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.
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 (
< 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.
Oxycodone (OC) is an opioid with strong analgesic effects widely used to treat acute and chronic pain. Interpretation of OC concentrations in postmortem cases is complicated due to tolerance and ...overlapping concentrations for fatal and non-fatal levels. In this study, our aim was to develop and validate a method for OC and its three metabolites: noroxycodone (NOC), oxymorphone (OM) and noroxymorphone (NOM) in postmortem femoral blood. Our goal was to define reference concentrations for intoxications and non-intoxications and investigate metabolic ratios in different causes of death. A rapid LC-MS-MS method using protein-precipitated postmortem blood was developed. Lower limit of quantitation was 0.005 μg/g blood for all analytes; upper limit of quantitation was 1.0 μg/g for OC and NOC and 0.25 μg/g for OM and NOM. The method displayed high precision (3.3-7.7%) and low bias (-0.3 to 12%). In total, 192 cases were analyzed and concentrations ranged from 0.005 to 13 μg/g for OC, 0.005 to 2.0 μg/g for NOC, 0.005 to 0.24 μg/g for OM, and 0.005 to 0.075 μg/g for NOM. We found a significant difference in OC concentration between the cases where OC contributed and those where it did not. In spite of that, we do not recommend the use of a specific blood concentration to distinguish fatal intoxications. Instead, the percentiles from our data set suggest that concentrations >0.2 μg/g are likely to have contributed to toxicity, but that concentrations as high as 0.3 might be tolerated without toxic effects. In addition, we also found that a low NOC/OC ratio could point toward an acute fatal intoxication. In conclusion, the OC concentration alone may not be sufficient to diagnose a fatal intoxication.
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. Co-administered 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 pharmaco-epidemiological 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.
Genetic disposition can cause variation in oxycodone pharmacokinetic characteristics and decrease or increase the expected clinical response. In forensic medicine, determination of cause of death or ...assessing time between drug intake and death can be facilitated by knowledge of parent and metabolite concentrations. In this study, the aim was to investigate if CYP2D6 genotyping can facilitate interpretation by investigating the frequency of the four CYP2D6 phenotypes, poor metabolizer, intermediate metabolizer, extensive metabolizer, and ultra-rapid metabolizer in postmortem cases, and to study if the CYP2D6 activity was associated with a certain cause of death, concentration, or metabolic ratio. Cases positive for oxycodone in femoral blood (n = 174) were genotyped by pyrosequencing for CYP2D6*3, *4, and *6 and concentrations of oxycodone, noroxycodone, oxymorphone, and noroxymorphone were determined by LC-MS/MS (LLOQ 0.005 µg/g). Digital droplet PCR was used to determine the copy number variation for CYP2D6*5. Cases were categorized by cause of death. It was found that poor and intermediate CYP2D6 metabolizers had significantly higher oxycodone and noroxycodone concentrations compared to extensive and ultra-rapid metabolizers. CYP2D6 phenotype were equally distributed between cause of death groups, showing that no phenotype was overrepresented in any of the cause of death groups. We also found that the concentration ratio between oxymorphone and oxycodone depended on the CYP2D6 activity when death was unrelated to intoxication. In general, a low metabolite to parent ratio indicate an acute intake. By using receiver operating characteristic (ROC) analysis, we conclude that an oxymorphone/oxycodone ratio lower than 0.075 has a high sensitivity for separating intoxications with oxycodone from other intoxications and non-intoxications. However, the phenotype needs to be known to reach a high specificity. Therefore, the ratio should not be used as a biomarker on its own to distinguish between different causes of death but needs to be complemented by genotyping.
•Postmortem concentrations of OC and NOC are significantly higher in PM and IM compared to EM and UM.•Concentration ratios between OM and OC depend on the genotype when death is unrelated to intoxication.•An OM/OC ratio lower than 0.075 was associated with intoxication but specificity depend on genotype.
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-3β-D-glucuronide, morphine-6β-D-glucuronide, normorphine, codeine-6β-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 μg/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-3β-D-glucuronide/morphine and significant differences were observed between codeine vs morphine (p=0.005) and codeine vs heroin (p≤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-3β-D-glucuronide results suggested a period of abstinence prior to death in 25% of the heroin intoxications.
•Oxycodone and its major and minor metabolites were quantified in whole blood.•LC-MS/MS enabled high sensitivity and selectivity.•Results from a pilot study showed comparable result to ...pharmacokinetic studies.
