Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
The 2,5-Dimethoxyamphetamines – A new Class of Designer Drugs
Studies on the Metabolite Identification, Toxicological Analysis, Involvement of Cytochrome P450 Isoforms in
Main Metabolic Steps, and Inhibition Potentials on Cytochrome P450 2D6
Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III -
Chemie, Pharmazie und Werkstoffwissenschaften
der Universität des Saarlandes
von
Andreas Heinrich Ewald Saarbrücken
2008
The 2,5-Dimethoxyamphetamines – A new Class of Designer Drugs
Studies on the Metabolite Identification, Toxicological Analysis, Involvement of Cytochrome P450 Isoforms in
Main Metabolic Steps, and Inhibition Potentials on Cytochrome P450 2D6
Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III -
Chemie, Pharmazie und Werkstoffwissenschaften
der Universität des Saarlandes
von
Andreas Heinrich Ewald
Saarbrücken
2008
Tag des Kolloquiums: 05. 12. 2008
Dekan: Univ.-Prof. Dr. U. Müller
Berichterstatter: Univ.-Prof. Dr. Dr. h.c. H. H. Maurer
Univ.-Prof. Dr. R. W. Hartmann
Die folgende Arbeit entstand unter der Anleitung von Herrn Professor Dr. Dr. h.c.
Hans H. Maurer in der Abteilung Experimentelle und Klinische Toxikologie der
Fachrichtung 2.4 Experimentelle und Klinische Pharmakologie und Toxikologie der
Universität des Saarlandes in Homburg/Saar von Januar 2004 bis November 2007.
Mein besonderer Dank gilt:
Herrn Prof. Dr. Dr. h.c. Hans H. Maurer für die Aufnahme in seinen Arbeitskreis, die
Vergabe eines interessanten und abwechslungsreichen Dissertationsthemas, die
Möglichkeit des selbstständigen Arbeitens, die Möglichkeit der aktiven Teilnahme
und Präsentation auf internationalen Fachkongressen und die
Diskussionsbereitschaft zu jeder Tages- und Nachtzeit,
Herrn Prof. Dr. Rolf Hartmann für die Übernahme des Koreferats,
Frau Prof. Dr. Dorothea Ehlers von der Firma cc chemical consulting, Herrn Dr.
Giselher Fritschi vom Hessischen Landeskriminalamt, Herrn Dr. Michael Pütz vom
Bundeskriminalamt und Herrn Dr. Wolf-Rainer Bork vom Landeskriminalamt Berlin
für die Bereitstellung eines Teils der untersuchten Substanzen und für ihre
Unterstützung bei speziellen Fragestellungen,
Herrn Dr. Frank T. Peters für seine moralische Unterstützung und für seine
wissenschaftliche Expertise und ständige Diskussionsbereitschaft,
Herrn Dr. Denis S. Theobald für die Einführung in die Isoenzym-Bestimmungen und
die kritische Diskussion,
meinen Kollegen für die freundschaftliche Atmosphäre, gute Zusammenarbeit und
die Unterstützung in schwierigen Situationen in der Dienstbereitschaft,
Herrn Armin Weber für seine ständige Einsatzbereitschaft, Wartung und Reparatur
der Messgeräte sowie Rat bei technischen Fragestellungen,
Frau Gabriele Ulrich für gewissenhaft ausgeführte Laborarbeiten,
meiner Frau Julia, die mir den Rücken frei gehalten und immer zu mir gehalten hat,
meiner Familie, insbesondere meinen Eltern, die mich in meinem Tun stets gefördert
und unterstützt haben,
meinen Freunden und Bekannten, die in den letzten Jahren des Öfteren ohne mich
auskommen mussten.
