Kristian Linnet - Academia.edu (original) (raw)

Papers by Kristian Linnet

Drug testing and analysis, Jan 11, 2015

In recent years, synthetic cannabinoids have emerged in the illicit drug market, in particular vi... more In recent years, synthetic cannabinoids have emerged in the illicit drug market, in particular via the Internet, leading to abuse of these drugs. There is currently limited knowledge about the specific enzymes involved in the metabolism of these drugs. In this study, we investigated the cytochrome P450 (CYP) enzymes involved in the metabolism of the two synthetic cannabinoids (1-pentyl-1H-indol-3-yl)-(2,2,3,3-tetramethylcyclopropyl)methanone (UR-144) and [1-(5-fluoropentyl)-1H-indol-3-yl)](2,2,3,3-tetramethylcyclopropyl)methanone (XLR-11). This study extends previous studies by identifying the specific CYP enzymes involved in the metabolism of UR-144 and XLR-11 utilizing a panel of nine recombinant enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 3A4, and 2E1). This is followed by an investigation of the effect of specific inhibitors targeted against CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 in human liver microsomes (HLM). Incubations of UR-144 and XLR-11 with recombinant CYP enzymes rev...

Journal of Analytical Toxicology, 2015

Brain tissue is a useful alternative to blood in postmortem forensic investigations, but scarcity... more Brain tissue is a useful alternative to blood in postmortem forensic investigations, but scarcity of information on reference concentrations in brain tissue makes interpretation challenging. Here we present a study of 43 cases where the antipsychotic drug quetiapine was quantified in brain tissue and related to concentrations in postmortem blood. For cases, where quetiapine was unrelated to the cause of death (N = 36), the 10-90 percentiles for quetiapine concentrations in brain tissue were 0.030-1.54 mg/kg (median 0.48 mg/kg, mean 0.79 mg/kg). Corresponding blood 10-90 percentile values were 0.007-0.39 mg/kg (median 0.15 mg/kg, mean 0.19 mg/kg), giving brain-blood ratio 10-90 percentiles of 2.31-6.54 (median 3.87, mean 4.32). Both correspond well to the limited amount of data found in the literature. For cases where quetiapine was a contributing factor to death (N = 5), the median value in brain tissue of 8.02 mg/kg (range 2.69-22.98 mg/kg) was more than 15 times higher than the median of the nontoxic values, and about the same relationship occurred for blood with a median of 3.19 mg/kg (range 1.00-6.90 mg/kg). The brain-blood ratios for toxic concentrations were in the range of 2.08-6.05, which correspond to those of the nontoxic concentrations. A single case, where quetiapine was ruled as the sole cause of death, a suicide by quetiapine overdose, had an even higher value of 25.74 mg/kg in brain tissue. The blood concentration was 8.99 mg/kg, giving a brain-blood ratio of 2.86. Thus, on average the brain concentrations were about four times the blood concentrations. The brain concentrations of quetiapine observed in cases, where quetiapine was unrelated to death, may serve as a reference, when evaluating postmortem cases with no blood available. The recorded concentrations, where quetiapine was contributing to death, give an indication of likely toxic concentrations.

Forensic Toxicology, 2009

The metabolism of the tricyclic antidepressant amitriptyline was studied in vitro in the presence... more The metabolism of the tricyclic antidepressant amitriptyline was studied in vitro in the presence of the main metabolite nortriptyline to simulate the steady state with amitriptyline and nortriptyline present in the ratio 1:1. The metabolism of the active metabolite nortriptyline in the presence of amitriptyline was also studied. The contribution of cytochrome P450 (CYP) 2D6, CYP2C19, and CYP3A4 was assessed

Clinical pharmacology and therapeutics, 1996

Steady-state serum concentration to dose ratios of the neuroleptic agent perphenazine were relate... more Steady-state serum concentration to dose ratios of the neuroleptic agent perphenazine were related to CYP2D6 metabolizer status for 96 psychiatric inpatients: 88 extensive metabolizers and eight poor metabolizers. The median concentration per dose of the poor metabolizer group (0.195 nmol/L per milligram) was about twice the median (0.098 nmol/L per milligram) of the 56 extensive metabolizers without interacting medicine (p < 0.01). The rest of the extensive metabolizers (n = 32), who were comedicated with drugs that compete with perphenazine for metabolism by CYP2D6, had an intermediate median value of 0.140 nmol/L per milligram. The range of concentration/dose values for the total extensive metabolizer group extended from 0.025 to 0.688 nmol/L per milligram, that is, an almost thirtyfold variation. The concentration/dose range of the eight poor metabolizer subjects was 0.096 to 0.750 nmol/L per milligram. Serum levels not corrected for dose overlapped to a large degree among th...

