Benzylpiperazine: “A messy drug”

Drug and Alcohol Dependence 164 (2016) 1–7

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Review

Benzylpiperazine: “A messy drug” D.P. Katz, J. Deruiter, D. Bhattacharya, M. Ahuja, S. Bhattacharya, C.R. Clark, V. Suppiramaniam, M. Dhanasekaran ∗ Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, United States

a r t i c l e

i n f o

Article history: Received 8 October 2015 Received in revised form 1 April 2016 Accepted 3 April 2016 Available online 6 May 2016 Keywords: Piperazine derivatives Benzylpiperazine Designer drugs Monoaminergic toxicity Toxicokinetics Substances of abuse

a b s t r a c t Designer drugs are synthetic structural analogues/congeners of controlled substances with slightly modified chemical structures intended to mimic the pharmacological effects of known drugs of abuse so as to evade drug classification. Benzylpiperazine (BZP), a piperazine derivative, elevates synaptic dopamine and serotonin levels producing stimulatory and hallucinogenic effects, respectively, similar to the wellknown drug of abuse, methylenedioxymethamphetamine (MDMA). Furthermore, BZP augments the release of norepinephrine by inhibiting presynaptic autoreceptors, therefore, BZP is a “messy drug” due to its multifaceted regulation of synaptic monoamine neurotransmitters. Initially, pharmaceutical companies used BZP as a therapeutic drug for the treatment of various disease states, but due to its contraindications and abuse potential it was withdrawn from the market. BZP imparts predominately sympathomimetic effects accompanied by serious cardiovascular implications. Addictive properties of BZP include behavioral sensitization, cross sensitization, conditioned place preference and repeated selfadministration. Additional testing of piperazine derived drugs is needed due to a scarcity of toxicological data and widely abuse worldwide. © 2016 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Perception of safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Patterns of use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Toxicokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 BZP/TFMPP combination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Health impact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Addictive properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Detection and quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1. Introduction

∗ Corresponding author. E-mail addresses: [email protected] (D.P. Katz), [email protected] (J. Deruiter), [email protected] (D. Bhattacharya), [email protected] (M. Ahuja), [email protected] (S. Bhattacharya), [email protected] (C.R. Clark), [email protected] (V. Suppiramaniam), [email protected] (M. Dhanasekaran). http://dx.doi.org/10.1016/j.drugalcdep.2016.04.010 0376-8716/© 2016 Elsevier Ireland Ltd. All rights reserved.

Piperazine derivatives are a group of chemically modified designer drugs derived from piperazine, a six-membered ring with two oppositely positioned nitrogen atoms (Fig. 1a). The name designer drug was first created in the mid-1980s by Dr. Gary Henderson at the University of California for psychoactive compounds which are suitable for educational purposes. Piperazinic derivatives are divided into two classes, benzylpiperazines and phenylpiper-

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D.P. Katz et al. / Drug and Alcohol Dependence 164 (2016) 1–7

Fig. 1. Chemical structures of (a) piperazine, (b) benzylpiperazine, (c) TFMPP, (d) 1(3,4-methylenedioxybenzyl)piperazine (MDBP), (e) 1-(3-chlorophenyl)piperazine (mCPP) and (f) 1-(4-methoxyphenyl)piperazine (MeOPP).

