Post-mortem SNP analysis of CYP2D6 gene reveals correlation between genotype and opioid drug (tramadol) metabolite ratios in blood

Tramadol is an opioid drug metabolised in phase I by cytochrome P450 (CYP) enzymes, of which CYP2D6 is mainly responsible for the O-demethylation of tramadol, but is not involved in N-demethylation. Defects in the genes encoding drug metabolising enzymes (DMEs) may lead to adverse drug effects, even to death. To aid interpretation of the forensic toxicology results, we studied how the genetic variation of the CYP2D6 gene is reflected in tramadol metabolite ratios found in post-mortem samples. In 33 Finnish autopsy cases where tramadol was found, we analysed both the CYP2D6 genotype and the concentrations of tramadol and its metabolites O- and N-demethyltramadol. As expected, we found a correlation between the number of functional CYP2D6 alleles and the ratio of tramadol to O-demethyltramadol. We also found a correlation between the number of functional alleles and the ratio of tramadol to N-demethyltramadol. This can be explained by the complementary nature of the two main tramadol demethylation pathways. No known CYP2D6 inhibitors were associated with exceptional metabolic ratios. Furthermore, no accidental tramadol poisonings were associated with a defective CYP2D6 gene. Our results on the tramadol are among the first to demonstrate that genetic variation in drug metabolising enzymes can be analysed in post-mortem blood, and that it correlates well with the parent drug to metabolite ratios. The results also suggest that genetic factors play, in general, a dominant role over other factors in the metabolism of individual drugs.     

A fatal doxepin poisoning associated with a defective CYP2D6 genotype

It has been suggested that the polymorphism of the CYP2D6 gene can contribute to occurrence of fatal adverse effects. We therefore investigated postmortem toxicology cases of fatal drug poisonings related to CYP2D6 substrates, with the manner of death denoted as accidental or undetermined. CYP2D6 genotypes were determined in 11 consecutive cases with samples available for DNA analysis. A case of fatal doxepin poisoning with an undetermined manner of death was found to coincide with a completely nonfunctional CYP2D6 genotype (*3/*4), indicating a total absence of CYP2D6 enzyme and suggesting a poor metabolizer phenotype. The doxepin concentration was 2.4 mg/L, the concentration of nordoxepin 2.9 mg/L, and the doxepin/nordoxepin ratio 0.83, the lowest found among the 35 nordoxepin-positive postmortem cases analyzed during the same year. No alcohols or other drugs were detected in the case. The CYP2C19 genotype was determined as that of an extensive metabolizer. The high N-desmethylmetabolite concentration is not consistent with acute intoxication. It is therefore probable that the defective genotype has contributed to the death, possibly involving repeated high dosage of doxepin. Our case strongly emphasizes that a pharmacogenetic analysis in postmortem forensic setting may reveal new insight to the cause or manner of death.     

Cytochrome P450 2D6 (CYP2D6) genotyping on postmortem blood as a supplementary tool for interpretation of forensic toxicological results

Debrisoquine hydroxylase (CYP2D6) is involved in the metabolism of many toxicologically important drugs. The gene encoding for this enzyme displays a polymorphic distribution in all populations examined. We report a study on 46 cases, where analyses of the CYP2D6 gene were conducted on postmortem femoral blood in order to investigate the occurrence of poor metabolizers (PM). A polymerase chain reaction (PCR) method, designed and routinely used for therapeutic drug monitoring, was employed, only slightly modified. Samples from 22 cases, where the parent drug to metabolite ratio was unexpectedly high were analyzed as well as samples from 24 control cases. Genotyping could be carried out in all but one case. Previous freezing or addition of potassium fluoride as preservative did not prevent analysis. Only one PM (from the control group) was discovered, implying an occurrence of only 2.2% as compared to the reported frequency of approx. 7% in Sweden. Among the extensive metabolizers (EM), however, a number of individuals with mutated genes were identified. Although it seems reasonable to suspect a PM genotype in cases with a high concentration of a drug metabolized by CYP2D6, but without suspicion of acute overdose, our study does not support the opinion that this interpretation pitfall is particularly common. This study rather indicates that drug interactions in EMs constitute a more frequent and important problem.     