Julia
und meinen Eltern
TABLE OF CONTENTS
1 GENERAL PART 1
1.1 Introduction 1
1.1.1 The 2,5-Dimethoxyamphetamine Derived Designer Drugs 1
1.1.2 The Cytochrome P450 System 3
1.2 Aims and Scopes 8
2 PUBLICATIONS OF THE RESULTS 9
2.1 Designer drugs 2,5-dimethoxy-4-bromo-amphetamine
(DOB) and 2,5-dimethoxy-4-bromo-methamphetamine
(MDOB): Studies on their metabolism and toxicological
detection in rat urine using gas chromatographic/mass
spectrometric techniques50
(DOI:10.1002/jms.1007) 9
2.2 Designer drug 2,4,5-trimethoxy-amphetamine (TMA-2):
Studies on its metabolism and toxicological detection
in rat urine using gas chromatographic/mass
spectrometric techniques51
(DOI:10.1002/jms.1059) 23
2.3 Metabolism and toxicological detection of the designer
drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat
urine using gas chromatography-mass spectrometry52
(DOI:10.1016/j.jchromb.2007.06.027) 35
2.4 Designer drug 2,5-dimethoxy-4-methyl-amphetamine
(DOM, STP): Involvement of the cytochrome P450
isoenzymes in formation of its main metabolite and
detection of the latter in rat urine as proof of a drug
intake using gas chromatography-mass spectrometry53
(DOI:10.1016/j.jchromb.2007.11.042) 43
2.5 Metabolism and toxicological detection of the designer
drug 4-chloro-2,5-dimethoxyamphetamine in rat urine
using gas chromatography-mass spectrometry54
(DOI:10.1007/s00216-008-1917-z) 51
2.6 2,5-Dimethoxyamphetamine-derived designer drugs:
Studies on the identification of cytochrome P450 (CYP)
isoenzymes involved in formation of their main
metabolites and on their capability to inhibit CYP2D655
(DOI:10.1016/j.toxlet.2008.09.014) 59
3 CONCLUSIONS 87
4 SUMMARY 89
5 REFERENCES 91
6 ABBREVIATIONS 95
7 ZUSAMMENFASSUNG 97
1 GENERAL PART
1.1 INTRODUCTION
1.1.1 The 2,5-Dimethoxyamphetamine Derived Designer Drugs
Consumption of drugs of abuse is a widespread problem in societies all over the
world. Especially, so-called designer drugs are becoming more and more popular
among young people. The most frequently abused drugs are amphetamine,
methamphetamine and their derivatives, such as 3,4-methylenedioxyamphetamine
(MDA), 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”), para-methoxy-
amphetamine (PMA), and para-methoxymethamphetamine (PMMA). However,
during the 1990s, the illicit drug market for recreational drugs changed considerably
with several new types of drugs appearing on the drug scene. Information about
these new drugs is readily available and they can even easily be purchased via the
internet.1
One of these new classes of drugs of abuse are the 2,5-dimethoxyamphetamines.
Typical drugs of this class are 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-chloro-
2,5-dimethoxyamphetamine (DOC), 4-iodo-2,5-dimethoxyamphetamine (DOI), 2,5-
dimethoxy-4-methyl-amphetamine (DOM), 4-bromo-2,5-dimethoxymethamphetamine
(MDOB), and 2,4,5-trimethoxyamphetamine (TMA-2). Their chemical structures are
shown in Fig. 1.
Fig. 1: Chemical structures of amphetamine-derived designer drugs.
- 1 -
They all have in common an amphetamine structure containing two methoxy groups
in positions 2 and 5 of the aromatic ring and a different lipophilic 4-substituent.
MDOB as the methamphetamine analogue of DOB contains an additional methyl
moiety at the nitrogen.
Introducing new substituents allows the drug abusers to create “legal” products which
are not scheduled as controlled substances. Most of the 2,5-
dimethoxyamphetamines were synthesized by Alexander Shulgin and described in
his compilation “PIHKAL”.2 This work contains the structures of the 2,5-
dimethoxyamphetamines, their hallucinogenic potency, effects, dosage and their
synthesis. Because of this it is relatively easy for the illicit drug producers to
manufacture a new 2,5-dimethoxyamphetamine entity when another one is
scheduled. The first synthesized of the studied drugs was TMA-2 in 19473 and it was
sold under the name “Zerox”. In 1967 DOM appeared on the illicit drug market
especially in the “hippie scene” in San Francisco under the name STP, which was
said to stand for Serenity, Tranquility, and Peace. Street names of DOB were
“Golden Eagle” or “LSD-25”. Although many 2,5-dimethoxyamphetamines were first
synthesized during the 1970s, they gained increasing popularity in the late 1990s and
the beginning of this century after the publication of “PIHKAL”. They were sold in so-
called “smart shops” alone or in mixture with other designer drugs in form of tablets,
powder, liquids or blotters. This trend was accompanied by seizures of tablets
containing 2,5-dimethoxyamphetamines or combinations of them with other drugs by
the police. Because of the high abuse potential of the 2,5-dimethoxyamphetamines,
many of them were scheduled in most countries and by the UN in the convention on
psychotropic substances in 19714. DOB became quite popular as a drug of abuse,
especially in Australia, Italy, and the USA5-10 At present, DOI and DOC, which are not
scheduled in the USA, and TMA-2 are entering the illicit drug market, as indicated by
seizures11-19 and experience reports on so-called drug information web sites
(http//:www.erowid.org, http//:www.lycaeum.org; September 2008). Some information
is available on pharmacological properties of the 2,5-dimethoxyamphetamines. They
show affinity to 5-HT2 receptors and act as agonists or antagonists at different
receptor subtypes.20-25 Because of the high potency and selectivity of DOI as 5-HT2
receptor agonist and the fact that it was not scheduled until now and is commercially
available, it was used in research when a selective 5-HT2 receptor agonist was
needed.26,27
- 2 -
The chemical structure responsible for the hallucinogen-like activity comprises a
primary amine functionality separated from the phenyl ring by two carbon atoms, the
presence of methoxy groups in position 2 and 5 of the aromatic ring, and a
hydrophobic 4-substituent (alkyl, halogen, alkylthio, etc.).25 The methyl moiety in
α-position to the nitrogen atom is reported to be responsible for increased in vivo
potency and duration of action.25
For some 2,5-dimethoxyamphetamines, analytical data are available.28,29 A GC/MS
procedure was described for detection of DOB and DOM (parent compounds), and
other amphetamine derivatives but not for the other studied drugs.30
However, for developing toxicological screening procedures, especially in urine, it is
a prerequisite to know the metabolism of the compounds in question, especially if
they are excreted in urine primarily or even exclusively in form of metabolites.