azines. The benzylpiperazines include N-benzylpiperazine (BZP) (Fig. 1b) and 1-(3,4-methylenedioxybenzyl)-piperazine (MDBP), the methylenedioxy analogue (Fig. 1d). Common phenylpiperazines abused are 1-(3-trifluoromethylphenyl) piperazine (TFMPP, Fig. 1c), 1-(3-chlorophenyl) piperazine (mCPP, Fig. 1e), and 1-(4-methoxyphenyl) piperazine (MeOPP) (Fig. 1f). Chemical modification of piperazine compounds enables clandestine manufacturers to avoid governmental bans and promotes widespread distribution under the pseudonyms “Rapture”, “Frenzy”, “Bliss”, “Charge”, “Herbal ecstasy”, “A2”, “Legal X”, and “Legal E” (Arbo et al., 2012). Other than the piperazines derivatives, the designer drugs also include cathinones (MDPV, mephedrone, methylone), synthetic cannabinoids, tryptamines and other botanical formulations. To make things much worse, numerous unrestricted and autonomous internet sites are significantly devoted to reveal the “fun/delightful” actions of these designer drugs. The information’s provided are very attractive and inquisitive to the common public, which increases the interest in these designer substances leading to the abuse. However, the data provided are dubious and misjudged by the public. The word “legal” used in the designer drugs has been misunderstood by the common public as a harmless substance that gives pleasure. This misinterpreted concept on designer drugs can lead to the destruction of the future generation (Corazza et al., 2014; Iversen et al., 2014). Substances were deliberately manufactured as designer drugs to induce the abuse potential similar to MDMA and amphetamines. Primarly their pharmacological actions were targeting monoamine release, transporters, reuptake and the receptors. Major monoaminergic neurotransmitters affected are dopamine, norepinephrine and serotonin. Based on their structure, the designer drugs have differential and selective effect on dopamine, norepinephrine and serotonin release and neurotransmission. Originally, BZP was synthesized by Burroughs, Wellcome & Co. of Wellcome Research Laboratories in the United Kingdom. BZP was tested as an anti-helminthic agent for the treatment of intestinal roundworm infestations (Haroz and Greenberg, 2006), but piperazine was preferred because of greater efficacy and fewer side effects (Gee et al., 2005; Johnstone et al., 2007). In the 1970’s, BZP was examined as a potential antidepressant, but was not pursued due to abuse potential (Bye et al., 1973). In late 1990s, New Zealand youth popularized the legal party drug, seeking its stimulatory effects (confidence, talkativeness, euphoria, vigor, activity and enhanced socialization), and as a result, its use spread rampantly

among New Zealand residents, due to a failure of regulation. In 2007, an estimated 5 million BZP pills were sold in New Zealand (Gee and Fountain, 2007). The majority of epidemiological and pharmacotoxicological data, including patterns of use, motivations and positive and adverse effects, pertaining to BZP use, emanates from New Zealand during 2000–2008 (Cohen and Butler, 2011). Students and workers, such as shift workers and truck drivers, abused the drug to increase alertness and enhance their physical and mental performance (Butler and Sheridan, 2007). Also, because of its anorectic properties, BZP was abused as an appetite suppressant among young women (Wilkins et al., 2006). BZP was also exploited in the performance enhancement arena, particularly the horse racing industry (Barclay, 2003) and athletics (Molly, 2005), but has since been prohibited. Based on various incidences, European Union formally and publicly warned regarding the new designer drugs exploitation particularly among youths. The warning clearly expressed its concerns regarding the use in humans, retail trade by means of an alternate but a fabricated and deceitful label, no formal and validated scientific background. Similarly, in the United States, on September 20, 2002, BZP was temporarily scheduled, in accordance with the controlled substances act (CSA) of the United States, as a schedule I drug, a drug with a high liability for abuse with no recognized medical use (Drug Enforcement Administration, 2014a). This scheduling resulted from an inaccurate report by the Drug Enforcement Administration (DEA); BZP displays 10–20 times greater potency than amphetamine, when actually BZP is 10 times less potent than dexamphetamine (Stargate International, 2004). Based on the European Union report constructed largely on abuse potential, on March 18, 2004, BZP was permanently placed among schedule I drugs by DEA. Identified BZP cases reported to federal, state and local forensic laboratories peaked at 15,174 in 2009, while in 2013 there were 2548 reports, according to DEA’s System to Retrieve Information from Drug Evidence (STRIDE) and the National Forensic Laboratory Information System (NFLIS) (Drug Enforcement Administration, 2014b). 2. Perception of safety Due to its psychoactive properties, legal status in many countries, and false reputation of safety, the recreational use of piperazine derivatives has gained popularity as an alternative to amphetamine, in spite of a plethora of experimental, clinical, and epidemiological studies linking its use with the development of severe serotonin syndrome, hepatotoxicity, neurotoxicity, psychopathology, and potential for abuse (Schep et al., 2011). New Zealand users believed that legality protected the quality and purity of BZP, when manufacturers synthesized without controls. Product labels gave consumers false impressions that they knew exactly what they were buying. Many users underestimated the strength of the pills and characterized the effects as moderate. Moreover, BZPparty pills were socially accepted and widely available due to a lack of legislation. It has since been proposed that BZP may entice users into using other illicit drugs (“gateway”) or it may provide illicit drug users a legal alternative (Sheridan and Butler, 2010). However, in New Zealand it is prohibited and the accessibility has declined immediately following its prohibition (Wilkins et al., 2014). 3. Patterns of use Administered orally in capsule, tablet, pill, powder, or liquid form (Gee et al., 2005), the piperazine designer drugs frequently appear as adulterants of, or additives to, ecstasy, cocaine, amphetamine and ketamine products (EMCDDA, 2009). Other reported routes of administration include inhalation, insufflation