Journal of chromatography. B, Biomedical sciences and applications, Jan 15, 2000

A fully automated on-line method for determination of nortriptyline in human serum was developed ... more A fully automated on-line method for determination of nortriptyline in human serum was developed using an ASPEC XL (Gilson) solid-phase extraction apparatus in combination with high-performance liquid chromatography. Solid phase extraction was performed on cyanopropyl cartridges. HPLC was carried out using a C18 column with a mobile phase of acetonitrile-0.01 M triethylamine (34:66 v/v) buffer, pH 3.0. UV detection was at 242 nm. The Inter-day CV% was <5%. Comparison with liquid-liquid extraction of serum from patients treated with nortriptyline showed good agreement. Studies of analytical interference from coadministered psychoactive drugs revealed that only imipramine and a methotrimeprazine metabolite interfered.

Journal of chromatography. B, Biomedical sciences and applications, Jan 4, 1998

A method for determination of the atypical neuroleptic drug olanzapine in serum was developed. Af... more A method for determination of the atypical neuroleptic drug olanzapine in serum was developed. After a single-step liquid-liquid extraction, the compound was separated from other constituents on a normal-phase silica gel column using a buffer-methanol mobile phase and measured by UV absorption at 270 nm. Addition of 0.25% ascorbic acid to serum protects olanzapine against oxidation during extraction and stabilizes the easily oxidised compound during storage. Inter-day variation was <8% at serum levels found in olanzapine treated patients. Analytical interference from coadministered psychoactive drugs and their metabolites were studied. Only risperidone, also a relatively newly developed antipsychotic drug, interfered, but the most commonly used antidepressants and traditional antipsychotics and their metabolites did not interfere.

Drug testing and analysis, Jan 24, 2014

Synthetic cannabimimetic agents are a large group of diverse compounds which act as agonists at c... more Synthetic cannabimimetic agents are a large group of diverse compounds which act as agonists at cannabinoid receptors. Since 2004, synthetic cannabinoids have been used recreationally, although several of the compounds have been shown to cause severe toxicity in humans. In this study, the metabolism of two indazole carboxamide derivatives, AB-PINACA and AB-FUBINACA, was investigated by using human liver microsomes (HLM). For both compounds, a major metabolic pathway was the enzymatic hydrolysis of the primary amide, resulting in the major metabolites AB-PINACA-COOH and AB-FUBINACA-COOH. Other major metabolic pathways were mono-hydroxylation of the N-pentyl chain in AB-PINACA and mono-hydroxylation of the 1-amino-3-methyl-1-oxobutane moiety in AB-FUBINACA. To identify the enzyme(s) responsible for the amide hydrolysis, incubations with recombinant carboxylesterases and human serum, as well as inhibition studies in HLM and human pulmonary microsomes (HPM) were performed. Carboxylester...

Forensic Science International, 2015

In Denmark, fatal poisoning among drug addicts is often related to methadone. The primary mechani... more In Denmark, fatal poisoning among drug addicts is often related to methadone. The primary mechanism contributing to fatal methadone overdose is respiratory depression. Concurrent use of other central nervous system (CNS) depressants is suggested to heighten the potential for fatal methadone toxicity. Reduced tolerance due to a short-time abstinence period is also proposed to determine a risk for fatal overdose. The primary aims of this study were to investigate if concurrent use of CNS depressants or reduced tolerance were significant risk factors in methadone-related fatalities using segmental hair analysis. The study included 99 methadone-related fatalities collected in Denmark from 2008 to 2011, where both blood and hair were available. The cases were divided into three subgroups based on the cause of death; methadone poisoning (N=64), poly-drug poisoning (N=28) or methadone poisoning combined with fatal diseases (N=7). No significant differences between methadone concentrations in the subgroups were obtained in both blood and hair. The methadone blood concentrations were highly variable (0.015-5.3, median: 0.52mg/kg) and mainly within the concentration range detected in living methadone users. In hair, methadone was detected in 97 fatalities with concentrations ranging from 0.061 to 211ng/mg (median: 11ng/mg). In the remaining two cases, methadone was detected in blood but absent in hair specimens, suggesting that these two subjects were methadone-naive users. Extensive poly-drug use was observed in all three subgroups, both recently and within the last months prior to death. Especially, concurrent use of multiple benzodiazepines was prevalent among the deceased followed by the abuse of morphine, codeine, amphetamine, cannabis, cocaine and ethanol. By including quantitative segmental hair analysis, additional information on poly-drug use was obtained. Especially, 6-acetylmorphine was detected more frequently in hair specimens, indicating that regular abuse of heroin was common among the deceased. In conclusion, continuous exposure of methadone provide by segmental hair analysis suggested that reduced tolerance of methadone was not a critical factor among methadone-related fatalities. In contrast, a high abundance of co-ingested CNS depressants suggested that adverse effects from drug-drug interactions were more important risk factors for fatal outcome in these deaths.

