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Home , Adinazolam, Bromazepam, Bromazolam, Clonazolam, Deschloroetizolam, Desmethylflunitrazepam

Neurotoxicology 73 (2019) 8–16

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Neurotoxicology

journal homepage: www.elsevier.com/locate/neuro

Review An expanding world of new psychoactive substances—designer T ⁎ Jolanta B. Zawilska , Jakub Wojcieszak

Department of , Medical University of Łódź, 90-151 Łódź, Muszynskiego 1, Poland

ARTICLE INFO ABSTRACT

Keywords: The abuse of new psychoactive substances (NPS) has been increasing dramatically since the late 2000s world- Benzodiazepines wide. Between 2009 and 2017, a total of 803 individual NPS were reported to the United Nations Office of Drugs New psychoactive substances and Crime by 111 countries and territories. Although the most popular compounds are synthetic cannabino- mimetics and psychostimulatory derivatives of cathinone (so-called β-keto-amphetamines), novel benzodiaze- pines have recently emerged on the recreational drug market. The misuse/abuse of “designer benzodiazepines” Impaired driving (DBZD), a common name for the class NPS, has become an increasing problem in many coun- tries. The DBZD group includes pharmaceutical drug candidates that have never been approved for medical use, compounds that were synthesized by a simple structural modification of a registered drug, and some active metabolites of registered benzodiazepines. This survey presents members of the DBZD group, describes the epidemiological trends and clinical effects associated with DBZD use, and discusses available data on their metabolism. Special emphasis is given to cases of intoxications involving these compounds.

1. Introduction the influence of drugs (e.g., Bertol et al., 2018; Drummer et al., 2012; Kriikku et al., 2012; Valen et al., 2017; Xiang et al., 2015). Benzodiazepines were introduced as therapeutic drugs in the early Over the last decade, an increasing number of new benzodiazepines 1960s. They act as positive allosteric modulators of γ-aminobutyric acid that are not registered as medical products have been introduced into (GABA)-A receptors, which are composed of heteropentameric multi- the recreational drug market (see Table 1). Currently, new psychoactive subunit proteins. The drugs bind to a specific high affinity binding site substances (NPS) belonging to the benzodiazepine class are commonly of GABA-A receptors, located at the α/γ subunit interface. The phar- referred to as “designer benzodiazepines” (DBZD), and their misuse or macological activity of the benzodiazepines is determined by the type abuse has become an increasing problem in many countries. This survey of GABA-A α subunit to which they bind. Thus, the , presents members of the DBZD group, describes the epidemiological anterograde amnesic and actions, as well as the addic- trends and clinical effects associated with DBZD use, and discusses tive potential of these drugs, require the presence of α1-containing available data on their metabolism. GABA-A receptors, while the effects are mediated by GABA-A receptors containing α2 subunits, and the myorelaxant actions by 2. Methodology GABA-A receptors containing α2, α3, and α5 subunits (Tan et al., 2011). Benzodiazepines are nowadays widely applied in the therapy of Review of the literature was based on the exhaustive search carried psychiatric and neurological disorders, including and panic out in PubMed (U.S. National Library of Medicine), using “designer attacks, , muscle spasms, epilepsy and withdrawal. benzodiazepines” and each of the compound name listed in Table 1 as Some are also used as a premedication prior to surgery and intra-op- keywords. Only papers written in English and with full texts available erative . In addition, benzodiazepines are commonly used by December 2018 were included. Additionally, official reports pub- in self-, and misused in combination with other psychoactive lished by the United Nations O ffice on Drugs and Crime (UNODOC), substances, namely , psychostimulants and alcohol (EMCDDA, European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) 2018a; Griffin et al., 2013; UNODC, 2017a). There are also reports on and World Health Organization (WHO), and governmental acts were the use of benzodiazepines in drug-facilitated crime and driving under reviewed. Furthermore, in each article and report retrived, references

⁎ Corresponding author. E-mail address: [email protected] (J.B. Zawilska). https://doi.org/10.1016/j.neuro.2019.02.015 Received 4 January 2019; Received in revised form 12 February 2019; Accepted 21 February 2019 Available online 23 February 2019 0161-813X/ © 2019 Elsevier B.V. All rights reserved. J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

Table 1 Table 1 (continued) Chemical structures and names of designer benzodiazepines. Chemical structure Names Chemical structure Names

1,4-BENZODIAZEPINES Cloniprazepam A likely prodrug of . IUPAC name: 5-(2-Chlorophenyl)-1-(cyclopropylmethyl)-7- nitro-1,3-dihydro-2H-benzo[e][1,4]diazepin-2- one