Furthermore, data on the metabolism are needed for toxicological risk assessment,
because the metabolites may play a major role in the toxicity of a drug.
One metabolism study in humans after intoxication related to DOB has been
reported.31
Besides the direct toxicity of a drug or a metabolite, inhibition of metabolizing
enzymes could also lead to toxic effects due to possible overdoses after co-
administration of different substances which were metabolized by the same enzyme.
Some studies about the inhibition properties of amphetamine analogues on CYP2D6
have been published.32-36
1.1.2 The Cytochrome P450 System
Most drugs are metabolized by a variety of enzymes, and these metabolic processes
can generally produce metabolites that are usually less toxic than the parent
compound. The metabolites may also be more reactive, producing toxic effects. The
metabolic profiling of drugs is, therefore, necessary to assess their effects and
toxicity.37 Cytochrome P450 (CYP) enzymes are responsible for oxidative and, to a
- 3 -
minor extent, reductive metabolic transformations of drugs, environmental chemicals
and natural compounds. Over its long history of more than 3.5 billion years, the CYP
superfamily of enzymes has developed remarkable versatility. The primary catalytic
function of CYPs was identified as transfer of one oxygen atom from molecular
oxygen into various substrates (Fig. 2). A coenzyme, cytochrome P450
oxidoreductase (OR), is essential for CYP catalytic function, and cytochrome b5 can
stimulate catalytic activities of some enzymes.38
Fig. 2: The cytochrome P450 redox cycle.
Single electron shifts are frequently responsible for the formation of reactive
intermediates or allow the leakage of free radicals capable of causing toxicity. When
a CYP enzyme activity is modified by induction or inhibition, the biological activity of
the xenobiotic substrate can be altered considerably. Such effects are called drug-
drug, drug-chemical or chemical-chemical-interactions. Such interactions can modify
the disposition of xenobiotics.39-41 CYPs are heme-containing, membrane-bound
enzymes (“heme-thiolate proteins”) detected in both prokaryotes and eukaryotes.
The enzymes were given their names because their complexes with carbon
monoxide under reductive conditions show an absorbance maximum at about
- 4 -
450 nm. In mammals the enzymes can be identified in nearly every tissue, being
most abundantly present in the liver. The CYP superfamily has been classified in
different families in accordance to the degree of homology of amino acid sequence in
their protein structures. CYP enzymes having ≤ 40% homology in their amino acid
sequence are classified in different families which are designated by Arabic numbers,
for example, CYP1. Each family is further divided into subfamilies of enzymes. The
enzymes within a mammalian subfamily have ≥ 55% sequence homology and are
designated by capital letters, for example, CYP1A. An Arabic number is used for
designating individual enzymes within a subfamily, for example, CYP1A2.39 In
humans, 18 CYP families with 43 subfamilies and 57 CYP isoenzymes are known so
far, of which only 3 families with 7 subfamilies and 12 CYP isoenzymes are relevant
for drug metabolism (Fig. 3),42 namely CYP1A1, CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and
CYP3A5.43
Fig. 3: Cytochrome P450s found in humans and their relevance in xenobiotic metabolism. Modified according to ref.43
Polymorphisms of clinical relevance
- 5 -
The remainder is responsible for the transformation of endogenous biomolecules, for
which reason they are called “housekeeping enzymes”. Fig. 4 illustrates the
abundances of CYPs in human liver and their importance in xenobiotic metabolism.
Some CYP genes are polymorphically expressed, leading to variabilities in patterns
of drug metabolism
Fig. 4: Relative quantities of CYPs in human liver and their relevance in drug metabolism. Left side: human CYP-expression in the liver. Right side: involvement in xenobiotics
metabolism.
Human liver derived enzyme preparations, e.g. human liver microsomes (HLM)
contain a natural mixture of CYPs. Chemical inhibitors, immunochemical inhibitors,
and/or correlation analyses with marker activities must be used to obtain information
on which enzymes are performing specific biotransformations. In contrast, only a
single active CYP is present in preparations of cDNA-expressed enzymes. Inhibitors
and correlation analyses are not needed, because the mentioned assignments can
be performed by direct incubation of the drug with a panel of individual CYPs.
