Investigating the correlation between genotypes and hepatic protein expression of CYP2C9, CYP2C19, CYP2D6, and CYP3A5 using postmortem tissue from a Danish population.

The cytochrome (CYP) P450 family of enzymes plays a central role in the metabolism of many drugs. CYP genes are highly polymorphic, which is known to affect protein levels, but for some low frequent CYP genotypes the correlation between genotype and CYP protein expression is less established. In this study, we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes of 250 Danish individuals included in a postmortem study. For 116 of the individuals, the hepatic CYP protein levels were investigated by a proteomics approach. Overall, we found the postmortem genetic and proteomic data to be in agreement with those of other studies performed on fresh hepatic tissue, showing the usability of postmortem hepatic tissue for this type of investigation. For less investigated genotypes we could corroborate previously found results: 1) statistically significantly lower levels of hepatic CYP2C9 protein in individuals carrying the CYP2C9*3 variant compared to individuals with two wild type (wt) alleles, 2) comparable levels of CYP2C19 in CYP2C19*2/*17 and CYP2C19*1/*2 individuals, 3) reduced CYP2D6 protein levels in heterozygous individuals with the CYP2D6*3, CYP2D6*4, and CYP2D6*5 gene deletion variants, and 4) significantly lower levels of CYP3A5 protein in CYP3A5*3 homozygous individuals compared to individuals that were heterozygous for the CYP3A5*3 allele or homozygous individuals for the wt alleles. In conclusion, the use of postmortem tissue significantly increases the access to human specimens for research purposes, and postmortem proteomics can be used to investigate the link between CYP genotypes and hepatic protein expression. Significance Statement In tissue from a large postmortem cohort (n=250) we determined the CYP2C9, CYP2C19, CYP2D6, and CYP3A5 genotypes. Hepatic CYP protein levels were investigated in 116 individuals using a proteomics approach. For common genotypes, we found results similar to previous knowledge, pointing towards the usability of postmortem tissue. For the less investigated genotypes, we were able to corroborate genotype / protein expression correlations. It is a novel approach to use a large postmortem cohort to investigate genetic / protein expression correlations.

Combined in vivo metabolic effects of quetiapine and methadone in brain and blood of rats.

Changes in pharmacokinetics and endogenous metabolites may underlie additive biological effects of concomitant use of antipsychotics and opioids. In this study, we employed untargeted metabolomics analysis and targeted analysis to examine the changes in drug metabolites and endogenous metabolites in the prefrontal cortex (PFC), midbrain, and blood of rats following acute co-administration of quetiapine and methadone. Rats were divided into four groups and received cumulative increasing doses of quetiapine (QTP), methadone (MTD), quetiapine + methadone (QTP + MTD), or vehicle (control). All samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). Our findings revealed increased levels of the quetiapine metabolites: Norquetiapine, O-dealkylquetiapine, 7-hydroxyquetiapine, and quetiapine sulfoxide, in the blood and brain when methadone was present. Our study also demonstrated a decrease in methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in the rat brain when quetiapine was present. Despite these findings, there were only small differences in the levels of 225-296 measured endogenous metabolites due to co-administration compared to single administrations. For example, N-methylglutamic acid, glutaric acid, p-hydroxyphenyllactic acid, and corticosterone levels were significantly decreased in the brain of rats treated with both compounds. Accumulation of serotonin in the midbrain was additionally observed in the MTD group, but not in the QTP + MTD group. In conclusion, this study in rats suggests a few but important additive metabolic effects when quetiapine and methadone are co-administered.

Enhanced autopsy triage (EA-Triage) in drug-related deaths: integrating quick toxicological analysis and postmortem computed tomography.

