Interpretation of Postmortem Toxicology Results

INTERPRETATION OF POSTMORTEM TOXICOLOGY RESULTS

Pharmacogenetics and Drug-Alcohol Interaction

Anna Koski

Department of Forensic Medicine University of Helsinki Finland

ACADEMIC DISSERTATION

To be publicly discussed, with the permission of the Medical Faculty of the University of Helsinki, in the auditorium of the Department of Forensic Medicine on September 23rd 2005, at 12 noon.

Helsinki 2005 SUPERVISORS Professor Antti Sajantila Department of Forensic Medicine University of Helsinki Helsinki, Finland

Docent Ilkka Ojanperä Department of Forensic Medicine University of Helsinki Helsinki, Finland

REVIEWERS Docent Eero Mervaala Institute of Biomedicine University of Helsinki Helsinki, Finland

Docent Kari Poikolainen Finnish Foundation for Alcohol Studies National Research and Development Centre for Welfare and Health Helsinki, Finland

OPPONENT Professor Jørg Mørland Division of Forensic Toxicology and Drug Abuse Norwegian Institute of Public Health Oslo, Norway

ISBN 952-91-9214-2 (paperback) ISBN 952-10-2662-6 (pdf) http://ethesis.helsinki.fi

Helsinki University Printing House Helsinki 2005

Obtaining a quantitative result is not the endpoint of the analytical process.

Irving Sunshine

CONTENTS

ABBREVIATIONS...... 6

LIST OF ORIGINAL PUBLICATIONS ...... 7

ABSTRACT...... 8

INTRODUCTION...... 9

REVIEW OF THE LITERATURE ...... 10 1 Interpretation of Postmortem Forensic Toxicology Results...... 10 1.1 Blood Samples ...... 10 1.1.1 Toxicological Tables ...... 11 1.2 Other Matrices ...... 12 1.3 Postmortem Changes ...... 12 1.4 Metabolites...... 13 1.5 Alcohol Toxicity...... 13 1.6 Drug Toxicity...... 13 1.6.1 Fatal Toxicity Index ...... 14 1.6.2 Other Measures of Toxicity...... 15 1.6.3 Sources of Bias in Toxicity Indices...... 15 2 Pharmacogenetics...... 16 2.1 Drug-Metabolizing Enzymes...... 16 2.1.1 Cytochrome P450 System ...... 16 CYP2D6 ...... 17 CYP2C19 ...... 19 2.2 Studies on Drug Metabolism ...... 19 2.2.1 Tramadol Metabolism ...... 19 2.2.2 Amitriptyline Metabolism...... 20 2.3 Clinical Pharmacogenetics...... 21 2.4 Postmortem Pharmacogenetics...... 22 3 Drug-Alcohol Interaction ...... 23 3.1 Alcohol Effects and Anesthetic Action ...... 23 3.1.1 γ-Aminobutyric Acid Receptor Type A ...... 24 3.2 Animal Studies...... 24 3.3 Postmortem Studies ...... 25

AIMS OF THE STUDY...... 26

4 CONTENTS

MATERIALS AND METHODS...... 27 1 Autopsy cases ...... 27 1.1 Blood Samples ...... 27 1.2 Database...... 27 2 Analysis of Drug Concentrations ...... 27 2.1 Screening...... 27 2.2 Metabolite Analysis ...... 28 3 Genotyping ...... 28 3.1 Long Polymerase Chain Reaction...... 29 3.2 Restriction Fragment Length Polymorphism Analysis...... 29 3.3 Multiplex Single-Base Extension Reaction...... 29 4 Case Selection Criteria ...... 29 5 Statistical Methods ...... 30

RESULTS...... 31 1 Pharmacogenetics ...... 31 1.1 CYP2D6 and Tramadol (I) ...... 31 1.2 CYP2D6 and Amitriptyline (II)...... 31 1.3 CYP2C19 and Amitriptyline (II)...... 32 1.4 Allele and Genotype Frequencies (I, II)...... 33 2 Fatal Toxicity Indices (IV, V) ...... 33 3 Drug-Alcohol Interaction...... 34 3.1 Alcohol and Benzodiazepines (III) ...... 34 3.2 Alcohol and Other Common Drugs (IV-VI)...... 34

DISCUSSION...... 38 1 Methodological Considerations...... 38 2 Pharmacogenetics ...... 39 3 Drug-Alcohol Interaction...... 41 3.1 Alcohol and Benzodiazepines...... 41 3.2 Alcohol and Other Common Drugs ...... 41 4 Drug Safety...... 42 4.1 Newer Antidepressants ...... 42 5 Implications for Interpretation...... 43

CONCLUSIONS ...... 45

ACKNOWLEDGMENTS ...... 46

REFERENCES...... 47

CONTENTS 5 ABBREVIATIONS

ADR adverse drug reaction BAC blood alcohol concentration BDZ benzodiazepine bp base pair CI confidence interval CNS central nervous system CYP cytochrome P450 CYP2C19 cytochrome P450 enzyme 2C19 CYP2C19 gene encoding CYP2C19 CYP2D6 cytochrome P450 enzyme 2D6 CYP2D6 gene encoding CYP2D6 DDD defined daily dose DME drug-metabolizing enzyme EHAT (E)-10-hydroxyamitriptyline EHNT (E)-10-hydroxynortriptyline EM extensive metabolizer (phenotype) FTI fatal toxicity index GC gas chromatography gEM genetically extensive metabolizer gPM genetically poor metabolizer gUM genetically ultra-rapid metabolizer ICD-10 International Classification of Diseases, 10th Revision IM intermediate metabolizer (phenotype) kb thousand base pairs LC/MS-MS liquid chromatography – tandem mass spectrometry

LD50 median lethal dose, the dose required to kill 50% of the given population LIMS laboratory information management system M1 O-demethyltramadol M2 N-demethyltramadol, nortramadol M3 N,N-didemethyltramadol M4 O,N,N-tridemethyltramadol M5 O,N-didemethyltramadol MR metabolite ratio MRM multiple reaction monitoring MS mass spectrometry NNT N-demethylnortriptyline p significance level OPLC overpressured layer chromatography PCR polymerase chain reaction PM poor metabolizer (phenotype) RFLP restriction fragment length polymorphism SSRI selective serotonin reuptake inhibitor TCA tricyclic antidepressant TLC thin layer chromatography UM ultra-rapid metabolizer (phenotype) ZHAT (Z)-10-hydroxyamitriptyline ZHNT (Z)-10-hydroxynortriptyline

6 ABBREVIATIONS

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following articles, which are referred to as I-VI in the text:

I Levo A, Koski A, Ojanperä I, Vuori E, Sajantila A (2003) Post-mortem SNP analysis of CYP2D6 gene reveals correlation between genotype and opioid drug (tramadol) metabolite ratios in blood. Forensic Sci Int 135(1):9-15.

II Koski A, Sistonen J, Ojanperä I, Gergov M, Vuori E, Sajantila A (2005) CYP2D6 and CYP2C19 genotypes and amitriptyline metabolite ratios in a series of medicolegal autopsies. Forensic Sci Int (in press, published online July 14, DOI: 10.1016/j.forsciint.2005.05.032).

III Koski A, Ojanperä I, Vuori E (2002) Alcohol and benzodiazepines in fatal poisonings. Alcohol Clin Exp Res 26(7):956-9.

IV Koski A, Ojanperä I, Vuori E (2003) Interaction of alcohol and drugs in fatal poisonings. Hum Exp Toxicol 22(5):281-7.

V Koski A, Vuori E, Ojanperä I (2005) Newer antidepressants: evaluation of fatal toxicity index and interaction with alcohol based on Finnish postmortem data. Int J Legal Med (in press, published online March 1, DOI: 10.1007/s00414-005-0528-x).

VI Koski A, Vuori E, Ojanperä I (2005) Relation of postmortem blood alcohol and drug concentrations in fatal poisonings involving amitriptyline, propoxyphene and promazine. Hum Exp Toxicol 24(8):389-96.

The original publications are reproduced with the permission of the copyright holders.

LIST OF ORIGINAL PUBLICATIONS 7 ABSTRACT

Postmortem forensic toxicology annually reveals more than a thousand fatal poisonings in Finland. Both alcohol and drugs are found in the vast majority of cases, with certain drugs more often involved than others. Some of the drugs commonly causing fatal poisonings are polymorphically metabolized.

In this thesis, a retrospective, statistical approach was taken to elucidate the role of pharmacogenetics and drug-alcohol interaction in fatal poisonings. More specifically, the objective was to investigate whether certain genetic variants associated with abnormal drug metabolism can be correlated with metabolite ratios in postmortem material, and whether an interaction between alcohol and common toxic drugs is perceptible in fatal poisonings. Methods included genotyping and metabolite analysis using autopsy blood, as well as statistical analysis of the obtained metabolite ratios. Drug safety was evaluated as a function of alcohol and drug concentrations determined in postmortem blood and of fatality rates in relation to the sales of drugs.

Correlations between the CYP2D6 gene dose and the tramadol metabolite ratios were observed in 33 cases involving tramadol. In 195 cases involving amitriptyline, similar correlations were found between the CYP2D6 gene dose and the metabolite ratios related to stereospecific ring hydroxylation and between CYP2C19 gene dose and the metabolite ratios related to N-demethylation. Most importantly, the nonfunctional genotypes were significantly different from the corresponding fully functional genotypes with respect to several of the investigated metabolite ratios. However, no fatal poisonings with accidental or undetermined cause of death were associated with nonfunctional genotypes.

Regarding fatal poisonings involving two common benzodiazepines, blood alcohol concentrations were on average lower in cases involving temazepam than in those involving diazepam or alcohol alone. Diazepam therefore appeared safer in combination with alcohol than temazepam. Among the drugs most commonly causing fatal poisonings, promazine, doxepin, amitriptyline, and propoxyphene were the least safe in combination with alcohol, whereas zopiclone, diltiazem, and the newer antidepressants proved relatively safe. The selective serotonin reuptake inhibitors appeared the safest among the newer antidepressants. Interestingly, the least safe drugs in combination with alcohol were also found to cause more fatal poisonings with respect to their sales than the other drugs included in the study. A similar correlation was also observed within the group of newer antidepressants.

In conclusion, genetic factors seem to play a more dominant role in metabolite ratios than age, gender, or environmental factors, and postmortem genotyping may therefore provide useful information in poisoning cases where the manner of death is unclear. When determining the cause of death, the possibility of a fatal poisoning due to an interaction between alcohol and drugs should be considered seriously, especially when certain toxic drugs are involved. These results have implications not only for the interpretation of postmortem toxicology results but for drug safety in general.

8 ABSTRACT

INTRODUCTION

Approximately 50 000 Finns die each year. automation in analysis, reporting, and data Twenty percent of these deaths are investigated management. Forensic toxicology findings are in a medicolegal autopsy, and in about half of therefore stored in a database such as the the autopsy cases samples are taken for Laboratory Information Management System toxicological analysis. Forensic toxicology, i.e. (LIMS) in the Laboratory of Toxicology. the use of toxicology for legal purposes, today Interpretation of postmortem toxicology employs a wide variety of analytical methods results is based on a forensic pathologist’s producing a wealth of data. In postmortem experience and on previously reported findings investigation, the main purpose of these data is regarding toxicity of drugs, alcohols, and other to help the forensic pathologist to determine the common poisons. In Finland, it is the forensic cause of death, but the eventual significance of pathologist who determines the cause of death, the results can vary greatly. Besides confirming, although the forensic toxicologist may be revealing, or excluding fatal poisonings, the consulted on analytical findings. A strength of findings may yield important information on the Finnish forensic medicine is the practice of the contributing factors and general circumstances investigating pathologist to send a copy of the of the death. completed death certificate to the Laboratory of According to Act 169/1948, all postmortem Toxicology. Integrating the information on forensic toxicology in Finland is centralized to cause and manner of death to the LIMS creates a the Laboratory of Toxicology, Department of nation-wide databank enabling complex Forensic Medicine, at the University of research and acquisition of detailed statistics on Helsinki. Blood, urine, and liver are routinely Finnish fatalities. screened for drugs, alcohols, and drugs of abuse. There are several confounding factors in Upon request, samples are also analyzed for the interpretation of postmortem toxicology carbon monoxide, cyanide, and other suspected results. Besides the background information, poisons. Analytical methods include chromato- important issues to be taken into consideration graphy, spectrometry, and immunological include postmortem redistribution, individual assays. DNA analysis may also be employed for variation, and concomitant findings. further investigations. Broad drug screens and Theoretically, any two compounds that share a dedicated analyses are focused on reliable mechanism of action or produce a similar detection, identification, and quantitation of response may cause unwanted or pronounced potentially toxic compounds. Quality control is adverse effects. In practice, the most common extensively applied to ascertain the integrity of agent to interact with a drug is alcohol. Alcohol results. The laboratory was accredited by is a frequent finding in forensic toxicology, and the Finnish Accreditation Service (FINAS) in being a central nervous system (CNS) 1997. depressant, is often deemed to have played a Advances in pharmacology, resulting in part in causing death. A study was therefore new drugs and combinations of drugs, as well as undertaken to elucidate the role of alcohol in in novel indications for existing drugs, pose a fatal poisonings. In addition, the role of genetic challenge to forensic toxicology. The discipline factors in drug-related deaths was investigated. is further complicated by illegal drugs, designer The general purpose of this thesis was to drugs, and changing local and global trends in assess the importance of pharmacodynamic drug abuse. To keep current on what is drug-alcohol interactions and pharmacogenetic happening ‛on the street’, the analytical methods variation affecting drug metabolism in a in the laboratory must be continuously postmortem context. The findings can be developed. Modern-day forensic toxicology expected to support the interpretation of produces a wealth of data, demanding postmortem toxicology results.