However, the balance of enzymes, present in vivo, is lost.38 Bacteria, yeast,
baculovirus and several mammalian cells have been used to produce a wide range of
catalytically active CYPs. The baculovirus system offers high-level expression of both
the CYP and OR, and are therefore advantageous for metabolism studies of all
kinds, especially for low turnover substrates. The development of the cDNA-bearing
virus is relatively time-consuming and labor-intensive, but baculovirus infected insect
cell microsomes are commercially available. However, because the enzymes are
produced transiently in the insect host cells, exact harvest time can have a
pronounced effect on the activity of the final preparation.44
Identification of the human enzymes involved in the metabolism of specific drugs is
becoming increasingly important for drug development. Such identifications should
- 6 -
consider two processes involving the new drug: metabolism and inhibition. The
identification of enzymes involved in metabolism of the new drug allows prediction,
based on knowledge of the ability of co-administered drugs to inhibit the same
enzymes, of which co-administered drugs may inhibit the metabolism of the new
drug. This information can also be used to predict individual variability based on
known metabolic polymorphisms.38 However, also the new drug can act as an
inhibitor what may lead to interactions with co-administered drugs.
- 7 -
1.2 AIMS AND SCOPES
In clinical cases where an unknown substance was ingested (e.g. poisonings), the
identity of this substance has to be clarified to be able to start suitable medical
treatment and to make statements on the clinical outcome. Also in forensic cases,
intake of an illegal drug has to be proven. Usually, a general unknown screening is
performed in urine, where the concentrations of the parent compound/and or its
metabolites are higher than in blood or plasma and the taken drugs or toxicants can
be detected for several hours or even days after ingestion, in contrast to blood
analysis which covers only a few hours.45,46 Knowledge about metabolic steps is a
prerequisite for developing toxicological screening procedures, especially, if the
compounds are excreted in urine only in form of their metabolites.
The knowledge of the involvement of particular CYP isoenzymes in the
biotransformation of a new drug is a prerequisite to predict possible drug-drug-
interactions, inter-individual variations in pharmacokinetic profiles and increased
appearance of side effects and serious poisonings.47,48 Inhibition capabilities for CYP
isoenzymes by new drugs are also sources for drug-drug-interactions which should
be examined. However, such risk assessment is typically performed for substances
intended for therapeutic use, but not for drugs of the illicit market. In addition, there is
good evidence that genetic variations in drug metabolism have important behavioral
consequences that can alter the risk of drug abuse and dependence.49 The 2,5-
dimethoxyamphetamines were not yet investigated in any of these respects, so that
the aims of the presented studies were:
• Identification of the metabolites
• Development of a urine screening procedure
• Identification of the cytochrome P450 isoforms involved in the main metabolic
steps
• Determination of the inhibition potentials on cytochrome P450 2D6
- 8 -
2 PUBLICATIONS OF THE RESULTS
The results of the studies were published in the following papers:
2.1 DESIGNER DRUGS 2,5-DIMETHOXY-4-BROMO-AMPHETAMINE (DOB) AND
2,5-DIMETHOXY-4-BROMO-METHAMPHETAMINE (MDOB): STUDIES ON
THEIR METABOLISM AND TOXICOLOGICAL DETECTION IN RAT URINE USING
GAS CHROMATOGRAPHIC/MASS SPECTROMETRIC TECHNIQUES50 (DOI:10.1002/JMS.1007)
- 9 -
2.2 DESIGNER DRUG 2,4,5-TRIMETHOXY-AMPHETAMINE (TMA-2): STUDIES ON
ITS METABOLISM AND TOXICOLOGICAL DETECTION IN RAT URINE USING GAS
CHROMATOGRAPHIC/MASS SPECTROMETRIC TECHNIQUES51 (DOI:10.1002/JMS.1059)
- 23 -
2.3 METABOLISM AND TOXICOLOGICAL DETECTION OF THE DESIGNER DRUG 4-IODO-2,5-DIMETHOXY-AMPHETAMINE (DOI) IN RAT URINE USING GAS
CHROMATOGRAPHY-MASS SPECTROMETRY52 (DOI:10.1016/J.JCHROMB.2007.06.027)
- 35 -
2.4 DESIGNER DRUG 2,5-DIMETHOXY-4-METHYL-AMPHETAMINE (DOM, STP): INVOLVEMENT OF THE CYTOCHROME P450 ISOENZYMES IN FORMATION OF
ITS MAIN METABOLITE AND DETECTION OF THE LATTER IN RAT URINE AS
PROOF OF A DRUG INTAKE USING GAS CHROMATOGRAPHY-MASS
SPECTROMETRY53 (DOI:10.1016/J.JCHROMB.2007.11.042)
- 43 -
2.5 METABOLISM AND TOXICOLOGICAL DETECTION OF THE DESIGNER DRUG
4-CHLORO-2,5-DIMETHOXYAMPHETAMINE IN RAT URINE USING GAS
CHROMATOGRAPHY-MASS SPECTROMETRY54 (DOI:10.1007/S00216-008-1917-Z)
- 51 -
2.6 2,5-DIMETHOXYAMPHETAMINE-DERIVED DESIGNER DRUGS: STUDIES ON
THE IDENTIFICATION OF CYTOCHROME P450 (CYP) ISOENZYMES
INVOLVED IN FORMATION OF THEIR MAIN METABOLITES AND ON THEIR
CAPABILITY TO INHIBIT CYP2D655 (DOI:10.1016/J.TOXLET.2008.09.014)
- 59 -
3 CONCLUSIONS
The studies presented here show that the 2,5-dimethoxyamphetamine designer
drugs DOB, DOC, DOI, MDOB, and TMA-2 were mainly metabolized by O-
demethylation, and in case of DOM by hydroxylation. Further steps were the side
chain hydroxylation and the oxidative deamination. As metabolic phase II reactions
partial glucuronidation and sulfation were observed. Furthermore, combinations of
these steps as well as minor metabolites were also detected.50-53,55
The developed screening procedures allowed the detection of the studied 2,5-
dimethoxyamphetamine in rat urine after administration of common drug abusers'
doses mainly via their metabolites.
