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

Forensic Science International 99 (1999) 25–34

Cytochrome P450 2D6 (CYP2D6) genotyping on postmortem blood as a supplementary tool for interpretation of forensic toxicological results ¨ Carlsson c , Johan Ahlner b ,c Henrik Druid a , *, Per Holmgren b , Bjorn a

Division of Forensic Medicine, Faculty of Health Sciences, Linkoping University, S-581 85 Linkoping, Sweden b Division of Human Toxicology, Faculty of Health Sciences, Linkoping University, S-581 85 Linkoping, Sweden c Division of Clinical Pharmacology, Faculty of Health Sciences, Linkoping University, S-581 85 Linkoping, Sweden Received 15 May 1998; accepted 22 September 1998

Abstract 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.  1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: CYP2D6; PCR method; Poor metabolizer; Drug interaction; Toxicology; Forensic medicine

*Corresponding author. Tel.: 146-13-130-485; fax: 146-13-101-182; e-mail: [email protected] 0379-0738 / 99 / $ – see front matter  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0379-0738( 98 )00169-8

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1. Introduction Forensic toxicology may often contribute substantially to the determination of the cause and manner of death, and may also provide valuable information for the interpretation of circumstances around death. Particularly, in overdose cases, it is essential to determine the drug(s) responsible for the intoxication, both for providing evidence in the particular case, and for identification of drugs, which could, over time, prove especially dangerous. Much effort has been put on the estimation of fatal concentrations of various drugs in postmortem material [1–4]. Most fatal intoxications concern suicidal overdoses, but during recent years, chronic high dosage has attracted increased interest [5–7]. In order to differentiate between an acute overdose and a chronic poisoning, the distribution of the drug in different samples can be of assistance, since in an acute overdose, the drug may not have reached high levels in samples like vitreous humor, CSF, or hair, when death ensues. For a number of drugs, the parent drug to metabolite ratio, expected to be high in acute overdose cases, may provide even more convincing evidence [8]. The parent drug to metabolite ratio may, however, provide false clues; a low ratio may be encountered in acute overdoses, preceded by a high chronic dosage of the same drug. Conversely, a high ratio could also be due to a reduced metabolism of the parent drug, either because of interaction with other drugs, or genetically low metabolic capacity. Most pharmaceutical drugs are metabolized by enzymes belonging to the cytochrome P450 family. Over 30 different P450 isoenzymes have been identified [9]. One of the most important and best studied enzymes in this family is CYP2D6 (debrisoquine hydroxylase), responsible for degradation of many drugs, e.g. demethylation of several antidepressants [9–14] In therapeutic drug monitoring, analyses of both the CYP2D6 genotype and phenotype have been used to optimize dosing of various antidepressants and neuroleptics [15,16]. The purpose of the present study was to find out if CYP2D6 genotyping could be carried out on postmortem material of various age and quality, and if so, whether the toxicological results in some uncertain, but suspected overdose cases, could be due to reduced enzymatic capacity in poor metabolizers (PM).

2. Materials and methods From the Swedish forensic pathology and toxicology databases [17], 22 uncertain, but supposedly overdose cases with high parent drug to metabolite ratios of drugs metabolized by CYP2D6 were selected (group A). For comparison, 24 consecutive cases were explored, regardless of cause and manner of death, and regardless of toxicological findings (group B). Apart from perusing the autopsy protocols, the police reports and available medical records were also studied. In all cases, postmortem femoral whole blood was used, both for analysis of alcohols and drugs, and for CYP2D6 genotype analysis. The samples were collected at autopsy according to the procedure previously described [4], and potassium fluoride was added