Influence of CYP2D6 genotype on the disposition of the enantiomers of venlafaxine and its major metabolites in postmortem femoral blood

Venlafaxine (VEN) is an antidepressant drug mainly metabolized by the cytochrome P450 (CYP) enzyme CYP2D6 to the active metabolite O-desmethylvenlafaxine (ODV). VEN is also metabolized to N-desmetylvenlafaxine (NDV) via CYP3A4. ODV and NDV are further metabolized to N,O-didesmethylvenlafaxine (DDV). VEN is a racemic mixture of the S- and R-enantiomers and these have in vitro displayed different degrees of serotonin and noradrenaline reuptake inhibition. The aim of the study was to investigate if an enantioselective analysis of VEN and its metabolites, in combination with genotyping for CYP2D6, could assist in the interpretation of forensic toxicological results in cases with different causes of deaths. Concentrations of the enantiomers of VEN and metabolites were determined in femoral blood obtained from 56 autopsy cases with different causes of death. The drug analysis was done by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the CYP2D6 genotyping by PCR and pyrosequencing. The mean (median) enantiomeric S/R ratios of VEN, ODV, NDV and DDV were 0.99 (0.91), 2.17 (0.93), 0.92 (0.86) and 1.08 (1.03), respectively. However, a substantial variation in the relationship between the S- and R-enantiomers of VEN and metabolites was evident (S/R ratios ranging from 0.23 to 17.6). In six cases, a low S/R VEN ratio (mean 0.5) was associated with a high S/R ODV ratio (mean 11.9). Genotyping showed that these individuals carried two inactive CYP2D6 genes indicating a poor metabolizer phenotype. From these data we conclude that enantioselective analysis of VEN and ODV can predict if a person is a poor metabolizer genotype/phenotype for CYP2D6. Knowledge of the relationship between the S- and R-enantiomers of this antidepressant drug and its active metabolite is also important since the enantiomers display different pharmacodynamic profiles.     

Analysis of 50 SNPs in CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2 by MALDI-TOF mass spectrometry in Chinese Han population

One of the major challenges in the near future is the identification of genes that affect the metabolism of different drugs. Large scale association studies that utilise single nucleotide polymorphisms (SNPs) have been considered a valuable tool for this purpose. CYP2D6, CYP2C19, CYP2C9, CYP3A4 and CYP1A2 were found to be involved in the majority of hepatically cleared drugs. To determine the allele frequencies of some SNPs that may have great potential value in forensic science, we screened 50 SNPs in these 5 CYP genes in Chinese Han people using an accurate, high-throughput, cost-effective method. Primers were designed using the MassARRAY Assay Design software. Genomic DNA was prepared from blood samples obtained from individuals of Chinese Han origin. Multiplex PCR was performed to amplify the relevant gene fragments, and the polymorphisms were analysed by allele-specific primer extension followed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). A panel of genomic DNA samples previously genotyped by other methods were analysed simultaneously for quality control, and the results demonstrated that this assay was 100% accurate. A total of 17 of the analysed SNPs were polymorphic. Of these 17 SNPs, 8 (rs16947, rs28371725, rs1800754, rs4244285, rs4986893, rs12248560, rs3758580, rs2242480) had an allele frequency that was significantly different between this Chinese Han population and Caucasians (p<0.01). In addition, the frequencies of two of these SNPs (rs1800754, rs3758581) in our Chinese Han population differed significantly from the existing Chinese frequency data (p<0.01). The described method thus provides reliable results and enables the genotyping of up to thousands of samples by taking advantage of the high-throughput MALDI-TOF technology. The results herein are now included as a supplement to the P450 database.     

