Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain

Non-Peptide Opioids Differ in Effects on Mu-Opioid (MOP) and Serotonin 1A (5-HT1A) Receptors Heterodimerization and Cellular Effectors (Ca2+, ERK1/2 and p38) Activation

The importance of the dynamic interplay between the opioid and the serotonin neuromodulatory systems in chronic pain is well recognized. In this study, we investigated whether these two signalling pathways can be integrated at the single-cell level via direct interactions between the mu-opioid (MOP) and the serotonin 1A (5-HT1A) receptors. Using fluorescence cross-correlation spectroscopy (FCCS), a quantitative method with single-molecule sensitivity, we characterized in live cells MOP and 5-HT1A interactions and the effects of prolonged (18 h) exposure to selected non-peptide opioids: morphine, codeine, oxycodone and fentanyl, on the extent of these interactions. The results indicate that in the plasma membrane, MOP and 5-HT1A receptors form heterodimers that are characterized with an apparent dissociation constant Kdapp = (440 ± 70) nM). Prolonged exposure to all non-peptide opioids tested facilitated MOP and 5-HT1A heterodimerization and stabilized the heterodimer complexes, albeit to a different extent: Kd, Fentanylapp = (80 ± 70) nM), Kd,Morphineapp = (200 ± 70) nM, Kd, Codeineapp = (100 ± 70) nM and Kd, Oxycodoneapp = (200 ± 70) nM. The non-peptide opioids differed also in the extent to which they affected the mitogen-activated protein kinases (MAPKs) p38 and the extracellular signal-regulated kinase (Erk1/2), with morphine, codeine and fentanyl activating both pathways, whereas oxycodone activated p38 but not ERK1/2. Acute stimulation with different non-peptide opioids differently affected the intracellular Ca2+ levels and signalling dynamics. Hypothetically, targeting MOP–5-HT1A heterodimer formation could become a new strategy to counteract opioid induced hyperalgesia and help to preserve the analgesic effects of opioids in chronic pain

Adaptations to Climate-Mediated Selective Pressures in Humans

Humans inhabit a remarkably diverse range of environments, and adaptation through natural selection has likely played a central role in the capacity to survive and thrive in extreme climates. Unlike numerous studies that used only population genetic data to search for evidence of selection, here we scan the human genome for selection signals by identifying the SNPs with the strongest correlations between allele frequencies and climate across 61 worldwide populations. We find a striking enrichment of genic and nonsynonymous SNPs relative to non-genic SNPs among those that are strongly correlated with these climate variables. Among the most extreme signals, several overlap with those from GWAS, including SNPs associated with pigmentation and autoimmune diseases. Further, we find an enrichment of strong signals in gene sets related to UV radiation, infection and immunity, and cancer. Our results imply that adaptations to climate shaped the spatial distribution of variation in humans