INTRODUCTION 9 REVIEW OF THE LITERATURE

1 Interpretation of Postmortem Forensic are often present in overdoses of other drugs Toxicology Results [16,17]. Moreover, tolerance (and cross- A forensic toxicological investigation consists of tolerance) to drugs or alcohol may have three main steps: obtaining the case history and developed prior to death. Since the extent and suitable specimens, performing toxicological duration of exposure are seldom precisely or analyses, and interpreting the findings. In a post- reliably known, concentrations of certain mortem case, toxicological specimens are analytes, e.g. lead, barbiturates, BDZs, and collected at autopsy or external examination and several drugs of abuse, are not necessarily very subjected to chemical analysis, which is a meaningful [6,10,14]. In addition to appreciating process of extraction, detection, identification, the pharmacodynamic properties of drugs, and quantitation of the analytes [1,2]. knowledge of pharmacokinetics, of drug bio- Comprehensive treatises on interpretation transformation in particular, is essential in of forensic toxicology results have most recently interpreting the results, especially when the appeared by Jones [3], Richardson [4], and parent compound is either rapidly or poly- Holmgren [5], but throughout the years the morphically metabolized or is not readily subject has inspired numerous reviews, essays, detected. Yet another phenomenon to be and book chapters [6-15]. Although the considered is idiosyncrasy: although most principles are globally applicable, some of the people react to a drug predictably, a few will discussion reflects professional experience, react differently [6]. personal opinions, and local medicolegal What is not found also influences the systems of the authors. In the Finnish system, assessment. Negative findings allow exclusion interpretation of postmortem toxicology results of many relevant poisons [2], but require involves both the forensic toxicologist, who comprehension of the limitations of analytical estimates the relevance of the findings and the methods. Even the most modern equipment is need for further analysis, and the forensic patho- able to detect only a part of the vast array of logist, who eventually assesses the contribution pharmacological agents in use today. Due to the of the findings to the cause of death. great number of possible toxicants, general Several aspects are considered in the unknown screening [18] and substance interpretation. First of all, the numerical results identification [19] are currently key problems in obtained in the laboratory are meaningful only state-of-the-art forensic toxicology. Moreover, in context with the individual case and such once a chemical entity has been detected and background information as acute/chronic identified, quantitation may prove impossible exposure, emergency treatment, and length of because certified reference materials are survival [6,8,15]. Besides individual variation, unavailable or difficult to obtain [20]. thought should be given to the site of specimen collection, the methods of collection and 1.1 Blood Samples analysis, findings in the other matrices Of the matrices available at autopsy, blood is investigated, autopsy findings, and possible essential for evaluating whether the deceased postmortem changes [8,10]. was under the influence of a drug at the time of Multiple substances are commonly death. The specimen of choice for quantitative involved in overdoses, hence the difficulty of purposes is femoral venous blood because it is attributing the fatality to any single one. the least susceptible to postmortem changes Whenever several drugs are found, the [3,15,21]. The recommended method of possibility of additive effects, synergism, or collection is to draw blood from a ligated or antagonism ought to be considered [6]. severed femoral vein into a plastic tube. A Especially benzodiazepines (BDZs) and alcohol supplementary sample of central blood is often

10 REVIEW OF THE LITERATURE

collected, especially when femoral blood is [48], are therefore those in which the lethal unavailable or scant, but should be reserved for concentrations in postmortem material are cited qualitative purposes only [15]. The drug [33,34,37]. Some of these tables also include concentrations in cardiac blood are often higher statistical information on concentration than in femoral blood [11,22], which may distributions in different types of fatalities facilitate detection. When time has elapsed [34,37]. between trauma and eventual death, analysis of However, how concentrations measured subdural and epidural blood clots may provide postmortem relate to those measured in life information pertaining to the time of injury. [10,13,15] remains obscure. Drawing In toxicological case work, blood drug correlations between these two situations is concentrations are today reliably determined therefore not straightforward. Firstly, post- using mass spectrometry (MS) [19], although mortem blood is not the same as circulating older tools, such as gas chromatography (GC) blood in a living person [13]. Secondly, in a coupled with flame ionization detection or with living body the pharmacodynamic response to a a nitrogen-phosphorus selective detector remain drug is dictated by the concentration of a free valuable in the analysis of alcohols [23] and drug, whereas a postmortem result represents the nitrogen-containing drugs [24], respectively. To sum of the free and protein-bound drug. Thirdly, ensure the quality of analytical results and their peak concentrations of the drug are likely to validity in courts of law, method validation, have been higher than what is seen postmortem, internal quality assurance, and external since the peak concentration may cause proficiency testing are widely employed in irreversible damage but not immediate death forensic toxicology laboratories [25-28]. [49]. It may take hours for the intoxicated person to succumb, with the drug being 1.1.1 Toxicological Tables metabolized in the meanwhile. Blood alcohol To aid interpretation of the results, therapeutic, concentration (BAC), for instance, decreases toxic, and lethal concentrations of drugs in 0.10-0.25‰/h in moderate drinkers and even human blood, plasma, and serum have been faster in alcoholics [50]. There are also instances compiled into various handbooks [22,29,30], where peak drug concentrations have been reviews, and original articles [31-44]. reported to be higher postmortem; in two cases Providing a useful addition to the in which intoxication had led to hospitalization traditional handbooks and other printed prior to death, higher amitriptyline and references, the internet can be employed to propoxyphene concentrations in blood were access many toxicology-related resources, found postmortem than antemortem [20]. recently reviewed by Goldberger and Polettini Finally, when a drug has one or more chiral [45]. These resources include two extensive centers, the pharmacodynamic and pharmaco- compilations of therapeutic and toxic levels of kinetic behavior usually differs between the iso- drugs in biological specimens [46,47]. mers. In the hopes of minimizing the expensive A major problem with using such reference production of ineffective isomers, avoiding the values in postmortem toxicology is that post- side-effects caused by harmful isomers (e.g. (–)- mortem drug concentrations are determined in thalidomide), and splitting the drug load on an hemolyzed whole blood, whereas clinical individual in general, drug development is now studies usually provide information on plasma aimed at enantiospecific drugs [51]. When a or serum concentrations [11]. To extrapolate drug may be present in toxicological samples postmortem results to the antemortem state, either as a racemate or as an enantiomer, as in whole blood/plasma concentration ratios [15] the cases of citalopram and amphetamine, and whether they stay the same in a interpretation of blood concentrations can be decomposing body [3] should be known. The considered confounded by yet another factor, compilations most relevant to forensic unless enantioselective analysis has been applied toxicology, as discussed by Druid and Holmgren to the forensic samples.

REVIEW OF THE LITERATURE 11 1.2 Other Matrices 1.3 Postmortem Changes Other common matrices of interest in The quality of a postmortem specimen is often postmortem toxicology include urine, liver, poor; it can be watery, putrefied, degraded, or gastric contents, bile, blood clots, and vitreous burned. The stability of drugs in postmortem humor [15,52]. Muscle tissue, bone marrow, and samples is another concern [15,52,58]. Changes hair can be used in severely putrefied cases, and in concentration are generally not sufficiently even larvae feeding on the corpse may be large to affect interpretation, especially when examined [8,52,53]. In all, alternative matrices femoral blood is used [58], but such drugs as can constitute valuable specimens for nitrobenzodiazepines and cocaine may disappear postmortem toxicology, as recently reviewed by gradually due to bacterial action [59] and Drummer and Gerostamoulos [52] and Skopp hydrolysis [60], respectively. It has also been [15]. suggested that, due to reformation of the parent Due to site and temporal dependence of drug from metabolites [58], e.g. by hydrolysis drug concentrations [54,55], the toxicological of conjugated entities [15], parent drug results obtained in other matrices differ in concentrations may even increase. significance from those determined in femoral Another type of postmortem change is the blood. A positive finding in urine, for instance, production of ethanol from carbohydrates by shows that the detected substance was present in certain microorganisms in a putrefying body or the body some time before death, but the during storage [61]. To prevent further physiological effects exerted by the compound conversion in the autopsy sample, refrigeration on the body may not be readily deduced from and addition of potassium or sodium fluoride to the concentration in urine [8,15]. a final concentration of 1-5% are generally Liver is a highly valuable specimen since recommended. Microbial activity may produce many substances are present in higher significant ethanol concentrations, in some cases concentrations in the liver and are thus more in excess of 1‰, and a positive BAC should easily detected than in femoral blood [11,22]. therefore be verified by analysis of urine or Utilization of hair samples has been recently vitreous humor whenever possible [62]. recapitulated by Kintz [53]. Hair is of The most relevant alteration occurring exceptional value in exhumed bodies, but can between death and autopsy is, however, post- also be used to detect exposure to drugs over a mortem redistribution, i.e. the migration of period of several months prior to death [15]. drugs between tissues and blood in a cadaver Detection of a compound in gastric contents [63,64]. Literature on postmortem redistribution does not necessarily indicate recent uptake, for has recently been summarized in a brief review drugs can be re-excreted into gastric juices [3]. by Leikin and Watson [11], and the mechanisms Vitreous humor is another valuable involved have been the subject of a more specimen in postmortem toxicology. It is a extensive review by Pélissier-Alicot et al. [65]. relatively well-isolated, sterile compartment Drugs that undergo postmortem redistribution protected from trauma and putrefaction, with are typically lipophilic, weakly basic drug concentrations typically following the compounds with a relatively large volume of concentrations in blood with a certain delay distribution or preferential binding to the [15]. Glucose, lactate, and potassium are myocardium [21,65,66]. conveniently determined in vitreous humor [56]. The most important mechanism of Alcohol concentrations in vitreous humor redistribution has been estimated to be drug closely follow BACs, when the difference in diffusion from the gastrointestinal tract, lungs, water content is taken into account [57]. Thus and other drug-rich tissues, such as the vitreous humor provides the only other matrix, myocardium, into surrounding tissues and blood besides peripheral blood, capable of yielding a [67]. Recent ingestion of a large amount of meaningful quantitative result [11]. Its use as an drugs may result in postmortem diffusion of the alternative specimen is, however, limited by the unabsorbed drug from the gastric contents to the small sample size of only 3-6 ml [3]. surrounding organs and vessels [65]. The extent

12 REVIEW OF THE LITERATURE

of redistribution depends on the drug study in which alcohol was found in two-thirds concentration and possible resuscitation of patients presenting with acute poisoning to an attempts, probably shifting cardiac blood emergency department [74]. towards the periphery [55,65], but also on the The BAC level causing death is often cited length of the delay between death and autopsy as >3.5‰ [42] or ≥4‰ (92mM) [31,33,38], but and the conditions during the delay [68]. lower estimates have also been presented [34,75]. Depending on the source, the given 1.4 Metabolites value may refer to either a BAC determined In intoxications, a large part of the toxic action postmortem or a peak BAC estimated to have may derive from the metabolites of the caused an irreversible event leading to death. consumed substances. When the metabolite is However, death may ensue already from lower pharmacologically active (e.g. amitriptyline/ BACs, especially in alcoholics with a weakened nortriptyline, codeine/morphine/morphine-6- physiological status (heart disease, malnutrition, glucuronide, methanol/formic acid), it may liver cirrhosis, ketoacidosis), as well as when contribute to death to the same or even to a aspiration of stomach content, postural asphyxia, greater extent than the parent drug. Knowledge or hypothermia is involved [75,76]. Old age and of metabolite levels is therefore of great concurrent CNS depression from other causes interpretative value in postmortem toxicology. are further factors that may lower the lethal Quantitative determination of known BAC [76]. Tolerance, however, is also an metabolites along with the parent drug may important aspect of alcohol toxicity. People have further help to determine the time of intake and survived – even driven motor vehicles [77] – at the type of poisoning (acute vs. chronic or concentrations much higher than 4‰, with a therapeutic exposure [69]), and possibly arouse BAC of 15‰ (340mM) probably being the suspicions of metabolic anomalies [70]. highest reported in a living person [78]. Qualitative identification of metabolites is often Reported mean and median BACs found in used to corroborate the finding of a parent drug. fatal alcohol poisonings usually range from 3‰ Furthermore, certain metabolites are present in to 4‰ [34,49,79-81]. Cumulative frequency the body in higher concentrations, thus being distributions have also been published [79-81]. easier to detect than the parent drug. Screening The curves in Figure 1 show the cumulative for metabolites is particularly important when proportion of fatal alcohol poisonings in which a the suspected parent compound is metabolized certain BAC was found. extremely fast (e.g. cocaine or heroin [14]), is not excreted in urine, or is not readily 1.6 Drug Toxicity detected. Unfortunately, few metabolites are Drugs exert their therapeutic effects by various commercially available [71]. mechanisms. Each drug usually has a specific mechanism of action, which may also mediate 1.5 Alcohol Toxicity the toxic effects produced by higher Alcohol (ethyl alcohol, ethanol) is a frequent concentrations, but no single mechanism can be finding in postmortem toxicology, with pointed out as the cause of drug toxicity in approximately 400 fatal poisonings attributed to general. Toxicity is therefore thoroughly it annually in Finland [72]. Alcohol was investigated in the process of drug development, detected in 47.7% of Swedish fatal poisonings in with each new drug having to pass an extensive 1992-2002 [73], and BACs of 0.50‰ or higher series of preclinical and clinical tests before were detected in 50.4% of Finnish fatal drug being approved for sale. Even so, virtually all poisonings in 2000-2001 [72]. Of all of the drugs on the market have some degree of Finnish postmortem cases analyzed for alcohol toxicity. Postmarketing research on drug toxicity in 2000-2004, BACs of 0.20‰ or higher were is therefore conducted as well, with the purpose detected in 45.5% (Vuori et al., unpublished of further improving drug safety. Identification results). The frequent involvement of alcohol in of particularly toxic drugs can lead to poisonings in general is illustrated by a Finnish restrictions or recommendations intended to

REVIEW OF THE LITERATURE 13

Figure 1. Cumulative frequency distribution of blood alcohol concentrations in fatal alcohol poisonings in Finland in 1983-1985. Modified from a report by Vuori et al. [80].

prevent future adverse events. Restricted a certain time period and area to the prescription of barbiturates since the late consumption of the drug over the same period 1960s, for instance, was a successful measure and area. Consumption can be measured either in reducing barbiturate poisonings [82], by number of prescriptions, kilograms, or and similarly, dispensing regulations for defined daily doses (DDDs) dispensed, with propoxyphene were tightened in the 1980s, after DDD being the assumed average maintenance many reports of propoxyphene-related fatalities dose per day for a drug used for its main [83-85]. The conventional method of assessing indication in adults [88]. This approach has been acute lethal toxicity, i.e. determining the amount used to compare both individual drugs and of drug required to kill 50% of a given classes of drugs, often in the UK [86,87,89-97]. population (LD50), is obviously not appropriate It must be noted, however, that the FTIs for people. Various methods have thus been calculated using prescription data are valid for developed for assessing acute drug toxicity in prescription medications only and cannot be humans in overdose situations, usually by taking applied to over-the-counter drugs such as an epidemiological approach. aspirin, paracetamol (acetaminophen), and ibuprofen. This limitation is not an issue when 1.6.1 Fatal Toxicity Index consumption data in DDDs or kilograms is The frequency of poisonings caused by a drug available. depends to a large extent on its availability and Death rate per millions of prescriptions was inherent toxicity [86,87]. Controlling for drug used to demonstrate that nitrazepam is a safer availability should thus enable us to compare the hypnotic than barbiturates [89,91]. By degree of inherent drug toxicity for man. From a estimating BDZ death rates per diazepam forensic toxicologist’s point of view, a practical equivalents, temazepam and flurazepam measure of relative drug toxicity is the fatal appeared more toxic than average hypnotics, and toxicity index (FTI). It is calculated by relating diazepam more toxic than average anxiolytics the number of fatalities attributed to a drug over [98]. The latter finding, however, was attributed

