Marie Luise Rao

Analysis of eighteen antidepressants, four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring

Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile-potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9-10.2% and 0.9-9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics.

A genetic variation of the inflammatory cytokine interleukin‐6 delays the initial onset and reduces the risk for sporadic Alzheimer's disease

Local inflammatory processes surrounding the amyloid plaques contribute to the progression and acceleration of the Alzheimers disease (AD)–related neurodegeneration. Interleukin‐6 (IL‐6) is an inflammatory cytokine with possible involvement in the local immune response occurring in the central nervous system of AD patients. We tested the hypothesis as to whether a genetic polymorphism of the IL‐6 gene (IL‐6) modifies the age at onset and risk for sporadic AD. Our results support an association of the C allele of the IL‐6 genotype with a delayed initial onset and reduced disease risk and indicate that genetically determined alterations of the immune response may modify the course of AD. Ann Neurol 1999;45:666–668

Impact of polymorphisms of cytochrome-P450 isoenzymes 2C9, 2C19 and 2D6 on plasma concentrations and clinical effects of antidepressants in a naturalistic clinical setting.

ObjectiveThis evaluation focuses on polymorphisms of the cytochrome-P450 (CYP) isoenzymes 2C9, 2C19 and 2D6 and their association with plasma concentrations within a typical clinical setting. Side effects and treatment response were analysed in an exploratory approach in poor and ultra-rapid metabolisers.Patients and methodsWe analysed 136 Caucasian depressed inpatients treated with amitriptyline, citalopram, clomipramine, doxepin, fluvoxamine, mirtazapine, paroxetine, sertraline and venlafaxine, who underwent weekly plasma concentration measurements, assessment of the severity of illness and side effects during their stay in the hospital. Patients were genotyped with respect to CYP2C9 alleles *1 and *2, the CYP2C19 alleles *1, *2 and *3 and the CYP2D6 alleles *1 to *9 and CYP2D6 gene duplication.ResultsCYP2D6 poor metaboliser genotype and co-medication with inhibitors of CYP2D6 were associated with higher plasma concentrations than the drug-specific median plasma concentration when normalised to dose; plasma concentrations of CYP2C19 extensive metabolisers and smokers were significantly lower than the drug-specific median. Five of the six CYP2D6 poor metabolisers experienced side effects. Response was not associated with plasma concentrations above or below the lower limit of a presumed therapeutic range.ConclusionThese data indicate a significant influence of the CYP2D6 genotype, minor influence of the CYP2C19 genotype and no influence of the CYP2C9 genotype on plasma concentrations of patients taking mainly second-generation antidepressants. Because of the good tolerability of the latter and the flat dose–response relationship, genotyping should only be considered in cases of suspected side effects.

Polymorphism in the cholesterol 24S-hydroxylase gene is associated with Alzheimer's disease

Cholesterol and 24S-hydroxycholesterol are involved in the pathogenesis of Alzheimers disease (AD). Increased serum cholesterol concentrations have been detected in patients with AD. 24S-Hydroxycholesterol is the primary cholesterol elimination product of the brain and possesses neurotoxic properties in vitro. The enzyme catalyzing the conversion of cholesterol to 24S-hydroxycholesterol, cholesterol 24S-hydroxylase (CYP46), is mainly expressed in neurons. Concentrations of 24S-hydroxycholesterol in cerebrospinal fluid (CSF) and serum differ significantly between AD patients and non-demented subjects. To test the hypothesis if polymorphisms in the CYP46 gene might influence the function of the respective enzyme and thus cholesterol metabolism in the human brain, we screened for polymorphisms in 114 AD patients and 144 healthy controls. Two intronic single nucleotide polymorphisms were observed and their allelic distribution was investigated. In our study sample, carriers of the C allele of the IVS3+43C → T polymorphism were more prevalent in the group of AD patients than in healthy controls, while another IVS2-150A → G polymorphism did not show a significant association with AD. The CC genotype of the IVS3+43C → T polymorphism was associated with an increased 24S-hydroxycholesterol/cholesterol ratio in the CSF of AD patients. Our results indicate that the CYP46 gene locus may predispose to AD by increasing the 24S-hydroxycholesterol/cholesterol ratio in the brain.

The neurotoxic effect of 24-hydroxycholesterol on SH-SY5Y human neuroblastoma cells

Neurodegenerative disorders are characterized by a massive loss of nerve cells. The neuronal cell death is accompanied by an increased cholesterol release and conversion of cholesterol into the polar metabolite, 24-hydroxycholesterol (24-OH-Chol), appears to be an important mechanism in the central nervous system for eliminating cholesterol from the brain. We tested the influence of 24-OH-Chol on SH-SY5Y human neuroblastoma cells by recording cell morphology, Trypan blue exclusion, LDH-release into the culture medium, intracellular calcium and reactive oxygen species (ROS). The exposure of SH-SY5Y human neuroblastoma cells to 50 microM 24-OH-Chol led to a 90% loss in cell viability within 30 h, the LDH-release into the medium increased rapidly after 24 h, and after 24 to 30 h we found an elevation in intracellular calcium. These results show that, in a physiological concentration range, 24-OH-Chol damages neuronal cells, thus we speculate that this oxysterol may be involved in the etiology of neurodegenerative disease.

