Determination of amitriptyline and nortriptyline in human liver microsomes with reversed-phase HPLC in vitro.
AIM: To develop a method for simultaneous determinations of amitriptyline (Ami) and its metabolite nortriptyline (Nor) in human liver microsomes. METHODS: An incubation buffer containing microsomes, NADPH-generating system, and Ami, after termination of enzyme reaction and desipramine (Des) as internal standard (IS), was extracted with diethy ether and separated on a reversed-phase ODS column. Detection was achieved at 242 nm by ultraviolet detector. RESULTS: No potential interfering peaks were found. Ami and Nor gave rapid elution and baseline resolution. The linear curves of both analyses ranged 0.02-10 nmol and the limit of detection was 0.01 nmol. The recovery (94%-101%) had good precision with relative s of < 8.3%. CONCLUSION: This method is rapid, sensitive, and simple for studying the metabolism of Ami and Nor. (+info)
Relaxant effects of antidepressants on human isolated mesenteric arteries.
AIMS: The therapeutic action of tricyclic agents may be accompanied by unwanted effects on the cardiovascular system. The evidence for the effects on vascular and nonvascular smooth muscle comes from animal studies. Whether these studies can be extrapolated to human vessels remains to be determined. Therefore, the present study was designed to investigate the influence of amitriptyline, nortriptyline and sertraline on the contractile responses of human isolated mesenteric arteries to electrical field stimulation, noradrenaline and potassium chloride. METHODS: Arterial segments (lumen diameter 0.8-1.2 mm) were obtained from portions of the human omentum during the course of 41 abdominal operations (22 men and 19 women), and rings 3 mm long were mounted in organ baths for isometric recording of tension. In some artery rings the endothelium was removed mechanically. RESULTS: In precontracted artery rings amitriptyline, nortriptyline and sertraline (3x10(-7)-10(-4) m ) produced concentration-dependent relaxation that was independent of the presence or absence of vascular endothelium. Incubation with indomethacin (3x10(-6) m ) reduced the pD2 values thus indicating the participation of dilating prostanoid substances in this response. Amitriptyline and nortriptyline inhibited both the neurogenic-and noradrenaline-induced contractions. In contrast, only the highest concentration of sertraline reduced the adrenergic responses. Amitriptyline, nortriptyline and sertraline inhibited contractions elicited by KCl and produced rightward shifts of the concentration-response curve to CaCl2 following incubation in calcium-free solution. CONCLUSIONS: These results indicate that amitriptyline and nortriptyline could act as adrenoceptor antagonists and direct inhibitors of smooth muscle contraction of human mesenteric arteries, whereas sertraline might principally exert its action only as direct inhibitor of smooth muscle contraction. This relaxant mechanism involves an interference with the entry of calcium. (+info)
Nonspecific binding of drugs to human liver microsomes.
AIMS: To characterize the nonspecific binding to human liver microsomes of drugs with varying physicochemical characteristics, and to develop a model for the effect of nonspecific binding on the in vitro kinetics of drug metabolism enzymes. METHODS: The extent of nonspecific binding to human liver microsomes of the acidic drugs caffeine, naproxen, tolbutamide and phenytoin, and of the basic drugs amiodarone, amitriptyline and nortriptyline was investigated. These drugs were chosen for study on the basis of their lipophilicity, charge, and extent of ionization at pH 7.4. The fraction of drug unbound in the microsomal mixture, fu(mic), was determined by equilibrium dialysis against 0.1 M phosphate buffer, pH 7.4. The data were fitted to a standard saturable binding model defined by the binding affinity KD, and the maximum binding capacity Bmax. The derived binding parameters, KD and Bmax, were used to simulate the effects of saturable nonspecific binding on in vitro enzyme kinetics. RESULTS: The acidic drugs caffeine, tolbutamide and naproxen did not bind appreciably to the microsomal membrane. Phenytoin, a lipophilic weak acid which is mainly unionized at pH 7. 4, was bound to a small extent (fu(mic) = 0.88) and the binding did not depend on drug concentration over the range used. The three weak bases amiodarone, amitriptyline and nortriptyline all bound extensively to the microsomal membrane. The binding was saturable for nortriptyline and amitriptyline. Bmax and KD values for nortriptyline at 1 mg ml-1 microsomal protein were 382 +/- 54 microM and 147 +/- 44 microM, respectively, and for amitriptyline were 375 +/- 23 microM and 178 +/- 33 microM, respectively. Bmax, but not KD, varied approximately proportionately with the microsome concentration. When KD is much less than the Km for a reaction, the apparent Km based on total drug can be corrected by multiplying by fu(mic). When the substrate concentration used in a kinetic study is similar to or greater than the KD (Km >/= KD), simulations predict complex effects on the reaction kinetics. When expressed in terms of total drug concentrations, sigmoidal reaction velocity vs substrate concentration plots and curved Eadie Hofstee plots are predicted. CONCLUSIONS: Nonspecific drug binding in microsomal incubation mixtures can be qualitatively predicted from the physicochemical characteristics of the drug substrate. The binding of lipophilic weak bases is saturable and can be described by a standard binding model. If the substrate concentrations used for in vitro kinetic studies are in the saturable binding range, complex effects are predicted on the reaction kinetics when expressed in terms of total (added) drug concentration. Sigmoidal reaction curves result which are similar to the Hill plots seen with cooperative substrate binding. (+info)
Effects of antidepressants on weight and on the plasma levels of leptin, TNF-alpha and soluble TNF receptors: A longitudinal study in patients treated with amitriptyline or paroxetine.