The frequencies of mutated alleles of CYP2D6 gene in a Turkish population

Allele and genotype frequency distribution of CYP2D6*3, *4, *5, *6 and *10 variants were analyzed in blood samples of 100 unrelated healthy individuals by Real-Time PCR. The allele frequencies of CYP2D6*3(A2549del), *4(G1846A), *6(T1707del) and *10(C100T) were 1%, 10%, 2.5% and 14.5% respectively, while allele frequency of CYP2D6*5 was 3% of the subjects tested. Extensive, poor and intermediate metabolizer (EM, PM, IM) genotype frequencies were 63%, 4% and 12%, respectively. CYP2D6 gene duplication was 4%. Our results show that the frequencies of the mutated alleles of CYP2D6 in Turkish populations are similar to some European populations. 4% of Turkish people who have two nonfunctional defective allele are a high risk group and 12.5% of Turkish people who have two decreased functional defective allele or one normal and one non functional defective allele were also in the risk group. Findings of this study demonstrate the importance of genetic variation in drug intoxicants.     

Interaction of 3,4-methylenedioxymethamphetamine and methamphetamine during metabolism by in vitro human metabolic enzymes and in rats

Illicit amphetamine-type stimulant (ATS) tablets commonly contain one or more active ingredients, which have hallucinogenic and/or stimulant effects. Because components such as 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (MA) in ATS tablets have similar chemical structures, they could be metabolized by common metabolic enzymes. To investigate potential metabolic interactions of ATS tablet components, we studied the in vitro metabolism of MDMA and MA using human metabolic enzymes. MDMA and MA were mainly metabolized by cytochrome P450 2D6 (CYP2D6) and mutually inhibited the production of their main metabolites. In vivo experiments were also performed using intravenous administration of MDMA, MA, or their mixture to rats. The plasma concentrations of MDMA and MA after co-administration were higher than those after administration of MDMA or MA alone. The results in this study imply that multiple components in ATS tablets can interact to mutually inhibit their metabolism and potentially enhance the toxicity of each component.     

The Ratio of 6β‐Hydroxycortisol to Cortisol in Urine as a Measure of Cytochrome P450 3A Activity in Postmortem Cases

Poisoning can occur with chronic accumulation of a drug due to reduced metabolic capacity; conversely, under‐treatment may occur due to an increased metabolic rate. Over half of all drugs are metabolized by the cytochrome P450 3A complex (CYP3A). The activity of CYP3A can be assessed by the urinary ratio of 6β‐hydroxycortisol to cortisol. The aim of this study was to determine the usefulness of this ratio as a postmortem marker for determining whether altered CYP3A enzyme activity occurred prior to death. In a series of 244 postmortem cases, this ratio ranged from 0.014 to 78.6 (median 3.50). The median was significantly higher (5.14) in a subgroup of 28 cases that exhibited the presence of CYP3A‐inducing drugs. In cirrhosis, the median ratio was 1.69. This pointed to a reduced metabolic capacity of CYP3A. Thus, the ratio may constitute a rough indicator of the CYP3A metabolic capacity, which could be of value in special cases.     

Analysis of 4‐Bromo‐2,5‐DimethoxyphenethylamineAbuser's Urine: Identification and Quantitation of Urinary Metabolites

The metabolites of 4‐bromo‐2,5‐dimethoxyphenethylamine (2C‐B), a psychoactive drug with hallucinogenic activity, were investigated in a urine sample from a user of 2C‐B. The urine sample was deconjugated enzymatically and the metabolites were recovered by liquid–liquid extraction. The extract was analyzed by gas chromatography/mass spectrometry after derivatization, and the results were used to identify and quantitate the metabolites. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid was the most abundant metabolite of 2C‐B in human urine and accounted for 73% of the total amount of detected metabolites, followed by 4‐bromo‐2‐hydroxy‐5‐methoxyphenylacetic acid (13%) and 4‐bromo‐2,5‐dimethoxyphenylethyl alcohol (4.5%). According to the literature, the main metabolites of 2C‐B in rat urine are N‐(4‐bromo‐2‐methoxy‐5‐hydroxyphenylethyl)acetamide and N‐(4‐bromo‐2‐hydroxy‐5‐methoxyphenylethyl)acetamide. However, these metabolites accounted for only a small proportion of the total amount of detected metabolites in human urine, which indicates that there are significant species‐specific differences in the metabolism of 2C‐B. 4‐Bromo‐2,5‐dimethoxyphenylacetic acid, which was the most abundant metabolite in human urine, is thought to be generated by deamination of 2C‐B by monoamine oxidase (MAO) followed by oxidation by aldehyde dehydrogenase. Our results suggest that MAO plays a crucial role in the metabolism of 2C‐B in humans.     