Phenazepam1 IUPAC name: 7-Bromo-5-(2-chlorophenyl)-1,3-dihydro-2H- fl Desmethyl unitrazepam (also known as 1,4-benzodiazepin-2-one fl nor unitrazepam, Ro-4435 and fonazepam) Trade names: BD98, Elzepam, Fenazepam, The active metabolite of the potent drug Phenazef, , Phezipam, fl unitrazepam. Phenorelaxan, Trankvezipam IUPAC name: Street names: Bonsai, Bonsai Supersleep, Fenaz, fl 5-(2- uorophenyl)-7-nitro-1,3-dihydro-2H-1,4- Soviet Benzo, Panda benzodiazepin-2-one 3-Hydroxyphenazepam The active metabolite of phenazepam. IUPAC name: (2-Chlorodiazepam, Ro5-3448) 7-Bromo-5-(2-chlorophenyl)-3-hydroxy-1,3- IUPAC name: dihydro-2H-1,4-benzodiazepin-2-one 7-Chloro-5-(2-chlorophenyl)-1-methyl-1,3- dihydro-2H-1,4-benzodiazepin-2-one

TRIAZOLOBENZODIAZEPINES (Deracyn®, Adinazolamum) IUPAC name: 1-(8-Chloro-6-phenyl-4H-[1,2,4]triazolo[4,3-a] [1,4]benzodiazepin-1-yl)-N,N- 4’-Chlorodiazepam (Ro5-4864) dimethylmethanamine IUPAC name: 7-Chloro-5-(4-chlorophenyl)-1-methyl-3H-1,4- benzodiazepin-2-one

Bromazolam IUPAC name: 8-Bromo-1-methyl-6-phenyl-4H-[1,2,4]triazolo [4,3-a][1,4]benzodiazepine IUPAC name: 7-Bromo-5-(2-fluorophenyl)-1,3-dihydro-2H- 1,4-benzodiazepin-2-one

Clonazolam (Clonitrazolam) IUPAC name: [(S)-3-methylclonazepam] 6-(2-Chlorophenyl)-1-methyl-8-nitro-4H-[1,2,4] IUPAC name: triazolo[4,3-a][1,4]benzodiazepine (3S)-5-(2-Chlorophenyl)-3-methyl-7-nitro-1,3- dihydro-2H-1,4-benzodiazepin-2-one

Flualprazolam (3- IUPAC name: fl hydroxydesmethylflunitrazepam, DP 370) 8-Chloro-6-(2- uorophenyl)-1-methyl-4H-benzo IUPAC name: [f][1,2,4]triazolo[4,3-a][1,4]diazepine 5-(2-Fluorophenyl)-3-hydroxy-7-nitro-2,3- dihydro-1H-1,4-benzodiazepin-2-one

Nitemazepam IUPAC name: IUPAC name: 3-Hydroxy-1-methyl-7-nitro-5-phenyl-2,3- 8-Bromo-6-(2-fluorophenyl)-1-methyl-4H- dihydro-1H-1,4-benzodiazepin-2-one (continued on next page)

9 J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

Table 1 (continued) Table 1 (continued)

Chemical structure Names Chemical structure Names

[1,2,4]triazolo[4,3-a][1,4]benzodiazepine Street name: liquid Xanax

Flunitrazolam IUPAC name: (desmethyletizolam) 6-(2-Fluorophenyl)-1-methyl-8-nitro-4H-[1,2,4] A metabolite of . triazolo[4,3-a][1,4]benzodiazepine IUPAC name: 4-(2-Chlorophenyl)-2-ethyl-6H-thieno[3,2-f] [1,2,4]triazolo[4,3-a][1,4]diazepine

Nitrazolam () IUPAC name: 1-Methyl-8-nitro-6-phenyl-4H-[1,2,4]triazolo Fluclotizolam [4,3-a][1,4]benzodiazepine IUPAC name: 2-Chloro-4-(2-fluorophenyl)-9-methyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a]diazepine

Pyrazolam IUPAC name: 8-Bromo-1-methyl-6-(pyridin-2-yl)-4H-[1,2,4] triazolo[4,3-a][1,4]benzodiazepine 1 Phenazepam is used as a prescription medicine in the Russian Federation, Estonia, Latvia, Lithuania and Belarus (WHO, 2015). 2 Etizolam is used as a prescription medicine in Japan, India and Italy (WHO, 2017).

were checked carefully in order to find possible additional publications, missed during the initial search. IUPAC name: 8-Chloro-6-(2-chlorophenyl)-4H-pyrido[2,3-f] [1,2,4]triazolo[4,3-a][1,4]diazepine 3. Legal status of designer benzodiazepines