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to a concentration of 1%. All but a few of the samples were, after toxicological analysis, frozen, until genotyped. The toxicological analyses of the pharmaceutical drugs were performed by gaschromatography as described by Druid and Holmgren [4]. Alcohols were analyzed by head-space gas-chromatography and opioids by a gas-chromatography, mass-spectrometry method. Carbon dioxide was analyzed by a spectrophotometric method. The genotyping was carried out according to the following method. Whole blood samples were extracted with Chelex as previously described [18]. Three microliters of the whole blood sample was added to 1 ml sterile water, vortex-mixed, and incubated for 15 min at room temperature. The sample was then centrifuged for 2 min. The pellet was dissolved in 980 ml of Chelex, mixed and incubated for 30 min at 568C, again vortex-mixed for 30 s and incubated for 8 min in boiling water. Finally, after centrifugation for 2 min, 100 ml of the supernatant was transferred to a 0.5 ml Eppendorf tube. The samples were frozen at 2208C until analyzed. The polymerase chain reaction (PCR) amplification of DNA for the identification of the CYP2D6 wild type gene and two mutated alleles, CYP2D6A (29A) and CYP2D6B (29B), was carried out as described by Heim and Meyer [19,20] after some important modifications. Twenty microliters of the Chelex extract were used in the first PCR and the Mg 21 concentration was increased to 25 mM as compared to only 9 mM used in the original method. The buffer was a GeneAmp 103PCR (Perkin Elmer) with Mg 21 (15 mM) to which Mg 21 was added to a final concentration of 25 mM. In the second PCR, 1.5 ml from the first amplification and GeneAmp 103PCR without extra Mg 21 were used. Normally, the Chelex extraction and the PCR method are applied to fresh EDTA whole blood from patients on antidepressant medication as a complement to the therapeutic drug monitoring. To process these samples, we used 20 mM of Mg 21 in the first PCR, but with the post-mortem femoral whole blood, the Mg 21 concentration had to be raised to 25 mM to obtain sharp bands. The reason for this might be that an increased level of hemoglobin degradation products like hematine reduce the Mg 21 concentration by complex binding. The use of the Chelex method for the extraction of DNA results in a less laborious extraction procedure for routine PCR analysis. Prior to investigation of the selected autopsy cases, a set of pilot whole blood samples were studied. EDTA, heparin or heparin1sodium fluoride were added to these samples, and CYP2D6 genotyping was carried out. The samples were subsequently frozen, and reanalyzed.

3. Results The age, sex, genotype, toxicology results, and the cause and manner of death of the cases in groups A and B are displayed in Table 1. It should be emphasized that the cause and manner of death refer to the statement by the responsible forensic pathologist. The high parent drug to metabolite ratio of substances metabolized by CYP2D6 could convincingly be explained by an intentional acute overdose in five cases in group A (cases 2, 6, 14, 20, 21). In the remainder of the group A cases, there was no

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Table 1 Age, sex, genotype, toxicological findings, cause of death and manner of death in cases with unexpectedly high parent to metabolite ratios of agents metabolized by CYP2D6 and in controls Case

Sex/age

Genotype

Toxicology

Cause of death

Manner of death

GROUP A 1

F 62

wt/wt, wt/D

1a) Choking (aspiration) 1b) Intox ethanol1clomipramine

Uncertain

2

M 41

wt/wt, wt/D

1a) Intox clomipramine1citalopram

Suicide

3

M 43

wt/B

1a) Anoxic brain damage 1b) Intox ethanol1clomipramine

Accident

4

M 41

wt/wt, wt/D

1a) Traumatic rupture of spleen

Homicide

5

M 52

wt/B

1a) Multiple drug intox

Uncertain

6

M 53

wt/B

1a) Intox clomipramine1flunitrazepam

Suicide

7

M 29

wt/B

1a) Intox remoxipride

Suicide

8

M 74

wt/wt, wt/D

0.9 clomipramine 0.5 desmethylclomipramine 2.5 ethanol 2.9 clomipramine 1.0 desmethylclomipramine 1.1 citalopram 0.3 desmethylcitalopram 1.4 clomipramine 0.4 desmethylclomipramine 0.07 midazolam 1.4 phenytoin 5.0 ketamine 0.1 propoxyphene 0.5 clomipramine 0.1 desmethylclomipramine 2 paracetamol d. metronidazole 2.0 ethanol 2.0 clomipramine 0.4 desmethylclomipramine 0.1 propoxyphene 11 carbamazepine 19 paracetamol 0.02 zopiclone 4.3 clomipramine 0.3 desmethylclomipramine 0.17 7-aminoflunitrazepam 46 remoxipride 12 caffeine 9.8 remoxipride