Investigation of morphine and morphine glucuronide levels and cytochrome P450 isoenzyme 2D6 genotype in codeine-related deaths

Compared to morphine and morphine-6-glucuronide (M6G), codeine and its other major metabolites codeine-6-glucuronide and norcodeine have weak affinity to opioid μ-receptors. Analgesic effects of codeine are thus largely dependent on metabolic conversion to morphine by the polymorphic cytochrome P450 isoenzyme 2D6 (CYP2D6). How this relates to toxicity and post-mortem whole blood levels is not known. This paper presents a case series of codeine-related deaths where concentrations of morphine, M6G and morphine-3-glucuronide (M3G), as well as CYP2D6 genotype, are taken into account. Post-mortem toxicological specimens from a total of 1444 consecutive forensic autopsy cases in Central Norway were analyzed. Among these, 111 cases with detectable amounts of codeine in femoral blood were identified, of which 34 had femoral blood concentrations exceeding the TIAFT toxicity threshold of 0.3mg/L. Autopsy records of these 34 cases were retrieved and reviewed. In the 34 reviewed cases, there was a large variability in individual morphine to codeine concentration ratios (M/C ratios), and morphine levels could not be predicted from codeine concentrations, even when CYP2D6 genotype was known. 13 cases had codeine concentrations exceeding the TIAFT threshold for possibly lethal serum concentrations (1.6 mg/L). Among these, 8 individuals had morphine concentrations below the toxic threshold according to TIAFT (0.15 mg/L). In one case, morphine as well as M6G and M3G concentrations were below the limit of detection. A comprehensive investigation of codeine-related fatalities should, in addition to a detailed case history, include quantification of morphine and morphine metabolites. CYP2D6 genotyping may be of interest in cases with unexpectedly high or low M/C ratios.     

Postmortem tissue concentrations of venlafaxine

Venlafaxine is a phenethylamine antidepressant which inhibits both serotonin and norepinephrine reuptake and is structurally unrelated to the serotonin reuptake inhibitors (SSRIs). Its major metabolite, O-desmethylvenlafaxine (ODV), also inhibits serotonin reuptake. Although metabolized by the cytochrome P-450 (CYP) system, venlafaxine inhibits CYP 2D6 and 3A4 to a far lesser extent than do the SSRIs. Mechanisms of drug action are reviewed and evaluated in the investigation of 12 fatalities occurring over a 6-month-period where venlafaxine was detected.Venlafaxine and ODV were identified by liquid chromatography-mass spectrometry (LC-MS) using atmospheric pressure ionization (API) electrospray in positive mode following an n-butyl chloride extraction. Postmortem tissue concentrations studied in each of 12 postmortem cases for venlafaxine and ODV, were 0.1-36 and <0.05-3.5mg/l (peripheral blood), <0.05-22 and <0.05-9.9mg/kg (liver), <0.05-10 and <0.05-1.5mg/l (vitreous), <0.05-53 and <0.05-6.8mg/l (bile), <0.05-55 and <0.05-21mg/l (urine), respectively, and 0.1-200mg of venlafaxine in the gastric contents. Venlafaxine was typically present with other drugs, including other antidepressants, alcohol, and benzodiazepines. The potential for interaction with each drug is discussed. Over the 6-month-period of this study, there were no deaths ascribed solely to venlafaxine intoxication.     