FcεRI Induces the Tryptophan Degradation Pathway Involved in Regulating T Cell Responses

FcεRI is suspected to play a pivotal role in the pathophysiology of atopic disorders such as atopic dermatitis. In search for genes differentially regulated by FcεRI on APCs, a differential cDNA bank of receptor-stimulated and unstimulated monocytes was established. By means of suppression subtractive hybridization, we identified kynurenine 3-monooxygenase and subsequently indoleamine 2,3-dioxygenase (IDO) to be overexpressed in FcεRI-activated monocytes. IDO is the rate-limiting enzyme in the catabolism of the essential amino acid tryptophan. We show that cross-linking of FcεRI on monocytes results in low tryptophan concentrations associated with impaired T cell stimulatory capacity. Importantly, T cell suppression could be prevented by the addition of tryptophan or inhibition of IDO. Moreover, stimulation of T cells by FcεRI-activated monocytes was increased compared with T cell stimulation by nonactivated monocytes if exogenous supply of tryptophan was available. We speculate that the expression of IDO by FcεRI+ APCs in vivo allows these cells to regulate T cell responses in atopic disorders by inhibiting or stimulating T cell proliferation, depending on the metabolic environment.

Neurotoxicity of 24-hydroxycholesterol, an important cholesterol elimination product of the brain, may be prevented by vitamin E and estradiol-17β

Summary. 24-Hydroxycholesterol, the main cholesterol elimination product of the brain is increased in serum of Alzheimer patients. This oxysterol behaves neurotoxic towards the human neuroblastoma cell line, SH-SY5Y. Here we demonstrate, that 24-hydroxycholesterol-induced neurotoxicity in differentiated SH-SY5Y cells was due to apoptosis, as indicated by DNA-fragmentation, caspase-3 activation and a decrease of the mitochondrial membrane potential. Free radicals were generated, resulting in the death of 75% of the cells within 48 h; neurotoxicity in differentiated SH-SY5Y cells was partially prevented by physiological concentrations of vitamin E (50–100 μM) in that 75% of the cells survived. Physiological concentrations of estradiol-17β (1–100 nM) elicited a protective effect in differentiated cells, which was not significant; however, in undifferentiated cells a significant protection was noted by this steroid hormone. Vitamin C and melatonin did not prevent 24-hydroxycholesterol-induced neurotoxicity. These in vitro data support the in vivo observed beneficial effects reported as circumstantial evidence of vitamin E and estradiol-17β treatment in the prevention and therapy of neurodegenerative disease.

Genetic polymorphism of cathepsin D is strongly associated with the risk for developing sporadic Alzheimer's disease

The beta amyloid peptide derives from its precursor protein via proteolytic cleavage of yet unidentified proteases (beta- and gamma-secretases). Cathepsin D is an intracellular protease with in-vitro beta-secretase-like features. An exonic polymorphism of the cathepsin D gene (alanine to valine transition at position 224, exon 2) has been associated with altered enzyme function. We tested the hypothesis that this polymorphism is associated with an increased risk for Alzheimers disease in 102 demented patients, 191 healthy subjects, and 160 depressed patients. There was a highly significant overrepresentation of the cathepsin D*T allele in demented patients (14.2%) compared to non-demented controls (6.7%, P = 0.0012). Carriers of the cathepsin D*T allele had a 2.4-fold increased risk for developing AD than non-carriers. Carriers of the apolipoprotein E epsilon 4 allele had a 4.1 -fold increased risk than non-carriers. The odds ratio for subjects with the apolipoprotein E epsilon 4 and the cathepsin D*T allele was 5.9. Our data suggest that the cathepsin D genotype is strongly associated with the risk for Alzheimers disease.

Early-Onset and Late-Onset Depression Are Independent of the Genetic Polymorphism of Apolipoprotein E

The recently shown association between apolipoprotein E (APOE) genotype and depressive illness has been challenged by subsequent studies. However, controversial results may derive from the different diagnostic criteria used for depression and from the small numbers of depressed patients included in the studies. We examined the association between depression and the genetic polymorphism of APOE in a large sample of depressed patients, Alzheimer’s disease (AD) patients, and healthy controls following clear definitions for late-life depression. The cumulative incidence of depression depending on the age at onset of the first episode was examined by survival analysis. Our data do not disconfirm the hypothesis of depression sharing some common pathophysiologic features with AD, however, it seems very unlikely that the APOE genotype will elucidate the assumed common mechanisms.

Circadian rhythm of vital signs, norepinephrine, epinephrine, thyroid hormones, and cortisol in schizophrenia

Changes in the circadian rhythmicity in vital signs, catecholamines, thyroid hormones, and cortisol have been observed in psychiatric disorders, most notably in depression. With respect to schizophrenia, the literature is scanty. We report here on the circadian parameter estimates of the vital signs, epinephrine, norepinephrine, triiodothyronine, thyroxine, thyroid stimulating hormone, and cortisol in the blood of 34 healthy subjects, 89 drug-free schizophrenic patients, and 25 neuroleptic-treated schizophrenic patients. The analyses are based on the cosine model to fit the experimental data. The circadian profiles of heart rate, blood pressure, and oral temperature are similar among schizophrenic patients and healthy subjects. Neuroleptic-treated patients have significantly higher MESORs (the daily mean) of serum norepinephrine and epinephrine than healthy subjects. The TSH MESOR is significantly lower in schizophrenic patients; the MESOR of triiodothyronine also shows a tendency to be nonsignificantly lower in schizophrenic patients compared with control subjects. The circadian serum thyroxine and cortisol profiles are similar in the three groups. The data show that the circadian profiles of vital signs in drug-free chronic schizophrenic patients who are not chronically hospitalized are similar to those of healthy subjects and that the increase in serum catecholamines and the apparent lowering in some thyroid indices might induce a down-regulation in the noradrenergic receptor system that could contribute to the pathophysiology of schizophrenia.