The objective was to assess the diagnostic accuracy of an enhanced autopsy triage (EA-Triage) setup consisting of postmortem computed tomography (PMCT), simulated quick toxicological analysis (sQTA), external examination, and case information in determining cause of death (COD) in persons with past or current use of illegal drugs (drug-related deaths). Information on drug-related deaths selected for medico-legal autopsy in 2020-2021 at the Department of Forensic Medicine, Aarhus University, Denmark, was analyzed retrospectively. The included cases underwent conventional autopsy, PMCT, and systematic toxicological analysis. A board-certified forensic pathologist, who was blinded to the internal examination and COD from the medico-legal autopsy, determined COD based on the EA-Triage setup. 154 cases with a median age of 40.6 years (range 17-70 years, 82% males) were included. The COD determined by medico-legal autopsy and that determined by EA-Triage matched in 113 cases (73%), including those with an unknown COD. EA-Triage and medico-legal autopsy determined unknown COD in 45 (29%) and 5 cases (3%), respectively. Excluding cases with an unknown COD, EA-Triage predicted COD in 109 cases (71%); of those, 72 (66%) had no unexplained case circumstances or suspicion of a criminal act. In these 72 cases, the CODs determined by EA-Triage and medico-legal autopsy matched in 71 cases (99%), and the sensitivity and specificity for detecting lethal intoxication were 100% and 90%, respectively. EA-Triage showed strong diagnostic accuracy for determining COD in drug-related deaths. This method may be suitable for enhancing preautopsy triage and guiding police investigations at an early stage.

Sex- and Lifestyle-Related Factors are Associated with Altered Hepatic CYP Protein Levels in People Diagnosed with Mental Disorders.

In this study, we used human postmortem tissue to investigate hepatic protein expression levels of cytochrome P450 (CYP) 1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 by LC-MS/MS in a population of people suffering from mental disorders (n = 171). We report hepatic protein levels of these six CYP isoforms in 171 individuals in total, and define a focused population dataset of 116 individuals after excluding 55 samples due to low microsomal protein per gram of liver (MPPGL) yield. Postmortem decay was most likely the reason for the low MPPGL yield in the 55 samples. In the focused population, we found women to have significantly higher protein levels of CYP3A4 than men in addition to decreased CYP3A4 protein levels among obese individuals. Furthermore, MPPGL was negatively correlated with body mass index (BMI). An increase in CYP1A2 protein levels was observed among smokers, and increased CYP2E1 protein levels were observed among individuals with a history of alcohol abuse. Finally, individuals who received phenobarbital (CYP3A4 inducer) had significantly higher CYP3A4 levels. In conclusion, lifestyle-related factors prevalent among people suffering from mental disorders are associated with altered CYP protein levels, which may alter drug metabolism and affect the efficacy of commonly prescribed drugs. Furthermore, this investigation demonstrates that postmortem hepatic tissue can be used to study how lifestyle and effectors affect hepatic CYP-levels in a large cohort of patients. SIGNIFICANCE STATEMENT: Using a large number of postmortem hepatic tissue specimens (n=116) originating from the autopsy of individuals diagnosed with mental disorders, we were able to show that hepatic CYP-levels were affected by alcohol, smoking, BMI, and sex and that MPPGL was affected by BMI. These lifestyle-related changes may alter drug metabolism and affect the efficacy of commonly prescribed drugs. It is a novel approach to use a large postmortem cohort to investigate how lifestyle and effectors affect hepatic CYP-levels.

Senicapoc treatment in COVID-19 patients with severe respiratory insufficiency-A randomized, open-label, phase II trial.