D.P. Katz et al. / Drug and Alcohol Dependence 164 (2016) 1–7

Fig. 2. Benzylpiperazine powder.

and intravenous use for a faster onset of action. BZP’s high alkalinity deters users from administering it intravenously because solutions are caustic and cause significant pain upon injection (Gee et al., 2005). Available as a pale, yellowish-green free-base or white hydrochloride salt (Fig. 2), BZP dosing ranges between 50 and 200 mg (Sheridan and Butler, 2007), with some pills containing up to 1000 mg (Gee et al., 2008, 2005). New Zealand “party-goers” consumed roughly two to three pills per experience, with some consuming eight or more (Butler and Sheridan, 2007). Manufacturers recommend taking two pills, waiting two hours, then taking another two if tolerable (Thompson et al., 2006). Users actually preferred to consume party pills with other psychoactive substances (alcohol, ecstasy, cannabis, amphetamines and nitrous oxide) for several reasons. First, amphetamines can increase BZP’s stimulatory effects. Second, greater amounts of alcohol could be consumed because of the sobering effects of party pills. Third, the negative effects of party pills could be tolerated well along with consumption of BZP’s. For example, concurrent use with cannabis reportedly engenders relaxation, stimulates appetite and fosters sleep (Butler and Sheridan, 2007). Caffeine, herbal extracts, electrolyte blends and amino acids are also commonly taken with BZP. l-Tyrosine, the amino acid precursor of dopamine, provides an auxiliary supply of dopamine to counteract dopamine depletion, often a consequence of overconsumption (Butler and Sheridan, 2007; Nikolova and Danchev, 2008; Sheridan and Butler, 2007). 4. Toxicokinetics Piperazine derivatives are rapidly absorbed in the gastrointestinal tract (Antia et al., 2009a; Schep et al., 2011) and chiefly metabolized by the liver; the phenylpiperazines, TFMPP and MeOPP, are more extensively metabolized than the benzylpiperazines, BZP and MDBP (Maurer et al., 2004). BZP, TFMPP, 1-(4-methoxyphenyl)piperazine (MeOPP) and 1-(3,4methylenedioxybenzyl) piperazine (MDBP) have shown to induce significant in vitro hepatotoxicity in primary rat hepatocytes and in human hepatic (HepaRG & HepG2) cells (Dias-da-Silva et al., 2015). Piperazine designer compounds (BZP, TFMPP, MeOPP, MDBP) upregulated the main hepatic enzymes associated with the synthesis of cholesterol in the hepatocytes and this can escalate the cause of phospholipidosis and steatosis (Arbo et al., 2016a,b). BZP’s CNS effects occur approximately two hours after oral ingestion (Bye et al., 1973; Gee et al., 2005), and usually last between four and eight hours (Nikolova and Danchev, 2008; Wikstrom et al., 2004). Pharmacokinetic studies have detected BZP in blood up to 30 h after oral intake (Antia et al., 2009a). Piperazine derivatives are known