Journal of analytical toxicology

A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/ep... more A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, sugges...

Drug Metabolism and Drug Interactions, 2006

The impact of P-glycoprotein (P-gp) on the distribution of nortriptyline (NT) and its metabolites... more The impact of P-glycoprotein (P-gp) on the distribution of nortriptyline (NT) and its metabolites between brain, liver and serum was studied experimentally. The interaction of NT with P-gp in vitro was confirmed by measurement of P-gp stimulated ATPase activity (Km = 257.6 microM, Vmax = 51.0 nmol phosphate released/mg protein-min). Administration of NT (5 mg/kg, s.c.) to mdrla knockout mice resulted in enhanced brain-serum (1.6-fold, p = 0.012) and liver-serum (1.4-fold, p = 0.019) ratios, as compared to the wild-type mice. For a series of NT doses (2.5, 5, 10, 25, 30 mg/kg, i.p.) inhibition of P-gp with cyclosporine A (CsA, 200 mg/kg, i.p.) in rats led to NT brain- and liver-serum ratios that were on average 1.3- (p = 0.005) and 2.1- (p = 0.001) fold higher than those of the controls, respectively. Verapamil (50 mg/kg) (NT, 5 mg/kg) increased the ratios by a factor of 1.6 (p &amp;amp;amp;amp;amp;amp;amp;lt;0.001) and 10.3 (p &amp;amp;amp;amp;amp;amp;amp;lt;0.001) for brain and liver, respectively. Finally, co-administration of methadone (1 mg/kg) did not alter the brain-serum ratio of NT, but in the liver a slight increase (1.5-fold, p = 0.035) was observed. In conclusion, verapamil yielded complete inhibition of P-gp at the blood-brain barrier and CsA had an effect corresponding to about 50% inhibition. The results show that P-gp influences the penetration of NT into the brain, and that drug-drug interactions may take place.

Therapeutic Drug Monitoring, 2001

A method is presented for unattended fully automated extraction and on-line determination of the ... more A method is presented for unattended fully automated extraction and on-line determination of the atypical antipsychotic drug olanzapine in serum. An ASPEC automatic sample-preparing apparatus with Isolute cyanopropyl-bonded silicagel cartridges was used for solid-phase extraction of the drugs from serum. The adsorbed drugs were eluted with methanol and an aliquot injected into a high-performance liquid chromatograph (HPLC) apparatus. Trifluoperazine was used as internal standard, and the analytes were separated on an unmodified silicagel column using methanol-ammonium acetate buffer pH 9.9 (85:15) as mobile phase. Ultraviolet detection at 257 nm was used for quantitation. Within the therapeutic range for the serum concentration of olanzapine, the interday variations for the quantitative determinations were <8%. Comparisons between concentrations measured using liquid-liquid extraction and the present on-line extraction method showed good agreement. Other drugs often used in combination with olanzapine did not interfere with the quantitative determinations. The method has been in routine use for more than 1 year for therapeutic drug monitoring.

Therapeutic Drug Monitoring, 2002

The influence of carbamazepine on the glucuronidation of the antipsychotic olanzapine was studied... more The influence of carbamazepine on the glucuronidation of the antipsychotic olanzapine was studied in a group of psychiatric patients. Steady-state serum concentrations of free and glucuronidated olanzapine were measured in 31 psychiatric patients in monotherapy (dose range, 2.5-30 mg/d; median, 15 mg/d) and in 16 patients being comedicated with carbamazepine (dose range, 5-50 mg/d; median, 20 mg/d). The concentrations were determined by HPLC with and without acid hydrolysis of glucuronidated olanzapine. For the monotherapy group, the concentrations of free and glucuronidated olanzapine ranged from 0 nmol/L to 292 nmol/L (median, 94 nmol/L) and from 0 nmol/L to 180 nmol/L (median, 27 nmol/L), respectively. The serum concentrations of the carbamazepine-treated group ranged from 21 nmol/L to 310 nmol/L (median, 81 nmol/L) and from 0 to 376 nmol/L (median, 57 nmol/L) for free and glucuronidated olanzapine, respectively. Two patients with outlying values were excluded from further analysis. The median concentration-to-daily dose ratios (C/D) of free and glucuronidated olanzapine in the monotherapy group were 5.8 nmol/L/mg and 2.2 nmol/L/mg, respectively (n =30). The corresponding values for the group comedicated with carbamazepine were 3.6 and 3.1 nmol/L/mg (n =15). Thus, the median C/D of free olanzapine in the carbamazepine group was 38% lower than that of the monotherapy group (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.01), confirming that carbamazepine accelerates the metabolism of olanzapine. Further, for the carbamazepine group the median glucuronidated olanzapine fraction constituted 79% of the free fraction compared with 43% for the monotherapy group (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.01), which suggests that an increased rate of olanzapine glucuronidation contributes to the increased rate of metabolism of olanzapine induced by carbamazepine.