Uncertain

9

F 58

wt/B

1a) Drowning 2a) Ischemic heart disease 1a) Multiple drug intox

10

F 56

wt/wt, wt/D

1a) Intox trimipramine1perphenazine

Uncertain

11

F 59

wt/wt, wt/D

1a) Chronic alcohol abuse

Natural

12

F 49

wt/wt, wt/D

1a) Intox codeine

Uncertain

2.1 trimipramine n.d. desmethyltrimipramine 30 moclobemide 2.5 oxomoclobemide 4.1 fluvoxamine 3.1 trimipramine 0.3 desmethyltrimipramine 1.0 orphenadrine 0.1 codeine 0.07 perphenazine 0.6 trimipramine nd. desmethyltrimipramine 0.03 zopiclone 0.4 trimipramine n.d. desmethyltrimipramine 0.3 diazepam 0.9 nordazepam 0.12 7-aminonitrazepam 10 carisoprodol 29 meprobamate 44 paracetamol 0.08 morphine 1.2 codeine

Suicide

H. Druid et al. / Forensic Science International 99 (1999) 25 – 34

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Table 1. Continued Case

Sex/age

Genotype

Toxicology

Cause of death

Manner of death

13

M 45

wt/B

1a) Intox fluoxetine 2a) Ischemic heart disease

Suicide

14

F 52

wt/wt, wt/D

1a) Intox citalopram1clomipramine

Suicide

15

F 55

wt/wt, wt/D

1a) Multiple drug intox

Suicide

16

M 38

wt/wt, wt/D

1a) Multiple drug intox

Uncertain

17

F 44

wt/B

1a) Hanging

Suicide

18

F 30

wt/B

1a) Hanging

Suicide

19

M 56

wt/B

1a) Intox ethanol1fluoxetine

Uncertain

20

M 38

wt/wt, wt/D

1a) Intox venlafaxine

Suicide

21

M 34

wt/B

1a) Intox ethanol1fluoxetine

Suicide

22

M 49

–

0.1 propoxyphene 1.5 fluoxetine na. norfluoxetine 4.7 citalopram 0.1 desmethylcitalopram 1.7 clomipramine 0.2 desmethylclomipramine 0.2 venlafaxine 0.2 desmethylvenlafaxine 1.5 amitriptyline 0.1 nortriptyline 0.6 propiomazine 0.2 dihydropropiomazine 0.18 nitrazepam 0.6 7-aminonitrazepam 0.03 alprazolam 0.4 zopiclone 15 venlafaxine 1.6 desmethylvenlafaxine 2.0 propoxyphene 0.9 clomipramine n.d. desmethyiclomipramine 0.1 7-amino-flunitrazepam 5 paracetamol 0.1 nordazepam 0.4 ephedrine 1.7 venlafaxine 0.7 desmethylvenlafaxine 1.4 venlafaxine 0.9 desmethylvenlafaxine 0.16 zolpidem 0.2 diazepam 0.3 nordazepam 0.3 dihydropropiomazine 0.5 oxazepam 5.3 fluoxetine n.a. norfluoxetine 0.2 thioridazine 0.03 alprazolam 2.0 ethanol 54 venlafaxine 3.5 desmethylvenlafaxine 0.5 fluconazole 0.1 diazepam 0.1 nordazepam 1.4 fluoxetine n.d. norfluoxetine 0.3 propoxyphene 0.2 alprazolam 0.3 zopiclone 1.6 ethanol 1.9 clomipramine 0.5 desmethylclomipramine 0.1 nordazepam 65% CO