14 REVIEW OF THE LITERATURE

to the concurrent use of alcohol. Tricyclic drugs drugs, as pointed out on several occasions as a group had a FTI (expressed as deaths per [16,109,110], a reported fatal poisoning caused million prescriptions) higher than the average by citalopram alone involved ingestion of a dose for all the drugs studied [86,92]. equal to more than a six-month therapeutic Among antidepressants, tricyclic anti- supply [111]. In this case, the citalopram depressants (TCAs) were associated with a concentration determined in femoral blood was higher FTI and antidepressants introduced after approximately 40 times greater than the highest 1973 with a lower FTI than the average for all concentration considered therapeutic [37]. antidepressants [86,93]. Mianserin, on the other In a British study, median fatal hand, was found early on to have a lower FTI concentrations of certain drugs, namely anti- than the TCAs [86,87,94,99], and the selective depressants, hypnotics, and volatile anesthetics, serotonin reuptake inhibitors (SSRIs) were later were shown to correlate with their aqueous shown to cause significantly less fatal solubility [112]. Inversely, drugs with high FTIs intoxications than the TCAs in proportion to were reported to show more lipophilic character their consumption [17,100-102]. Moreover, than the least toxic drugs. These drugs were most SSRI fatalities appear to involve co- thought to act via a nonspecific mechanism ingestion of other substances [16]. Among the disrupting physiological processes in the lipo- TCAs, amitriptyline, desipramine, doxepin, and protein membranes of the brain [112]. However, dothiepin have been estimated to be the most this approach was not applicable to nonnarcotic toxic in several FTI studies [17,92,93,99]. poisons which exert their lethal effects by very Among the newer antidepressants, a higher FTI specific mechanisms. Nonspecific membrane- has been reported for venlafaxine than for the stabilizing activity, also termed a quinidine-like other serotoninergic agents [96]. effect, was nevertheless offered as a cause of fatal poisoning and a mechanism of additive 1.6.2 Other Measures of Toxicity interactions [113]. Correlations were also been Toxicity indices may also be calculated for reported between antidepressant rank orders by ranking purposes by substituting the number of FTI and LD50 in mice [95,100,114]. mentions on death certificates for the number of deaths attributed to the drug [103]. Drug toxicity 1.6.3 Sources of Bias in Toxicity Indices has also been estimated by comparing attempted Toxicity index measures do not necessarily and completed suicides [104]. Besides fatalities, directly represent the inherent toxicity of a drug overdose-related hospital admissions, clinical but can also be related to the indications and representation, and outcome have been manner of use. Antidepressants, for instance, are compared, as have seriousness of side-effects consumed by people who have suicidal and possibility of drug interactions [105-107]. tendencies and who are thus at an elevated risk Yet another measure is related to the therapeutic of death compared with nondepressed index; de Jonghe and Swinkels classified individuals [115]. It has also been suggested that antidepressants as ‛safe’ or ‛less safe’ according prescribing practices may result in biased to whether the amount of drug prescribed for perceptions of toxicity differences between anti- two weeks’ therapy can prove fatal. They depressants since dual-action antidepressants, considered an antidepressant safe when a two- such as venlafaxine, are prescribed to patients week supply was not life-threatening in already at a relatively high risk of suicide, i.e. overdose [105,106]. TCAs would therefore be patients whose depression has been resistant to considered ‛less safe’ since significant narrow-spectrum serotonergic agents or whose symptoms can result from ingestion of three to initial symptoms suggest use of something other four times the therapeutic daily dose, with a than a SSRI as a first-line drug [116,117]. lethal dose only eight to ten times greater Furthermore, some antidepressants may have [16,108]. several indications besides depression, e.g. Although depressed patients should be obsessive-compulsive behavior, bulimia, and allowed only limited access to antidepressant nocturnal enuresis, conditions which generally

REVIEW OF THE LITERATURE 15 are associated with lower risk of drug abuse or amount of a normal product can be expected to self-harm than depression. Yet another increase the likelihood of adverse drug reactions consideration is that TCAs may actually be (ADRs) or of inadequate therapeutic outcome at prescribed in subtherapeutic doses because in normal drug dosages. When two or more therapeutic doses, side-effects are common and variants of the same gene locus occur at a may lead to noncompliance. For these reasons, frequency of 1% or higher in a population, the TCAs may be ineffective in treating depression, gene is termed polymorphic [129]. Poly- whereas SSRIs, due to their mild side-effect morphisms may affect pharmacodynamics, e.g. profiles, can be taken in therapeutic amounts the structure of receptors, ion channels, and and with good compliance, resulting in carrier proteins [121,130], but most of the improvement of the condition, and thus, a lower currently available information concerns risk of suicide [109,118]. pharmacokinetics, especially enzymes involved On the other hand, fatal drug poisonings in drug metabolism. are not always suicides, but may also occur by accident, and even an intentional overdose can 2.1 Drug-Metabolizing Enzymes lead to an unintentional death. A Finnish study Drug-metabolizing enzymes (DMEs) act as a reported 26% of unintentional deaths among defense mechanism against foreign compounds. fatal drug poisonings, with 76% of fatal These enzymes have evolved in animals during antidepressant poisonings being suicides [102]. the course of interaction with plants [131]. Most Similarly, the proportion of suicides did not exogenous substances enter the body via the exceed 80% in a Danish study on lethal gastrointestinal tract, where they are absorbed antidepressant intoxications [119]. Another into the portal circulation, which transports them behavioral characteristic affecting poisoning to the liver. DMEs are predominantly located in statistics is that some prescription drugs, the liver, enabling efficient first-pass especially those with euphorigenic or anxiolytic metabolism of foreign entities and thus properties, are abused more often than others, constituting an important factor in the while some of the prescribed and acquired bioavailability of ingested drugs. Their body- medications are never ingested. Fast-acting protecting function comprises rendering a drugs with short-term effects have been compound more easily excretable; in Phase 1 estimated to have a higher abuse potential [120]. reactions, DMEs unmask or incorporate a polar, These aspects must be kept in mind when often oxygen-containing function in the compiling and interpreting toxicity index data. compound, thereby creating a site for a Phase 2 reaction, which conjugates the compound with a highly polar agent. Genetic variation in DMEs 2 Pharmacogenetics makes it difficult to predict dosage, efficacy, or Drug response as well as absorption, disposition, safety of a drug. Patients with an abnormal metabolism, and excretion are affected by enzymatic status are prone to be predisposed to individual variation. Pharmacogenetics, the ADRs (Table 1) [132]. study of the heritable component of this An individual’s response is also affected by variation, is a rapidly expanding field of science; several other factors, including age, gender, diet, in the 2000s, hundreds of review articles have concurrent medication, general health, lifestyle, appeared on the subject [for recent reviews, see and even education and socioeconomic status 121-128]. The increasing research interest is [133]. In pursuit of personalized medicine, explained by the powerful new tools available phenotyping panels have been devised for the for DNA analysis and by the observations that most common polymorphic DMEs [134-137]. even a single nucleotide change in a gene may, due to altered dose-response relationships, lead 2.1.1 Cytochrome P450 System to clinically significant differences between The superfamily of cytochrome P450 (CYP) individuals. More specifically, expression of a enzymes is the most important metabolic system functionally altered protein product or an altered in Phase 1 [138]. These enzymes are heme-

16 REVIEW OF THE LITERATURE

Table 1. Possible consequences of abnormal enzymatic status depending on the properties of substrate drugs and expected metabolites involved in the reaction. Enzymatic Substrate Expected product Consequence status Normal + Normal dose of Æ Normal metabolite Æ Expected response enzyme in drug (toxic effects rare) normal quantities

Lack of + Active parent drug Æ Inactive metabolite Æ Excessive response functional enzyme, + Toxic parent drug Æ Detoxified metabolite Æ Toxic effects inhibited enzyme + (Pro)drug Æ Active metabolite Æ Lack of response, undertreatment

Excess of + Active parent drug Æ Inactive metabolite Æ Lack of response, enzyme undertreatment

+ Active parent drug Æ Toxic metabolite Æ Toxic effects

+ (Pro)drug Æ Active metabolite Æ Excessive response

containing proteins that show a characteristic CYP2D6 absorption maximum at 450 nm in reduced The CYP2D6 enzyme was originally called microsomes treated with carbon monoxide. CYP sparteine hydroxylase or debrisoquine enzymes are located in the endoplasmic hydroxylase due to two separate clinical trials reticulum and expressed mainly in the liver, but where some of the subjects experienced ADRs also in extra-hepatic tissues such as the intestine, because they were unable to hydroxylate these brain, and lung [128,139]. There are 57 compounds [141,142]. CYP2D6 has been sequenced human CYP genes and 58 pseudo- estimated to participate in the metabolism of genes, the latter having mutated to such an more than 70 common drugs and 20-25% of all extent that all variants have lost the ability to drugs in clinical use [125,128,138]. Most produce functional enzymes [140]. importantly, CYP2D6 metabolizes many Human CYP forms are divided into psychoactive substances such as several families and subfamilies on the basis of antidepressants (TCAs, SSRIs, mianserin, similarities in amino acid sequence. The mirtazapine, venlafaxine) and various individual isozymes are very versatile and are antipsychotics (haloperidol, perphenazine, often capable of catalyzing several types of risperidone, thioridazine). CYP2D6 substrates oxidative reactions [138]. There is increasing also include opioids (codeine, dextro- evidence that CYPs are involved in chemical methorphan, ethylmorphine, methadone, carcinogenesis and chemical-induced toxicity oxycodone, tramadol), β-blockers (metoprolol, through metabolic activation, i.e. formation of propranolol, timolol), type 1 antiarrhythmics reactive metabolites [139]. Families CYP1, (flecainide, mexiletine, propafenone), and CYP2, and CYP3 participate extensively in drug methylenedioxymethamphetamine (MDMA, i.e. metabolism, with three of the major isozymes ‛ecstasy’) [138]. The major reaction types (CYP2C9, CYP2C19, and CYP2D6) being poly- catalyzed by CYP2D6 appear to be ring morphic to a clinically significant degree [125]. oxidation and O-demethylation. Substrates of The following sections focus on research CYP2D6 tend to be basic in character, with a involving the hepatic enzymes CYP2D6 and protonatable nitrogen atom at a distance of CYP2C19 and the polymorphic genes CYP2D6 5-7 Å from the site of the oxidative reaction and CYP2C19 encoding them. [143].

REVIEW OF THE LITERATURE 17

Figure 2. Chromosome 22, gene CYP2D6, and the sequence positions of the major CYP2D6 polymorphisms in the nine exons, with adjacent pseudogenes CYP2D7P and CYP2D8P shown.

According to current knowledge, CYP2D6 common in East Asian (38-70%) and Black is the most polymorphic CYP gene [144], with African (24%) populations [147]. more than 80 allelic variants documented to date Individuals carrying two functional copies [145,146]. In humans, the 4.2-kb region of CYP2D6, i.e. genotypically extensive containing the CYP2D6 gene (MIM*124030) metabolizers (gEMs), are predicted to have an resides on the long arm of chromosome 22 extensive metabolizer phenotype (EM). Since (22q13.1), with two pseudogenes, CYP2D7P the range of metabolic ratios (MRs) associated and CYP2D8P, in close proximity upstream with one functional gene generally overlaps with (Figure 2). that observed for gEMs [149,150], the carriers In addition to interindividual variation, the of one functional gene are typically also CYP genes show interethnic variation. considered EMs [127,128]. A nonfunctional Approximately 7% of the Caucasian population CYP2D6 allele in combination with a and 1% of Orientals carry a homozygously functionally deficient allele [128,151,152] is defective CYP2D6 genotype (gPM). They currently considered to predict an intermediate produce no active CYP2D6 enzyme, and thus, metabolizer phenotype (IM). Furthermore, regarding CYP2D6 substrates, exhibit a poor inhibitors or high-affinity substrates of metabolizer phenotype (PM). The major CYP2D6, such as quinidine, paroxetine, or nonfunctional (null) alleles *3 (frame shift), *4 fluoxetine, may temporarily convert gEMs to (splicing defect), and *5 (deletion of the entire IMs or PMs, thus constituting a source of gene) are responsible for approximately 90% of clinically significant drug interactions [128]. gPMs in Europeans [123]. Of the alleles Expression of CYP2D6, unlike many other associated with decreased CYP2D6 activity, *9, CYP genes, is noninducible, but during human *10, and *17 do not contribute significantly to evolution its metabolic capacity has been up- drug metabolism in Caucasians [147], whereas modulated by duplication and multiduplication the frequency of newly described allele *41 is of the entire gene. Some individuals may approximately 8% [148]. The functional alleles therefore carry extra copies of CYP2D6. Three *1 and *2 are common in European, African, or more copies of CYP2D6, constituting an and Asian populations, with a combined allele ultra-rapid metabolizer genotype (gUM), is frequency of ~71%, ~68%, and ~52%, considered to lead to ultra-extensive production respectively. Allele *4 is relatively frequent in of CYP2D6 protein, thus predicting an ultra- Europeans (20%), while alleles *10 and *17 are rapid metabolizer (UM) phenotype [153].

18 REVIEW OF THE LITERATURE

However, the sensitivity of genotyping to by CYP2C19 include dealkylation and ring predict the UM phenotype is low; common hydroxylation [143]. estimates of the frequency of duplication in CYP2C19 is a large gene of more than 90 European UMs range from 10% to 30% kb, including nine exons, on chromosome [128,132]. For instance, a duplicated copy of 10q24.1-q24.3 (MIM*124020). Approximately CYP2D6 has been found in 4 of 18 UMs (22%) 2-3% of the Caucasian population [162] and phenotyped with sparteine [150] and in 14 of 64 14-21% of East Asians [161] are CYP2C19 UMs (23%) phenotyped with debrisoquine gPMs. Allele *2 (splicing defect) is the only [154]. Moreover, the phenotypes exhibited by common defective mutation in Caucasians gUMs and gEMs overlap [128,152]. (15%) and Blacks (17%). In addition to a high Probe drugs used in CYP2D6 phenotyping frequency (30%) of allele *2 (splicing defect), include debrisoquine, sparteine, and dextro- allele *3 (premature stop codon) is present in the methorphan [124]. In genotyping, the poly- Chinese at a frequency of 5% [161]. The morphic positions of the CYP2D6 gene are not mutations corresponding to these alleles are detected directly in one step because of high 681G>A in exon 5 [163] and 636G>A in exon 4 sequence homology with neighboring pseudo- [164], respectively. They are readily detected by genes [155]. Therefore, a CYP2D6-specific first amplifying a fragment covering exons 4 and fragment is first amplified by polymerase chain 5 and then applying one of the various reaction (PCR) in parallel with two other techniques mentioned above (section CYP2D6). possible fragments identifying a deleted Of the CYP2C19 genotypes commonly observed CYP2D6 gene (*5) and a (multi)duplicated one in Caucasians, *1/*1 is considered to predict an (*1xN, *2xN, *4xN, *10xN, *35xN). The poly- EM, *1/*2 an IM, and *2/*2 a PM of CYP2C19 morphic positions are then identified using such substrates [126,165]. techniques as restriction fragment length poly- morphism (RFLP) analysis [155], multiplex 2.2 Studies on Drug Metabolism single-base extension reaction (e.g. SNaPshotTM) Human liver microsomes have been used [156], real-time fluorometric melting point extensively in studying metabolic poly- analysis [157], pyrosequencing [158], and oligo- morphisms, but the ‛well-characterized’ human nucleotide microarray technology (‛gene chips’) liver microsomes used in in vitro studies may [151,159]. contain enzyme variants that metabolize well the probe drug but not the drug being investigated CYP2C19 [166]. Therefore, in vitro studies are not The CYP2C19 polymorphism was originally reviewed in detail in the following sections, discovered as a deficiency in (S)-mephenytoin focusing instead on the role of CYP enzymes in 4'-hydroxylation [160]. In addition to the metabolism of the opioid drug tramadol and mephenytoin, omeprazole has been used as a TCAs, especially amitriptyline. probe drug in CYP2C19 phenotyping [161]. Other CYP2C19 substrates include anti- 2.2.1 Tramadol Metabolism depressants (TCAs, SSRIs, mianserin, Tramadol is administered as a racemic mixture moclobemide, venlafaxine), antipsychotics of (+)- and (–)-trans-tramadol, i.e. (R,R)- and (clozapine, perphenazine), BDZs (diazepam, (S,S)-tramadol, respectively. CYP2D6 has been flunitrazepam, temazepam), β-blockers shown to convert tramadol to O-demethyl- (metoprolol, propranolol), several proton pump tramadol (M1) in vitro [167,168] and in vivo inhibitors, dextromethorphan, phenytoin, and [169]. The formation of (+)-M1 is important for (S)-warfarin [138]. Substrates of CYP2C19 are the hypoalgesic effect because it has a higher often weakly basic in character and have two affinity for opioid receptors than the parent drug hydrogen bond donor/acceptor atoms. There are [170]. Demethylation of tramadol in vitro is typically seven or eight chain atoms between the stereoselective, with (+)-tramadol being site of metabolism and the site forming a preferentially O-demethylated by CYP2D6 and hydrogen bond. The major reactions catalyzed (–)-tramadol N-demethylated by CYP3A4 [167].