Discrepancy between CYP2D6 phenotype and genotype derived from post-mortem dextromethorphan blood level

OBJECTIVE: To describe the death of a toddler after a therapeutic dose of dextromethorphan and its investigation. STUDY DESIGN: Case report, cytochrome P450 phenotype and genotype determination in the victim and post-mortem drug redistribution study performed in rats. RESULTS: A 20-month Asian male who received 3 mg of dextromethorphan once at 09:00 h and again at 22:00 h was found dead at 04:35 h. Post-mortem examination showed signs of early bronchopneumonia (bacterial cultures were negative); dextromethorphan and dextrorphan blood concentrations taken from the heart cavity were 500 ng/ml (1. 84 micromol/l) and 200 ng/ml (0.78 micromol/l), respectively. Despite the dextromethorphan level being almost 100-fold higher than expected after therapeutic doses, intentional or unintentional overdose was extremely unlikely; other potential causes were investigated. Post-mortem drug redistribution study performed in rats showed that dextromethorphan does not undergo extensive redistribution after death (6+/-5-fold increase) and could not explain the observed dextromethorphan level. The dextromethorphan/dextrorphan concentration ratio of 2.5 found in this toddler was compatible with a slow CYP2D6 metabolizer phenotype. However, the toddler exhibited a fast metabolizer genotype. Potential reasons for this discrepancy are discussed. CONCLUSION: CYP450 phenotypes derived from post-mortem blood levels should be interpreted with caution and preferably confirmed by a genotype analysis.     

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.     

Pharmacogenomics as an aspect of molecular autopsy for forensic pathology/toxicology: does genotyping CYP 2D6 serve as an adjunct for certifying methadone toxicity?

Pharmacogenomics, applied as an aspect of molecular autopsy, may be used as an adjunct for certifying methadone fatalities. Methadone is metabolized by cytochrome P-450 (CYP) 1A2, 3A4, and 2D6. We hypothesized that methadone toxicity may be partially due to CYP 2D6 *3, *4, and *5 variant alleles, resulting in poor drug metabolism. A retrospective analysis was performed on covariables and risk factors of 21 methadone cases from the Milwaukee County Medical Examiner's Office (1998-2000). PCR genotyping showed: one heterozygous for 2D6*3, two homozygous for 2D6*4, five heterozygous for 2D6*4, and one heterozygous for both 2D6*3 and *4. This limited number of cases showed that the prevalence of poor metabolizer was higher but not significantly different from that of a control group (n = 23) (P > 0.05, Fisher Exact Test). Thus, CYP 2D6 mutations may not yet be directly associated with methadone toxicity. However, pharmacogenomics, complementing other case findings, served as an adjunct in interpreting methadone toxicity of poor and intermediate metabolizers.     

Screening for drugs in forensic blood samples using EMIT urine assays

A screening method for the detection of drugs in haemolysed whole blood has been evaluated. Methanolic extracts of 300 forensic blood samples known to be positive or negative for drugs were analysed with EMIT d.a.u. assay kits for amphetamine, cannabinoids, opiates and benzodiazepines (the latter to analyse for diazepam and the main metabolite N-desmethyldiazepam). There were very few false positive results, except for the amphetamine assay in postmortem blood samples, where 9% were false positive. For amphetamine and cannabinoids a few false negatives were found, these were from samples with very low drug concentrations. No false negatives were found for opiates and diazepam. The present modification of the EMIT d.a.u. method seems to be a good method for screening of drugs in forensic blood samples, except for amphetamine in postmortem samples. The method is simple and requires only 0.5 ml blood.     

