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| Journal of the New Zealand Medical Association,
24-April-2009, Vol 122 No 1293
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Party pills and drug-drug interactions
Meghan Murphy, Ushtana Antia, Hsin-Yao Chang, Jae Young Han,
Abir Ibrahim, Malcolm Tingle, Bruce Russell
Party pills are commonly used recreational drugs in New
Zealand.1 Benzylpiperazine (BZP) and
trifluoromethylphenylpiperazine (TFMPP) are amphetamine-like compounds and the
active ingredients of the majority of party pills sold in New Zealand (Figure
1).
Figure 1. Chemical structure of BZP (top left),
TFMPP (top right), MDMA (bottom left), and amphetamine (bottom
right)
 Together BZP and TFMPP have been reported to exert effects
similar to methyldioxymethamphetamine2 (MDMA,
Ecstasy) in rodents (Figure 1). Despite their availability and liberal use, the
metabolism of BZP and TFMPP, and their ability to alter the metabolism of other
drugs in the human body is not well understood.
CYP isoenzymes CYP2D6, CYP3A4, and CYP1A2 seem to play a
role in the metabolism of TFMPP.3 It has been
predicted that BZP would be metabolised in a similar manner due to the
structural similarities between the two compounds. Therefore, both TFMPP and BZP
might interact with substrates of CYP2D6, CYP3A4, and CYP1A2 in the clinical
setting.
It is important to investigate the possibility of drug-drug
interactions because severe adverse effects may arise secondary to increased
levels of a co-administered drug. For example, increased levels of the
anti-depressant fluoxetine (a CYP2D6 substrate) may be associated with the
serotonin syndrome.4
The substrates used as probes for a particular CYP reaction
were selected as they are selectively metabolised by a single CYP isoenzyme.
While a drug-drug interaction involving a probe substrate (such as
dextromethorphan for CYP2D6) is unlikely to be problematic in the clinical
setting, the data obtained can be extrapolated to include other CYP2D6
substrates. This approach has been used
previously5,6 to explore drug-drug interactions
and allows for multiple CYP isoenzyme reactions to be studied and produces
results applicable to several commonly used drugs.
The aim of this study is to determine the effects of BZP and
TFMPP on the metabolism of commonly used drugs. However, it is important to
consider that a drug-drug interaction may influence the pharmacokinetics not
only of the substrate, but also that of BZP/TFMPP. Hence, it is important to
take into account the potential adverse
effects.7
MethodMaterialsBZP, TFMPP, dextromethorphan, caffeine,
ethinyloestradiol, omeprazole, phenytoin, quinidine, troleandomycin (TAO),
tetrahydrofuran (THF), perchloric acid, and sodium dihydrogen phosphate were
obtained from Sigma-Aldrich, USA. Acetonitrile (ACN), sodium dihydrogen
phosphate, and methanol were obtained from Scharlau Chemie, Spain. NADPH was
sourced from Applichem, Denmark. Furafylline was purchased from Ultrafine
Chemicals, UK. Proguanil was obtained from Astra Zeneca, UK and ammonium formate
was obtained from Acros Organics, USA.
Human liver microsome
preparation—With approval from the Northern X Regional Ethics
Committee, six livers were collected from human donors at Auckland City Hospital
(Table 1). Portions of the livers were rinsed in ice-cold phosphate buffer and
blotted dry. Livers were then homogenised in 67 mM phosphate buffer containing
1.15% potassium chloride. The volume of the buffer used was three times the
weight of the liver to provide a 25% homogenate. This homogenate was centrifuged
at 10,000 g for 20 minutes and the supernatant removed and centrifuged at
105,000 g for a further hour. The pellet was gently rinsed with phosphate buffer
and re-suspended in a small volume (2–5 mL) of phosphate
buffer8 and aliquots were stored at
–80°C until use. An aliquot of the suspension (~200 µL) was
removed for protein determination.5
HPLC—The chosen HPLC system
included an Agilent 1100 series binary pump, an Agilent series 1100 Multiple
Wavelength detector (UV), an Agilent series 1100 Autosampler, and an Agilent
Extend reverse-phase C18 column (150×4.6 mm, 5 μm).