REVIEW OF THE LITERATURE 19 HO HO HO

CYP3A4 CYP3A4?

NMe2 NHMe NH2 OMe tramadol OMe M2 OMe M3

CYP2D6 CYP2D6? CYP2D6?

HO HO HO

CYP3A4? CYP3A4?

NMe2 NHMe NH2 OH M1 OH M5 OH M4

Figure 3. Outline of main pathways of tramadol metabolism in Phase 1, starting from (R,R)- tramadol, i.e. (+)-trans-tramadol, the isomer with the highest affinity for the µ-opioid receptor. Cyclohexyl oxidation is not shown.

Apparently, the N-demethylation product Nortriptyline is the N-demethylated N-demethyltramadol (M2, nortramadol) is metabolite of amitriptyline, but also a drug on its further N-demethylated to N,N-didemethyl- own. Studies on nortriptyline metabolism are of tramadol (M3) by CYP3A4 and O-demethylated great relevance here because nortriptyline to O,N-didemethyltramadol (M5) by CYP2D6, formation is quantitatively the most important possibly followed by formation of O,N,N-tri- pathway in amitriptyline metabolism [173], with demethyltramadol (M4) from M3 via CYP2D6 (E)-10-hydroxyamitriptyline (EHAT) formed to as well as from M5 via CYP3A4 (Figure 3) a lesser extent. [171]. At low tramadol concentrations, in vitro The major metabolite of nortriptyline is M1 formation predominates, while M2 is the (E)-10-hydroxynortriptyline (EHNT) [176], or major metabolite at higher concentrations [168]. more precisely, the (–)-enantiomer of EHNT [181]. The enzymes mediating (Z)-10-hydroxy- 2.2.2 Amitriptyline Metabolism metabolite formation are not known, but Metabolism of TCAs is well known [for (Z)-10-hydroxyamitriptyline (ZHAT) and reviews, see 123,172], for it was the subject of (Z)-10-hydroxynortriptyline (ZHNT) have been intensive research even before the discovery of detected in vitro [182] and in vivo [181]. CYP genes. In early studies, aliphatic ring Nortriptyline is further demethylated to hydroxylation of both amitriptyline [173,174] N-demethylnortriptyline (NNT) (Figure 4). and nortriptyline [175-177] in vivo correlated In vivo, the stereoselective formation of with polymorphic 4-hydroxylation of (–)-EHAT and (–)-EHNT in particular has been debrisoquine, whereas N-demethylation of shown to depend on the activity of CYP2D6 amitriptyline did not [178]. The 2-hydroxylation [181]. In amitriptyline demethylation to reactions of imipramine [179] and desipramine nortriptyline, several enzymes have been [180] have also been suggested to be under the implicated, namely CYP2C19, CYP3A4, same genetic control as sparteine hydroxylation. CYP1A2, and CYP2D6 [183,184]. The results In parallel with amitriptyline metabolism, suggest a dominant role of CYP2C19 at N-demethylation of imipramine to desipramine therapeutic concentrations and involvement of does not cosegregate with sparteine CYP3A4 at higher concentrations. polymorphism [179].

20 REVIEW OF THE LITERATURE

NHMe NMe2 NH2

CYP2C19 CYP2C19

HHH H amitriptyline HHHH nortriptyline HHH H NNT

CYP2D6 CYP2D6

NMe2 NHMe CYP2C19

EHAT EHNT HO HO

Figure 4. Outline of amitriptyline metabolism in Phase 1, with formation of (E)-hydroxy- metabolites shown. The major pathway is indicated with bold arrows. N-oxide formation is not shown.

2.3 Clinical Pharmacogenetics prescribed may eventually behave differently The discipline of clinical pharmacogenetics is [166]. aimed at individualized drug therapy, primarily Genotyping offers several advantages over by identifying patients at risk prior to initiating phenotyping: the patient is not exposed to probe treatment (improved risk prediction). When the drugs; drawing one blood sample takes little drug to be prescribed is exceptionally toxic time; genotyping can also be carried out post- or expensive, diagnostic phenotyping or mortem, when clinical phenotyping is no longer genotyping, in addition to therapeutic drug an option; and genotyping is very specific, with monitoring, may help to improve safety and no interference from comedication. On the other efficacy, i.e. to avoid ADRs and therapeutic hand, the latter is also a disadvantage since failure. This will maximize medical and interactions arising from comedications are not financial benefits and minimize the burden of taken into account. medication both on the individual and on public In evaluating the concordance between health. Furthermore, in nonresponsive patients, tramadol metabolism, dextromethorphan pheno- phenotyping or genotyping may allow type, and CYP2D6 genotype, only a modest differentiation between ultra-rapid metabolism correlation was found between the tramadol/M1 and noncompliance [185,186]. plasma ratio and the urinary dextromethorphan/ Phenotyping typically involves dextrorphan ratio in general, but when the measurements in plasma or urine. After giving subjects were segregated according to the the patient a single oral dose of a probe drug, the number of functional CYP2D6 genes, a much concentrations of the unchanged drug and a stronger relationship was observed in gEMs relevant metabolite are analyzed, and the [187]. The impact of the CYP2D6 genotype on obtained MR is compared with a reference value 10-hydroxylation of nortriptyline [188-191] and or distribution determined in a large population. amitriptyline [192], and the effect of the The metabolism of a probe drug cannot, CYP2C19 genotype on N-demethylation of however, accurately represent the metabolism of amitriptyline [192,193] have also been another drug because several enzymes are examined in volunteers and psychiatric patients. typically involved in the metabolism of a The nortriptyline/10-hydroxynortriptyline ratio drug, and the probe drug and the drug to be was shown to be influenced by CYP2D6 geno-

REVIEW OF THE LITERATURE 21 type and gender in one study [188], whereas in found. The patients had been comedicated with another, age and gender as factors did not reach thioridazine and chlorpromazine, both of which statistical significance, with only the number of inhibit the CYP2D6 enzyme responsible for mutated CYP2D6 alleles being significant [191]. desipramine metabolism [138]. However, Theoretically, PMs are at an elevated risk CYP2D6 is also involved in the metabolism of of developing excessive plasma concentrations thioridazine and chlorpromazine, and high of certain drugs, for instance, of nortriptyline or concentrations of imipramine and desipramine desipramine, when these are given as such or are can competitively inhibit CYP2D6. The formed from amitriptyline or imipramine, possibility of a drug interaction was therefore respectively. Such instances have in fact been also considered [200]. documented in several case reports [185,194- In 1999, CYP2D6 genotyping was 196]. Furthermore, a German study recently demonstrated to be feasible using autopsy blood, found that gPMs were overrepresented among with 22 suspected overdose fatalities and 24 28 patients reported to suffer from ADRs after controls successfully genotyped for CYP2D6 taking CYP2D6-dependent antidepressant drugs, alleles *1, *3, and *4. No gPMs were found with an observed frequency of 29% compared among the overdose cases, but CYP2D6 with a frequency of 7% in a random German inhibitors were present in eight cases. However, population. The same study also genotyped 16 the cases were not preselected according to nonresponders and found that duplication of known CYP2D6-catalyzed reactions, and the CYP2D6 was overrepresented, with an allele relevance of the investigated metabolic reactions frequency of 12.5% vs. 1.8% in a general to CYP2D6 was not discussed. Furthermore, no German population [132]. MRs allowing comparison between genotypes ADRs are, however, relatively rare, were calculated [201]. probably because most drugs can be A case report of a toddler, deceased at the metabolized by several enzymes, so that when age of two years and genotyped for CYP2D6 one is not fully active, complementary pathways postmortem, was published in 2000. The cause may compensate, thus preventing harmful of death was determined as dextromethorphan accumulation [187,192]. For instance, no poisoning following a therapeutic ingestion of evidence of an increased ADR rate was found in cough medicine. Although the dextro- gPMs treated with fluoxetine or nortriptyline methorphan/dextrorphan ratio of 2.5 suggested [197]. Nevertheless, on the basis of advanced slow O-demethylation, a reaction catalyzed by study settings and calculations, preliminary CYP2D6, the CYP2D6 genotype was that of an genotype-based dose recommendations for EM. No concurrent analytes were found in certain antidepressants have been published general drug screening [202]. [126,165,198]. Furthermore, since CYP2D6- In another case reported in 2000, the death inhibiting comedication may convert EMs to of a child was investigated in depth when high PMs, e.g. when paroxetine is combined with blood concentrations of both fluoxetine and desipramine [199], it may also constitute a norfluoxetine were found in postmortem favorable interaction by converting an toxicology. The parents were first accused of unresponsive UM to a responsive patient. homicide, but were vindicated by the results of genotyping, with DNA analysis revealing a 2.4 Postmortem Pharmacogenetics homozygously defective CYP2D6 genotype. The Even before the advent of postmortem geno- interpretation of the results was therefore typing, the possibility of a defective CYP2D6 chronic accumulation of fluoxetine and genotype leading to death was discussed in a norfluoxetine. In fact, the nine-year-old boy had case report presenting two fatal poisonings been prescribed fluoxetine at 100 mg/day, a involving imipramine and desipramine. Chronic dose five times the DDD, and his medical accumulation of imipramine and desipramine, history indicated several hospitalizations due to particularly of desipramine, was suspected since seizure episodes. The parents eventually filed a very low imipramine/desipramine ratios were malpractice suit against the neurologist who had

22 REVIEW OF THE LITERATURE

prescribed fluoxetine at an exceptionally high [72]. An overview of the current knowledge of dosage and yet failed to recognize the symptoms the mechanisms of drug-alcohol interactions will of toxicity in the patient [79,203]. be provided below and research on postmortem In a Swedish series of 242 fatal drug material summarized. Although most studies on intoxications, both CYP2D6 and CYP2C19 drug-alcohol interactions relate to animals or genotypes were determined and the genotype living persons, clinical studies pertaining to distributions compared with those in 281 psychomotor skills are beyond the scope of this controls (blood donors). The CYP2C19 geno- review. typing results in the autopsy cases were similar to those in the blood donors, but the prevalence 3.1 Alcohol Effects and Anesthetic Action of CYP2D6 gPMs in fatal intoxications was A pharmacologically active drug typically has found to be lower (4.7%) than expected from the one or more known mechanisms and sites of frequencies of these genotypes in the blood action, e.g. binding directly to a specific donors (8.5%), leading the authors to suggest proteinaceous receptor or enzyme. With regard that intoxication victims might exhibit a lower to alcohol, while its effects are well known, the frequency of CYP2D6 gPMs than the general mechanism is less clear. Knowledge of this population [204]. No explanation has been mechanism is, however, crucial in elucidating offered for this observation. the interaction between drugs and alcohol. Many In 15 fatalities involving oxycodone [205] aspects of ethanol, recently reviewed by Jones and in 21 involving methadone [206], CYP2D6 [208], differ from medicinal drugs. Ethanol is genotyping has been used to aid interpretation of often ingested in large quantities, has nutritional postmortem toxicology results, although without value, and is evenly distributed throughout the calculating the relevant MRs. body. The molecular structure of ethanol is In 53 Swedish autopsy cases involving small and simple, with several potential citalopram, genotyping of CYP2D6 and physiological targets. In alcohol-related CYP2C19 was combined with enantioselective fatalities, anesthesia and CNS depression analysis of citalopram enantiomers by chiral leading to respiratory failure are considered the liquid chromatography. No gPMs were found mechanisms of major importance [208]. for CYP2C19 and only 2 gPMs (3.8%) for General anesthesia can be produced by a CYP2D6. The authors suggested that wide variety of chemical entities, including pharmacokinetic interactions are likely to play a alcohols, alkanes, ketones, ethers, and inert more important role than pharmacogenetic gases, but the mechanism of action remains deficiencies in drug metabolism [207]. largely unresolved. An early effort to explain it, In summary, postmortem pharmaco- the Meyer-Overton hypothesis, based on the genetics is a relatively new area of research; the independent but similar findings of Meyer in extent to which it will contribute to medicolegal 1899 and Overton in 1901, states that there is a investigations remains to be seen. correlation between anesthetic potency and oil solubility (i.e. hydrophobicity) of a compound [209]. Anesthesia was then proposed to occur 3 Drug-Alcohol Interaction when a critical drug concentration is achieved in Drug-alcohol interactions have been widely the cell membrane, but the intramembrane investigated in animals and in clinical settings, volume was later found to be a better parameter especially with regard to psychomotor than the intramembrane concentration for equal performance, but studies on human postmortem degrees of narcosis produced by different agents material are scarce. Alcohol is, however, a [210]. In the 1970s, the anesthetic site of action frequent finding in fatal poisonings. For was concluded to be located within the neuronal instance, alcohol was detected in 47.7% of membranes [211], and the physiological site of Swedish fatal poisonings in 1992-2002 [73], and action of general anesthetics was thought to BACs of 0.50‰ or more were detected in 50.4% involve proteins rather than the lipid region of of Finnish fatal drug poisonings in 2000-2001 the membrane [212]. This proposition was based