1a) CO-intox

Suicide

H. Druid et al. / Forensic Science International 99 (1999) 25 – 34

30 Table 1. Continued GROUP B 23

M 59

wt/B

24 25 26 27

F 75 M 81 M 38 M 65

wt/B wt/wt, wt/D wt/B wt/B

28 29

M 87 F 49

wt/B wt/wt, wt/D

30 31 32

F 58 M 54 M 51

wt/B wt/B wt/wt, wt/D

33 34

M 58 M 81

wt/B wt/wt, wt/D

35

F 52

wt/wt, wt/D

36

M 38

wt/wt, wt/D

37

F 54

B/B, B/D

38 39

F 81 F 68

wt/B wt/B

40

M 23

wt/wt, wt/D

41

M 27

wt/wt, wt/D

42

F 48

wt/wt, wt/D

43 44

M 57 F 74

wt/B wt/B

45

M 38

wt/wt, wt/D

46

F 75

wt/A

11 phenazone 0.26 acetone 0.18 isopropanol 0.77 ethanol – – 1 paracetamol 0.3 melperone 0.1 dihydromelperone 0.01 flunitrazepam 83 phenazone 1.6 propoxyphene 0.4 7-amino-flunitrazepam 0.3 paroxetine 0.1 paroxetine – – 3.0 ethanol – – 3.1 ethanol 0.8 citalopram 0.1 desmethylcitalopram d. sulphapyridine 2.4 ethanol 0.44 ethanol 1.0 propoxyphene 0.02 7-amino-flunitrazepam 190 paracetamol 1.6 ethanol – 0.1 propranolol 4 paracetamol 0.15 morphine 5 paracetamol 0.3 diazepam 0.1 nordazepam 0.14 morphine 0.01 6-acetyl-morphine 0.07 codeine 4.0 carbamazepine 0.7 verapamil 1.8 ethanol – 1.0 quinine 0.6 thiopental 0.2 morphine 0.02 6-acetyl-morphine 0.02 codeine 0.91 ethanol –

1a) Chronic alcohol abuse 2a) Gastric bleeding

Natural

1a) Traumatic aortic rupture 1a) Traumatic aortic rupture 1a) Acute myocardial infarction 1a) Drowning

Accident Accident Natural Uncertain

1a) Intrathoracic injuries 1a) Multiple drug intox

Accident Suicide

1a) Blunt injuries 1a) Ischemic heart disease 1a) Asphyxia 1b) Thoracic compression 1a) Ischemic heart disease 1a) Chronic alcohol abuse 2a) Ischemic heart disease 1a) Gunshot wound to the head

Homicide Natural Accident

1a) 2a) 1a) 2a)

Myocardial fibrosis Ischemic heart disease Intox ethanol1paracetamol SLE

Natural Natural Suicide

Natural Suicide

1a) Ischemic heart disease 1a) Ischemic heart disease

Natural Natural

1a) Septicemia 1b) Intox heroin 1a) Intox heroin 2a) Chronic drug abuse

Accident Accident

1a) Chronic alcohol abuse

Natural

1a) Chronic alcohol abuse 1a) Brain injuries

Natural Accident

1a) Intox heroin1ethanol 2a) Chronic drug abuse 2b) Hepatitis

Accident

1a) Traumatic aortic rupture

Accident

Group A (cases 1–22), cases selected because of high parent drug to metabolite ratio of substance(s) metabolized by CYP2D6; group B (cases 23–46), consecutive autopsy cases serving as controls. All genotype and toxicology data refer to analyses on postmortem femoral blood. The cause and manner of death are based on the statement by the responsible pathologist. Abbreviations: 1a, immediate cause of death; 1b, underlying cause of death; 2a, contributory cause of death; d., detected, not quantified; n.d., not detected; n.a., not analyzed; Intox, intoxication. All genotypes including a wt (wild type) gene are considered to be extensive metabolizers. All drug concentrations are given in mg/g femoral blood. Ethanol, isopropanol and acetone concentrations are expressed in promille.

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circumstantial evidence of an acute overdose, yet another three A-cases (cases 7, 9, 15), had been considered to be suicidal overdoses, although the diagnosis was mainly based on the toxicology results. No genetic defects suggesting a PM phenotype were found among the group A cases. One of the group B cases (case 37) showed a deletion / splice-site mutation, implying a PM, but in this case, no drugs interacting with CYP2D6 were found. However, among the extensive metabolizers (EMs) of both groups, a substantial number of cases showed mutations of the CYP2D6 gene, possibly implying reduced enzymatic capacity in some cases. None of the additives in the pilot samples affected the PCR genotyping. Freezing did not prevent analysis either. In all autopsy cases but one, the CYP2D6 genotype analyses were successful. The toxicology revealed the presence of more than one substrate for or inhibitor of CYP2D6 in eight of the group A cases (cases 2, 10, 13, 14, 15, 16, 19, 21). In some of these cases, pharmacokinetic interactions between the different substances seem likely, and may explain the high metabolic ratio of the substance with the weakest affinity for CYP2D6. Besides, in another two cases (cases 9 and 42), interactions may have played a role for the drug concentrations detected, although probably not involving CYP2D6.