BACKGROUND: The aim of the current study was to determine if treatment with senicapoc, improves the PaO2 /FiO2 ratio in patients with COVID-19 and severe respiratory insufficiency. METHODS: Investigator-initiated, randomized, open-label, phase II trial in four intensive care units (ICU) in Denmark. We included patients aged ≥18 years and admitted to an ICU with severe respiratory insufficiency due to COVID-19. The intervention consisted of 50 mg enteral senicapoc administered as soon as possible after randomization and again after 24 h. Patients in the control group received standard care only. The primary outcome was the PaO2 /FiO2 ratio at 72 h. RESULTS: Twenty patients were randomized to senicapoc and 26 patients to standard care. Important differences existed in patient characteristics at baseline, including more patients being on non-invasive/invasive ventilation in the control group (54% vs. 35%). The median senicapoc concentration at 72 h was 62.1 ng/ml (IQR 46.7-71.2). The primary outcome, PaO2 /FiO2 ratio at 72 h, was significantly lower in the senicapoc group (mean 19.5 kPa, SD 6.6) than in the control group (mean 24.4 kPa, SD 9.2) (mean difference -5.1 kPa [95% CI -10.2, -0.04] p = .05). The 28-day mortality in the senicapoc group was 2/20 (10%) compared with 6/26 (23%) in the control group (OR 0.36 95% CI 0.06-2.07, p = .26). CONCLUSIONS: Treatment with senicapoc resulted in a significantly lower PaO2 /FiO2 ratio at 72 h with no differences for other outcomes.

Utilizing postmortem drug concentrations in mechanistic modeling and simulation of cardiac effects: a proof of concept study with methadone.

Methadone-related poisoning has been found to be the leading and increasing cause of death among intoxication cases in several countries. Aside from respiratory depression, methadone is known to cause QT-prolongation, which may lead to sudden cardiac death. Concentrations in heart tissue should be more accurate for estimating cardiotoxic effects. The aim of this study was to investigate whether the effect of methadone on the QT-interval could be simulated and whether the concentrations in heart tissues allowed for better prediction of the Bazett corrected QT-interval (QTcB). A predictive performance study was conducted using the simulation platform Cardiac Safety Simulator to mimic five literature studies using their described study conditions. Both free and total plasma and heart concentrations were investigated using two different in silico models: the O'Hara-Rudy (ORD) model and the 10 Tusscher (TNNP) model. The results showed that the QTcB of methadone was best predicted either with total plasma using the TNNP model or with free plasma using the ORD model. The ORD model was highly sensitive to the total heart concentrations, resulting in overprediction of the QTcB. The TNNP model also overpredicted the QTcB, but to a lesser degree than the ORD model. Furthermore, due to a low baseline QTcB, the ORD model underpredicted the QTcB for both the free plasma and free heart concentrations. In conclusion, it is possible to simulate the cardiac effects of methadone, yet several elements influence the approach uncertainty including but not limited to biophysically details model of cardiac electrophysiology, exposure data, and input parameters.

A Metabolomics Study of Retrospective Forensic Data from Whole Blood Samples of Humans Exposed to 3,4-Methylenedioxymethamphetamine: A New Approach for Identifying Drug Metabolites and Changes in Metabolism Related to Drug Consumption.

The illicit drug 3,4-methylenedioxymethamphetamine (MDMA) has profound physiological cerebral, cardiac, and hepatic effects that are reflected in the blood. Screening of blood for MDMA and other narcotics are routinely performed in forensics analysis using ultra-performance liquid chromatography with high-resolution time-of-flight mass spectrometry (UPLC-HR-TOFMS). The aim of this study was to investigate whether such UPLC-HR-TOFMS data collected over a two-year period could be used for untargeted metabolomics to determine MDMA metabolites as well as endogenous changes related to drug response and toxicology. Whole blood samples from living Danish drivers' positive for MDMA in different concentrations were compared to negative control samples using various statistical methods. The untargeted identification of known MDMA metabolites was used to validate the methods. The results further revealed changes of several acylcarnitines, adenosine monophosphate, adenosine, inosine, thiomorpholine 3-carboxylate, tryptophan, S-adenosyl-l-homocysteine (SAH), and lysophospatidylcholine (lysoPC) species in response to MDMA. These endogenous metabolites could be implicated in an increased energy demand and mechanisms related to the serotonergic syndrome as well as drug induced neurotoxicity. The findings showed that it was possible to extract meaningful results from retrospective UPLC-HR-TOFMS screening data for metabolic profiling in relation to drug metabolism, endogenous physiological effects, and toxicology.