REVIEW OF THE LITERATURE 23 on the observed correlation of anesthetic GABAA receptor, prolonging the channel open potency and the n-octanol/water partition time and thereby enhancing the flow of chloride coefficient [212]. ions into the cell [217,220,222]. As a result, the The potency of structurally diverse neuronal excitability is reduced more than by anesthetic agents was thereafter shown to GABA alone, which may lead to incoordination, correlate with their ability to partition into the sedation, and even anesthesia, typical of mild to phospholipid bilayer (instead of oil) [213]. moderate or severe alcohol intoxication [220]. In Furthermore, the membrane-disordering potency moderate drinkers, BACs above 2.0‰ cause of various aliphatic alcohols was found to be hypnotic and anesthetic effects [222]. Lower closely related to their oil/water partition concentrations of alcohol cause sedative and coefficients and thus to their membrane motor-incoordinating effects, which seem to solubilities [214]. Aqueous solubility was also involve the GABAA system. Anesthetic offered as a theoretical basis for the synergism concentrations also seem to potentiate GABAA observed in barbiturate-ethanol poisonings receptor responses, but possibly with a different [79,215]. Nevertheless, the protein theory mechanism [223]. eventually gained ground when the activity of a The GABAA receptor (Figure 5) is a large pure soluble protein (firefly luciferase) was protein consisting of five subunits with different shown to be inhibited by general anesthetics at specificities and sensitivities to modulatory concentrations which induce anesthesia [216]. agents such as BDZs, barbiturates, alcohols, Disordering or fluidization of the lipid neurosteroids, and volatile anesthetics membrane is therefore no longer considered to [220,221]. This is currently believed to explain the commonly observed effects and constitute the physiological basis of interaction toxicity of alcohol [217,218]. However, the between alcohol and BDZs or barbiturates, membrane-related effects of alcohols and although alcohol is known to affect the function anesthetics are still being researched to explain of several brain receptors and enzymes, e.g. the Meyer-Overton correlation [209,219]. acutely inhibiting N-methyl-D-aspartate In current research into the mechanism of (NMDA) receptors of glutamate [221]. general anesthesia, and therefore also into the Development of tolerance is typical of GABAA mechanism of alcohol effects, proteins – substrates, and evaluating concentration- membrane-embedded receptors, in particular – response relationships, including acute toxicity, are favored over the lipid bilayer as probable and interactions with other CNS-affecting agents anesthetic targets in the CNS. Members of the is therefore difficult. Fatal poisonings by BDZs ligand-gated superfamily of ion channels, for are, however, known to predominantly occur in instance, seem sensitive to general anesthetics combination with other CNS depressants, [217,218]. This superfamily includes the usually alcohol [22,98,225]. receptors for the inhibitory neurotransmitters glycine and γ-aminobutyric acid (GABA). 3.2 Animal Studies Within these receptors, putative sites of alcohol The interaction of ethanol with other CNS- and volatile anesthetic action have been depressing substances has been investigated in discovered, as reviewed by Mihic and Harris animals in terms of psychomotor performance, [220] and Harris et al. [221]. sleep time, and lethality, with the latter being the most relevant to this study. The effects of 3.1.1 γ-Aminobutyric Acid Receptor Type A ethanol on drug lethality have been examined in GABA is the major inhibitory messenger in the mice and rats. In one study, oral ethanol up to vertebrate CNS, and the GABA type A receptor 4.0 g/kg had no effect on the lethality caused by

(GABAA) has been established as a prime chlordiazepoxide in mice, whereas 2 g/kg anesthetic target [217]. General anesthetics, reduced the LD50 of pentobarbital by 13% including alcohol, potentiate the GABA- and 4 g/kg by 41% [226]. In rats, ethanol in mediated inhibition of neuronal transmission by sedative doses was not – contrary to the study binding to a specific modulatory site in the hypothesis – observed to increase lethality after

24 REVIEW OF THE LITERATURE

Figure 5. Schematic illustration showing the pentameric structure of a GABAA receptor with suggested binding sites for γ-amino butyric acid (GABA), ethanol, barbiturates, benzodiazepines, and neurosteroids. Modified from reviews by Mihic and Harris [220] and McKernan and Whiting [224].

propoxyphene overdoses [227]. However, as was found in postmortem blood either alone or doses are often reported instead of BACs and as together with alcohol, and the methods involved the pharmacokinetics of alcohol in animals may comparing the average (mean or median) differ from that in people, the results of animal concentrations and the cumulative frequencies studies are not directly generalizable to humans between different types of cases [79,228-230]. and will not be discussed in more detail here. The major findings were that when alcohol was present the concentrations of propoxyphene 3.3 Postmortem Studies [228-230], amitriptyline [229], and barbiturates Alcohol is often found in drug-related fatalities [79,229] were lower. The greatest differences [72], especially in accidental antidepressant were found in amitriptyline poisonings with and poisonings [119]. In Frey et al. [17], for without alcohol, leading to the interpretation instance, fatal citalopram intoxications occurred that amitriptyline potentiates the toxic effects of only in combination with alcohol. Although the alcohol to a relatively large extent [229]. Major role of drug-alcohol interaction in fatal shortcomings in these studies were small sample poisonings has often been discussed, empiric size, unknown site and method of blood sample evaluation using quantitative measures on collection, and lack of statistical analysis, but postmortem material has been carried out on these studies, nevertheless, provide useful only a few occasions. The material consisted of examples for modern research of drug-alcohol toxicologically examined cases in which a drug interactions in postmortem material.

REVIEW OF THE LITERATURE 25 AIMS OF THE STUDY

Previous observations suggest that fatal poisonings involving both drugs and alcohol may exhibit analyte concentrations lower than those found in single-substance poisonings. Moreover, clinical studies have shown the genetic regulation of some DMEs, producing variable drug response.

In this thesis, a retrospective, statistical approach was undertaken to investigate drug-alcohol interactions and pharmacogenetics in postmortem material in a medicolegal setting. In addition, the safety of newer antidepressants, especially in combination with alcohol, was evaluated based on postmortem material.

Specifically, the studies sought to answer the following questions:

Are there correlations between the CYP2D6 gene dose and tramadol metabolite ratios (MRs) in a postmortem sample population? Can accidental or undetermined tramadol poisonings be attributed to a genetic inability to produce functional CYP2D6 enzyme? (I)

Do CYP2D6 and/or CYP2C19 gene doses correlate with amitriptyline MRs in a large postmortem sample population? Can accidental or undetermined amitriptyline poisonings be attributed to a genetic inability to produce functional CYP2D6 or CYP2C19 enzyme? (II)

Do BACs in fatal poisonings involving certain BDZs differ from those found in cases involving alcohol alone? Does the amount of the difference depend on the identity of the BDZ? (III)

Do BACs in fatal poisonings involving certain common drugs – i.e. those most often found in fatal poisonings – differ from those found in cases involving alcohol alone? What are the fatal toxicity indices of the drugs in question? Do these two measures correlate? (IV)

Do BACs in fatal poisonings involving newer antidepressants differ from those found in cases involving alcohol alone? What are the fatal toxicity indices for the newer antidepressants? Do these measures correlate? (V)

Do blood concentrations of common toxic drugs in fatal poisonings involving alcohol differ from those found in cases involving the drug alone? (VI)

26 AIMS OF THE STUDY

MATERIALS AND METHODS

1 Autopsy cases Table 2. Therapeutic ranges of selected drugs in All of the cases were autopsied in Finland whole blood. during 1995-2003. The cases included in Study I Drug Therapeutic Reference were autopsied at the Department of Forensic range (mg/l) Medicine, University of Helsinki. All autopsy Amitriptyline 0.04-0.2 [34] samples taken for forensic toxicology were Citalopram 0.01-0.4 [37] analyzed at the Laboratory of Toxicology, Diazepam 0.1-2.5 [33] Department of Forensic Medicine, University of Diltiazem 0.05-0.3 [36] Helsinki. The results of chemical analyses and Doxepin 0.03-0.15 [33] the eventual death certificate information were Levomepromazine 0.05-0.14 [34] coded into the laboratory database. Promazine 0.1-0.4 [36] Propoxyphene 0.1-0.75 [36] 1.1 Blood Samples Temazepam 0.4-0.9 [34] All concentration data used in these studies were Tramadol 0.1-0.6 [42] acquired from femoral venous blood taken at Zopiclone 0.01-0.1 [36] autopsy into plastic tubes containing a small amount of sodium fluoride to prevent microbial degradation. The samples were stored at 4°C (except during transport) until analysis, after which they were preserved at –20°C. 2 Analysis of Drug Concentrations The Laboratory of Toxicology received official 1.2 Database accreditation from the Finnish Accreditation For storing pertinent information on the forensic Service (FINAS) in 1997. A vast array of toxicology cases, the Laboratory of Toxicology validated, stable methods, covering a broad utilized dBase (Microsoft Corp., Redmond, WA, range of analytes and producing a wealth of USA) until the end of the year 1999. At the reliable and commensurate data, is used. beginning of 2000, Access 2000 (Microsoft Screening and dedicated methods are performed Corp., Redmond, WA, USA) was inaugurated as according to pathologists' request or when the new Laboratory Information Management otherwise deemed. System (LIMS). Descriptive information stored The drug concentrations considered in this in the database includes name, age, gender, site study to indicate therapeutic use when found in of residence, and known occupation. Analytical postmortem blood are listed in Table 2. entries identify the analyte, the matrix, and the qualitative (positive/negative) or quantitative 2.1 Screening (concentration) result, among others. The codes Each postmortem case was submitted to a broad denoting the cause of death and the manner of drug screen. The major screening methods used death are later entered into the database from a to detect toxicologically significant analytes copy of the completed death certificate provided included gas chromatography (GC), thin-layer by the pathologist. In fatal poisonings, the most chromatography (TLC), overpressured layer important toxicological finding is indicated on chromatography (OPLC), and immunological the death certificate by a code stating the assays, such as EMIT. Screening was always underlying cause of death. The 10th revision of carried out in blood (GC, GC-MS) and urine or the International Classification of Diseases liver (TLC, OPLC, GC). Separate GC screens (WHO, Geneva, Switzerland), ICD-10, has been were used for acidic and basic drugs, which used for this purpose since 1996. The manner of were also quantitated directly in the screening death in poisoning cases is generally accidental, analysis. Alcohol screening by head-space GC suicide, or undetermined. was performed in practically all cases in blood

MATERIALS AND METHODS 27 and urine. The BACs were reported in mass per 3 Genotyping mass units as parts per thousand (‰). The limit DNA was isolated from an autopsy bloodstain of quantitation for ethanol was 0.20‰. (I) dried on dedicated paper (FTA® GeneCard, Tramadol and amitriptyline were Invitrogen Life Technologies, Carlsbad, CA, determined in 1 ml of blood submitted to routine USA) or from blood (II). The stains on FTA® drug screening. After extraction into ethyl paper were processed according to acetate at pH 9, the samples were injected into a manufacturer’s recommendations, whereas gaschromatograph equipped with a nitrogen- liquid blood samples were subjected to the phosphorus-selective detector [24]. For both following standard procedure: 0.5-3 ml of blood tramadol and amitriptyline, the method was was mixed with 10-12.5 ml of lysis buffer linear up to 10 mg/l, with a limit of quantitation (10mM Tris-HCl, pH 7.5, 5mM MgCl2, of 0.1 mg/l. 0.32M sucrose, 1% Triton X-100) in a screw- cap plastic tube. After centrifugation, the 2.2 Metabolite Analysis supernatant was decanted and 4 ml of lysis The major metabolites of tramadol (I) and buffer was added to the precipitate. After amitriptyline (II) were analyzed by liquid mixing, centrifugation, and decantation, the chromatography coupled with tandem mass leukocyte pellets were digested in 2 ml of spectrometry (LC-MS/MS). digestion buffer (10mM Tris-HCl, pH 8.0, The tramadol metabolites O-demethyl- 10mM EDTA, 100mM NaCl, 2% sodium tramadol (M1) and N-demethyltramadol (M2) dodecyl sulfate) and 20 µl of proteinase K were obtained from Grünenthal GmbH (Aachen, (20 mg/ml) at 56°C overnight. The incubated Germany) and determined in blood samples to sample was then transferred to a Phase Lock Gel a limit of quantitation of 0.01 mg/l. MR1 and Light™ tube (Eppendorf AG, Hamburg, MR2 were calculated as concentration of Germany). The next step consisted of adding 1 tramadol per concentration of metabolite. The ml of phenol and 1 ml of chloroform/3-methyl- analytes were extracted into a mixture of 1-butanol (96:4), mixing, and centrifugation. dichloromethane and 2-propanol, followed by This step was repeated first with a similar liquid chromatographic separation on a addition and then with 2 ml of chloroform/ C18 column and detection by tandem MS using 3-methyl-1-butanol. In the end, the supernatant multiple reaction monitoring (MRM). Linear was decanted into another screw-cap plastic tube calibration was used from 0.0025 mg/l to containing 10 ml of cold (-20°C) ethanol and 0.3 mg/l and quadratic calibration above 400 µl of 3M sodium acetate (pH 7.0). After 0.3 mg/l. mixing and centrifugation at 4°C, ethanol was The amitriptyline metabolites nortriptyline, decanted and the precipitate was washed with NNT, EHAT, ZHAT, EHNT, and ZHNT were 5 ml of 70% cold ethanol. After mixing and obtained from H. Lundbeck A/S (Copenhagen, centrifugation (4°C), ethanol was decanted and Denmark). Imipramine was used as an internal the precipitate was dried at room temperature standard. The analytes were extracted into a overnight. In the morning, the precipitate was mixture of ethyl acetate and 2-propanol (97:3). dissolved in 100 µl of 1xTE buffer A sufficient separation was achieved with (10mM Tris-HCl, pH 7.5, 10mM EDTA). The gradient elution on a C18 column, and the DNA stocks were stored frozen (-20°C) in analytes were detected by MS using MRM. For buffer solution. amitriptyline metabolites, the limit of The regions of interest were amplified quantitation was 0.001 mg/l; quadratic using PCR and the relevant polymorphic calibration was used from 0.001 mg/l to 5 mg/l, positions were detected with an RFLP (I) or and concentrations above 5 mg/l were SNaPshotTM (II) method (see Figure 2 on p. 18). quantitated in 1:10 dilutions. For primer specifications, see the Materials and Methods section in Studies I and II. CYP2D6 allele nomenclature and nucleotide numbering are according to the CYP Allele Nomenclature

28 MATERIALS AND METHODS

Committee [146]. The genotyping methods used Table 3. CYP2D6 positions genotyped in Studies I take into account all of the common CYP2D6 and II (highlighted) and their known occurrence in various alleles [146]. See also Figure 2 on p. 18. and CYP2C19 mutations in Caucasians Mutation Allele [147,161]. 100C>T *4, *10, *14

124G>A *12 3.1 Long Polymerase Chain Reaction 138insT *15 For CYP2D6 genotyping (I, II), three parallel 843T>G *2, *4 long PCRs were performed including 1) a 883G>C *11 fragment covering the whole CYP2D6 gene 974C>A *4 (4.7 kb in I, 5.1 kb in II), 2) a duplication- 984A>G *4 specific fragment (3.6 kb in I, 3.2 kb in II) and 997C>G *4 an internal control fragment (5.2 kb in I, 3,8 kb 1023C>T *17 in II), and 3) a deletion-specific fragment 1661G>C *2, *4, *8, *10-*12, *14, *39 (3.5 kb) identifying allele *5 and an internal 1707delT *6 control fragment (3.0 kb). For CYP2C19 1758G>T/A *8, *14 genotyping (II), a 1.9-kb fragment covering 1846G>A *4 exons 4 and 5 was amplified. 2549delA *3