Scandinavian Journal of Clinical & Laboratory Investigation, 2007

Four different mixed-mode cation exchange solid-phase extraction columns were compared for extrac... more Four different mixed-mode cation exchange solid-phase extraction columns were compared for extraction of basic drugs from urine using HPLC-DAD analysis: Isolute HCX-3, ABN, Bond Elut Certify and Oasis MCX. With one exception, all the studied drugs attained recoveries exceeding 50 % for all columns. We considered the following drugs: amphetamine, amlodipine, chlorprothixene, fentanyl, haloperidol, ketobemidone, methadone, mirtazapine, olanzapine, quetiapine, sertraline and zopiclone. The average recoveries for the four column types ranged from 68.5 % to 92.1 % in the experiments. In conclusion, the basic compounds performed reasonably well in all columns, and other factors, such as availability and price, may be decisive in regard to choice of column.

Pharmacology, 1997

The metabolism of amitriptyline was studied in vitro using cDNA-expressed human cytochrome P450 (... more The metabolism of amitriptyline was studied in vitro using cDNA-expressed human cytochrome P450 (CYP) enzymes 1A2, 3A4, 2C9, 2C19, 2D6 and 2E1. CYP 2C19 was the most important enzyme with regard to the demethylation of amitriptyline, the quantitatively most important metabolic pathway. CYP 1A2, 3A4, 2C9 and CYP 2D6 also participated in the demethylation of amitriptyline. CYP 2D6 was the sole enzyme mediating the hydroxylation of amitriptyline, and (E)-10-OH-amitriptyline was exclusively produced. CYP 2E1 did not metabolize amitriptyline. Concerning the quantitative relations, CYP 2C19 and 2D6 exhibited high affinities with Km values in the range of 5-13 mumol/l, whereas the affinities of 1A2, 3A4 and 2C9 were somewhat lower with Km values ranging from 74 to 92 mumol/l. CYP 2C19 displayed the highest reaction capacity per mole with Vmax equal to 475 mol h-1 (mol CYP)-1. The other enzymes had Vmax values in the range of 90-145 mol h-1 (mol CYP)-1. Allowing for the typical relative distribution of amounts of CYP enzymes in the liver, a simulation study suggested that, at therapeutic doses, on average about 60% of the metabolism depended on CYP 2C19. At toxic doses, CYP 2C19 is expected to be saturated, and CYP 3A4 may now play a dominant role in the metabolism.

Pharmacology, 1999

The involvement of CYP enzymes in the metabolism of citalopram was studied, inclusive the convers... more The involvement of CYP enzymes in the metabolism of citalopram was studied, inclusive the conversion of demethylcitalopram to didemethylcitalopram and the formation of citalopram N-oxide, which both have not been considered previously. Using human mixed liver microsomes and cDNA-expressed CYP enzymes, we confirmed that CYP3A4, 2C19 and 2D6 are involved in the first demethylation step of citalopram, all favouring conversion of the biologically active S-enantiomer. Inhibitor studies indicated that at therapeutic citalopram concentrations CYP3A4 was responsible for 40-50% of demethylcitalopram formation, while the contribution of CYP2C19 increased and that of CYP2D6 tended to decrease with increasing drug concentration. CYP2D6 exclusively mediated the second demethylation step, and citalopram N-oxide was also exclusively formed by CYP2D6. None of the studied CYP enzymes mediated deamination to the propionic acid derivative.