Medicinal cannabis tea contains variable doses of cannabinoids and no terpenes.

Tea is a recommended way of administration of prescribed cannabis plant products in Denmark. We aimed to investigate the cannabinoid and terpene doses contained in different teas. We analysed tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and terpene concentrations in three repeated preparations of each type of tea, and in plant material. In standard tea, concentrations of THC were [median (min-max)] 9.5 (2.3-15), 19 (13-34), and 36 (26-57) µg/mL for products with a labelled content of 6.3%, 14%, and 22% total THC (THC + THCA), respectively. The CBD concentration in tea from a product labelled with 8% total CBD (CBD + CBDA) was 7.5 (1.9-10) µg/mL. Based on this, the recommended starting amount of 0.2 L of the different teas would contain between 0.46 and 11.3 mg THC, and 0.38 to 2.0 mg CBD. Adding creamer before, but not after boiling, increased the THC and CBD concentration 2.3-4.4 and 2.1-fold, respectively. Terpenes were detected in plant material, but not in tea. The study elucidates THC and CBD doses in different teas, which may assist the clinician's choice of cannabis product. Moreover, it underscores the need for caution as administration as tea can result in exposure to different doses, even when the same cannabis product is used.

Exploring death scenes and circumstances in fatal opioid poisonings: Insights for preventive strategies using forensic autopsy cases in Western Denmark.

INTRODUCTION: Fatal opioid poisoning is a growing global issue. This study aims to describe circumstances surrounding fatal opioid poisonings by examining death scenes, demographics, and information from bystanders with the goal of informing prevention efforts. METHODS: We extracted data from the autopsy reports of 327 forensic autopsy cases with fatal poisoning involving methadone and/or morphine from 2013-2020. RESULTS: Fatal opioid poisonings occurred in both rural and urban areas. Death scene was the decedent's own home and a relative's or friend's home in 62% and 21%, respectively. The decedent died alone in 64% of the cases while other people were staying at the same address while death occurred in 30%. Decedents aged 15-34 years were more likely to die with other people staying at the same address than persons aged > 44 years (OR±SD: 2.3 ± 0.9, p = 0.005), and had lower postmortem blood methadone concentrations compared to persons > 34 years (Median [interquartile range]: 0.36 [0.23-0.62] vs 0.63 [0.28-1.2] mg/kg, p = 0.002). Female sex was more prevalent, and persons using illegal drugs were less prevalent in decedents aged > 44 years compared to those with age 15-44 years (29% vs 20%, p = 0.05% and 67% vs 89%, p < 0.001, respectively). Other psychoactive drugs were detected in 97% of decedents, mainly benzodiazepines (80%). CONCLUSIONS: Preventive strategies based on our findings include the need for harm reduction initiatives in both urban and rural areas, recognizing symptoms of fatal poisoning, and awareness of low tolerance among younger age groups. Urgent attention should be given to avoiding opioid use alone, particularly among older individuals, including women using prescribed opioids. Conveying the risks of polydrug use to all age groups is essential, especially co-use of sedative drugs.

Pharmacokinetics and pharmacodynamics of cannabis-based medicine in a patient population included in a randomized, placebo-controlled, clinical trial.