2613–5delAGA *9 3.2 Restriction Fragment Length 2850C>T *2, *8, *11, *12, *14, *17 Polymorphism Analysis 2935A>C *7 In Study I, 18 selected positions with known 4180G>C *2, *4, *8, *10-*12, *14, *17, *39 mutations (100C>T, 124G>A, 138insT,

843T>G, 883G>C, 974C>A, 984A>G, 997C>G, 1023C>T, 1661G>C, 1707delT, 1758G>T/A, 1846G>A, 2549delA, 2613– 15delAGA, 2850C>T, 2935A>C, 4180G>C; An application of this method was used to detect Table 3) were detected by reamplifying eight the positions 636G>A and 681G>A in separate fragments of 186–471 bp from the CYP2C19, allowing identification of CYP2C19 4.7-kb amplificate and digesting them with a set alleles *3 and *2, respectively. Alleles not of 15 restriction enzymes (HphI, MspI, BspMI, carrying these mutations were classified as *1. PstI, BsmAI, BstNI, BsaAI, MboII, HinP1I, FokI, BanII, BstEII, HaeII, SacII, EagI). The RFLP panel allowed identification of alleles *2- 4 Case Selection Criteria *4, *6-*12, *14, *15, *17, and *39. For details, The cases investigated in Study I were autopsy see Study I, Table 1. Alleles not carrying any of cases from June 1998 to June 2000 in which the above mutations were classified as *1. tramadol was found, a bloodstain on FTA® paper was available, and a sufficient amount of 3.3 Multiplex Single-Base Extension Reaction blood remained for metabolite analysis. In Study II, nine selected polymorphic positions The cases investigated in Study II included (100C>T, 1023C>T, 1661G>C, 1707delT, a consecutive series of autopsy cases from 1999 1846G>A, 2549delA, 2613-5delAGA, to 2001 in which amitriptyline was found at 2850C>T, 4180G>C; Table 3) in CYP2D6 were concentrations ≥0.2 mg/l in blood and a detected using a multiplex single-base extension sufficient amount of blood was available. reaction with nine detection primers and an ABI The cases investigated in Study III PRISM SNaPshot™ Multiplex Kit (Applied included fatal poisonings from 1995 to 2000 Biosystems, Foster City, CA, USA). The involving alcohol alone or in combination with SNaPshot method allowed the identification of the BDZ anxiolytic diazepam or the BDZ CYP2D6 alleles *3, *4, *6, *9, *10, and *17 hypnotic temazepam. [156]. Alleles with 1661G>C, 2850C>T, and The cases investigated in Study IV 4180G>C were classified as *2. included fatal poisonings from 1995 to 2000

RESULTS 29 involving alcohol alone or in combination with a ethanol-positive cases (III-VI), diazepam drug commonly causing fatal poisonings in metabolites nordiazepam and chlordiazepoxide Finland, i.e. the phenothiazine antipsychotic (III), and therapeutic concentrations of BDZs promazine or levomepromazine (methotri- (IV-VI). meprazine), the TCA doxepin or amitriptyline, the opioid analgesic propoxyphene (dextro- propoxyphene), the BDZ hypnotic temazepam, 5 Statistical Methods the non-BDZ hypnotic zopiclone, the SSRI Statistical analysis was performed using citalopram, or the calcium channel inhibitor MINITAB 13 (Minitab Inc., State College, PA, diltiazem. USA) and SPSS 10 (SPSS Inc., Chicago, IL, The cases investigated in Study V included USA) software for calculation of confidence fatal poisonings from 1995 to 2002 involving intervals (CIs) for means and medians and for newer antidepressants available in Finland performing univariate analysis of variance, test during this period, i.e. mianserin, mirtazapine, of two proportions, Student’s t-test, and Mann- venlafaxine, milnacipran, reboxetine, Whitney test. A p-value of <0.05 was nefazodone, trazodone, the SSRIs citalopram, considered to indicate a statistically significant fluoxetine, fluvoxamine, paroxetine, and difference. The 95% CIs for the number of sertraline, and the monoamineoxidase type A observed deaths were taken from the Poisson inhibitor moclobemide. distribution. The cases investigated in Study VI When average drug concentrations were included fatal poisonings from 1995 to 2002 compared between groups, medians were used involving amitriptyline, propoxyphene, or instead of means. Drug concentrations are not promazine either alone or in combination with normally distributed because pharmacological alcohol. response generally exhibits a logarithmic In addition to the selection findings, relation to a substance concentration. Medians allowed coincidental findings were caffeine and are therefore more appropriate for describing nicotine (III-VI), acetone and 2-propanol in these distributions.

30 MATERIALS AND METHODS

RESULTS

1 Pharmacogenetics tramadol O-demethylation, and a negative Genotyping of postmortem samples was correlation between the CYP2D6 gene dose and generally successful despite the often poor the rate of tramadol N-demethylation (Figure 6). quality of cadaveric blood specimens and the More specifically, the median metabolite ratio large fragments required for CYP2D6 analysis. MR1 was significantly higher and the median MR2 significantly lower in the cases with no 1.1 CYP2D6 and Tramadol (I) functional genes than in those with two or more. All of the 33 tramadol cases were successfully No fatal poisonings coincided with a genotyped. A positive correlation was found homozygous nonfunctional genotype. between the CYP2D6 gene dose and the rate of

Number of functional CYP2D6 genes Number of functional CYP2D6 genes

Figure 6. Metabolite ratios MR1 (O-demethylation) and MR2 (N-demethylation) of tramadol plotted against the number of functional CYP2D6 genes. Logarithmic transformations of median MRs are shown with 95% confidence intervals. See also Figure 3 on p. 20. = p<0.05, = p<0.01.

1.2 CYP2D6 and Amitriptyline (II) significantly different between the genotype Of the amitriptyline-related cases, 195 groups with zero, one, or two functional genes. individuals of 202 were successfully genotyped. Only one of the fatal poisonings included Gene dose correlated with several MRs, with in the material coincided with a homozygous expected correlations found between the number nonfunctional genotype (*4/*4). The case was a of functional CYP2D6 genes and the MRs suicide: a 56-year old female had ingested a related to the rate of trans-hydroxylation, i.e. large amount of her husband’s medication. EHNT/ZHNT, EHAT/ZHAT, nortriptyline/ Amitriptyline was found in postmortem blood at EHNT, amitriptyline/EHAT, and nortriptyline/ a concentration of 60 mg/l, when the upper limit EHAT (Figure 7). Several of the MRs were of the therapeutic range is 0.2 mg/l [36].

RESULTS 31 1.3

) 0.6

) 1.1 HNT

Z 0.4 /NT 0.9

0.2 g(AT EHNT/ lo

log( 0 0.7

-0.2 0.5 0.7 0 ) 0.5 HAT /EHNT) -0.2 0.3 EHAT/Z ( g(EHAT -0.4

0.1 lo log

-0.1 -0.6 0.9 0.1 T) ) 0.6 -0.1 /ZHN /EHNT 0.3 -0.3 ZHAT g( log(NT o

0 l -0.5

-0.3 -0.7

2 0.6 ) T

A 1.7 H

E 0.4 /EHAT)

AT/ 1.4 ( log(NT log 0.2 1.1

0.8 0 1.6 1.1 ) ) 1.2 0.9 HAT /EHAT /Z

0.8 T 0.7 NT N ( log( 0.4 log 0.5

0 0.3 0123 012 Number of functional CYP2D6 genes Number of functional CYP2C19 genes

Figure 7. Relevant metabolite ratios in amitriptyline metabolism plotted against the number of functional CYP2D6 and CYP2C19 genes. Logarithmic transformations of median metabolite ratios are shown with 95% confidence intervals. AT = amitriptyline, NT= nortriptyline. See also Figure 4 on p. 21. = p<0.05, = p<0.01, = p<0.001.

1.3 CYP2C19 and Amitriptyline (II) of functional CYP2C19 genes and MRs related Regarding CYP2C19, 177 individuals of 195 to the rate of N-demethylation (amitriptyline/ were successfully genotyped. The CYP2C19 nortriptyline, EHAT/EHNT, ZHAT/ZHNT, gene dose correlated with several MRs, with nortriptyline/EHAT, and nortriptyline/ZHAT; expected correlations found between the number Figure 7). Several of the MRs were significantly

32 RESULTS

different between the genotype groups with Table 4. CYP2D6 allele frequencies in the 228 zero, one, or two functional genes, but none of successfully genotyped cases. the fatal poisonings coincided with a Allele n % *1 151 33.1 homozygous nonfunctional genotype. *2 165 36.2 *3 17 3.7 1.4 Allele and Genotype Frequencies (I, II) *4 64 14.0 In Studies I and II, altogether 228 individuals *5 12 2.6 were successfully genotyped for CYP2D6. The *6 14 3.1 allele frequencies in this population are shown *9 30.7 in Table 4. *10 81.8 Most of this population, i.e. 124 *1xN 92.0 individuals (54.4%), had two functional *2xN 13 2.9 CYP2D6 genes, but 69 (30.3%) had only one Total 456 100 and 17 (7.5%) none. Three or more functional CYP2D6 genes were found in 18 cases (7.9%). Overall, the frequency of duplicated alleles in the Finnish population appears to be 5% and that 2 Fatal Toxicity Indices (IV, V) of null alleles (*3-*6) 23.4%. According to the FTIs were calculated for nine newer anti- Hardy-Weinberg principle, this would depressants (V) and eight other common drugs correspond to approximately 5% of CYP2D6 (IV), with citalopram included in both FTI gPMs and 7% of CYP2D6 gUMs in the Finnish studies. Furthermore, the number of fatal population. poisonings caused by each of the newer anti- In the population of 177 cases successfully depressants was compared with the number of genotyped for CYP2C19, the observed expected poisonings (V), revealing that venla- CYP2C19 allele frequencies were 0.836 for faxine, mianserin, and mirtazapine caused more *1 and 0.164 for *2. Allele *3 was not found. and fluoxetine, sertraline, and moclobemide less The majority, i.e. 130 cases (73.4%), fatal poisonings than expected from the sales of revealed two functional CYP2C19 genes, but newer antidepressants in 1995-2002 (Table 5). 36 (20.3%) carried only one and 11 (6.2%) FTIs were highest for the phenothiazine none. antipsychotics promazine and levomepromazine,

Table 5. Sales and observed and expected deaths due to newer antidepressants. Drug Salesa Fatal poisonings p-valuec Observed Expectedb Citalopram (SSRI) 84.0 104 114 >0.05 Fluoxetine (SSRI) 48.9 16 66 <0.001 Sertraline (SSRI) 15.7 6 21 0.003 Mirtazapine 14.5 37 20 0.017 Paroxetine (SSRI) 11.2 16 15 >0.05 Mianserin 10.9 33 15 0.006 Moclobemide 10.9 29 15 0.027 Venlafaxine 6.80 30 9 <0.001 Fluvoxamine (SSRI) 5.26 8 7 >0.05 Othersd 1.12 5 2 >0.05 Total 209.1 284 a) In DDD/1000 inhabitants/day (National Agency for Medicines) b) Calculated for each drug by dividing the total number of poisonings by the corresponding proportion of total sales c) p-value for the difference between the proportions of observed and expected deaths d) Includes trazodone, nefazodone, milnacipran, and reboxetine

RESULTS 33 the opioid analgesic propoxyphene, and the median BAC was lower in suicides than in TCAs doxepin and amitriptyline (Table 6). accidental poisonings (2.55‰ (n=20) vs. 2.1‰ (n=7)), with a point estimate of difference of 0.6‰ (p=0.02). A difference was also observed 3 Drug-Alcohol Interaction in temazepam concentrations, with a median of Striking differences were observed in median 2.6 mg/l in suicides and 0.3 mg/l in accidental BACs between fatal poisonings involving poisonings (p<0.001). different drugs. 3.2 Alcohol and Other Common Drugs (IV-VI) 3.1 Alcohol and Benzodiazepines (III) In addition to diazepam and temazepam, median Median BAC in fatal accidental alcohol BACs in fatal drug-alcohol poisonings were poisonings where no other analytes were calculated for six newer antidepressants (V) and detected was 3.3‰ (n=615). In fatal poisonings seven other common drugs (IV). The median involving diazepam it was 3.5‰ (n=161), but in BACs (Table 6) were lower than that in pure those involving temazepam it was only 2.45‰ alcohol poisonings (3.3‰), with the exception (n=32) (III). of poisonings involving fluoxetine. In drug- The major difference between the alcohol poisonings involving moclobemide characteristics of these two groups was that (n=9), the difference did not reach statistical manner of death was accidental in 99.8% and significance. However, the drugs associated with 98.8% of ethanol- and diazepam-related low median BACs (IV) were also the ones more fatalities, respectively, but in only 62.5% of often associated with suicides and less often temazepam-related poisonings. In the latter, the with accidental manner of death (Figure 9).

Table 6. Fatal toxicity indices (FTIs) and median blood alcohol concentrations (BACs) in fatal drug-alcohol poisonings. Drug FTI (95% CI)a Median BAC (95% CI) n BDZ+ (deaths/DDD/1000 (‰) (%) inhabitants/year) Fluoxetine* 0.33 (0.19-0.53) 3.4 (3.0-3.9) 21 62 Citalopram* 1.2 (1.0-1.5) 2.9 (2.5-3.2) 80 63 Diltiazem 1.4 (1.1-1.8) 2.8 (2.0-3.3) 23 30 Zopiclone 0.96 (0.8-1.1) 2.7 (2.2-3.1) 38 24 Moclobemide* 2.7 (1.8-3.8) 2.7 (1.1-3.9) 9 56 Mirtazapine° 2.6 (1.8-3.5) 2.7 (2.3-3.2) 16 88 Levomepromazine 48 (42-53) 2.6 (2.0-2.9) 64 45 Temazepam 0.90 (0.7-1.1) 2.5 (2.2-2.7) 57 (100) Mianserin* 3.0 (2.1-4.3) 2.4 (1.7-2.9) 16 63 Venlafaxine° 4.4 (3.0-6.3) 2.4 (0.4-2.7) 8 75 Propoxyphene 33 (29-37) 1.7 (1.5-1.8) 67 25 Doxepin 21 (18-24) 1.6 (1.2-1.9) 27 33 Amitriptyline 12 (11-14) 1.6 (1.4-1.9) 50 46 Promazine 120 (110-140) 1.3 (1.1-1.6) 31 29 Fluvoxamine* 1.5 (0.66-3.0) nd - - - Sertraline* 0.38 (0.14-0.83) nd - - - Paroxetine* 1.4 (0.82-2.3) nd - - - nd = not determined; BDZ+ = proportion of benzodiazepine-positive cases in drug-alcohol poisonings a) Data from 1997 to 2002 for venlafaxine and mirtazapine (°), from 1995 to 2002 for the other newer antidepressants (*), and from 1995 to 2000 for the other common drugs. Confidence intervals (CI) calculated using the Poisson distribution.

