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their alpha- and beta-subunits migrated with significantly different retention times (t(R)) in the following order of increasing hydrophobicity: alpha-hCGAssuntos
Gonadotropina Coriônica/análise
, Cromatografia Líquida de Alta Pressão/métodos
, Cromatografia de Fase Reversa/métodos
, Hormônio Luteinizante/análise
, Preparações Farmacêuticas/análise
, Gonadotropina Coriônica Humana Subunidade beta/análise
, Subunidade alfa de Hormônios Glicoproteicos/análise
, Humanos
, Proteínas Recombinantes/análise
RESUMO
Complete dissociation into subunits was attained by incubating Chinese hamster ovary (CHO)-derived or native human thyrotropin, follitropin and lutropin overnight at 37 degrees C in acetic acid. The alpha-and beta-subunits of the pituitary glycoprotein hormones were rapidly and quantitatively isolated by reversed-phase high-performance liquid chromatography (RP-HPLC). A dissociation efficiency of > 98% was obtained on the basis of mass determinations of the heterodimers and subunits carried out via mass spectrometry. CHO-derived or native subunits were isolated on a C4 column (80-90% total recovery) and characterized comparatively for purity, hydrophobicity, molecular mass and charge distribution by HPLC, mass spectrometry, sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. Thyrotropin was used as a model for showing that, after subunit reassociation, the in vivo bioactivity of the hormone was completely restored. The method described is mild, practical, flexible, and can be adapted to dissociate microgram amounts of native or recombinant glycoprotein hormones, allowing characterization of each subunit.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Subunidade alfa de Hormônios Glicoproteicos/isolamento & purificação , Hormônios Adeno-Hipofisários/isolamento & purificação , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Focalização Isoelétrica , Hormônios Adeno-Hipofisários/metabolismo , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
O presente trabalho objetivou determinar o efeito do líquido folicular bovino tratado com carväo ativado (LFb) na secreçäo do hormônio folículo estimulante (FSH) de novilhas pré-púberes ovariectomizadas ou intatas. A aplicaçäo de LFb (quatro injeçöes de 10 ml com intervalo de 8 horas) provocou uma queda de aproximadamente 44 por cento na concentraçäo plasmática de FSH nas novilhas ovariectomizadas, mas näo teve efeito nas novilhas intatas. Näo foi observada hipersecreçäo de FSH após o término da aplicaçäo do LFb. Esses resultados sugerem que proteínas presentes no LFb atuam ao nível hipofisiário para inibir a secreçäo de FSH e, diferentemente das intatas, as novilhas ovariectomizadas constituem um modelo adequado para evidenciar esse efeito, particularmente quando o LFb possui reduzida atividade supressora do FSH
Assuntos
Bovinos , Hormônio Foliculoestimulante , Líquido FolicularRESUMO
Dez vacas multíparas, secas, foram distribuídas aleatoriamente em dois grupos de cinco animais cada. Nos dias 8 a 12 do diestro, o primeiro grupo recebeu 100 ml de anti-soro contra líquido folicular livre de esteróides (anti-LFb) produzido em ovelhas ovariectomizadas. O segundo grupo (controle) recebeu 100 ml de soro de ovelhas näo-imunizadas. Seis horas após a aplicaçäo, os dois grupos foram superovulados com FSH (18 NIH-FSH-S1 unidades) e LH (0,29 NIH-LH-S1 unidades) administrados em quantidades decrescentes durante quatro dias. Na manhä do terceiro dia, foi administrada uma dose luteolítica de cloprostenol. Duas inseminaçöes foram realizadas 48 e 60 horas após. Os embriöes foram recuperados pelo método cervical 7 dias após a primeira inseminaçäo. Amostras de sangue foram coletadas durante todo o período experimental para determinar, por radioimunoensaio, as concentraçöes plasmáticas de FSH, LH e progesterona. Todas as vacas do grupo imunizado e 3 do grupo controle apresentaram mais de 2 CL. Näo existiu diferença significativa (p>0,05) na taxa de ovulaçäo entre os grupos imunizado e controle (14,4 e 9,9, respectivamente). O número de embriöes recuperado näo foi significativamente diferente (p>0,05) entre os grupos, embora o grupo imunizado tenha apresentado maior número de embriöes transferíveis (3,4 ñ 1,0 versus 0,8 ñ 0,4, p<0,05). As concentraçöes de gonadotrofinas plasmáticas näo foram correlacionadas com a taxa de ovulaçäo ou com o número de embriöes recuperados. As concentraçöes de progesterona plasmática foram positivamente correlacionadas (r = 0,88, p<0,01) com a taxa de ovulaçäo. Os resultados sugerem que o anti-LFb, aplicado antes da superovulaçäo, näo reduz a variabilidade da resposta ovariana