Information on the pharmacokinetics (PK) and pharmacodynamics (PD) of orally administered cannabis-based medicine (CBM) in capsule formulation in patient populations is sparse. In this exploratory study, we aimed to evaluate the PK and PD in a probable steady state of CBM in neuropathic pain and spasticity in a population of patients with multiple sclerosis (MS). Of 134 patients participating in a randomized, double-blinded, placebo-controlled, trial, 23 patients with MS (17 female) mean age 52 years (range 21-67) were enrolled in this substudy. They received oral capsules containing Δ9 -tetrahydrocannabinol (THC, n = 4), cannabidiol (CBD, n = 6), a combination (THC&CBD, n = 4), or placebo (n = 9). Maximum doses were 22.5 mg (THC) and 45 mg (CBD) a day divided into three administrations. PD parameters were evaluated for pain and spasticity. Blood samples were analyzed using an ultra-high-performance liquid chromatography-tandem mass spectrometer after protein precipitation and phospholipid removal. PK parameters were estimated using computerized modeling. The variation in daily dose and PK between individuals was considerable in a steady state, yet comparable with previous reports from healthy controls. Based on a simulation of the best model, the estimated PK parameters (mean) for THC (5 mg) were Cmax 1.21 ng/mL, Tmax 2.68 h, and half-life 2.75 h, and for CBD (10 mg) were Cmax 2.67 ng/mL, Tmax 0.10 h, and half-life 4.95 h, respectively. No effect was found on the PD parameters, but the placebo response was considerable. More immediate adverse events were registered in the active treatment groups compared with the placebo group.

Stability of Lisdexamfetamine in Sampled Whole Blood-Implications of Sampling Tube Additives and Storage Temperature for Interpretation of Impairment.

Lisdexamfetamine (LDX) is a prodrug that is enzymatically converted into dextroamphetamine (d-AMP), a central nervous system stimulant. The stability of LDX in sampled whole blood is an important issue that may be crucial in the assessment of impaired driving caused by d-AMP. This study investigated the stability of LDX in whole blood collected in two different tubes containing a fluoride oxalate (FX) mixture and a fluoride citrate (FC) mixture. Without additives, LDX was unstable. LDX was also unstable in FX blood stored at ambient temperature or 4°C. After 3 days of storage at ambient temperature, an initial LDX concentration of 47 ± 1 ng/g (mean ± SD) was no longer detectable in the samples (n = 3). Instead, 19 ± 0.6 ng/g d-AMP was formed. The stability was improved at 4°C. After 7 days of storage at 4°C, 88 ± 5% of an initial LDX concentration of 50 ± 0.4 ng/g was recovered and 3.8 ± 0.3 ng/g d-AMP was formed. The stability of LDX was greater in FC blood than in FX blood; 79 ± 10% and 93 ± 4% of initial LDX concentrations of 48 ± 2 and 51 ± 0.5 ng/g were recovered from FC blood after 7 days of storage at ambient temperature and 4°C, respectively, and the corresponding formation of d-AMP was 5.8 ± 0.6 and 0.5 ± 0.3 ng/g, respectively. When FX and FC blood were stored at -20°C or -80°C, no detectable degradation of LDX or formation of d-AMP was observed after 3 weeks of storage.

Deaths caused by medication in persons not using illicit narcotic drugs: An autopsy study from Western Denmark.

Information regarding deaths caused by poisoning or adverse effects of medication in Danish persons not using illicit narcotic drugs (PNUIDs) is sparse. To characterize aetiology, demographics, and death scene, we reviewed all legal autopsies performed at Aarhus University from 2017 to 2019 and isolated 96 deaths caused by medications in PNUIDs. Suicides caused by medication overdose accounted for 38%. Opioids and psychotropic medications were the main cause of death in 48% and 35% of the 96 cases, respectively. Morphine, tramadol, and quetiapine were the most commonly involved individual medications. A single medication caused death in 50% of cases, and multiple substances were involved in 50%. The median total number [interquartile range] of detected medications was 5 [4-6], with a higher number in females (5 [4-7]) than males (4 [2-5]), p = 0.009. Median age was 51 [42.5-61.5] years, and 57% were female. Scene of death most frequently involved a body on a bed or couch in the decedent's own home (72%). In conclusion, opioids and psychotropic medications dominated by morphine, tramadol and quetiapine most frequently caused medication-related deaths in PNUIDs. Monitoring this type of death may yield important knowledge to direct prophylactic initiatives regarding medication use and prescription.

Large-Scale metabolomics: Predicting biological age using 10,133 routine untargeted LC-MS measurements.