In vitro studies showed that CYP2D6 was the only isoform catalyzing the
demethylation of the DOB, DOC, DOI, MDOB, and TMA-2 and the hydroxylation of
DOM. Besides the drugs were substrates of CYP2D6 inhibition properties on
CYP2D6 could also be observed.53,55
This detailed knowledge of the metabolic steps of designer drugs and the inhibition
potential on CYP2D6 is an important prerequisite for assessing possible interaction
with other drugs or food ingredients as well as inter-individual pharmacogenetic
differences. These pharmacogenetic differences due to poor or ultra rapid
metabolizers are shown especially by CYP2D6 which is polymorphically expressed.
Besides CYP2D6 is the only enzyme catalyzing the studied metabolic steps and is
also inhibited by the drugs, this enzyme is involved in metabolism of many
pharmaceuticals so the possibility of drug-drug interaction is given but unlikely due to
the high inhibition constants of the drugs. Whether these possible interactions are of
clinical relevance can not be concluded at the moment. However, further clinical
studies would be necessary to predict the risk of interactions. Also studies on the
pharmacology and toxicology of the metabolites together with well documented
clinical data will be necessary.
- 87 -
4 SUMMARY
In the presented studies, the metabolism and the toxicological analysis in urine of the
amphetamine-derived designer drugs DOB, DOC, DOI, DOM, MDOB and TMA-2
were investigated. Furthermore, CYP isoform dependence of the main metabolic
steps and the inhibition potential of the parent drugs on CYP2D6 were elucidated to
predict possible drug-drug interactions and influence of genetic polymorphisms. The
2,5-dimethoxyamphetamines were mainly metabolized by O-demethylation in
position 2 and 5 of the ring, respectively, and by deamination followed by reduction to
the corresponding alcohol. A further metabolic step was the hydroxylation of the side
chains. Phase II reactions consisted of partial glucuronidation and/or sulfation.
Combinations of these steps could also be detected. The target analytes for the
toxicological analysis were the derivatized hydroxy metabolite of DOM or the O-
demethyl metabolites of DOB, DOC, DOI, MDOB and TMA-2. CYP2D6 was identified
to be the only CYP isoform involved in the main metabolic steps. In addition, the
studied drugs act also as inhibitor of CYP2D6. It could be shown, that the mode of
inhibition of none of the studied drugs was irreversible, but competitive for all drugs.
- 89 -
5 REFERENCES
1. Wax PM. Just a click away: recreational drug Web sites on the Internet. Pediatrics 2002; 109: e96
2. Shulgin A, Pihkal, A Chemical Love Story, 1st Transform Press: Berkley (CA) 1991; 3. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Report on the
risk assessment of TMA-2 in the framework of the joint action on new synthetic drugs. http://www. emcdda. europa. eu/attachements. cfm/att_33358_EN_Risk7. pdf. Accessed September 30, 2008. 2003
4. United Nations. UN Conventions - List of substances in the schedules. 1971; E.78.XI.3. 5. Drug Enforcement Administration - Office of Forensic Sciences. LSD Blotter Acid Mimic
Containing 4-Bromo-2,5-Dimethoxy-Amphetamine (DOB) Seized Near Burns, Oregon. Microgram 2005; 38: 147
6. Drug Enforcement Administration - Office of Forensic Sciences. LSD Blotter Acid Mimics (Containing 4-Bromo-2,5-Dimethoxy-Amphetamine (DOB)) In Ames, Iowa. Microgram 2006; 39: 145
7. Drug Enforcement Administration - Office of Forensic Sciences. Blotter Acid Mimic (Containing 4-Bromo-2,5-Dimethoxy-Amphetamine (DOB)) In Concord, California. Microgram 2006; 39: 136
8. Drug Enforcement Administration - Office of Forensic Sciences. Blotter Acid Mimic (Containing 4-Bromo-2,5-Dimethoxy-Amphetamine (DOB)) In Concord, California. Microgram 2007; 40: 24
9. Buhrich N, Morris G, Cook G. Bromo-DMA: the Australasian hallucinogen? Aust. N. Z. J. Psychiatry 1983; 17: 275.
10. Furnari C, Ottaviano V, Rosati F. [Determination of 4-bromo-2,5-dimethoxyamphetamine (DOB) found in illicit tablets seized in Italy]. Ann. Ist. Super. Sanita 2001; 37: 297.