Journal of Separation Science, 2013

A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE ... more A broad forensic screening method for 256 analytes in whole blood based on a fully automated SPE robotic extraction and ultra-high-performance liquid chromatography (UHPLC) with TOF-MS with data-independent acquisition has been developed. The limit of identification was evaluated for all 256 compounds and 95 of these compounds were validated with regard to matrix effects, extraction recovery, and process efficiency. The limit of identification ranged from 0.001 to 0.1 mg/kg, and the process efficiency exceeded 50% for 73 of the 95 analytes. As an example of application, 1335 forensic traffic cases were analyzed with the presented screening method. Of these, 992 cases (74%) were positive for one or more traffic-relevant drugs above the Danish legal limits. Commonly abused drugs such as amphetamine, cocaine, and frequent types of benzodiazepines were the major findings. Nineteen less frequently encountered drugs were detected e.g. buprenorphine, butylone, cathine, fentanyl, lysergic acid diethylamide, m-chlorophenylpiperazine, 3,4methylenedioxypyrovalerone, mephedrone, 4-methylamphetamine, p-fluoroamphetamine, and p-methoxy-N-methylamphetamine. In conclusion, using UHPLC-TOF-MS screening with data-independent acquisition resulted in the detection of common drugs of abuse as well as new designer drugs and more rarely occurring drugs. Thus, TOF-MS screening of blood samples constitutes a practical way for screening traffic cases, with the exception of ␦-9-tetrahydrocannabinol, which should be handled in a separate method.

Journal of Forensic Sciences, 2010

We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity... more We investigated toxicological and pharmacogenetic factors that could influence methadone toxicity using postmortem samples.

Journal of Chromatography B, 2003

A quantitative method for determination of quetiapine (QTP) in human serum is presented. The meth... more A quantitative method for determination of quetiapine (QTP) in human serum is presented. The method is fully automated and based on high performance liquid chromatography (HPLC) with on-line solid phase extraction (SPE). The extraction procedure is based on a C2 cartridge, which is eluted with methanol. The eluate is injected onto a silica column with a mobile phase consisting of methanol:20 mM NH(4)CH(3)COO, pH 5.0 (99:1). Quetiapine is quantified by ultra-violet (UV) absorbance at 257 nM with trifluoperazine as the internal standard (I.S.). The extraction recoveries for quetiapine and trifluoperazine were 69 and 57%, respectively. The total inter day coefficient of variation was 11.1, 3.8 and 3.1% at 20, 500 and 1000 nM, respectively. The detection limit was 10.3 nM quetiapine. The method has been used in our therapeutic drug monitoring (TDM) laboratory where co-administered drugs often are observed. In an investigation of analytical interference from co-administered drugs, demethyl-mianserine was the only drug which interfered with the internal standard. There was no interference with quetiapine itself. The method showed good agreement with mass spectrometric quantification of quetiapine.

Journal of Agricultural and Food Chemistry, 1983

A simplified method for the determination of racemic citalopram and its main metabolite desmethyl... more A simplified method for the determination of racemic citalopram and its main metabolite desmethylcitalopram in serum using HPLC was developed. The compounds were extracted with heptane-isoamyl alcohol (98:2) and subsequently transferred into phosphate buffer pH 2.5 for direct injection into the HPLC apparatus. The analytes were separated with an acetonitrile-phosphate buffer, pH 2.5-tetraethylamine mobile phase on a C18 column and measured by UV detection at 240 nm. Within the typical range of serum concentrations (30-100 ng/ml) the inter-day variation was < 6% for both compounds. Possible analytical interference from a number of commonly coadministered psychoactive drugs and their metabolites was studied by extracting sera from patients receiving these drugs. Interference was not a problem for the developed method.

Human Psychopharmacology: Clinical and Experimental, 2005

The distribution of the antidepressant drug nortriptyline (NT) and its main metabolite E-10-hydro... more The distribution of the antidepressant drug nortriptyline (NT) and its main metabolite E-10-hydroxy-nortriptyline (E-10-OH-NT) across the blood-brain barrier was considered in relation to inhibition of the multidrug transporter P-glycoprotein (P-gp). Rats received NT in doses of 25 mg/kg orally, 10 mg/kg i.p. or 25 mg/kg i.p. Half the rats were treated with the Pglycoprotein inhibitor cyclosporine A (CsA) (200 mg/kg) 2 h prior to NT administration, and the other half served as a control group. NT and the metabolite were extracted from brain and serum by liquid-liquid extraction and analysed by HPLC with UV-detection. The brain to serum ratio of NT was increased in the CsA treated groups (22.3-26.8) compared with the control groups (16.5-22.7), the difference being statistically significant in two of the three experiments ( p < 0.05). Increased brain-serum ratios were also found for E-10-OH-NT, but the differences were not statistically significant. These results suggest that inhibition of P-gp by CsA increases the accumulation of NT in the brain. Administration of the antipsychotic drug risperidone (0.5 mg/kg s.c.), which is a P-gp substrate, instead of CsA did not exert any measurable influence on the blood-brain ratio of NT concentrations.