Oxycodone in Forensic Toxicology : Analytical Strategies and Interpretation

Oxycodone is a common finding in forensic casework and widely used as an analgetic. Oxycodone’s pharmacokinetic and pharmacodynamic properties make the interpretation of post mortem oxycodone blood concentrations complicated. Coadministered substances and inter-individual differences in metabolic capacity can alter the oxycodone blood concentration and thereby cause an unexpected pharmacological effect and possibly lead to negative side-effects, respiratory depression, and death. As the level of tolerance often is unknown in post mortem cases the correlation between blood concentration and effect is weak. In this thesis, the overall aim was to increase the ability to determine cause and manner of death in suspected oxycodone intoxications, by studying concentrations of oxycodone and its metabolites, CYP2D6 phenotype, as well as endogenous substances in post mortem cases. Moreover, trends and patterns in prescription and post mortem findings of substances causing pharmacodynamic and pharmacokinetic interactions with oxycodone were studied. In paper I, concentrations of oxycodone, noroxycodone, oxymorphone and noroxymorphone were determined in femoral blood from 192 post mortem cases by LC-MS/MS. Concentrations and the metabolite-to-parent drug ratio were studied in groups separated by cause of death, A) intoxication by oxycodone, B) intoxication caused by oxycodone and additional substance/s, C) intoxication where oxycodone did not contribute, D) other causes of death than intoxication. It was found that concentrations above 0.2 μg/g indicate an oxycodone intoxication but that concentrations up to 0.3 μg/g can be seen in tolerant individuals. The results also demonstrated that a low noroxycodone/oxycodone ratio indicates an oxycodone intoxication. Paper II included LC-MS/MS analysis as in paper I, and in addition, genotyping for CYP2D6 enzyme activity in 174 cases. The metabolite-to-parent drug ratios were compared between poor, intermediate, extensive, and ultra-rapid metabolizers. It was concluded that with knowledge of CYP2D6 activity the oxymorphone/oxycodone ratio could distinguish between oxycodone-related deaths and other causes of deaths. Paper III was a pharmacoepidemiological study where post mortem findings were investigated in combination with prescription records in 1081 cases to evaluate the presence of interacting drugs in oxycodone-related intoxications. One of the main findings was that pharmacodynamically interacting drugs were twice as often prescribed, and five times more common as a co-finding in oxycodone-related intoxications compared to other causes of death. An oxycodone prescription was missing in 34% of all cases, with a trend that individuals, 35 years or below, more often lacked an oxycodone prescription. In paper IV, the post mortem metabolome was explored in 934 cases to reveal possible biomarkers correlated with oxycodone intoxications. The results showed that levels of acylcarnitines, a group of endogenous substances involved in mitochondrial metabolism, were significantly decreased in oxycodone-related intoxications compared to a control group, revealing post mortem metabolome analysis as a possible complemental approach of interpretation in post mortem toxicology. In conclusion, this thesis emphasizes the importance of including metabolites in the toxicological analytical strategy to improve the interpretation in post mortem case work. Also, the applicability of pharmacogenetic analysis is highly useful in certain cases. Furthermore, the use of post mortem metabolomics is a possible future promising strategy to the early identification of oxycodone-related deaths.Updates:2022-09-13 The thesis was first published online. The onlinepublished version reflects the printed version.2022-10-21 The thesis was updated with an errata list which isalso downloadable from the DOI landing page. Before this date the PDF was downloaded 35 times.2022-10-27 The thesis was updated with an new errata list. Before this date the PDF was downloaded 12 times.</p

Postmortem Metabolomics Reveal Acylcarnitines as Potential Biomarkers for Fatal Oxycodone-Related Intoxication

Postmortem metabolomics has recently been suggested as a potential tool for discovering new biological markers able to assist in death investigations. Interpretation of oxycodone concentrations in postmortem cases is complicated, as oxycodone tolerance leads to overlapping concentrations for oxycodone intoxications versus non-intoxications. The primary aim of this study was to use postmortem metabolomics to identify potential endogenous biomarkers that discriminate between oxycodone-related intoxications and non-intoxications. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry data from 934 postmortem femoral blood samples, including oxycodone intoxications and controls positive and negative for oxycodone, were used in this study. Data were processed and evaluated with XCMS and SIMCA. A clear trend in group separation was observed between intoxications and controls, with a model sensitivity and specificity of 80% and 76%. Approximately halved levels of short-, medium-, and long-chain acylcarnitines were observed for oxycodone intoxications in comparison with controls (p &amp;lt; 0.001). These biochemical changes seem to relate to the toxicological effects of oxycodone and potentially acylcarnitines constituting a biologically relevant biomarker for opioid poisonings. More studies are needed in order to elucidate the potential of acylcarnitines as biomarker for oxycodone toxicity and their relation to CNS-depressant effects

Heroin-Related Compounds and Metabolic Ratios in Postmortem Samples Using LC-MS-MS

Analysis of postmortem samples with the presence of morphine can sometimes be challenging to interpret. Tolerance complicates interpretation of intoxications and causes of death due to overlap in therapeutic and fatal concentrations. Determination of metabolites and metabolic ratios can potentially differentiate between abstinence, continuous administration, and perhaps time of administration. The purpose of this study was to (a) develop and validate a method for quantitation of morphine-3 beta-D-glucuronide, morphine-6 beta-D-glucuronide, normorphine, codeine-6 beta-D-glucuronide, norcodeine, codeine, 6-acetylmorphine, and ethylmorphine in urine using liquid chromatography-tandem mass spectrometry; (b) apply the method to opiate related deaths; (c) compare metabolic ratios in urine in different causes of death (CoD) and after different drug intakes and (d) compare heroin intoxications in rapid and delayed deaths. Validation parameters such as precision, bias, matrix effects, stability, process efficiency, and dilution integrity were assessed and deemed acceptable. Lower limits of quantitation ranged from 0.01-0.2 mu g/mL for all analytes. Autopsy cases (n=135) with paired blood and urine samples were analyzed. Cases were divided into three groups based on CoD; opiate intoxication, intoxication with other drugs than opiates, and other CoD. The cases were classified by intake: codeine (n=42), heroin (n=36), morphine (n=49), and ethylmorphine (n=3). Five cases were classified as mixed intakes and excluded. Heroin intoxications (n=35) were divided into rapid (n=15) or delayed (n=20) deaths. Parent drug groups were compared using metabolic ratio morphine-3 beta-D-glucuronide/morphine and significant differences were observed between codeine vs morphine (p=0.005) and codeine vs heroin (p &amp;lt;= 0.0001). Urine and blood concentrations, and metabolic ratios in rapid and delayed heroin intoxications were compared and determined a significant difference for morphine (p=0.001), codeine (p=0.009), 6-acetylmorphine (p=0.02) in urine, and morphine (p=0.02) in blood, but there was no significant difference (p=0.9) between metabolic ratios. Morphine-3 beta-D-glucuronide results suggested a period of abstinence prior to death in 25% of the heroin intoxications