K2 Toxicity: Fatal Case of Psychiatric Complications Following AM2201 Exposure

Limited forensic and clinical experience and the lack of confirmatory testing strategies for synthetic cannabinoids (SC) prevent adequate characterization of SC toxicity and the potential impact on public health. A statewide surveillance system identified a fatality involving a 23‐year‐old man found with a large stab wound to the neck following use of a SC product suspected of containing AM2201. Analytical testing for common SCs, SC metabolites, routine drugs of abuse, and over‐the‐counter medications was performed on heart blood obtained at autopsy. Additionally, assays were performed on the SC raw material and drug paraphernalia found on the decedent. High concentrations of AM2201 were detected in all samples. AM2201 metabolites were detected in postmortem blood. Other than a trace amount of JWH‐073 found in smoke residue, no other substances were detected. Psychiatric complications including self‐induced, lethal trauma can occur after the use of SC products.     

Detection of amitriptyline, citalopram, and metabolites in porcine bones following extended outdoor decomposition

Skeletal remains of a domestic pig were assessed for relative distribution of amitriptyline, citalopram, and metabolites. Following acute exposure and outdoor decomposition for 2 years, drugs and metabolites were analyzed in 13 different bones. Bones were pulverized following a simple wash procedure, and drugs were extracted by passive incubation in methanol, followed by solid-phase extraction. Samples were analyzed by ultra-high performance liquid chromatography (UHPLC) and confirmed with gas chromatography-mass spectrometry. The Kruskall-Wallis test showed that bone type was a main effect with respect to drug level for all analytes, with levels varying from 33- to 166-fold. Ratios of levels of drug to that of the corresponding metabolite were less variable, varying roughly one- to eightfold. This suggests limitations in the interpretive value of drug measurements in bone and that relative levels of drug and metabolites should be further investigated in terms of forensic value.     

The rise and rise of fentanyl in postmortem casework

Forensic toxicology laboratories are navigating a period of time with increasing drug overdose deaths, an opioid epidemic, the impact of the COVID-19 pandemic, and the illicit drug market flooded with novel psychoactive substances. In New York City, the Department of Forensic Toxicology has experienced a 56% increase in postmortem casework in the past decade with fentanyl detected in 80% of all overdose deaths. Over a period of 2.5 years, 15,638 postmortem cases were tested for the presence of fentanyl and fentanyl analogs using liquid-chromatography tandem mass spectrometry (LCMSMS). Fentanyl was detected in approximately one third of cases and of these 4447 cases with femoral blood. A twofold increase in cases with high concentrations of fentanyl (>100 ng/mL) was observed between 2021 and 2022. The minor metabolite and precursor chemical, 4-ANPP (4-anilino-N-phenethylpiperidine) may help differentiate between illicit and licit fentanyl. 4-ANPP blood concentrations were <10 ng/mL in 98% of the cases and the 4-ANPP:fentanyl ratio was <0.67 for 99.1% of blood specimens. Only six cases had 4-ANPP concentrations higher than the corresponding fentanyl blood concentration. This study also highlights, the changing fentanyl analogs found in postmortem cases since 2016 in NYC with the emergence of fluorofentanyl initially identified in 2020 and continuing to dominate in comparison with the prevalence of other analogs, many of which are no longer detected in casework. The detection of one of the latest drugs to be mixed with fentanyl, namely xylazine, has also increased in prevalence by 36.7% in 2022 compared with 2021.     

Postmortem stability and interpretation of beta 2-agonist concentrations.

This paper describes a series of stability and redistribution studies aimed at understanding the presence and significance of beta 2-agonists in asthma deaths. Salbutamol and terbutaline were shown to be stable in postmortem blood at 23 degrees C for 1 week, 4 degrees C for 6 months and -20 degrees C for 1 to 2 years. However, fenoterol was shown to degrade at 23 degrees C (83% loss), 4 degrees C (93% loss) and -20 degrees C (66% loss) over the same time. Salbutamol concentrations detected in blood taken at the time of body admission to the mortuary were not significantly different from the concentrations detected in blood taken from the same cases at the time of autopsy (45 h later). This suggests that significant postmortem redistribution of salbutamol is unlikely to occur during this period. Postmortem blood concentrations of at least salbutamol are likely to reflect the concentration of these drugs in the body at the time of death.