The objective of this study was to develop and validate a highly sensitive method for the detection of oxycodone, noroxycodone, 6β-oxycodol, 6α-oxycodol, oxymorphone, and noroxymorphone in blood by liquid chromatography tandem mass spectrometry. The analytes were extracted from blood (0.5 mL) using Bond Elut Certify Solid Phase Extraction columns, evaporated to dryness and reconstituted before analysis was performed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD. Academy Standards Board Standard Practices for Method Development in Forensic Toxicology were used for the validation of this method. The limit of quantitation for all analytes was established at 0.5 ng/mL. Calibration range for noroxymorphone, oxymorphone, 6α-oxycodol and 6β-oxycodol was 0.5–25 ng/mL and 0.5–100 ng/mL for noroxycodone and oxycodone. Precision (2.90–17.3%) and bias studies resulted in a ±15% deviation. There were no interferences observed from internal standard, matrix, or common drugs of abuse. Stability of all analytes at two concentrations at 24, 48, and 72 h in the autosampler did not exceed ±20% difference from the initial T0. Dilution integrity at a ten-fold dilution was acceptable as analyte concentrations ranged between (±18%) of the target concentration. Once validated, the method was used in a pilot dosing study of one male subject after taking a 10 mg immediate release tablet of oxycodone. Blood samples were collected at 0.25, 0.50, 0.75, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 9, and 24 h after ingestion. Oxycodone and noroxycodone both reached Tmax at 1.5 h and had Cmax values of 25.9 and 12.8 ng/mL, respectively. Oxycodone, 6α-oxycodol, and 6β-oxycodol were detectable up to 9 h, while noroxymorphone and noroxycodone were still detected at 24 h.
Oxycodone (OC) is a schedule II semisynthetic opioid in the USA that is prescribed for its analgesic effects and has a high potential for abuse. Prescriptions for OC vary based on the dosage and ...formulation, immediate release (IR) and controlled release (CR). Monitoring OC metabolites is beneficial for forensic casework. The limited studies that involve pharmacokinetics of the urinary excretion of OC metabolites leave a knowledge gap regarding the excretion of conjugated and minor metabolites, pharmacokinetic differences by formulation, and the impact of CYP2D6 activity on the metabolism and excretion of OC. The objectives of this study were to compare urinary excretion of phase I and II metabolites by formulation and investigate if ratio changes over time could be used to predict the time of intake. Subjects (n = 7) received a single 10 mg IR tablet of Oxycodone Actavis. A few weeks later the same subjects received a single 10 mg CR tablet of Oxycodone Actavis. During each setting, urine was collected at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 9, 10, 12, 14, 24, 48 and 72 h. Urine samples (100 µL) were diluted with 900 µL internal standard mixture and analyzed on an Acquity UPLC® I-class coupled to a Waters Xevo TQD using a previously validated method. The CYP2D6 phenotypes were categorized as poor metabolizers (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM). Comparisons between IR and CR were performed using two-tailed paired t-test at a significance level of P = 0.05. The metabolite ratios showed a general increase over time. Four metabolite to parent ratios were used to predict the time of intake showing that predictions were best at the early time points.
Abstract
The purpose of this research was to develop and validate an analytical method for the detection and quantification of noroxymorphone-3β-D-glucuronide (NOMG), oxymorphone-3β-D-glucuronide ...(NOMG), noroxymorphone (NOM), oxymorphone (OM), 6α-oxycodol (αOCL), 6β-oxycodol (βOCL), noroxycodone (NOC) and oxycodone (OC) in urine by liquid chromatography tandem mass spectrometry to be used in a human study. The method was validated according to the Academy Standards Board Standard Practices for Method Development in Forensic Toxicology. The method was then applied to a single-dose pilot study of a subject. Urine samples were collected from the subject after ingesting 10-mg OC as an immediate-release tablet. Additionally, urine specimens (n = 15) that had previously been confirmed positive for OC were analyzed using the validated method. The calibration range for NOMG and OMG was 0.05–10 μg/mL; for all other analytes, it was 0.015–10 μg/mL. Validation parameters such as bias, precision, carryover and dilution integrity, all met the validation criteria. After the method was validated, urine samples from the first subject in the controlled dose study were analyzed. It was observed that OC, NOC and OMG contained the highest concentrations and were present in either the 0.5 or 1 h void. NOC and OMG were detected until the 48 h collection, while OC was detectable till the 24 h collection. Time to reach maximum concentration (Tmax) in the urine was achieved within 1.5 h for OC and within 3 h for NOC and OMG. Maximum concentration (Cmax) in the urine for OC, NOC and OMG was 3.15, 2.0 and 1.56 μg/mg, respectively. OC concentrations in authentic urines ranged from 0.015 to 12 μg/mL. Ranges for NOMG and OMG were 0.054–9.7 μg/mL and 0.14–67 μg/mL, respectively. A comprehensive method for the quantification of NOMG, OMG, NOM, OM, αOCL, βOCL, NOC and OC in urine was optimized and met the validation criteria. The concentrations of NOMG and OMG presented in this study provide the details needed in the forensic community to better comprehend OC pharmacokinetics.
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