In Canada, all benzodiazepines are classified as schedule IV drugs (Ministry of Justice, Canada, 2018). In the United Kingdom, broma- zolam, 4-chlorodiazepam, , , diclazepam, etizolam, flubromazepam, flubromazolam, fonezepam, 3-hydro- xyphenazepam, meclonazepam, metizolam, nifoxipam, nitrazolam and THIENOTRIAZOLOBENZODIAZEPINES 2 have been classified as Class C drugs by the May 2017 Etizolam IUPAC name: amendment to The (Advice Council on Misuse 4-(2-Chlorophenyl)-2-ethyl-9-methyl-6H-thieno of Drugs, 2017). Several DBZD have been placed under national control [3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine in other countries (UNODC, 2017c; WHO, 2015, 2017): International trade names: Arophalm, Capsafe, Depas, Dezolam, Eticalm, Etidrale, Etisedan, Etizolan, Guperies, Medipeace, Mozun, • China: phenazepam; Nonnerv, Palgin, Pasaden, Sedekopan, • Denmark: clonazolam, deschloroetizolam, diclazepam, flu- Sylazepam , flubromazolam, metizolam, nifoxipam, phenazepam Street names: Etiz, Eitizzy, Etizest and pyrazolam; • Finland: clonazolam, deschloroetizolam, diclazepam, etizolam, flu- Deschloroetizolam bromazepam, flubromazolam, meclonazepam, nifoxipam, phena- IUPAC name: zepam and pyrazolam; 4-Phenyl-2-ethyl-9-methyl-6H-thieno[3,2-f] [1,2,4]triazolo[4,3-a][1,4]diazepine • Germany: etizolam and phenazepam; Street names: Etizolam-2, Etiz-dechlorinate, • Ireland: phenazepam; Legal-Eti • Netherlands: phenazepam; • Poland: etizolam; • The Republic of Korea: diclazepam; • Sweden: adinazolam, cloniprazepam, desmethylflunitrazepam,

10 J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

flubromazepam, flunitrazolam, phenazepam, pyrazolam and zapi- natural extracts (Couch and Madhavaram, 2012). Additionally, there zolam; are reports of phenazepam being sold in combination with stimulant • Switzerland: clonazolam, deschloroetizolam, diclazepam, etizolam, recreational drugs, such as dimethocaine (distributed under the brand flubromazepam, flubromazolam, nifoxipam and pyrazolam; name of “Dimethocaine Phenazepam Legal Powder”)(Advisory Council • Turkey: adinazolam, deschloroetizolam, diclazepam, flu- on the Misuse of Drugs, 2011). bromazepam, flubromazolam, meclonazepam and pyrazolam; At present, limited information is available regarding the pre- • The United Arab Emirates: diclazepam, etizolam, flubromazepam valence and pattern of DBZD use, and existing studies should be re- and pyrazolam; garded with caution as many are based on online surveys and self-re- • The United States: etizolam is not currently listed under the ports on user websites. The common route of administration for DBZD Controlled Substances Act, but has been declared a controlled sub- is oral consumption, such as by pills, or powder inserted into gel cap- stance in some of the states, i.e., Alabama, Arkansas, Arizona, sules or dissolved in or propylene glycol. Alternatively, rectal, Florida, Georgia, Indiana, Mississippi, and Virginia. sublingual, intravenous or intranasal routes can be used, as well as inhalation (smoking and vaporization). Intravenous administration was 4. Designer benzodiazepines – historical background, appearance reported to give a “rush” (Anderson and Kjeligren, 2017; Carpenter of products, patterns of consumption et al., 2018).