Table 1. Summary of liver donor details used is
the HLM (human liver microsome) preparation. Each liver sample was received
after liver resection for colorectal cancer metastasis
Study designDue to the paucity of information about drug
interactions with BZP and TFMPP, this study aimed to screen a wide range of
potential drug interactions in vitro. To confirm that specific
isoenzymes are involved in a drug interaction, probe drugs (or substrates whose
metabolism is carried out chiefly by one isoenzyme) were co-incubated with
selective isoenzyme inhibitors and BZP or TFMPP. Furthermore, the drugs chosen
are commonly used drugs relevant to the demographic of people consuming party
pills. For example ethinyloestradiol is present in numerous combined oral
contraceptives.
In the inhibition assays, the substrate, inhibitor (BZP
or TFMPP), NADPH, buffer, and the protein (pooled liver microsomes) were
co-incubated at 37°C for 20 minutes, after which the reaction was stopped.
Excess NADPH was added to each inhibition assay to ensure that any reduction in
metabolism was due to inhibition and not simply due to lack of an energy
generating source.
The inhibitors (BZP or TFMPP) were dissolved in
phosphate buffer which was pH adjusted to improve solubility. Organic solvents
were avoided due to the potential for confounding inhibitory effects. The liver
microsome concentrations used were based on methods found in the literature and
optimised for the metabolism of each probe
substrate.8–11
All microsomal incubations without inhibitors resulted
in a limited amount of metabolism (10–20%) so that inhibitory effects of
any additional compounds could be observed. The rate of disappearance of each
substrate was measured at the end of the 20-minute incubation.
There are no published studies demonstrating plasma or
liver concentrations of BZP and TFMPP. Therefore, a wide range of concentrations
was used (2, 20, and 200 μM), with the highest concentration of inhibitor
corresponding to the substrate concentration. Results of inhibition studies were
confirmed using pooled human liver microsomes (n=6). Positive and negative
controls were included in each set of the inhibition assays.
Incubation conditionsIncubation mixtures (n=6) contained pooled liver
microsomes (20 μL; 2 mg/mL), NADPH (20 μL; 10 mM), the substrate (20
μL; 1 mM), the proposed inhibitor (20 μL; BZP, or TFMPP 0, 2, 20, or
200 μM, final concentration), which was made up to volume using phosphate
buffer (to 100 μL; 67 mM, pH 7.4). The samples were then incubated at
37°C in a water bath for 20 minutes and the reactions were terminated by
the addition of perchloric acid (5 μL) and vortex-mixing. The samples were
stored at -20ºC for 1 hour, thawed, then centrifuged at 10,000 rpm for 5
minutes and the supernatant used for analysis. Enzyme selective positive and
negative controls were included (Table 2).
Analysis methodDextromethorphan (CYP2D6)—10 mM
formate buffer (pH 4.5) and ACN were used as the mobile phase with a flow rate
of 1 mL/min. A gradient of 90:10 v/v (formate buffer: ACN) to 20:80 v/v was
used. Substrate detection was carried out at 231 nm (UV) and the injection
volume was 20 μL.
Caffeine (CYP1A2)—Formate buffer
(pH 4.0, 1% methanol, 1% ACN, 1.6% THF) and 100% ACN were used as the mobile
phase with a flow rate of 1 mL/min. A gradient of 100:0 v/v (formate buffer:
ACN) to 20:80 v/v was used. Detection of substrate was carried out at 224 nm
(UV) and the injection volume was 10 μL.
Ethinyloestradiol
(CYP3A4)—Milli-Q water and 100% ACN were used as the mobile phase
with a flow rate of 1 mL/min. A gradient of 55:45 v/v (milli-Q water: ACN) to
20:80 v/v was used. Detection of substrate was carried out at 212 nm and the
injection volume was 10 μL.
Omeprazole (CYP2C19)—Formate
buffer (pH 4.0, 1% methanol, 1% ACN, 1.6% THF) and 100% ACN were used as the
mobile phase with a flow rate of 1.100mL/min. A gradient of 90:10v/v (formate
buffer: ACN) to 20:80v/v was used. Detection of the substrate was carried out at
224nm and the injection volume was 10μL.
Table 3. Calculated precision and accuracy for
each substrate assay; data produced from six repeats
Statistical analysis—Single
factor ANOVA was used to compare differences in the metabolism of the substrate
between the various inhibitors and control (no inhibitor). P<0.05 was
statistically significant.