Evaluation of new guidelines for ROP screening in Sweden using SWEDROP - a national quality register

PurposeTo investigate whether recent Swedish guidelines for Retinopathy of Prematurity (ROP) screening, that is, a gestational age (GA) at birth of <31weeks (w), are applicable in a new national cohort of prematurely born infants. MethodsSWEDROP is a national register for ROP, initiated in 2006. The present paper reports on data from the register on various aspects of screening for ROP in infants born between 2010 and 2011 and compares the results with those for a previously published cohort born between 2008 and 2009. ResultsDuring the study period, 1744 infants were screened for ROP. Mean GA was 28.4w (22-31), and mean birth weight was 1239g (382-2615). Screening started at postnatal age (PNA) 5.4w (0.4-13.3) and postmenstrual age (PMA) 33.8 w (24.9-50.1) Mean number of examinations was 5.4 per infant (1-38). Mild (stages 1-2) and severe ( stage 3) ROP was found in 15.4% and 8.7%, respectively. Treatment was performed in 4.2% (73/1744) of the infants, but in none with a GA of 30weeks or more. The first treatment was performed at a mean PNA and PMA of 12.7 w (7.7-25.4) and 37.4 w (32.1-51.4), respectively. ConclusionsRecently introduced new guidelines for ROP screening in Sweden remain applicable. Reassuringly, in infants born between 2010 and 2011, incidence of ROP, frequency and timing of treatment, frequency and timing of examinations and national coverage of ROP screening remained almost identical to those for a previous cohort from 2008 to 2009. The two SWEDROP cohorts provide a basis for discussion among Swedish ophthalmologists and neonatologists on the question of further lowering the upper screening limit with 1week

Oxycodone-Related Deaths : The Significance of Pharmacokinetic and Pharmacodynamic Drug Interactions

Background and Objectives Oxycodone is frequently prescribed as well as detected in postmortem cases. Concurrent use of pharmacodynamically or pharmacokinetically interacting drugs can cause adverse effects or even fatal intoxication. The aims of this study were to investigate differences in prescriptions for and toxicological findings of pharmacodynamically and pharmacokinetically interacting drugs in fatal oxycodone-related intoxications and other causes of death. We also aimed to investigate the differences in prevalence of oxycodone prescriptions, and the detected postmortem oxycodone concentrations between fatal oxycodone-related intoxications and other causes of death. Methods Forensic autopsy cases (2012-2018) where oxycodone was identified in femoral blood (n = 1236) were included. Medical history and prescription data were retrieved from national databases and linked to the forensic toxicology findings. Results Oxycodone-related deaths were found to have higher blood concentrations of oxycodone (median 0.30 mu g/g vs. 0.05 mu g/g) and were less likely to have a prescription for oxycodone (OR 0.62) compared to nonintoxication deaths. Pharmacodynamically interacting drugs were prescribed in 79% and found in blood in 81% of the cases. Pharmacokinetically interacting drugs were rarely prescribed (1%). Oxycodone-related deaths were more likely to have prescriptions for a pharmacodynamically interacting drug (OR 1.7) and more often have co-findings of one or multiple pharmacodynamically interacting drugs (OR 5.6). Conclusion The results suggest that combined use of oxycodone and pharmacodynamically interacting drugs is associated with oxycodone-related death and that non-medical use of oxycodone is a potential risk factor for oxycodone-related intoxication.Funding Agencies|National Board of Forensic Medicine</p