11. Drug Enforcement Administration - Office of Forensic Sciences. LSD blotter acid mimics (containing 2,5-dimethoxy-4-chloro-amphetamine (DOC)) in Boca Raton, Florida. Microgram 2006; 39: 72
12. Drug Enforcement Administration - Office of Forensic Sciences. LSD Blotter Acid Mimic (Containing 4-Iodo-2,5-Dimethoxy-Amphetamine (DOI)) In Orlando And Winter Springs, Florida. Microgram 2006; 39: 55
13. Drug Enforcement Administration - Office of Forensic Sciences. Intelligence alert: 4-chloro-2,5-dimethoxyamphetamine (DOC) and 4-iodo-2,5-dimethoxyamphetamine (DOI) in Berkley, Michigan. Microgram 2006; 39: 137
14. Drug Enforcement Administration - Office of Forensic Sciences. LSD Blotter Acid Mimics Containing 4-Chloro-2,5-Dimethoxy-Amphetamine (DOC)) In Concord, California. Microgram 2007; 40: 110
15. Drug Enforcement Administration - Office of Forensic Sciences. 4-Iodo-2,5-Dimethoxy-Amphetamine (DOI) In Madison, Wisconsin. Microgram 2007; 40: 89
16. Drug Enforcement Administration - Office of Forensic Sciences. 4-Chloro-2,5-dimethoxyamphetamine (DOC) in Santa Fe, New Mexico. Microgram 2007; 40: 93
17. Drug Enforcement Administration - Office of Forensic Sciences. Aqueous solutions of 4-chloro-2,5-dimethoxyamphetamine (DOC) in Tampa, Florida. Microgram 2007; 40: 42
18. Drug Enforcement Administration - Office of Forensic Sciences. LSD Blotter Mimic (Actually Containing A Mixture Of 4-Chloro-2,5-Dimethoxy-Amphetamine (DOC) And 4-Iodo-2,5-Dimethoxy-Amphetamine (DOI)) In Paducah, Kentucky. Microgram 2008; 41: 28
19. Drug Enforcement Administration - Office of Forensic Sciences. LSD Blotter Mimics (Actually Containing A Mixture Of 4-Chloro-2,5-Dimethoxy-Amphetamine (DOC) And 4-Iodo-2,5-Dimethoxy-Amphetamine (DOI)) In Lantana, Florida. Microgram 2008; 41: 53
- 91 -
20. Acuna-Castillo C, Villalobos C, Moya PR, Saez P, Cassels BK, Huidobro-Toro JP. Differences in potency and efficacy of a series of phenylisopropylamine/phenylethylamine pairs at 5-HT(2A) and 5-HT(2C) receptors. Br. J. Pharmacol. 2002; 136: 510.
21. Glennon RA, Titeler M, McKenney JD. Evidence for 5-HT2 involvement in the mechanism of action of hallucinogenic agents. Life Sci. 1984; 35: 2505.
22. Glennon RA, McKenney JD, Lyon RA, Titeler M. 5-HT1 and 5-HT2 binding characteristics of 1-(2,5-dimethoxy-4-bromophenyl)-2-aminopropane analogues. J. Med. Chem. 1986; 29: 194.
23. Glennon RA, Titeler M, Seggel MR, Lyon RA. N-methyl derivatives of the 5-HT2 agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane. J. Med. Chem. 1987; 30: 930.
24. Glennon RA, Raghupathi R, Bartyzel P, Teitler M, Leonhardt S. Binding of phenylalkylamine derivatives at 5-HT1C and 5-HT2 serotonin receptors: evidence for a lack of selectivity. J. Med. Chem. 1992; 35: 734.
25. Monte AP, Marona-Lewicka D, Parker MA, Wainscott DB, Nelson DL, Nichols DE. Dihydrobenzofuran analogues of hallucinogens. 3. Models of 4-substituted (2,5-dimethoxyphenyl)alkylamine derivatives with rigidified methoxy groups. J. Med. Chem. 1996; 39: 2953.
26. Body S, Cheung TH, Bezzina G, Asgari K, Fone KC, Glennon JC, Bradshaw CM, Szabadi E. Effects of d-amphetamine and DOI (2,5-dimethoxy-4-iodoamphetamine) on timing behavior: interaction between D1 and 5-HT2A receptors. Psychopharmacology (Berl) 2006; 189: 331.