34 RESULTS

3.2 3.2 R2 = 0.70 9 9 R2 = 0.70 2.8 5 2.8 5 ] 6 6 7 8 7 2.4 8 2.4 BAC [‰ n

2.0 an BAC [‰] 2.0 dia e M 1 Medi 1 1.6 3 4 1.6 4 3

2 2 1.2 1.2 0% 20% 40% 60% 80% 0% 20% 40% 60% Manner of death accidental Manner of death suicide

Figure 9. The correlation of median blood alcohol concentration (BAC) with the manner of death in drug-alcohol poisonings. The drugs involved are propoxyphene (1), promazine (2), doxepin (3), amitriptyline (4), diltiazem (5), zopiclone (6), levomepromazine (7), temazepam (8), and citalopram (9).

Therapeutic concentrations of some common combination with amitriptyline, propoxyphene, BDZs were present in 45% of drug-alcohol and promazine from Study VI are shown in poisonings included in Studies IV and V Figure 11. In the cumulative distributions, a (excluding citalopram and temazepam cases notable shift occurs towards lower BACs in all from Study IV) (Table 6). of the combinations. The most prominent shift is The combined results of FTI and median observed in promazine-related cases, with BAC analyses are shown in Figure 10. The almost no overlap with the curve for fatal drugs appearing the safest are located in the poisonings by alcohol alone. lower left-hand corner (fluoxetine, etc.), and the Combined drug-alcohol concentration ones appearing the least safe in the upper right- curves (isobolograms) were constructed in Study hand corner (promazine, etc.). VI (Figure 12). They illustrate the concentration Median drug concentrations in fatal distributions seen in fatal poisonings involving poisonings were calculated for amitriptyline, alcohol alone (y-axis), drug alone (x-axis), and propoxyphene, and promazine (VI). Median their combination (connecting lines). The lines amitriptyline and propoxyphene concentrations connect concentration pairs equally effective in were lower in drug-alcohol poisonings than in causing 10%, 30%, 50%, 70%, and 90% of the pure drug poisonings. Amitriptyline fatalities. The drug concentrations generally concentrations were on average lower also when increase as alcohol concentrations decrease, BDZs were present. with the exception of promazine concentrations BAC distributions in fatal poisonings being relatively low in fatal poisonings by involving alcohol alone and alcohol in promazine alone.

RESULTS 35

Figure 10. Fatal toxicity index (FTI) plotted against the deviation of median blood alcohol concentration (BAC) in drug-alcohol poisonings from that found in pure alcohol poisonings (IV, V). The bars represent the 95% confidence intervals for the difference in BAC. * = n<10.

90 %

80 %

70 %

aths 60 %

De 50 % tive 40 %

30 % Cumula

20 %

10 %

0 % 0.5 ‰ 1 ‰ 2 ‰ 3 ‰ 4 ‰ BAC

Figure 11. Concentration-response curves in cases of fatal poisoning involving alcohol alone (—) and alcohol in combination with amitriptyline (–z–), propoxyphene (–▲–), and promazine (–■–).

36 RESULTS

4

3 ] ‰

C [ 2 BA

1

0 02468101214 a Amitriptyline concentration [mg/l]

4

3 ] ‰

C [ 2 BA

1

0 0 5 10 15 20 25 30 35 40 b Propoxyphene concentration [mg/l]

4

3

2 BAC [‰]

1

0 0 3 6 9 12 15 18 21 24 c Promazine concentration [mg/l]

Figure 12. Concentration-concentration curves illustrating the concentration distributions seen in fatal poisonings involving alcohol and a drug. The lines connect concentration pairs which would account for 10% (––), 30% (–■–), 50% (—), 70% (–z–), and 90% (–▲–) of cases.

RESULTS 37 DISCUSSION

1 Methodological Considerations In fatal poisonings due to alcohol alone and Finland is an excellent site for research on without complicating factors, the maximum forensically interesting postmortem material antemortem alcohol concentrations can be because the medicolegal autopsy rate is high, assumed to have been much higher, with an recently 20% of all annual deaths, with forensic estimated mean of 4.63 g/l (4.4‰), and a toxicology involved in approximately one-half subsequent decrease during survival time [76]. of cases [94,231]. A high autopsy rate (or a large However, this does not preclude statistical population) is essential for achieving sufficient analysis based on postmortem concentrations. sample sizes for statistical studies, such as the On the contrary, a statistical approach may be ones presented here. A small number of cases assumed to compensate for possible postmortem would result in low statistical power and hence changes – such as redistribution of alcohol (or in low interpretative value in any study. drugs) in the body or alcohol formation by Furthermore, with postmortem blood collection microbial activity – in individual cases. from the femoral vein being the standard The drugs investigated in Studies III and procedure throughout the country from 1995 IV were chosen because they were among the onwards and with validated methodologies most common causes of fatal poisonings employed in the Laboratory of Forensic investigated in Finland during 1995-2000. In Toxicology, the blood concentrations obtained addition, they are the most common drugs in in Finnish postmortem toxicology constitute Finnish hospitalizations due to intoxication, reliable, commensurate data. two-thirds of which also involve alcohol [74]. Although the relevance of proper collection However, a problem with retrospective research and preservation of autopsy samples was not in forensic toxicology is that the poisoning assessed in the experimental part of this thesis, it panorama is ever-changing and only a part of must be noted that they are the basis for all of the results is applicable to the present situation. the research presented here, as well as for all For instance, since 1995, the use of results reported by any laboratory of propoxyphene has diminished in Finland, most postmortem forensic toxicology. If sample likely due to published warnings on its abuse collection is in some way compromised, it potential and to the introduction of tramadol. cannot be compensated for at a later stage. Alcohol-propoxyphene poisonings are therefore Therefore, the origin of the sample should less common today than during the study period. always be known to the forensic toxicologist and Despite the introduction of newer should also be stated in the methods section of a antidepressants, amitriptyline and doxepin publication, especially if the reported value is to remain on the market and are frequently be used as a reference in interpretation. encountered in fatal poisonings [94]. Likewise, As discussed by King more than 20 years promazine, predominantly prescribed to alcohol ago [229], most studies on drug-alcohol abusers to relieve withdrawal symptoms, still interactions are conducted on animals or living constitutes a serious problem, reflected in the persons, with little data available on human number of fatal poisonings involving this postmortem investigations. This is a problem neuroleptic agent [94]. Therefore, in spite of the because drug concentrations can be markedly retrospective approach the results presented here higher in fatal poisonings than concentrations can be considered relevant in forensic medicine produced in a clinical study. In fact, a massive today. overdose can result in such concentrations that In a department of forensic medicine, they are no longer in proportion with the end identification of unknown dead bodies using result, i.e. death would have resulted from a genetic profiling is common practice. The concentration many times lower than that found detection methods used for genotyping in this in the samples. study were originally chosen according to the

38 DISCUSSION

existing equipment, which was the one in place extent of DNA degradation, precluding for genetic profiling. An alternative genotyping amplification of the large CYP2D6 fragments, approach could also be applied to postmortem and the presence of impurities that probably CYP genotyping [204]. However, the techniques inhibited the enzyme used for the amplification employed here worked relatively well, as of CYP2C19 fragments. discussed below, and significant progress was Despite these limitations, postmortem made in CYP genotyping by replacing the pharmacogenetics may provide important laborious RFLP method with the moderate information in individual cases and shed light on throughput SNaPshot method [156]. the cause and mode of death in otherwise unclear forensic cases. Postmortem samples are routinely taken for toxicological analysis in 2 Pharmacogenetics cases where the autopsy findings or background Postmortem determinations are inevitably information indicate poisoning. In Finland, these limited to one-time sampling at a random time- samples are stored for one year after analysis, point after an unknown intake instead of timed making them available for later re-examination, sampling after controlled ingestion, followed by if necessary. Genetic factors could explain those calculation of the area under the curve, as in cases where intentional overdose can reasonably clinical settings. This is one of the most be ruled out but toxicological analysis reveals important factors complicating interpretation of either an unexpectedly high concentration of the postmortem toxicology results. parent drug or an exceptional parent drug/ Drug pharmacokinetics may be affected by metabolite ratio. This is best exemplified by a several other factors besides genetic variation, case report of an incident where parents were especially by metabolic drug interactions, age, absolved from homicide charges as a result of a and renal or liver malfunction. This is of homozygously defective CYP2D6 gene being particular interest in a medicolegal context, detected in the postmortem investigation of a where polypharmacy and various pathophysio- fluoxetine-related death [70]. logical conditions are common findings. In our We examined the possibility of a fatal case series, gender was not observed to affect poisoning occurring due to a combination of the MRs, whereas age, due to age-associated drug treatment and a defective genotype. physiological status, may have played a role Amitriptyline and tramadol are toxic drugs [133]. However, due to the large number of known to cause fatal intoxications, and they known inhibitors of CYP2C19 or CYP2D6, the were therefore selected as candidate drugs in varying degree of inhibition, and the wide range this study. Among the tramadol cases, there was of inhibitor concentrations present in the one fatal drug poisoning due to tramadol alone material, taking these factors into account in a (9 mg/l), but the CYP2D6 genotype was fully reasonable manner was not possible. functional (*1/*2) and MR1 and MR2 were in Another limitation of the study was the the ranges typical of cases with two functional quality of the autopsy samples. Postmortem genes. Among the amitriptyline cases, none of DNA can be difficult to amplify because of the accidental fatal poisonings was associated extensive degradation. The process of with either a homozygously defective decomposition may also give rise to impurities, CYP2D6 genotype or a homozygously defective such as free metal cations, which may inhibit the CYP2C19 genotype. Even though Studies I and enzymes used in PCR. Thus, analysis of II did not reveal fatal poisonings which could be degraded samples will not always yield an attributed to genetic defects, we have recently unequivocally interpretable result. Problems identified a case of fatal doxepin poisoning were encountered here both with CYP2D6 where the manner of death is accidental and the genotyping (II), unsuccessful in 7 of 202 cases, CYP2D6 genotype is completely nonfunctional and with CYP2C19 genotyping, unsuccessful in (Koski et al., unpublished results). 18 of 195 cases (II). Probable reasons for the With tramadol, however, it was evident lack of result in these cases may have been the that when the number of functional genes

DISCUSSION 39 increased, the median MR1 decreased. The [128]. It was recently suggested that allele median MR2 also correlated with the number of CYP2D6*41, also associated with decreased functional genes, but in the reverse direction, as expression of a functional protein product [233], was expected based on the complementary might be differentiated from CYP2D6*2 by nature of O- and N- demethylation pathways, genotyping the position 2988G>A (*41:2988A, respectively. A clinical study on children found *2:2988G) [148]. In this study, allele *41 was that the CYP2D6-mediated metabolite (M1) was not included in the genotyping methods. With an formed to a lesser extent, and the formation of estimated allele frequency of 8.4% [148], *41 is the non-CYP2D6 product (M2) was more more common in Caucasian population than the extensive in subjects carrying one functional other known alleles associated with lower CYP2D6 gene than in those carrying two CYP2D6 expression, but its importance is functional genes [187]. This would implicate unclear [152]. Alleles *9 and *10 have a total that when one metabolic pathway is absent or allele frequency of 3-4% in Europeans blocked, the metabolism is shunted towards an [149,232], with our result of 2.5% agreeing well alternative route. with earlier reports. Subjects homozygous for Even a single metabolic reaction may be *10 have been found to show a doubled catalyzed by different enzymes, thus having nortriptyline plasma half-life compared with several complementary pathways. Other subjects homozygous for *1 [190], and thus enzymes besides CYP2C19 have been suggested carrying two alleles associated with decreased to participate in N-demethylation of activity might be considered equal to carrying amitriptyline, especially at high amitriptyline one functional gene. Having only one functional concentrations [184]. Therefore, less correlation gene has been suggested to constitute a risk can be expected between amitriptyline MRs and factor for CYP2D6 substrate toxicity [206]. Any CYP2C19 genotypes than between amitriptyline CYP2D6 alleles associated with diminished metabolites and CYP2D6 genotypes. Judging activity and present in a relevant frequency in a from the correlations between the observed ami- population should therefore be taken into triptyline metabolite patterns and the determined account in future work. CYP2D6 and CYP2C19 genotypes, amitriptyline Finally, it is highly unlikely that the metabolism appeared more dependent on metabolism and excretion of a drug would CYP2D6 than on CYP2C19. depend on only one type of enzyme. Of the The total allele frequency of the CYP2D6 extensive field of pharmacogenetics, only two null alleles (*3-*6) observed here, 23.4%, is metabolic enzymes and two substrates were somewhat lower than previously reported in targeted here, and although differences in Caucasians, e.g. 25.2% in Germans [149] and metabolite ratios were observed between 27% in the Swiss/French [232]. Moreover, genotype groups, they were relatively small and frequencies of 31.0% and 23.1% have been often overlapping. However, the finding of reported in Swedish blood donors and in fatal differences in this material, unadjusted for age, intoxications, respectively [204]. The allele gender, or dosage, suggests that in the context frequency of CYP2C19*2 in this study, 16.4%, studied the genes play a dominant role over agrees well with those reported in the Swedish other factors. The effects of a drug obviously study, i.e. 14.3% in blood donors and 15.5% in also depend on the genes associated with fatal intoxications [204], and the frequency transporter proteins and receptors, but the estimated by Wedlund, 14.7% [161]. polymorphic metabolic enzymes are today No distinction was made between alleles considered of such importance that the associated with normal CYP2D6 enzyme pharmaceutical companies abandon a activity (*1 and *2) and those associated with polymorphically metabolized candidate drug decreased activity (*9 and *10). By making this molecule fairly early on in the development if a distinction, a category corresponding to an pharmacologically equipotent alternative exists approximately 0.5 functional gene predicting the [125]. This can be a change for the better also IM phenotype could have been established from the Finnish point of view, since altogether