Leptin, tumor necrosis factor-alpha (TNF-alpha), and soluble TNF receptors are involved in weight regulation. Antipsychotic agents, such as clozapine, induce weight gain and increase circulating levels of these cytokines. To assess whether obesity-inducing antidepressants have a similar effect, we measured plasma cytokine levels in depressive inpatients during the first six weeks of treatment with tricyclic agents (amitriptyline or nortriptyline, n = 12), with paroxetine (n = 10), or without medication (n = 14). There was an increase in the body mass index at week 6 of treatment with the tricyclics, which was preceded by a significant increase in soluble TNF receptor p75 plasma levels. Circulating levels of leptin were not affected. Paroxetine and drug-free treatment did not affect any of these parameters. We conclude that weight gain induced by psychotropic agents may occur without increased circulating levels of leptin. However, activation of the TNF-alpha system might be an early and sensitive marker of ensuing weight gain. (+info)
Treatment of cognitive impairment after poststroke depression : a double-blind treatment trial.
BACKGROUND AND PURPOSE: Patients with poststroke major depression have a greater severity of cognitive impairment than nondepressed patients even when matched for size and location of stroke lesion. Prior treatment studies have consistently failed to show an improvement in cognitive function even when poststroke mood disorders responded to antidepressant therapy. We examined the response of cognitive function to treatment with nortriptyline or placebo in a double-blind trial. METHODS: Patients with major (n=33) or minor (n=14) depression participated in a double-blind treatment study with nortriptyline or placebo. They were examined for change in depressive mood, measured by the Hamilton Rating Scale for Depression (HAM-D), and change in cognitive impairment, assessed by the Mini-Mental State Examination (MMSE), after treatment with nortriptyline or placebo. Cognitive treatment response, as measured by the MMSE, was compared between patients whose depression did and did not respond to treatment. RESULTS: Patients whose poststroke depression remitted (predominantly associated with nortriptyline treatment) had significantly greater recovery in cognitive function over the course of the treatment study than patients whose mood disorder did not remit (predominantly associated with placebo treatment). CONCLUSIONS: Our findings support the contention that poststroke major depression leads to a "dementia of depression." Prior studies failed to show an effect of treatment because the effect size was too small. Successful treatment of depression may constitute one of the major methods of promoting cognitive recovery in victims of stroke. (+info)
Allelic variation in the serotonin transporter promoter affects onset of paroxetine treatment response in late-life depression.
The relationship of the serotonin transporter gene promoter region polymorphism (5-HTTLPR) to antidepressant response was examined in 95 elderly patients receiving a protocolized treatment for depression with paroxetine or nortriptyline. Patients were treated for up to 12 weeks and assessed weekly with clinical ratings and measurements of plasma drug concentrations. Twenty-one of the paroxetine-treated subjects were found to have the ll genotype and 30 had at least one s allele. There were no baseline differences between these groups in pretreatment Hamilton Rating Scale for Depression (HRSD) scores or anxiety symptoms. During acute treatment with paroxetine, mean reductions from baseline in HRSD were significantly more rapid for patients with the ll genotype than for those possessing an s allele, despite equivalent paroxetine concentrations. Onset of response to nortriptyline was not affected. Allelic variation of 5-HTTLPR may contribute to the variable initial response of patients treated with a selective serotonin reuptake inhibitor. (+info)
Determination of residues in the norepinephrine transporter that are critical for tricyclic antidepressant affinity.
The norepinephrine (NET) and dopamine (DAT) transporters are highly homologous proteins, displaying many pharmacological similarities. Both transport dopamine with higher affinity than norepinephrine and are targets for the psychostimulants cocaine and amphetamine. However, they strikingly contrast in their affinities for tricyclic antidepressants (TCA). Previous studies, based on chimeric proteins between DAT and NET suggest that domains ranging from putative transmembrane domain (TMD) 5 to 8 are involved in the high affinity binding of TCA to NET. We substituted 24 amino acids within this region in the human NET with their counterparts in the human DAT, resulting in 22 different mutants. Mutations of residues located in extra- or intracytoplasmic loops have no effect on binding affinity of neither TCA nor cocaine. Three point mutations in TMD6 (F316C), -7 (V356S), and -8 (G400L) induced a loss of TCA binding affinity of 8-, 5-, and 4-fold, respectively, without affecting the affinity of cocaine. The triple mutation F316C/V356S/G400L produced a 40-fold shift in desipramine affinity. These three residues are strongly conserved in all TCA-sensitive transporters cloned in mammalian and nonmammalian species. A strong shift in TCA affinity (IC(50)) was also observed for double mutants F316C/D336T (35-fold) and S399P/G400L (80-fold for nortriptyline and 1000-fold for desipramine). Reverse mutations P401S/L402G in hDAT did not elicit any gain in TCA affinities, whereas C318F and S358V resulted in a 3- and 10-fold increase in affinity, respectively. Our results clearly indicate that two residues located in TMD6 and -7 of hNET may play an important role in TCA interaction and that a critical region in TMD8 is likely to be involved in the tertiary structure allowing the high affinity binding of TCA. (+info)
Comparison of noradrenergic and serotonergic antidepressants in reducing immobility time in the tail suspension test.
We examined the effects of two noradrenergic tricyclic antidepressants and two selective serotonin re-uptake inhibitors in the tail suspension test, with a suspension period of 30 min instead of the usual 10 min. Within the first 10 min, desipramine, nortriptyline and fluvoxamine significantly reduced the duration of immobility. Whereas desipramine and nortriptyline were also efficacious in the rest of the test period, fluvoxamine was not. Fluoxetine showed no significant effect throughout the study period. These results suggest that a prolonged tail suspension test results in functional changes in the noradrenergic and serotonergic systems and alters the sensitivity to antidepressants. (+info)