RESUMO
We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.
Assuntos
Fármacos Anti-HIV/análise , Fármacos Anti-HIV/imunologia , Anticorpos/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Nucleotídeos de Timina/análise , Nucleotídeos de Timina/imunologia , Zidovudina/análogos & derivados , Zidovudina/análise , Zidovudina/imunologia , Acetilcolinesterase/química , Animais , Fármacos Anti-HIV/química , Anticorpos/isolamento & purificação , Anticorpos/metabolismo , Calibragem , Didesoxinucleotídeos , Hemocianinas/química , Hemocianinas/imunologia , Estrutura Molecular , Coelhos , Sensibilidade e Especificidade , Nucleotídeos de Timina/química , Zidovudina/químicaRESUMO
Four new steroidal alkaloids, N20-formylbuxaminol E [(20S)-16alpha-hydroxy-20-(formylamino)-3beta-(dimethylamino)-9,10 -seco-buxa-9(11),10(19)-diene] (1), O16-syringylbuxaminol E [(20S)-16alpha-syringoyl-3beta-(dimethylamino)-20-(amino)-9, 10-seco-buxa-9(11),10(19)-diene] (2), N20-acetylbuxamine G [(20S)-20-(acetylamino)-3beta-(methylamino)-9,10-seco-buxa-9(11),1 0(19)-diene] (3) and N20-acetylbuxamine E [(20S)-20-(acetylamino)-3beta-(dimethylamino)-9,10-seco-buxa-9(11) ,10(19)-diene] (4) were isolated from the leaves of Buxus sempervirens. Their structures were determined mainly on the basis of 2D NMR studies.
Assuntos
Alcaloides/isolamento & purificação , Magnoliopsida/química , Esteroides/química , Alcaloides/química , Estrutura Molecular , Folhas de Planta/química , Análise EspectralRESUMO
Lamivudine or 3TC, the (-) eniantiomer of 2'-deoxy-3'-thiacytidine, is a prototype of a novel class of levogyre dideoxynucleosides analogues used in treatment of HIV and HBV infection. We describe a method corresponding to the first enzyme immunoassay for quantifying this antiviral drug. This technique use an enzyme conjugate that not require the use of radioactive labelling. In this study, anti-3TC antibodies were raised in rabbits by immunising with 3TC-HS-kelhoyle limpet hemocyanin (KLH) conjugate.
Assuntos
Fármacos Anti-HIV/análise , Imunoensaio/métodos , Lamivudina/análise , Inibidores da Transcriptase Reversa/análise , Fármacos Anti-HIV/imunologia , Reações Cruzadas , Lamivudina/imunologia , Inibidores da Transcriptase Reversa/imunologiaRESUMO
A new sensitive method for the measurement of lamivudine triphosphate (3TC-TP), the active intracellular metabolite of lamivudine in human cells in vivo, has been established. The procedure involves rapid separation of 3TC-TP by using Sep-Pak cartridges, dephosphorylation to 3TC by using acid phosphatase, and measurement by radioimmunoassay using a newly developed anti-3TC serum. The radioimmunoassay had errors of less than 21% and a cross-reactivity of less than 0.016% with a wide variety of other nucleoside analogs. The limit of quantitation of the assay for intracellular 3TC-TP was 0.195 ng/ml (0.212 pmol/10(6) cells), and a cell sample of only 4 million cells was ample for the assay. This procedure, combined with our previously developed method for measuring zidovudine (ZDV) metabolite levels, proved capable of measuring 3TC-TP, ZDV monophosphate (ZDV-MP) and ZDV triphosphate (ZDV-TP) in human immunodeficiency virus (HIV)-infected subjects treated with combination 3TC and ZDV therapy. In seven subjects, intracellular 3TC-TP levels ranged from 2.21 to 7.29 pmol/10(6) cells, while intracellular ZDV-MP and ZDV-TP levels ranged from <0. 01 to 1.76 and 0.01 to 0.07 pmol/10(6) cells, respectively. Concentrations of 3TC in plasma determined in these subjects ranged from 0.34 to 9.40 microM, which was about fivefold higher than ZDV levels in plasma of 0.04 to 1.4 microM. This is the first study to determine the intracellular levels of the active metabolites in HIV-infected subjects treated with this combination. These methods should prove very useful for in vivo pharmacodynamic studies of combination therapy.