27. Dimpfel W, Spuler M, Nichols DE. Hallucinogenic and stimulatory amphetamine derivatives: fingerprinting DOM, DOI, DOB, MDMA, and MBDB by spectral analysis of brain field potentials in the freely moving rat (Tele-Stereo-EEG). Psychopharmacology (Berl) 1989; 98: 297.
28. DeRuiter J, Clark CR, Noggle FT. LC and GC-MS analysis of 4-bromo-2,5-dimethoxyphenethylamine (Nexus) and 2-propanamine and 2-butanamine analogs. J. Chromatogr. Sci. 1995; 33: 583.
29. Boatto G, Nieddu M, Carta A, Pau A, Palomba M, Asproni B, Cerri R. Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2005; 814: 93.
30. Battu C, Marquet P, Fauconnet AL, Lacassie E, Lachatre G. Screening procedure for 21 amphetamine-related compounds in urine using solid-phase microextraction and gas chromatography-mass spectrometry. J. Chromatogr. Sci. 1998; 36: 1.
31. Balikova M. Nonfatal and fatal DOB (2,5-dimethoxy-4-bromoamphetamine) overdose. Forensic Sci. Int. 2005; 153: 85.
32. Heydari A, Yeo KR, Lennard MS, Ellis SW, Tucker GT, Rostami-Hodjegan A. Mechanism-based inactivation of CYP2D6 by methylenedioxymethamphetamine. Drug Metab. Dispos. 2004; 32: 1213.
33. de la Torre R, Farre M, Mathuna BO, Roset PN, Pizarro N, Segura M, Torrens M, Ortuno J, Pujadas M, Cami J. MDMA (ecstasy) pharmacokinetics in a CYP2D6 poor metaboliser and in nine CYP2D6 extensive metabolisers. Eur. J. Clin. Pharmacol. 2005; 61: 551.
34. Yang J, Jamei M, Heydari A, Yeo KR, de la Torre R, Farre M, Tucker GT, Rostami-Hodjegan A. Implications of mechanism-based inhibition of CYP2D6 for the pharmacokinetics and toxicity of MDMA. J. Psychopharmacol. 2006; 20: 842.
35. Ramamoorthy Y, Yu AM, Suh N, Haining RL, Tyndale RF, Sellers EM. Reduced(+/-)-3,4-methylenedioxymethamphetamine ("Ecstasy") metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro. Biochem. Pharmacol. 2002; 63: 2111.
36. Tucker GT, Lennard MS, Ellis SW, Woods HF, Cho AK, Lin LY, Hiratsuka A, Schmitz DA, Chu TY. The demethylenation of methylenedioxymethamphetamine ("ecstasy") by debrisoquine hydroxylase (CYP2D6). Biochem. Pharmacol. 1994; 47: 1151.
- 92 -
37. Ono S, Hatanaka T, Hotta H, Satoh T, Gonzalez FJ, Tsutsui M. Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 1996; 26: 681.
38. Crespi CL, Penman BW. Use of cDNA-expressed human cytochrome P450 enzymes to study potential drug-drug interactions. Adv. Pharmacol. 1997; 43: 171.
39. Rendic S, Di Carlo FJ. Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. Drug Metab. Rev. 1997; 29: 413.
40. Sesardic D, Boobis AR, Edwards RJ, Davies DS. A form of cytochrome P450 in man, orthologous to form d in the rat, catalyses the O-deethylation of phenacetin and is inducible by cigarette smoking. Br. J. Clin. Pharmacol. 1988; 26: 363.
41. Distlerath LM, Reilly PE, Martin MV, Davis GG, Wilkinson GR, Guengerich FP. Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J. Biol. Chem. 1985; 260: 9057.
42. Ortiz-de-Montellano PR, Cytochrome P450 - Structure, Mechanism, and Biochemistry, 3rd Kluwer Academic/Plenum Publishers: New York 2005
43. Aktories K, Förstermann U, Hofmann F, Starke K, Allgemeine und spezielle Pharmakologie und Toxikologie, 9 Urban & Fischer: München 2004;
44. Crespi CL, Miller VP. The use of heterologously expressed drug metabolizing enzymes-state of the art and prospects for the future. Pharmacol. Ther. 1999; 84: 121.
45. Maurer HH. Screening procedures for simultaneous detection of several drug classes used in the high throughput toxicological analysis and doping control [review]. Comb. Chem. High Throughput Screen. 2000; 3: 461.
46. Maurer HH. Screening for Drugs in Body Fluids by GC/MS. In Yinon J (ed). Advances in Forensic Applications of Mass Spectrometry, CRC Press LLC: Boca Raton (FL) 2003; 1.
47. Krishnan S, Moncrief S. An evaluation of the cytochrome p450 inhibition potential of lisdexamfetamine in human liver microsomes. Drug Metab. Dispos. 2007; 35: 180.
48. Evans WE, McLeod HL. Pharmacogenomics-drug disposition, drug targets, and side effects. N. Engl. J. Med. 2003; 348: 538.