ResultsMetabolism observed in the absence of an inhibitor (the
control) was set at 100%, with metabolism in the assays calculated as a
percentage of the control.
TFMPP significantly inhibited of the metabolism of
dextromethorphan in a concentration-dependent manner (Figure 2). Significant
inhibition (p<0.001) was only observed at the highest concentration (200
μM) of BZP used.
Figure 2. Effect of
BZP and TFMPP on the metabolism of 200 µM dextromethorphan (a CYP2D6
substrate) in pooled human liver microsomes
 Note: Furafylline was used as the
negative control; quinidine as the positive control. Values shown are mean
± standard deviation, n=6.
Caffeine metabolism was only inhibited by the two highest
concentrations of BZP and TFMPP (p<0.001) (Figure 3).
Figure 3. Effect of BZP and TFMPP on the
metabolism of 200 µM caffeine (a CYP1A2 substrate)
 Note: Quinidine was used as the
negative control; furafylline as the positive control. Values shown are mean
± standard deviation, n=6.
Only the highest concentration of TFMPP significantly
inhibited the metabolism of ethinyloestradiol (P<0.001) (Figure 4). Results
with the higher concentrations of BZP were also significant but, only a minor
reduction metabolism was observed. Neither BZP nor TFMPP inhibited the
metabolism of omeprazole. (Figure 5).
Figure 4. Effect of BZP and TFMPP on the
metabolism of 200 µM ethinyloestradiol (a CYP3A4 substrate)
 Note: Quinidine was used as the
negative control; TAO (troleandomycin) as the positive control. Values shown are
mean ± standard deviation, n=6.
 Note: Quinidine was used as the
negative control; proguanil as the positive control. Values shown are mean
± standard deviation, n=6.
DiscussionTFMPP has been reported to be predominantly metabolised by
CYP2D6 and to a lesser degree by CYP1A2 and CYP3A4.3
Therefore, TFMPP could potentially affect the metabolism of other CYP2D6,
CYP1A2, and CYP3A4 substrates.
The results obtained from the human microsomal incubation
assays used in this study revealed potentially significant interactions between
TFMPP and BZP with dextromethorphan (a known CYP2D6 substrate) and caffeine (a
known CYP1A2 substrate). TFMPP also had an inhibitory effect on the metabolism
of ethinyloestradiol (a known CYP3A4 substrate).
The results from this research consistently indicate that
TFMPP is a more potent inhibitor than BZP of the metabolism of dextromethorphan,
caffeine, and ethinyloestradiol. This is also in agreement with data from
published literature, suggesting that BZP is excreted mostly unchanged in the
urine, whereas the metabolism of TFMPP principally involves the CYP
isoenzymes.3,12
The magnitude of the inhibitory effects of BZP and TFMPP
must not be dismissed because CYP2D6, CYP1A2, and CYP3A4 are involved in the
metabolism of a wide range of drugs. Consequently many of the drugs frequently
co-ingested with party pills could be poorly metabolised and in turn lead to
higher than expected concentrations and consequences of clinical
significance.13
For example, because the metabolism of dextromethorphan was
altered, similar interactions with other CYP2D6 substrates such as paroxetine (a
selective serotonin re-uptake inhibitor), or imipramine (a tricyclic
antidepressant) can be predicted.10,11 Higher
than expected plasma concentrations of these drugs in the human body can have
severe adverse effects. Inhibiting the metabolism of paroxetine can effectively
produce an overdose leading to the serotonin syndrome—a potentially fatal
condition characterised by hypomania, confusion, hallucinations, agitation,
myoclonus, nausea, diarrhoea, tachycardia, and
coma.16
Interestingly the only fatalities associated with BZP-use
occurred with simultaneous consumption of MDMA
(Ecstasy).17 The finding that BZP can inhibit
CYP2D6 may account for toxicity due to increased MDMA levels because MDMA is
also metabolised by CYP2D6.18
Consideration must also be given to the possibility of
synergistic pharmacodynamic interactions secondary to pharmacokinetic
interactions. For example, BZP also has serotonergic
effects.2 Continuing with the example of
elevated paroxetine levels secondary to CYP2D6 inhibition, the potential for
adverse effects is greater again because both of compounds enhance serotonergic
activity.