Untargeted metabolomics is the study of all detectable small molecules, and in geroscience, metabolomics has shown great potential to describe the biological age-a complex trait impacted by many factors. Unfortunately, the sample sizes are often insufficient to achieve sufficient power and minimize potential biases caused by, for example, demographic factors. In this study, we present the analysis of biological age in ~10,000 toxicologic routine blood measurements. The untargeted screening samples obtained from ultra-high pressure liquid chromatography-quadruple time of flight mass spectrometry (UHPLC- QTOF) cover + 300 batches and + 30 months, lack pooled quality controls, lack controlled sample collection, and has previously only been used in small-scale studies. To overcome experimental effects, we developed and tested a custom neural network model and compared it with existing prediction methods. Overall, the neural network was able to predict the chronological age with an rmse of 5.88 years (r2  = 0.63) improving upon the 6.15 years achieved by existing normalization methods. We used the feature importance algorithm, Shapley Additive exPlanations (SHAP), to identify compounds related to the biological age. Most importantly, the model returned known aging markers such as kynurenine, indole-3-aldehyde, and acylcarnitines along with a potential novel aging marker, cyclo (leu-pro). Our results validate the association of tryptophan and acylcarnitine metabolism to aging in a highly uncontrolled large-s cale sample. Also, we have shown that by using robust computational methods it is possible to deploy large LC-MS datasets for metabolomics studies to reduce the risk of bias and empower aging studies.

Efficacy of Chronic Paroxetine Treatment in Mitigating Amyloid Pathology and Microgliosis in APPSWE/PS1ΔE9 Transgenic Mice.

BACKGROUND: Modulation of serotonergic signaling by treatment with selective serotonin reuptake inhibitors (SSRIs) has been suggested to mitigate amyloid-ß (Aß) pathology in Alzheimer's disease, in addition to exerting an anti-depressant action. OBJECTIVE: To investigate the efficacy of chronic treatment with the SSRI paroxetine, in mitigating Aß pathology and Aß plaque-induced microgliosis in the hippocampus of 18-month-old APPswe/PS1ΔE9 mice. METHODS: Plaque-bearing APPswe/PS1ΔE9 and wildtype mice were treated with paroxetine per os at a dose of 5 mg/kg/day, from 9 to 18 months of age. The per os treatment was monitored by recording of the body weights and serum paroxetine concentrations, and by assessment of the serotonin transporter occupancy by [3H]DASB-binding in wildtype mice. Additionally, 5,7-dihydroxytryptamine was administered to 9-month-old APPswe/PS1ΔE9 mice, to examine the effect of serotonin depletion on Aß pathology. Aß pathology was evaluated by Aß plaque load estimation and the Aß42/Aß40 ratio by ELISA. RESULTS: Paroxetine treatment led to > 80% serotonin transporter occupancy. The treatment increased the body weight of wildtype mice, but not of APPswe/PS1ΔE9 mice. The treatment had no effect on the Aß plaque load (p = 0.39), the number and size of plaques, or the Aß plaque-induced increases in microglial numbers in the dentate gyrus. Three months of serotonin depletion did not significantly impact the Aß plaque load or Aß42/Aß40 ratio in APPswe/PS1ΔE9 mice at 12 months. CONCLUSION: Our results show that chronic treatment with the SSRI paroxetine does not mitigate Aß pathology and Aß plaque-induced microgliosis in the hippocampus of APPswe/PS1ΔE9 mice.

A Retrospective Metabolomics Analysis of Gamma-Hydroxybutyrate in Humans: New Potential Markers and Changes in Metabolism Related to GHB Consumption.