4. Discussion The parent drug to metabolite ratio is now commonly used to support the diagnosis of acute poisoning, or at least, used as an indicator of a recent intake of certain drugs. However, drug interactions may pose problems since a number of drugs may be metabolized by the same enzyme, which could cause an accumulation of the drug with the weakest binding capacity. In addition, some individuals may be PMs of the particular enzyme. This latter alternative has attracted increasing interest, since the PMs may provide an interpretation pitfall. The worst scenario would be that a patient being a PM would follow the standard dosing for a particular drug and eventually be fatally intoxicated due to chronic accumulation of the drug. This possibility has been suggested by several authors, and recently has been thoroughly discussed by Swanson et al. [7], reporting on two cases where chronic poisoning seems very likely. The question whether a high metabolic ratio is due to poor enzyme capacity could be easily solved if the responsible gene is analyzed. This communication presents a method for analysis of CYP2D6 genotype on postmortem femoral blood, which allows for differentiation between EM and PM. The method could successfully be carried out even on material which was moderately decomposed, and stored for months. Addition of fluoride, heparin or EDTA did not affect the analysis, nor did freezing of the samples. As compared to the method used in clinical practice, a successful analysis of the postmortem blood required a higher concentration of Mg 21 , presumably due to increased levels of hemoglobin degradation products, which could reduce Mg 21 by complex formation. The genotyping failed in only one case (case 22), a suicidal carbon monoxide poisoning. We have not found any support for the assumption that high CO con-

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H. Druid et al. / Forensic Science International 99 (1999) 25 – 34

centrations would disturb the analysis, and the reason for the analytical failure in this case remains obscure. In the present study, we did not find any PMs among the 22 cases with unexpectedly high metabolic ratios of compounds known to be metabolized by CYP2D6. One PM was found among the control cases. This figure, either compared to the number of control cases or compared to the total number of cases indicates a lower frequency than reported in larger material of Caucasians (approx 7%) [21]. This might constitute a chance difference due to the small sample. Another possibility is that other genetic characteristics, implying certain personality features [22,23], associated with defect expression of the CYP2D6 would make the PMs less prone to end up at a department of forensic medicine. In particular, the frequency of PMs could be lower among depressed patients committing suicide. However, Chen et al. [16] did not find any difference in frequency of PMs among depressed outpatients and controls. Although the genotyping strongly indicates that all group A cases were EMs, this does not prove that they all had the same CYP2D6 activity. Ten of these individuals actually showed one mutated allele, which might suggest a somewhat lower metabolic capacity. In fact, the metabolic ratios of debrisoquine / 4-hydroxydebrisoquine among extensive metabolizers are normally distributed on a log scale [24]. This implies a considerable variation even among ‘‘normal’’ individuals. Evaluation of the toxicological results revealed that in eight of the group A cases, interactions between substances metabolized by or acting as inhibitors of CYP2D6 could have occurred. In four of these cases (cases 10, 13, 16 and 19), there was no circumstantial evidence of intentional overdose. In addition, interactions could have played a role for the toxicological result of cases 9 and 42, although probably chiefly involving CYP1A2 and CYP3A4 [9,25–27]. It should also be recognized that some substances may still inhibit a metabolizing enzyme a considerable time after the last intake of the drug. For example, the inhibition of CYP2D6 after discontinuation of fluoxetine treatment may require 5 weeks until it is completely reversed [28], and at that time point, fluoxetine and norfluoxetine may not be detectable. As opposed to the statement by Swanson [7], interactions in drug metabolism are only important among EMs. These may then be converted to phenocopies of PMs. The PMs do not produce the enzyme at all, hence there is no enzyme to be inhibited. This study has focused on the importance of CYP2D6, and the cases in group A were selected because of the presence of substances being metabolized by or inhibitors of this enzyme. However, a number of other cytochrome enzymes are involved in the metabolism of drugs, which may be of toxicological interest. CYP3A4, constituting 60% of the total P450 content of liver specimens [29], may prove valuable for interpretation of toxicological data, since it is involved in the metabolism of carbamazepine, tricyclic antidepressants, codeine, and several benzodiazepines. In conclusion, we have modified a PCR method, which can be used for genotyping of CYP2D6 on postmortem material. The method may be employed in cases where a PM phenotype is suspected. However, in our series of cases where an unexpected high parent drug to metabolite ratio of compounds metabolized by CYP2D6 was observed, no PMs were found. This suggests that the PM pitfall is not very frequent. On the other

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hand, a number of possible drug interactions were found, which could explain some of the unexpected high metabolic ratios for certain substances. We thus suggest that both interactions and genetically poor metabolic capacity should be considered and evaluated in obscure cases.

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