In view of the circumstances under which party pills are
consumed, caffeine becomes a relevant substrate. Many alcoholic drinks served at
bars and clubs are caffeinated. In addition, caffeine is an ingredient of many
medicines, for example Panadol Extra®.19
Caffeine is a useful probe substrate for CYP1A2 and results of this study
could also be used to predict interactions between BZP and TFMPP and other
CYP1A2 substrates such as theophylline (a bronchodilator used in chronic
obstructive pulmonary disease and asthma) and olanzapine (an atypical
antipsychotic), which are metabolised by this isoenzyme to a significant
degree.20
Caffeine overdose can lead to hypokalaemia and agitation;
however, very high levels of caffeine would need to be
achieved.21 Increased serum concentrations of
olanzapine can cause tachycardia, severe central nervous system depression, and
may be associated with the potentially fatal neuroleptic malignant
syndrome.22
Using ethinyloestradiol as a probe substrate, BZP and TFMPP
demonstrated inhibitory effects on CYP3A4. Increased serum concentrations of
ethinyloestradiol itself can lead to serious adverse effects such as an
increased risk of thromboembolism.23 Once again
this result is applicable to other CYP3A4 substrates such as carbamazepine which
even at therapeutic concentrations can cause adverse effects such as drowsiness
and leucopaenia, or in the case of an elevated plasma concentration,
agranulocytosis.24,22
Drug-drug interactions via CYP isoenzymes may partly explain
some of the adverse effects reported following ingestion of BZP such as
insomnia, tachycardia, agitation, and
psychosis.1,7,26 These adverse effects may not
be solely due to increased levels of BZP but might be due to inhibition of its
metabolism by co-ingested drugs.
Interactions observed with CYP2D6, CYP1A2, and CYP3A4 in
this study are in agreement with our previous work in which quinidine (a CYP2D6
inhibitor), furafylline (a CYP1A2 inhibitor), and TAO (a CYP3A4 inhibitor) also
reduced the metabolism of BZP.27
Taking these two studies together, it is apparent that BZP
and dextromethorphan inhibit the metabolism of each other and co-ingestion of
BZP and dextromethorphan may result in a variety of adverse effects because of
the increased plasma concentration of both drugs.
These results also demonstrate that TFMPP and BZP may have a
pharmacokinetic interaction with each other. This finding is of a great
importance because BZP and TFMPP are frequently taken together.
A significant limitation of this research is that the
expected plasma concentrations of BZP and TFMPP were unknown before these
experiments began because there were no published pharmacokinetic trials in
humans using either drug. Furthermore, variables such as the
Ki of BZP and TFMPP, and the degree of their
protein binding, are unknown which makes it difficult to use incubation
parameters that accurately reflect in vivo parameters.
It is feasible to obtain Cmax for most drugs from blood
samples, however free drug concentrations in the liver can only be obtained by
liver biopsies which are undesirable due to their invasive nature.
This study confirms the possibility of drug-drug
interactions involving party pills and concomitant medications metabolised by
CYP2D6, CYP1A2, and CYP3A4; the severity of these interactions warrants further
research. Despite the best efforts of clinicians to screen for and prevent
drug-drug interactions, these adverse events may still occur in patients using
party pills.
Many people might not consider party pills as illicit
substances and are unlikely to report their use if asked about illicit drugs
during the recording of a medication history. Therefore it is recommended that
recording a thorough drug history includes not only an assessment of alcohol
intake, smoking habits, and illicit drug-use, but also specific questions
targeting party pill-use.
Competing interests: None known.
Author information: Meghan Murphy,
Pharmacist and Masters Student, School of Pharmacy; Ushtana Antia, PhD Student,
School of Pharmacy; Hsin-Yao Chang, Pharmacy Student, School of Pharmacy; Abir
Ibrahim, Pharmacy Student, School of Pharmacy; Jae Young Han, Pharmacy Student,
School of Pharmacy; Malcolm Tingle, Senior Lecturer in Toxicology, Department of
Pharmacology; Bruce Russell, Senior Lecturer in Pharmacotherapy, School of
Pharmacy; University of Auckland
Correspondence: Bruce Russell, Senior
Lecturer in Pharmacotherapy, School of Pharmacy, University of Auckland. Private
Bag 92019, Auckland, New Zealand. Email: b.russell@auckland.ac.nz
Reference:
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