GHB is an endogenous short-chain organic acid presumably also widely applied as a rape and knock out drug in cases of drug-facilitated crimes or sexual assaults (DFSA). Due to the endogenous nature of GHB and its fast metabolism in vivo, the detection window of exogenous GHB is however narrow, making it challenging to prove use of GHB in DFSA cases. Alternative markers of GHB intake have recently appeared though none has hitherto been validated for forensic use. UHPLC-HRMS based screening of blood samples for drugs of abuse is routinely performed in several forensic laboratories which leaves an enormous amount of unexploited data. Recently we devised a novel metabolomics approach to use archived data from such routine screenings for elucidating both direct metabolites from exogenous compounds, but potentially also regulation of endogenous metabolism and metabolites. In this paper we used UHPLC-HRMS data acquired over a 6-year period from whole blood analysis of 51 drivers driving under the influence of GHB as well as a matched control group. The data were analyzed using a metabolomics approach applying a range of advanced analytical methods such as OPLS-DA, LASSO, random forest, and Pearson correlation to examine the data in depth and demonstrate the feasibility and potential power of the approach. This was done by initially detecting a range of potential biomarkers of GHB consumption, some that previously have been found in controlled GHB studies, as well as several new potential markers not hitherto known. Furthermore, we investigate the impact of GHB intake on human metabolism. In aggregate, we demonstrate the feasibility to extract meaningful information from archived data here exemplified using GHB cases. Hereby we hope to pave the way for more general use of the principle to elucidate human metabolites of e.g. new legal or illegal drugs as well as for applications in more global and large scale metabolomics studies in the future.

Stability investigations of cytochrome P450 (CYP) enzymes immediately after death in a pig model support the applicability of postmortem hepatic CYP quantification.

Quantification of drug-metabolizing cytochrome P450 (CYP) isoforms using LC-MS/MS has been proposed as a potential way of estimating antemortem CYP levels using postmortem tissue, but the postmortem stability of CYP proteins is incompletely investigated. If one can use data obtained from the analysis of postmortem specimens to inform physiologically based pharmacokinetic (PBPK) models this greatly increases the access to rare specimens among special subpopulations. In this study, we developed and validated an LC-MS/MS method for targeted CYP protein quantification in a porcine animal model to study postmortem stability. We measured 19.9-28.3 pmol CYP1A2, 50.3-66.2 pmol CYP2D25, 132.9-142.7 pmol CYP2E1, and 16.8-48 pmol CYP3A29 protein per mg PLM in nondegraded tissue. In tissue stored at 4°C, we found that the CYP protein levels were unaffected by degradation after 72 h. At 21°C CYP1A2, CYP2D25, and CYP2E1 protein levels were nearly unaffected by degradation after 24 h, whereas a loss of approximately 50% was seen after 48 h. At 21°C CYP3A29 had a loss of 50% at 24 h and 70% at 48 h exhibiting less postmortem stability. In vitro enzyme activity measurements in the same tissue stored at 21°C showed a 50% decrease after 24 h and a complete loss of enzyme activity after 48 h. When stored at 4°C, the in vitro enzyme activity decreased to 50% activity after 96 h. In conclusion, measuring CYP levels by an LC-MS/MS approach was clearly less affected by postmortem changes than an activity-based approach. The found postmortem stability for 24 h at 21°C for 3 out of 4 CYP isoforms supports the use of properly stored postmortem tissue to inform PBPK models.

Entrapment of drugs in dental calculus - Detection validation based on test results from post-mortem investigations.

For prospective investigation of drugs and metabolites in archaeological and contemporary dental calculus, a sensitive, broadly applicable ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) was developed. The dental calculus was treated with citric acid and the dissolution extracts were cleaned using weak and strong polymeric cation-exchange sorbents. The method was validated on hydroxyapatite for the analysis of 67 drugs and metabolites. Typically, the lower limits of quantification were in the range of 0.01-0.05ng for the sample mass extracted. The general applicability of the method was tested using dental calculus material sampled from 10 corpses undergoing forensic autopsy. The calculus material was washed several times before dissolution to remove residual substances originating from saliva, gingival crevicular fluid and blood. The wash extracts and the calculus samples (cleaned calculus material) were analysed using the same instrumental conditions. The dry mass of the calculus samples ranged from 1 to 10mg. The total number of drug detections was 131 in the dental calculus samples and 117 in the whole blood samples. From the analyses of the wash extracts and calculus samples, it was proven that drug residues were trapped in the interior of the calculus material. In 82 of the drug detections, the drug concentrations were higher in the dental calculus than in the blood. Among substances detected in the dental calculus but not in the blood were cocaine, heroin, 6-MAM and THCA-A.

