RESUMO
Methods for the quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay (FPIA) is described. One immunoassay allows for the accurate quantification of amitriptyline in the presence of nortriptyline while the second immunoassay allows for the accurate quantification of nortriptyline in the presence of amitriptyline.
Assuntos
Amitriptilina/sangue , Nortriptilina/sangue , Psicotrópicos/sangue , Amitriptilina/imunologia , Animais , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Imunoensaio de Fluorescência por Polarização , Corantes Fluorescentes , Humanos , Indicadores e Reagentes , Nortriptilina/imunologia , Coelhos/imunologia , SolventesRESUMO
Amitriptyline and nortriptyline were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin (BSA). Rabbits inoculated with these conjugates yielded polyclonal antisera with high selectivity and good titers. This approach required novel spacer arms and new conjugation methods. The antisera produced were characterized with respect to their cross-reactivity with amitriptyline, nortriptyline and their hydroxy metabolites as well as selected structurally related compounds.
Assuntos
Amitriptilina/análise , Anticorpos/imunologia , Nortriptilina/análise , Amitriptilina/química , Amitriptilina/imunologia , Animais , Especificidade de Anticorpos , Clorpromazina/química , Reações Cruzadas , Polarização de Fluorescência , Técnicas Imunológicas , Indicadores e Reagentes , Modelos Moleculares , Nortriptilina/química , Nortriptilina/imunologia , Coelhos , Sensibilidade e EspecificidadeRESUMO
Rabbits were actively immunized by a conjugate of nortriptyline (NT) to study the effect of specific anti-NT antibodies on toxicity and disposition of amitriptyline (AT). Control and immunized rabbits received 115 mg/kg AT intraperitoneally (i.p.). The lethality dose (LD) profile exhibited a gentle slope; LD100 and LD0 were separated by 100 mg/kg. Mortality was significantly reduced from LD67 to LD43 (P less than 0.05). Total plasma concentrations of the toxin were increased in the immunized group compared to the control group. AUC0.5-24 h value was 5-fold higher in the immunized group than in the control group. Moreover, a smaller fraction of unbound toxin in plasma was observed in the immunized group than in the control group. These observations indicate that AT was actively sequestered by antibodies. The intensity of this phenomenon was a function of both the antibody affinity constant (10(9) M-1) and the neutralizing capacity (varying from 0.005 to 0.2 mg/kg) of the circulating antibodies in each immunized rabbit. Results clearly show that anti-NT antibodies are able to effectively sequestrate AT.
Assuntos
Amitriptilina/toxicidade , Anticorpos/farmacologia , Amitriptilina/imunologia , Amitriptilina/farmacocinética , Animais , Anticorpos/imunologia , Reações Cruzadas , Imunização , Injeções Intraperitoneais , Injeções Intravenosas , Dose Letal Mediana , Masculino , Testes de Neutralização , Nortriptilina/imunologia , Coelhos , RadioimunoensaioRESUMO
Inhibition constants of tricyclic anti-depressants and related drugs determined for a monoclonal anti-nortriptyline antibody were close to those previously calculated with the same compounds for the brain acetylcholine muscarinic receptor. A highly significant correlation was found between these two series of inhibition constants when no correlation existed between the inhibition constants for the antibody and those for other receptors. This suggests that the binding site for tricyclic anti-depressants on the antibody mimics the binding site for these ligands on muscarinic receptors. Although nortriptyline reveals a noncompetitive inhibition of N-methyl-scopolamine binding to muscarinic receptors, muscarinic ligands display weak or no binding to the antibody. These findings indicate that the binding site for tricyclic anti-depressants on the receptor is distinct from that for the muscarinic ligands.
Assuntos
Anticorpos Monoclonais/metabolismo , Antidepressivos Tricíclicos/imunologia , Nortriptilina/imunologia , Receptores Muscarínicos/metabolismo , Animais , Antidepressivos Tricíclicos/metabolismo , Atropina/metabolismo , Sítios de Ligação , Ligação Competitiva , Carbacol/metabolismo , Bovinos , Epitopos , Nortriptilina/metabolismoRESUMO
After somatic cell fusion between splenocytes of immunized BALB/c mice and NS-1 myeloma cells, a clone was obtained that secreted an anti-nortriptyline antibody of the IgG1 kappa isotype. The association constant of this antibody for pharmacologically active tricyclic antidepressant drugs ranged from 0.6 X 10(7) to 3 X 10(7) M-1. From thermodynamic and binding studies as well as tridimensional structures of tested compounds, the epitope recognized by the monoclonal antibody appeared to include both a hydrophobic tricycle in which the two phenyl rings form an angle of 120 to 130 degrees, and a side chain in which the amino group is separated from the two lateral rings of the tricyclic structure by a distance of approximately 5.9 A and 7.5 A, respectively. This conformation seems to be the one interacting with muscarinic acetylcholine brain receptors.
Assuntos
Anticorpos Monoclonais , Epitopos , Nortriptilina/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Reações Antígeno-Anticorpo , Sítios de Ligação de Anticorpos , Ligação Competitiva , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores Muscarínicos , TermodinâmicaAssuntos
Antidepressivos Tricíclicos/imunologia , Adulto , Amitriptilina/efeitos adversos , Amitriptilina/imunologia , Antidepressivos Tricíclicos/efeitos adversos , Hipersensibilidade a Drogas/etiologia , Eritema/induzido quimicamente , Feminino , Humanos , Masculino , Nortriptilina/efeitos adversos , Nortriptilina/imunologiaRESUMO
1 Antisera to nortriptyline were prepared by immunizing rabbits with N-succinylnortriptyline--bovine serum albumin conjugate. 2 A sensitive radioimmunoassay has been developed for the tricyclic antidepressants amitriptyline and nortriptyline. 3 amitriptyline and nortriptyline are separated from each other and from interfering metabolites before assay. 4 Using [3H]-imipramine and [3H]-succinylnortriptyline as tracers the radioimmunoassay can measure amitriptyline and nortriptyline levels down to 2--3 ng/ml using 0.05 ml plasma sample. 5 Agreement between the radioimmunoassay and a gas-chromatographic assay was excellent for both amitriptyline and nortriptyline.
Assuntos
Amitriptilina/sangue , Nortriptilina/sangue , Amitriptilina/imunologia , Animais , Formação de Anticorpos , Especificidade de Anticorpos , Bovinos , Cromatografia Gasosa , Métodos , Nortriptilina/imunologia , Coelhos/imunologia , RadioimunoensaioRESUMO
A specific radioimmunoassay (RIA) for the determination in plasma of the widely used tricyclic antidepressant amitriptyline (AT) and its major metabolite nortriptyline (NT) has been developed employing 3H-AT as the radioligand and a rabbit antiserum to a bovine serum albumin conjugate of N-succinyl-nortriptyline. Although the antiserum cross-reacts almost equally well with AT and NT, specificity is achieved by selective extraction of each compound from plasma at a different pH. A unique aspect of the assay is that at no time during the entire extraction procedure is the AT or NT taken out of solution. Both compounds are back extracted from the organic phase into 0.1 N HC1 and the acid fraction subjected to RIA directly. The method has a limit of sensitivity of about 2 ng/ml using a 0.5 ml sample of plasma. Satisfactory agreement was obtained for plasma levels of AT and NT when determined by the RIA and a specific GC/MS procedure. The correlation coefficients were 0.89 and 0.98 for AT and NT, respectively. THE RIA has been used to measure steady-state levels of AT and NT in man after chronic administration of AT and following a single oral 75 mg dose. The method also lends itself for the specific determination of NT alone in subjects receiving therapeutic doses of NT.