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to readily cross the blood-brain barrier and the TFMPP brain-toblood concentration ratio was in excess of one order of magnitude demonstrating high tissue concentrations (Schep et al., 2011). In human subjects administered a 200 mg oral BZP dose, 12.5% of BZP and its metabolites were detected in urine after 24 h (Antia et al., 2009a). Peak plasma concentrations (Cmax ) were reached after 75 min (Tmax ) at a concentration of 262 ng/ml. The absorption halflife (t1/2 ) occurred after 6.2 min. The clearance was 58.3 L/h with a drug half-life of 5.5 h (Antia et al., 2009a). The two major metabolic pathways of BZP involve hydroxylation and N-dealkylation catalyzed by cytochrome P450 (CYP) enzymes (Antia et al., 2009b; Staack et al., 2002, 2003). Single or double hydroxylation of the aromatic ring, by CYP450, followed by COMT-mediated methylation to N-(4-hydroxy-4-methoxybenzyl) piperazine and subsequent phase 2 metabolism to glucuronic and/or sulphuric acid conjugates, promote BZP excretion. N-dealkylation at the benzylic carbon frees the piperazine ring (Fig. 3). Double N-dealkylation of the piperazine heterocycle yields N-benzylethylenediamine or benzylamine (Staack and Maurer, 2005). CYP450 isoenzymes that play a role in BZP and TFMPP metabolism include CYP2D6, CYP1A2 and CYP3A4. Cytochrome P450 enzymes are susceptible to genetic polymorphisms; therefore, inter-individual differences in metabolism may occur (EMCDDA, 2009). BZP and TFMPP may inhibit these enzymes and alter the metabolism of commonly used pharmaceuticals, supporting a contraindication for administration of piperazine drugs with other CYP substrates (Antia et al., 2009b; Murphy et al., 2009). Due to the structural features of BZP, Cytochrome P-450 system metabolizes and catalyzes BZP to N-oxide and N-,O-demethylation and/or oxidation. MeOP and N-oxide-MeOP (the major metabolites) and other minor metabolites (very minimal quantities) can be detected using gas chromatography-mass spectrometry and liquid chromatography coupled with multistage mass spectrometry standard urine screening approaches (Meyer et al., 2015). Unusually, isotopic profiling helps with the detection of the origination (place where synthesis occurred) and identification of the illegal drug makers. This recent method has a unique advantage because of its ability to get evidences regarding the sources which were inaccessible through the common chemical analysis (Beckett et al., 2015).

5. Pharmacology In vivo and in vitro studies suggest BZP is a “messy drug”, meaning it affects synaptic levels of dopamine (DA), serotonin (5-HT) and norepinephrine (NE). Benzylpiperazine causes nonexocytotic substrate release of dopamine by reversing transporter flux (Baumann et al., 2004), inhibiting dopamine re-uptake, blocking dopamine transporters (Meririnne et al., 2006) and acting as an agonist at postsynaptic dopamine receptor sites (Oberlander et al., 1979). Similar to MDMA, BZP modulates both dopamine and serotonin levels in the synaptic cleft (Baumann et al., 2004). Human embryonic kidney 293 (HEK 293) cells are a valid and good model to study the in vitro effect of various drugs on monoaminergic receptor binding and the transporters. These embryonic kidney 293 cells expressed the human NET, SERT, and DAT. BZP inhibits serotonin transporters preventing reuptake and binds as an agonist to 5-HT1 receptors (Simmler et al., 2014; Tekes et al., 1987). BZP was also shown to inhibit the reuptake of monoamines (DA and NE, but with lesser extent 5-HT) in rat brain synaptosomal preparations (Nagai et al., 2007). Monoamine-releasing activity, mostly of DA and NE, occurred after BZP administration (Nagai et al., 2007) in the same synaptosomes. The mild hallucinogenic effects experienced with high doses are due to BZP binding with the 5-HT2A subtype. The 5-HT2B receptor, localized in the gastrointestinal tract, is bound by BZP, inducing

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Fig. 3. Metabolic pathways of BZP.

