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- Karlsson, Louise
(författare)
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P-glycoprotein and chiral antidepressant drugs : Pharmacokinetic, pharmacogenetic and toxicological aspects
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The blood-brain barrier (BBB) is formed by the capillary endothelial cells, joined together by tight junctions, with transporter proteins. BBB acts to regulate the brain concentrations of substances including many drugs. Transport across the cells is necessary for a drug to ensure that the drug reaches the site of action and transport proteins such as P-glycoprotein (P-gp; ABCB1) can limit the entrance into various tissues, including the brain.Molecules that are not superimposable on their mirror images and thus exist in two enantiomeric forms (enantiomers) are said to be chiral. A racemic compound is one composed of a 50:50 mixture of two enantiomers, S- and R-enantiomers. Two examples of frequently prescribed racemic drugs are the chiral antidepressants venlafaxine (VEN) and citalopram (CIT). The enantiomers of VEN possess different pharmacodynamic profiles where the R-enantiomer is a potent inhibitor of both serotonin and noradrenaline reuptake (SNRI), while the S-enantiomer is more selective in inhibiting serotonin reuptake (SSRI). The SSRI effect of CIT resides in the S-enantiomer, whereas the R-enantiomer is considered to be therapeutically inactive, or even that it counteracts the effects. The S-enantiomer of CIT is now available as a separate SSRI (escitalopram, EsCIT). VEN and CIT are also among the most commonly found drugs in forensic autopsy cases.Few previous studies have examined a possible enantioselective activity of P-gp. Thus, the general aim of this thesis was to study the enantiomeric distribution of chiral antidepressant drugs, focusing on the role of P-gp in the BBB. For this purpose, a mouse model disrupted of the genes coding for P-gp (abcb1ab (-/-) mice) was used. Brain and serum concentrations of the enantiomers of VEN and CIT, and their major metabolites, were compared to the corresponding wild-type mice (abcb1ab (+/+) mice). The open-field locomotor and rearing activities were examined after chronic VEN administration. In addition to the animal studies, genetic and toxicological aspects of P-gp were studied in a forensic autopsy material, where intoxication cases were compared with cases that were not related to intoxications.The brain to serum concentration ratios for VEN, CIT and EsCIT differed between knockout mice and wild-type mice, with 2-3 fold higher brain concentrations in mice with no expression of P-gp. Hence, all studied drugs, and their major metabolites, were substrates for P-gp. There was no evidence for a stereoselective P-gp mediated transport. The P-gp substrate properties were reflected in the open-field behavior test where the knockout mice displayed increased center activity compared with wild-type mice following chronic VEN exposure. The genotype distribution of ABCB1 SNPs C1236T, G2677T and C3435T in VEN positive cases was significantly (or borderline) different between the intoxication cases and the non-intoxication cases. This difference in genotype distribution was not observed for the CIT positive cases.To conclude, the present work has led to an increased knowledge about how the enantiomers of VEN and CIT are affected by the BBB transporter P-gp. Using an animal model, VEN and CIT have proved to be actively transported out of the brain by P-gp and no difference was observed for the enantiomers with regard to P-gp transport. Further, the ABCB1 genotype distribution was different in intoxication cases compared with non-intoxication cases. Taken together, these findings offer the possibility that the expression of P-gp in humans may be a contributing factor for limited treatment response and increased risk of side-effects following antidepressant drug treatment.
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| 2. |
- Nilsson, Gunnel, 1950-
(författare)
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Zopiclone degradation in biological samples : Characteristics and consequences in forensic toxicology
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bio-analytical results are influenced by in vivo factors such as genetics, pharmacological and physiological conditions and in vitro factors such as specimen composition, sample additives and storage conditions. Zopiclone (ZOP) is a short-acting hypnotic drug (Imovane®) used for treatment of insomnia. ZOP is metabolized by three major pathways; oxidation to the active zopiclone N-oxide (ZOPNO), demethylation to the inactive N-desmethylzopiclone (NDZOP) and oxidative decarboxylation to other inactive metabolites. ZOP is increasingly being encountered in forensic cases and is a common finding in samples from drug-impaired drivers, users of illicit recreational drugs, victims of drug facilitated sexual assaults and forensic autopsy cases. ZOP is a notoriously unstable analyte in biological matrices and analytical results depend on pre-analytical factors, such as storage time and temperature. The overall aim of this thesis was to investigate the stability of ZOP and the factors of importance for degradation during storage in biological samples and to identify consequences for interpretation of results in forensic toxicology.In paper I, different stability tests in spiked samples were performed including short-term, longterm, freeze-thaw and processed stability. Analyses of ZOP were performed by gas chromatography with nitrogen phosphorous detection and ZOP concentrations were measured at selected time intervals. The degradation product 2-amino-5-chloropyridine (ACP) was identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The stability investigations showed