Determination of camostat and its metabolites in human plasma - Preservation of samples and quantification by a validated UHPLC-MS/MS method.

OBJECTIVES: Camostat mesilate is a drug that is being repurposed for new applications such as that against COVID-19 and prostate cancer. This induces a need for the development of an analytical method for the quantification of camostat and its metabolites in plasma samples. Camostat is, however, very unstable in whole blood and plasma due to its two ester bonds. The molecule is readily hydrolysed by esterases to 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) and further to 4-guanidinobenzoic acid (GBA). For reliable quantification of camostat, a technique is required that can instantly inhibit esterases when blood samples are collected. DESIGN AND METHODS: An ultra-high-performance liquid chromatography-tandem mass spectrometry method (UHPLC-ESI-MS/MS) using stable isotopically labelled analogues as internal standards was developed and validated. Different esterase inhibitors were tested for their ability to stop the hydrolysis of camostat ester bonds. RESULTS: Both diisopropylfluorophosphate (DFP) and paraoxon were discovered as efficient inhibitors of camostat metabolism at 10 mM concentrations. No significant changes in camostat and GBPA concentrations were observed in fluoride-citrate-DFP/paraoxon-preserved plasma after 24 h of storage at room temperature or 4 months of storage at -20 °C and -80 °C. The lower limits of quantification were 0.1 ng/mL for camostat and GBPA and 0.2 ng/mL for GBA. The mean true extraction recoveries were greater than 90%. The relative intra-laboratory reproducibility standard deviations were at a maximum of 8% at concentrations of 1-800 ng/mL. The trueness expressed as the relative bias of the test results was within ±3% at concentrations of 1-800 ng/mL. CONCLUSIONS: A methodology was developed that preserves camostat and GBPA in plasma samples and provides accurate and sensitive quantification of camostat, GBPA and GBA by UHPLC-MS/MS.

A validated UHPLC-MS/MS method for rapid determination of senicapoc in plasma samples.

The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-µL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100 %. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.

Increased risk of fatal intoxication and polypharmacy among psychiatric patients at death.

Patients suffering from psychiatric disorders have an excess mortality and a shorter life span expectancy compared to the general population. Furthermore, they are treated with multiple drugs and are known to have an increased risk of drug abuse. In this study, we aimed at investigating the pharmaceutical drug and drug of abuse profiles of the deceased included in the Danish prospective autopsy-based forensic study on psychiatric patients, SURVIVE. Using the postmortem systematic toxicological analysis results, we identified 129 different consumed compounds in our population (n = 443). Polypharmacy (≥5 compounds) was detected in 39.5% of the deceased. Deceased with a psychiatric diagnosis or who died from a fatal intoxication had significantly more compounds at the time of their death compared to having either no psychiatric diagnosis or another cause of death, respectively. Evidence of drug abuse was present, as 29.8% of our total population had consumed either methadone or illicit drugs of abuse, excluding tetrahydrocannabinol. Of those deceased with a psychiatric diagnosis, 33.6% had either consumed methadone or illicit drugs of abuse, a greater number than those without a psychiatric diagnosis. Fatal intoxication was the most frequent cause of death (40.6%) with methadone as the major intoxicant. Here, we found that those without a psychiatric diagnosis had fewer fatal pharmaceutical drug intoxications compared to the psychiatric diagnosis groups. Our findings add further context to understanding the excess mortality of psychiatric patents, since there is an increased occurrence of fatal intoxication, polypharmacy, and drug abuse in this population.