intense peripheral side effects, such as stomach pains and nausea. BZP-bound 5HT3 receptors are reported to be responsible for the development of migraine headaches (Nikolova and Danchev, 2008). The stimulatory effects of BZP are attributed to dopaminergic neurotransmission, while euphoria, hallucinations, alertness and sociability, all desired feelings at rave dance parties, are associated with serotonergic activation. BZP acts as an antagonist at alpha-2-adrenoreceptors, which inhibits negative feedback and augments the release of norepinephrine in peripheral sympathetic nerve fibers in vitro (Magyar et al., 1986). Elevated levels of circulating norepinephrine bind readily with central and peripheral and adrenergic receptors provoking an increase in blood pressure and heart rate. Yarosh et al. (2007) examined the stimulant-like behavioral patterns of BZP in mice (Yarosh et al., 2007). The head twitch response assay failed to elicit hallucinogen-like actions, a dose-dependent increase in locomotor activity was observed in the open field and BZP fully substituted for the S-(+)-enantiomer (stimulant-like) of MDMA. TFMPP decreased locomotor activity, induced hallucinations in the head twitch assay, but was unsuccessful in substituting for R(−)-MDMA (hallucinogenic-like). Increases in synaptic serotonin by TFMPP yield hallucinations, while BZP/TFMPP accounts for the dopaminergic stimulatory effects (Yarosh et al., 2007). There is a balance between the inhibitory and excitatory neurotransmission in the brain. GABA is the major inhibitory neurotransmitter that controls the excitatory pathway. Piperazines can bind to the GABAA (post-synaptic, ion channel) receptor without any intrinsic effect. This effect will antagonize the GABAergic neurotransmission resulting in an enhanced catecholaminergic neurotransmission which consequently results in excessive toxicity and adverse contraindications during overindulges (Hondebrink et al., 2015). When BZP was administered with TFMPP several brain regions such as caudate putamen, dorsal striatum and thalamus were affected. With regard to the mechanisms of cellular toxicity, piperazine derivatives 1-(3-trifluoromethylphenyl)piperazine, (N-benzylpiperazine, 1-(4-methoxyphenyl)piperazine and 1-(3,4methylenedioxybenzyl)piperazine) have shown to induce affect mitochondrial and calcium signaling leading to cell death (Arbo et al., 2016a,b). 6. BZP/TFMPP combination Typically, benzylpiperazine is taken in combination with other piperazines to enhance the positive reinforcing effects. A 2:1 combination of BZP and TFMPP is reported to strongly resemble the effects of MDMA in humans (de Boer et al., 2001), although combinations up to 10:1 have been reported (Lin et al., 2011). TFMPP, a serotonin-mimicking piperazine, blocks serotonin reup-

take, enhances serotonin release and is a nonselective agonist at serotonin receptors (Nikolova and Danchev, 2008). TFMPP does not possess significant noradrenergic actions (Fantegrossi et al., 2005; Herndon et al., 1992), but rather binds to 5-HT1 and 5-HT2 receptors with little affinity for 5-HT3 receptors (Baumann et al., 2005; Cunningham and Appel, 1986; Robertson et al., 1992). Baumann et al. (2005) observed MDMA-like dopamine and 5HT release in BZP/TFMPP treated mice (Baumann et al., 2005). They evaluated the transporter-mediated efflux of [3 H]MPP+ , a dopamine transporter substrate (DAT), and [3 H]5-HT, a serotonin transporter substrate (SERT), in rat synaptosomes. 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a meperidine analogue, is a highly lipophilic neurotoxin that readily crosses the blood-brain barrier. MPTP becomes oxidized to 1-methyl-4-phenylpyridinium (MPP+) via monoamine oxidase B (MAO-B) (Ramsay et al., 1986). BZP caused a DAT-dependent release of [3 H]MPP+ , but unlike TFMPP, BZP showed no effect on [3 H]5-HT release by SERT (Baumann et al., 2005). Remarkably, the BZP/TFMPP blend reacted synergistically, resulting in an increase in endogenously released dopamine and serotonin, similar to MDMA (Baumann et al., 2004, 2005; Yeap et al., 2010). Cumulative release of DA and 5-HT was greater in combination than when drugs were given individually (Hashimoto et al., 1992). TFMPP was 3-fold less potent than MDMA at elevating extracellular 5-HT levels (Baumann et al., 2005). Rats experience seizures and ataxia at higher level doses of BZP/TFMPP (Baumann et al., 2004, 2005). The combination of BZP with TFMPP blocked the caudate activation by an indirect mechanism on dopamine release through the serotonin (5-HT2c) receptors. However, the dorsal striatum and thalamus was stimulated by BZP and TFMPP (Curley et al., 2015). 7. Health impact BZP adverse side effects include insomnia, mild to severe hangover, dilated pupils, dryness of mouth, extreme alertness, pruritus, confusion, agitation, tremor, dystonia, headache, dizziness, anxiety, insomnia, vomiting, chest pain, tachycardia, hypertension, palpitations, collapse, hyperventilation, hyperthermia and urine retention (Nikolova and Danchev, 2008; Table 1), reflective of a sympathomimetic toxidrome. The adrenergic actions of BZP, in the peripheral nervous system, produce serious cardiovascular effects including vasoconstriction, ischemia, tachycardia and arrhythmia. Over activation leads to hypertension and potential stroke or myocardial infarct (Arbo et al., 2014). In women, a 200 mg single administration of BZP elevated systolic and diastolic blood pressure and heart rate when compared to placebo (Lin et al., 2009). Administration of single doses of BZP/TFMPP have shown the same cardiovascular effects (Lin et al., 2011).