a very poor short-term storage stability of ZOP.Therefore, in paper II, the influence of pre-analytical conditions was further investigated in dosed subjects. Whole blood from volunteers was obtained before and after oral administration of Imovane®. In this study, the influence from physiological factors such as drug interactions, matrix composition and plasma protein levels were minimized. The results showed that ZOP was stable in whole blood for only one day at room temperature, one week in a refrigerator and at least three months frozen in authentic as well as in spiked whole blood. The rapid degradation of ZOP at ambient temperature can cause an underestimation of the true concentration and consequently flaw the interpretation. However, by also analyzing the degradation product ACP the original concentration of ZOP may be estimated.In papers III and IV, two LC-MS-MS methods were validated for the quantitation of ACP, ZOP and NDZOP in blood and ACP, ZOP, NDZOP and ZOPNO in urine. These methods were used in a controlled pharmacokinetic study where whole blood and urine were obtained after oral administration of Imovane®. Samples of blood and urine were aliquoted, analyzed and stored under different conditions and the formation of ACP was monitored. Additionally, at each studied time point the pH of the blood and urine samples was measured using i-STAT® system. The results showed that ACP was formed in equimolar amounts to the degradation of ZOP and its metabolites.In urine samples, the formation of ACP occurred at elevated pH or temperature and mirrored the degradation of ZOP, NDZOP and ZOPNO. The high concentrations of metabolites, which also degraded to ACP, made it impossible to estimate the original ZOP concentration.The results from analysis of blood samples containing ACP were also used to develop mathematical models to estimate the original ZOP concentration. Both models showed strong correlation to the original ZOP concentration (r=0.960 and r=0.955) with p<0.01. This study showed that the equimolar degradation of ZOP and NDZOP to ACP could be used to estimate the original concentration of ZOP in blood samples.Absence of ACP in the blood or urine samples analysed strongly suggests that degradation has not occurred and that the measured concentration of ZOP is reliable. For proper interpretation in forensic cases, it is strongly recommended that ZOP and its metabolites as well as ACP are included in the analysis.
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| 3. |
- Kugelberg, Fredrik C., 1974-
(författare)
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Chiral and toxicological aspects of citalopram : an experimental study in rats
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Citalopram (CIT) is a selective serotonin reuptake inhibitor (SSRI), which is used for the treatment of different psychiatric disorders. The indications for prescription of OT are linked to high risks for intentional intoxications. CIT is one of the most commonly found drugs in Swedish forensic autopsy cases. CIT is a chiral compound, which exists as a racemic mixture (50:50) of the S-(+)-enantiomer (S-CIT) and the R-(-)-enantiomer (R-CIT). The main metabolites, demethylcitalopram (DCIT) and didemethylcitalopram (DCIT), are also chiral compounds. The SSRI effect of CIT is mediated by S-CIT. The routine toxicological screening is an achiral analysis, in which the total amount of the two enantiomers of CIT and metabolites are measured. An extended analysis of the disposition of the Sand R-enantiomers may provide additional information in interpreting forensic toxicological results. Hence, the blood and brain dispositions of the enantiomers of CIT, DCIT and DDCIT in vivo as well as postmortem were studied in an animal model, which also included studies of the behavioural activity.Rats underwent systemic CIT exposure of clinically relevant and high/toxic doses, which were administered acute, chronic or acute-on-chronic. Samples from serum/blood and two brain regions (cortex and mesencephalon-pons) were collected for analysis of the concentrations of the enantiomers of CIT and metabolites using a chiral high performance liquid chromatography (HPLC) method. The open-field locomotor and rearing activities were examined after the chronic CIT exposure.Following chronic CIT administration, R-CIT was present in an increased proportion compared with S-CIT when higher CIT concentration prevailed. Higher drug levels were observed in brain than in serum, and the drug levels between the two compartments correlated well. The rats treated with the high/toxic dose displayed lower behavioural activity during the first test hour as compared to controls and rats given clinically relevant doses. No major effects of CIT on the behavioural rhythm were observed. Shortly after the acute CIT administration, the ratio between S- and R-CIT was close to unity, whereas R-OT was found in higher amount than S-CIT at the end of the study period. The heart blood levels of CIT and metabolites increased postmortem in comparison with the levels observed antemortem after acute, chronic and acute-on-chronic administration. Irrespective of administered dose, the ratios between the S- and R-enantiomers of CIT and DCIT, as well as the CIT/DCIT ratios, were similar antemortem and postmortem.Chiral analysis provided additional information regarding the different administration procedures as compared to achiral analysis. The stereoselective in vivo disposition of the enantiomers of CIT and metabolites was found similar in blood and brain. An equal degree of postmortem redistribution was also seen regarding the enantiomers of CIT and metabolites. These findings may facilitate and improve the interpretation of forensic toxicological results in humans.
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