49. Howard LA, Sellers EM, Tyndale RF. The role of pharmacogenetically-variable cytochrome P450 enzymes in drug abuse and dependence. Pharmacogenomics 2002; 3: 185.
50. Ewald AH, Fritschi G, Bork WR, Maurer HH. Designer drugs 2,5-dimethoxy-4-bromoamphetamine (DOB) and 2,5-dimethoxy-4-bromomethamphetamine (MDOB): Studies on their metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques. J. Mass Spectrom. 2006; 41: 487.
51. Ewald AH, Fritschi G, Maurer HH. Designer drug 2,4,5-trimethoxyamphetamine (TMA-2): Studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric techniques. J. Mass Spectrom. 2006; 41: 1140.
52. Ewald AH, Fritschi G, Maurer HH. Metabolism and toxicological detection of the designer drug 4-iodo-2,5-dimethoxy-amphetamine (DOI) in rat urine using gas chromatography-mass spectrometry. J Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007; 857: 170.
53. Ewald AH, Puetz M, Maurer HH. Designer drug 2,5-dimethoxy-4-methyl-amphetamine (DOM, STP): Involvement of the cytochrome P450 isoenzymes in formation of its main metabolite and detection of the latter in rat urine as proof of a drug intake using gas chromatography-mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2008; 862: 252.
54. Ewald AH, Ehlers D, Maurer HH. Metabolism and toxicological detection of the designer drug 4-chloro-2,5-dimethoxyamphetamine in rat urine using gas chromatography-mass spectrometry. Anal. Bioanal. Chem. 2008; 390: 1837.
55. Ewald AH, Maurer HH. 2,5-Dimethoxyamphetamine-derived designer drugs: Studies on the identification of cytochrome P450 (CYP) isoenzymes involved in formation of their
- 93 -
main metabolites and on their capability to inhibit CYP2D6. Toxicol. Lett. 2008; DOI:10.1016/j.toxlet.2008.09.014
- 94 -
6 ABBREVIATIONS
DOB 4-bromo-2,5-dimethoxyamphetamine
DOC 4-chloro-2,5-dimethoxyamphetamine
DOI 4-iodo-2,5-dimethoxyamphetamine
DOM 2,5-dimethoxy-4-methylamphetamine
MDOB 4-bromo-2,5-dimethoxymethamphetamine
TMA-2 2,4,5-trimethoxyamphetamine
CYP cytochrome P450
GC-MS gas chromatography-mass spectrometry
LC-MS liquid chromatography-mass spectrometry
FAD flavin adenine dinucleotide
5-HT 5-hydroxytryptamine (serotonin)
cDNA copy deoxyribonucleic acid
Km substrate concentration at half of the maximal turnover rate
MDA 3,4-methylenedioxyamphetamine
MDMA 3,4-methylenedioxymethamphetamine
OR oxidoreductase
PMA para-Methoxyamphetamine
PMMA para-Methoxymethamphetamine
Vmax maximal turnover rate
- 95 -
7 ZUSAMMENFASSUNG
Im Rahmen dieser Dissertation wurden der Metabolismus und die Nachweisbarkeit
der neuen Designerdrogen des Amphetamin-Typs DOB, DOC, DOI, DOM, MDOB
und TMA-2 im Urin untersucht. Um mögliche Interaktionen oder Einflüsse
genetischer Polymorphismen abzuschätzen, wurde darüber hinaus untersucht,
welche Cytochrom P450 (CYP) Isoenzyme die Hauptmetabolismusschritte
katalysieren und ob die Drogen CYP2D6 inhibieren können. Die 2,5-
Dimethoxyamphetamine wurden hauptsächlich durch O-Demethylierung in Position 2
bzw. 5 des Ringes oder durch Deaminierung gefolgt von Reduktion zum
entsprechenden Alkohol metabolisiert. Weitere Metabolismusschritte waren die
Seitenkettenhydroxylierung. Als Phase-II-Reaktionen konnten partielle
Glucuronidierung oder Sulfatierung gefunden werden. Metabolite aus Kombinationen
dieser Schritte konnten ebenso detektiert werden. Die derivatisierten Metabolite der
Drogen aus den Hauptstoffwechselwegen (O-Demethylierung bei DOB, DOC, DOI,
MDOB und TMA-2; Hydroxylierung bei DOM) waren die Zielsubstanzen im
toxikologischen Nachweisverfahren. Für CYP2D6 konnte als einzige Cytochrom
P450 Isoform eine Beteiligung an den Hauptstoffwechselwegen nachgewiesen
werden. Neben der Verstoffwechselung der untersuchten Substanzen durch
CYP2D6 konnten auch eine Hemmung dieses Enzyms durch diese Substanzen
beobachtet werden. Es wurde gezeigt, dass keine der untersuchten Substanzen eine
irreversible sondern eine kompetitive Hemmung bewirkte.
- 97 -