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Table 1 Various effects of BZP. System

Effect of BZP

Serotonergic Adrenergic Dopaminergic. Cholinergic Immunologic Other

Agitation, confusion, dissociative states, hangover, headache, vomiting, hyperthermia Alertness, anxiety, dilated pupils, insomnia, dizziness, respiratory acidosis, hyperventilation, palpitations, chest pain, tachycardia, seizures Movement disorder (dystonia), tremor hypertension, vomiting, urine retention, acute paranoid psychosis Xerostoma, vomiting Hypersensitivity reactions (Pruritus) Acute renal injury multiple organ failure, metabolic acidosis, intravascular coagulation, hyponatremia, rhabdomyolysis

Serious toxic effects of BZP include metabolic and respiratory acidosis, hyponatremia, seizures, acute paranoid psychosis, dissociative states, worsening of mental illnesses, rhabdomyolysis, disseminated intravascular coagulation, acute renal injury and multiple organ failure (Alansari and Hamilton, 2006; Austin and Monasterio, 2004; Gee et al., 2008; Wood et al., 2007). The environment of a rave dance party, consisting of intense heat, increased physical activity, sleep deprivation, dehydration and excessive fluid intake, is believed to contribute to BZP-party pill toxicity (Berney-Meyer et al., 2012). After effects, occurring hours to days after BZP ingestion, reportedly include insomnia, lack of appetite, tiredness, dehydration, headache, xerostoma, aching/shaking body, depressed mood, tension and anxiety (Butler and Sheridan, 2007; Nicholson, 2006; Thompson et al., 2010). BZP and TFMPP have serotonergic effects that may lead to serotonin toxicity (Baumann et al., 2005). Combination of BZP and/or TFMPP with other recreational drugs (MDMA) or therapeutic serotonergic agents including selective serotonin reuptake inhibitors (SSRI’s), may increase the risk of developing serotonin syndrome. Treatment of serotonin toxicity centers on controlling agitation by managing symptoms with the benzodiazepines, cyproheptadine and chlorpromazine (Schep et al., 2011). Although fatalities due to consumption of BZP alone appear to be rare, there are several reported in the literature. For example, a 23-year-old woman ingested BZP, MDMA and a large quantity of water resulting in hyponatremia, cerebral edema and ultimately death (Balmelli et al., 2001). In most BZP-related fatality cases, BZP was detected along with other drugs of abuse, such as MDMA (Hartung et al., 2002). Therefore, it is difficult to establish the role, if any, that BZP plays in morbidity. 8. Addictive properties Previous users of amphetamine were unable to discriminate between equipotent intravenous doses of BZP and dexamphetamine (Campbell et al., 1973). The acute effects of BZP are similar to methamphetamine in rats, although methamphetamine displays greater potency, indicating potential abuse and drug dependency in BZP users (Herbert and Hughes, 2009). Dose-dependent hyperactivity and stereotypy were observed in Sprague-Dawley rats co-administered with BZP and methamphetamine. Behavioral sensitization, a phenomenon in which repeated doses of drug cause a long-lasting and progressive increase in effect, was noticed upon 5 day repeated exposure of BZP and methamphetamine. Furthermore, administration of a low dose of BZP in methamphetamine pre-treated rats, upon a 2 day withdrawal period, evoked cross-sensitization between BZP and methamphetamine (Brennan et al., 2007). Cross-sensitization occurs when sensitivity to one drug, in this case methamphetamine, predisposes the user to sensitivity to another drug because of similarities in their chemical structures. BZP-administered rats repeatedly returned to the location of administration, dose-dependently spending more time in the apartment associated with the drug, indicating a conditioned place-preference. The D1 receptor antagonist SCH and the 5-HT3 receptor antagonist MDL attenuated conditioned place-preference