40 DISCUSSION

12% of the individuals genotyped in this study 3.2 Alcohol and Other Common Drugs were either CYP2D6 gPMs or gUMs, and thus, For the other drugs commonly found in fatal at least one in eight Finns taking CYP2D6 poisonings, marked differences were present substrates could be expected to experience in alcohol concentration distributions. The ADRs due to genetically altered metabolism. median BACs in cases involving amitriptyline, Moreover, 6.2% of the cases investigated here doxepin, propoxyphene, and promazine were revealed a nonfunctional CYP2C19 genotype. conspicuously low compared with those in other cases. They might therefore be considered to interact with alcohol in a fatal manner, 3 Drug-Alcohol Interaction although the mechanism of interaction cannot be Clinical studies often focus on the pharmaco- deduced from this data. Whereas alcohol and kinetic interactions and psychomotor effects of BDZs are considered to exert their effects at drug-alcohol combinations, whereas in fatal least in part via the GABAA receptor and thus poisonings, the pharmacodynamic interactions have a rational basis for interaction, this are more important. In drug-related fatalities, mechanism is not likely to apply to the other the relevant questions are which findings drugs. All of them, however, exert their have contributed and by what mechanism. It is therapeutic effects via the CNS, a fact that also important to consider whether certain renders the idea of interaction with alcohol combinations of drugs and alcohol have plausible. greater effects than others in a manner The analgesic propoxyphene acts via reflected in concentrations found in postmortem opioid receptors. Stimulation of opioid receptors investigation. has several specific effects, including depression of the respiratory center. Propoxyphene can 3.1 Alcohol and Benzodiazepines therefore be expected to show additive or A striking difference is apparent in the outcome synergistic effects with alcohol [234]. The of ethanol poisonings depending on the nature of toxicity of amitriptyline has been attributed to concomitant drugs. With regard to the common the quinidine-like action by which it causes BDZs, lower ethanol concentrations appear to cardiac depression. The interactions of result in fatal poisonings when temazepam is propoxyphene and amitriptyline with alcohol involved than when diazepam or no other drug have therefore been hypothesized to involve is involved. Interestingly, no cases combining membrane-stabilizing activity [113,234]. The high concentrations of alcohol and diazepam phenothiazines promazine and levomepromazine were found in any of these studies, and thus, no exert their antipsychotic effects by acting on the conclusions about such combinations should be dopamine system, especially by blocking inferred. The relatively frequent occurrence of dopamine receptors, but they are also known to high temazepam concentrations further cause nonspecific sedation. Mianserin and accentuates the absence of high diazepam venlafaxine were associated with the lowest concentrations, which may reflect differences in BAC levels among cases of fatal poisonings tissue distribution of temazepam and diazepam. involving newer antidepressants, although the The proportion of suicides is, however, number of cases was relatively small (Table 6). relatively high in the temazepam group and These drugs also act via the CNS, their nonexistent in the fairly large diazepam group. It mechanism of action being based on inhibition therefore seems that those who have of monoamine reuptake. This does not, however, attained high blood concentrations of both explain why they would interact more strongly alcohol and diazepam simultaneously have with alcohol than the other newer recovered, with or without supportive treatment. antidepressants. The general public may also perceive the Another line of reasoning which may hypnotic temazepam as a toxic substance, but explain the relatively low BACs found in diazepam as a relatively mild and harmless combination with amitriptyline, doxepin, anxiolytic. propoxyphene, and promazine is that these four

DISCUSSION 41 drugs are the ones most often involved in magnitude of median BAC deviation in pure suicides (Figure 9). Whereas the relatively low alcohol poisonings, with promazine, BACs might be considered to result from amitriptyline, doxepin, and propoxyphene additive or synergistic interactions with these appearing the least safe (Figure 10). drugs, they might, alternatively, be considered to This division obviously reflects not only indicate intentional but moderate use of alcohol the acute toxicity but also the manner of use of during the suicide attempt, for instance, for the these drugs. Judging from the varying purpose of flushing down the pills or giving the proportions of suicides and blood concentrations attempter more courage to complete the act. It is exceeding therapeutic ranges, it is evident that also conceivable that impaired judgment and certain drugs are deliberately taken in overdoses dysphoric states provoked by alcohol may give for intoxication purposes or with suicidal intent. rise to impulsive and aggressive acts, even to However, Buckley and McManus [97] have suicide, with less serious intent [235]. concluded that the FTIs that they obtained for In addition to the lower BAC distributions anxiolytic and sedative drugs largely reflect the in fatal poisonings involving drugs, lower blood inherent toxicity of these drugs. Also Henry drug concentrations were found in fatal [101] and Farmer and Pinder [87] considered the poisonings in which amitriptyline or propoxy- inherent toxicity of the compound the crucial phene was present in combination with alcohol factor in the fatal toxicity of antidepressants and than in cases not involving alcohol (VI). This the prescribing practices and the popular would support the hypothesis of interaction. perception of toxicity of secondary importance. However, the median promazine concentrations Even if the ingested dose and the acute toxicity were similar in alcohol-positive and alcohol- of a drug are deemed the determining factors for negative cases, although the median BAC (IV) outcome, reaching hospital care certainly is was the lowest in promazine cases, and the another; most overdose fatalities occur outside isobologram constructed in Study VI did not hospitals, with very low overall mortality of differ significantly from those constructed for hospitalized overdose patients [74,237,238]. amitriptyline and propoxyphene. Interestingly, The drugs investigated in Studies IV and V none of the isobolograms were convex towards are to a large extent newer than those covered in the origin, as were the alcohol-barbiturate curves the 1980s. Amitriptyline and propoxyphene are created by Stead and Moffat [79]. The potential striking exceptions, although since the study interaction can therefore be considered additive period, the use of propoxyphene has decreased. at most and not synergistic. Amitriptyline Promazine is an older drug and its use is concentrations were also lower in BDZ-positive not very common worldwide. The results than in BDZ-negative cases, in parallel with the concerning promazine might, however, be results of an earlier study [236]. generalizable to chlorpromazine, a close relative of promazine and in more widespread use. The 4 Drug Safety absence of high alcohol concentrations among The BAC in drug-alcohol poisonings can be the promazine cases is especially remarkable, considered a reliable parameter for assessing with the maximum BAC in the promazine- drug-alcohol interaction since the distribution of alcohol poisonings equaling the median BAC in postmortem alcohol concentrations in poisoning pure alcohol poisonings. Furthermore, the FTIs caused by alcohol alone is essentially similar of promazine, and to a lesser extent levome- independent of the sample, as illustrated earlier promazine, are markedly higher than those of by Vuori et al. (see also Figure 1) [80] and the other common drugs included here. Study VI (Figure 11). By considering the median BAC as a relative measure of drug 4.1 Newer Antidepressants toxicity similar to FTI, these two measures can The newer antidepressants in general appeared be compared. Among the 18 drugs studied in the safer in combination with alcohol than context of FTIs and median BACs (IV, V), the amitriptyline and doxepin. Among the common FTI rank order largely agreed with the newer antidepressants, fluoxetine and sertraline

42 DISCUSSION

had caused significantly less and the non-SSRI 42% accidental, whereas of cases with alcohol agents significantly more deaths than expected. noted as the most important finding, 96% were The rare drugs caused almost no deaths: in accidental, with no suicides. Furthermore, the 1995-2002, trazodone caused three, nefazodone median BAC in accidental deaths was one, milnacipran one, and reboxetine none. Both significantly higher than the median in suicides the FTIs and the median BAC differences (3.1‰ vs. 2.0‰), but similar to the median indicate that among the newer antidepressants, found in a series of accidental alcohol the SSRIs, especially fluoxetine, are relatively poisonings caused by ethanol alone (3.3‰). safe and venlafaxine relatively unsafe, with Another confounding factor was that normal or mianserin, mirtazapine, and moclobemide therapeutic concentrations of BDZs were present somewhere in between (V). in 65% of cases included in the drug-alcohol Mianserin, which was released to the UK part of Study V, which is more than the 40% market in 1976, was relatively early on average for all BDZ findings in postmortem associated with a lower FTI than those of TCAs cases undergoing drug screening [240]. [93,94,99]. For mirtazapine and venlafaxine, In all, the newer antidepressants however, the FTIs obtained in this study yielded investigated here, starting with mianserin novel information since these drugs were introduced in 1976, appear safer than the older introduced in Finland after the study period ones, indicating both successful drug covered by Öhberg et al. [102] and since development and a shift from older to newer mirtazapine was represented by only a single drugs in prescription practices. This progress is fatality in the study of Buckley and McManus very welcome since antidepressants have [96]. The relatively high toxicity of venlafaxine become increasingly important in the Western and the relative safety of SSRIs have already world. In Finland, the consumption of been established by several studies [e.g. 96,107]. antidepressants has increased 600% in the past An important factor here might be the suggested 14 years (Figure 13), with SSRIs accounting for differences in prescribing practices, with 68% of current antidepressant consumption. venlafaxine allegedly prescribed to people Citalopram surpassed the TCAs amitriptyline already at a relatively high risk of suicide and doxepin in 1992 and is today the most (recurrent or treatment-resistant depression) common antidepressant in Finland, with the [116,117]. A Swedish study recently found that second most common being fluoxetine. It is not the SSRIs were underrepresented and other surprising therefore that citalopram-related modern antidepressants overrepresented in fatalities have also increased, although not to a suicides compared with a control group level comparable to the TCAs. In Austria, the consisting of accidental and natural deaths, with increasing use of newer antidepressants was TCAs occurring equally in both groups [239]. found not to result in an increase in suicidal The low mortality associated with SSRIs may in poisonings by these drugs [17]. In England, by part arise from their also being prescribed for contrast, although a decrease in the number of conditions other than depression. Moreover, the deaths involving antidepressants has been SSRIs are generally tolerated at effective doses, observed since 1996, an 8% rise was seen from resulting in good compliance and eventual 2002 to 2003, with the biggest proportional improvement of the condition. increase in SSRI-related deaths [241]. Besides inherent toxicity, perception of toxicity, and prescription practices, the manner of use is an important factor in postmortem drug 5 Implications for Interpretation toxicity evaluations. In drug-alcohol poisonings The forensic relevance of postmortem involving newer antidepressants (V), the manner toxicology results is most expediently of death, indicating the putative manner of drug exemplified by cases of suspected homicide or use, was associated with the cause of death; of in traffic accident fatalities, but the results may cases with a newer antidepressant noted as the also reveal unsuspected drug abuse, erroneous most important finding, 46% were suicides and medication, or noncompliance. A special case

DISCUSSION 43

50 Other antidepressants 45 Monoamine oxidase type A inhibitors 40 Selective serotonin reuptake inhibitors Nonselective monoamine reuptake inhibitors 35

30

25

20

15

10 Sales (DDD/1000 inhabitants/day) 5

0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 Figure 13. Sales of antidepressants in Finland in 1990-2004 (National Agency for Medicines).

relevant to this study is one where the forensic involving antidepressant poisonings may remain toxicology results indicate fatal poisoning, but undetermined [242]. where it is not clear, judging from the Nevertheless, the results presented here circumstances, whether the manner of death is indicate that the possibility of accidental accidental or suicide [242]. Most fatal poisoning should be considered seriously when antidepressant poisonings are indeed suicides, as alcohol is present or when the drug considered discussed above. In Finland, the proportion of responsible for the poisoning is polymorphically suicides in fatal antidepressant poisonings has in metabolized and the MR is inconsistent with recent years ranged from 63% to 67% (Vuori acute poisoning. The presence of any interacting et al., unpublished data), but as many as 80% of substances should also be taken into account. antidepressant poisonings were found to be Finally, it must be kept in mind that the suicides in a Danish study [119]. On the other forensic pathologist may more readily attribute hand, abuse of alcohol is often indicated in fatalities to drugs generally perceived as antidepressant poisonings, e.g. in 38% of the dangerous, resulting in drugs considered safer cases in the Danish study [119]. Deciding not being implicated as cause of death even between alcohol and antidepressants as a cause when found in high concentrations in blood. of death and between accident and suicide as a These perceptions are likely to affect the manner of death might therefore be difficult, and proportion of deaths attributed to a drug, and, thus, the manner of death in fatal poisonings consequently, the perceptions themselves.

44 DISCUSSION

CONCLUSIONS

These results on tramadol and amitriptyline are safer than temazepam, although combining any among the first to demonstrate that analysis of BDZs with alcohol should be avoided. Of the genetic variation of DMEs using postmortem most common drugs in the recent Finnish blood is possible. Although genetic factors in poisoning panorama, amitriptyline, propoxy- drug metabolism were observed to have a phene, and promazine appear to be especially dominant role over various pathological dangerous in combination with alcohol. conditions, interacting substances, and other Although the newer antidepressants are confounding factors, the study did not reveal increasingly common findings in postmortem any fatal poisonings to be associated with non- investigations, they cause fewer deaths than functional CYP2D6 or CYP2C19 genotypes. expected from the sales of antidepressants, and Furthermore, alternative metabolic pathways they can also be considered safer in combination appear to compensate for a defective route thus with alcohol than TCAs and other common toxic preventing accumulation of the parent drug in drugs. All in all, our results confirm that the gPMs. While genotyping does not seem newer antidepressants are significantly safer worthwhile in routine case work, it comprises a than other common drugs involved in fatal valuable tool in elucidating the manner of death intoxications. Furthermore, the differences in in suspicious fatal poisoning cases, where toxicity between the newer antidepressants are background information does not suggest small. suicidal intent. In the future, more attention should be Our findings on common toxic drugs offer given to the contention that some drug-alcohol a new viewpoint on fatal drug-alcohol combinations are less safe than others. A safer interactions with implications for drug safety. alternative could be chosen already at the Fatal drug poisonings often involve alcohol, prescription stage, especially when indications BDZs, or both, and an additive or synergistic of alcohol abuse or suicide risk are present. In interaction may occur between some of the interpretation of postmortem forensic toxicology components. An interaction appears to exist results, even a moderate concentration of between temazepam and alcohol, with alcohol should be considered seriously. pronounced effects at high temazepam However, in addition to the inherent drug concentrations. Diazepam, chlordiazepoxide, toxicity, behavioral aspects, prescribing and nordiazepam do not seem to affect ethanol practices, and the popular perception of toxicity lethality in the range of BDZ concentrations should be considered in evaluating the combined present in the data. In combination with alcohol, effect of alcohol and drugs in postmortem diazepam and chlordiazepoxide therefore appear forensic toxicology.

CONCLUSIONS 45 ACKNOWLEDGMENTS

This study was carried out at the Department of Forensic Medicine, University of Helsinki, in 2001-2004. Financial support from the Finnish Foundation for Alcohol Studies is gratefully acknowledged.

I am indebted to Professor Erkki Vuori, Head of the Department and of the Forensic Toxicology Division, for giving me the opportunity to prepare a doctoral thesis in Forensic Toxicology. I sincerely thank him for providing excellent working facilities and for creating an enthusiastic working atmosphere; he has always shown a genuine interest in the well-being of his personnel.

I am deeply grateful to my supervisors, Professor Antti Sajantila and Docent Ilkka Ojanperä, for novel ideas and expert scientific advice. Their patience and positive attitude never failed me.

The pre-examiners of this thesis, Docent Kari Poikolainen and Docent Eero Mervaala, are thanked for constructive and insightful comments. I also thank Carol Ann Pelli, HonBSc, for editing the language of the manuscript.

I sincerely thank Dr. Merja Gergov and Johanna Sistonen, MSc, for their collaboration and expertise, as well as Juhani Vartiovaara, MSc, and Jari Nokua, MSc, for everything related to data, computers, and other electronic media. Special thanks go to Helena Liuha for her kind help in numerous practical matters. I also wish to thank all of the personnel at the Forensic Toxicology Division for bearing with me. I especially enjoyed working in the GC-MS group.

I am also grateful to my friends and colleagues at the Departments of Chemistry and Applied Chemistry and Microbiology; they got me started in my graduate education and introduced me to the world of science, and their companionship and encouragement have followed me ever since. My friends from the world outside of work receive my heartfelt thanks for providing plenty of fun times and enough shoulders to cry on. My warmest thanks go to Jukka for believing in me and keeping me sane.

Finally, I would like to express my deepest gratitude to my mother for more than three decades of love, encouragement, and support.

Helsinki, August 2005

Anna Koski

46 ACKNOWLEDGMENTS

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