in BZP treated rats (Meririnne et al., 2006), suggesting the compound has rewarding properties and therefore abuse potential. Also, primates (rhesus monkeys) displayed repeated intravenous self-administration, a reinforcing activity of BZP. However, TFMPP alone did not maintain self-administration. Appropriately, the BZP/TFMPP revealed less reinforcing activity when compared to BZP alone (Fantegrossi et al., 2005). Heroin, cocaine and methamphetamine also exhibit conditioned place-preference and self-administration properties. A 1:1 BZP/TFMPP cocktail elevated DA and 5-HT in the nucleus accumbens (Baumann et al., 2005), the “pleasure center” in the mesocorticolimbic pathway. Dopaminergic input extending from the ventral tegmental area to the nucleus accumbens regulates rewarding experiences, such as drug addiction. 9. Detection and quantification Granted that BZP has been classified as a schedule I controlled substance, availability over the internet and underground drug trafficking has necessitated accurate and sensitive forensic toxicological identification techniques. Immunoassays and colorimetric tests are used to screen biological specimens in order to rapidly detect drugs (Arbo et al., 2012). However, colorimetric assays lack specificity and sensitivity because they target specific functional groups on a molecule as opposed to the molecule as a whole. Therefore, colorimetric assays frequently produce false positives and negative results (Elie et al., 2012; Flanagan et al., 2008). Immunoassays utilize antigen-antibody specific reactions, but are prone to cross-reactivity, particularly with amphetamines. In humans, functional magnetic resonance imaging (fMRI) was used to detect the effects of BZP and TFMPP on neuronal activation, receptor stimulation and release of neurotransmitters. BZP and TFMPP can stimulate 5HT2C receptors in the striatum (dorsal) and thalamus leading to dopamine release (Curley et al., 2015). Chromatographic techniques (LC–MS/MS-triple-quadrupole mass spectrometer) have been developed for identification and quantification of BZP and its hydroxylated metabolites, 3-OH-BZP and 4-OH-BZP (Tsutsumi et al., 2006; Tang et al., 2015). Gas and liquid chromatography coupled with mass spectrometry is the preferred system for detection, but diode array detector, fluorescence and ultraviolet detection have been employed. Sample preparation, including hydrolysis (acid or enzymatic), extraction (solid phase or liquid-liquid) and derivatization, along with internal standard are all considered for development of protocol 10. Conclusion Piperazine-derived designer drugs have gained popularity worldwide because of widespread distribution, legality and a false reputation of safety. Piperazine derivatives exert their stimulatory and hallucinogenic effects by elevating synaptic monoamine concentrations, similar to amphetamine, cocaine and MDMA. Concomitant use of BZP and TFMPP, usually in cocktails or blends, amplifies the amount of endogenously released dopamine and serotonin, resulting in powerful effects. Adverse effects of BZP are mostly adrenergic, leading to profound cardiovascular effects. BZP

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can also cause detrimental psychological, neurological and systemic effects, including severe serotonin syndrome and multiple organ failure. Human and animal studies reveal BZP and other piperazine derivatives have reinforcing effects and are therefore have addiction potential. A lack of existing literature and pharmacological and toxicological data on the piperazine drug family prompts further investigations into all aspects of drug development, formulation, mechanism of action, toxicity and detection. Conflict of interest None. Contributors Daniel P. Katz (First author): Mr. Katz was the person who mainly visualized, shaped and formatted the manuscript. Dwipayan Bhattacharya: Assisted with the reading and article preparation. Subhrajit Bhattacharya: Assisted with the reading and article preparation. Jack Deruiter: Assisted with the reading and article preparation. C. Randall Clark: Assisted with the funding, reading and article preparation. Vishnu Suppiramaniam: Assisted with the reading and article preparation. Muralikrishnan Dhanasekaran (Corresponding author): Assisted with the funding, plan, editing and article preparation. Acknowledgement We are thankful to the Harrison School of Pharmacy, Auburn University for the support of this study. References Alansari, M., Hamilton, D., 2006. Nephrotoxicity of BZP-based herbal party pills: a New Zealand case report. N. Z. Med. J. 119, U1959. Antia, U., Lee, H.S., Kydd, R.R., Tingle, M.D., Russell, B.R., 2009a. Pharmacokinetics of ‘party pill’ drug N-benzylpiperazine (BZP) in healthy human participants. Forensic Sci. Int. 186, 63–67. Antia, U., Tingle, M.D., Russell, B.R., 2009b. Metabolic interactions with piperazine-based ‘party pill’ drugs. J. Pharm. Pharmacol. 61, 877–882. Arbo, M.D., Bastos, M.L., Carmo, H.F., 2012. Piperazine compounds as drugs of abuse. Drug Alcohol Depend. 122, 174–185. Arbo, M.D., Silva, R., Barbosa, D.J., da Silva, D.D., Rossato, L.G., Bastos Mde, L., Carmo, H., 2014. Piperazine designer drugs induce toxicity in cardiomyoblast h9c2 cells through mitochondrial impairment. Toxicol. Lett. 229, 178–189. Arbo, M.D., Melega, S., Stöber, R., Schug, M., Rempel, E., Rahnenführer, J., Godoy, P., Reif, R., Cadenas, C., de Lourdes Bastos, M., Carmo, H., Hengstler, J.G., 2016a. Hepatotoxicity of piperazine designer drugs: up-regulation of key enzymes of cholesterol and lipid biosynthesis. Arch. Toxicol. [Epub ahead of print]. Arbo, M.D., Silva, R., Barbosa, D.J., da Silva, D.D., Silva, S.P., Teixeira, J.P., Bastos, M.L., Carmo, H., 2016b. In vitro neurotoxicity evaluation of piperazine designer drugs in differentiated human neuroblastoma SH-SY5Y cells. J. Appl. Toxicol. 36, 121–130. Austin, H., Monasterio, E., 2004. Acute psychosis following ingestion of ‘Rapture’. Australas. Psychiatry 12, 406–408. Balmelli, C., Kupferschmidt, H., Rentsch, K., Schneemann, M., 2001. Fatal brain edema after ingestion of ecstasy and benzylpiperazine. Dtsch. Med. Wochenschr. 126, 809–811. Barclay, L., 2003. Benzylpiperazine/trifluromethyl-phenylpiperazine. In: Blachford, S., Krapp, K. (Eds.), Drugs and Controlled Substances: Information for Students. Gale, Detroit, pp. 53–57. Baumann, M.H., Clark, R.D., Budzynski, A.G., Partilla, J.S., Blough, B.E., Rothman, R.B., 2004. Effects of Legal X piperazine analogs on dopamine and serotonin release in rat brain. Ann. N. Y. Acad. Sci. 1025, 189–197. Baumann, M.H., Clark, R.D., Budzynski, A.G., Partilla, J.S., Blough, B.E., Rothman, R.B., 2005. N-substituted piperazines abused by humans mimic the molecular mechanism of 3,4-methylenedioxymethamphetamine (MDMA, or ‘Ecstasy’). Neuropsychopharmacology 30, 550–560. Beckett, N.M., Cresswell, S.L., Grice, D.I., Carter, J.F., 2015. Isotopic profiling of seized benzylpiperazine and trifluoromethylphenylpiperazine tablets using ␦13C and ␦15N stable isotopes. Sci. Justice 55, 51–61.

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