The first recreationally-used benzodiazepine was phenazepam 5. Biological effects (2007) followed by etizolam (2011) (EMCDDA, 2018a). Phenazepam was originally developed in the in the 1970s, and pre- Information on the effects of DBZD are largely limited to self-re- scribed to treat anxiety and alcohol withdrawal (WHO, 2015). Etizolam ported experiences from user websites and case reports/series is an anxiolytic medicine, originally developed in Japan, where it was (Anderson and Kjeligren, 2017; Benerjee, 2018; Benesch and Iqbal, introduced under the brand name of Depas in 1984. Both compounds 2018; Corkery et al., 2012; Dargan et al., 2013; Ghazi and Mohmand, are used as prescribed medicines in a few countries (see Table 1). 2017; Gupta and Garg, 2014; Manchester et al., 2018; O’Connell et al., Since 2012, new benzodiazepines have been introduced on the re- 2015). creational drug market and distributed by online retailers mostly as By analogy to pharmaceutical benzodiazepines, the desired actions “research chemicals”. Between 2011 and 2016, the United Nations of DBZD include increased sociability, muscle relaxation, feelings of Office on Drugs and Crime (UNODC) Early Warning Advisory (EWA) well-being or even . The sleep-inducing effects, and the control received a total of 209 reports of DBZD, most of them from European of anxiety, nervousness, panic attacks and chronic pain are used for self- countries (UNODC, 2017a). In Europe, the number of countries re- medication. The drugs are often taken by users in order to in- porting the emergence of new benzodiazepines as NPS has increased crease the opioid high and to help to sedate the descent phase, as well from three in 2011 to 21 in 2015. The European Monitoring Centre for as by users of psychostimulants to facilitate a come-down after an ex- Drugs and Drug Addiction (EMCDDA) is currently monitoring 23 DBZD, tensive dose, and by individuals addicted to alcohol to augment its with more than half having been detected since 2015 (EMCDDA, 2018a, effect synergistically. In individuals addicted to benzodiazepines, 2018b). The most frequently reported drugs from this group are eti- opioids, stimulants and alcohol DBZD are used to alleviate withdrawal zolam, diclazepam, flubromazolam, phenazepam and pyrazolam or abstinence syndromes (Abouchedid et al., 2018; Anderson and (EMCDDA, 2018a; UNODC, 2017a). Notably, etizolam, diclazepam, Kjeligren, 2017; EMCDDA, 2018a; Jones et al., 2012; WHO, 2015, flubromazolam and phenazepam account for more than 80% of all ta- 2017). blets containing DBZD that have been seized in Europe since 2005 The common adverse effects of DBZD include , impaired (EMCDDA, 2018a). DBZD, mostly clonazolam, flubromazepam, nifox- balance, , loss of coordination, impaired thinking and self-as- ipam, , phenazepam and pyrazolam were also identified in sessment capability, muscle weakness, , slurred speech, South-East Asia (UNODC, 2017a, 2017b). blurred vision, , , drowsiness, lethargy, fatigue and Most of DBZD either derive from pharmaceutical drug candidates palpitations. At high doses, they could induce delirium, auditory and (e.g., clonazolam, deschloroetizolam, diclazepam, flubromazepam, visual hallucinations, , deep sleep and coma (Ali et al., 2015; meclonazepam and pyrazolam) that have never been approved for Anderson and Kjeligren, 2017; Benerjee, 2018; Benesch and Iqbal, medical use, or are synthesized by a simple structural modification of a 2018; Carpenter et al., 2018; Corkery et al., 2012; Dargan et al., 2013; registered drug (e.g., flubromazolam). Some active metabolites of re- Huppertz et al., 2018; Łukasik-Głębocka et al., 2016; Manchester et al., gistered benzodiazepines are also sold as NPS. For example, des- 2018; O’Connell et al., 2015; WHO, 2015, 2017). There are also reports methylflunitrazepam and 3-hydroxydesmethylflunitrazepam, the active of paradoxical excitement and increase in energy (Anderson and metabolites of the potent drug flunitrazepam, are marketed under the Kjeligren, 2017). Users of long-acting flubromazolam have described names of fonazepam and nifoxipam, respectively (Katselou et al., unpleasant night dreams, sleeping paralysis, and somnambulistic states 2017). that persisted for several days (Anderson and Kjeligren, 2017). Sus- DBZD are sold as tablets, pills, capsules, pellets, powders, blotters, tained involuntary closing of the eyelids () were spor- and recently liquids under their own names (Abouchedid et al., 2018; adically observed in patients who had used etizolam for at least one Corkery et al., 2012; Pope et al., 2018; US Department of Justice, month (Wakakura et al., 2004). The toxic effects of phenazepam and, in 2009). However, some of them are also sold on the illicit drug market as particular, flubromazolam may last for several days (e.g., Anderson and counterfeit forms of and , commonly prescribed Kjeligren, 2017; Dargan et al., 2013). Like pharmaceutical benzodia- anxiolytic medicines, which increases the risk of unintentional overdose zepines, DBZD can have severe toxicity when concomitantly used with and intoxication. For instance, tablets sold on the illicit drug market in other CNS drugs, especially opioids and alcohol, which in- Europe as alprazolam were found to contain flubromazolam, and those creases the risk of respiratory depression and death (EMCDDA, 2018a). sold as diazepam contained various other DBZD: phenazepam, eti- Chronic use of DBZD results in the development of tolerance, as well zolam, diclazepam and flubromazepam (EMCDDA, 2016; UNODC, as psychological and . Abrupt cessation may lead 2017a). In California, etizolam, together with fentanyl, was found in to withdrawal symptoms such as anxiety, insomnia, restlessness, sei- serum samples of intoxicated subjects who took counterfeit alprazolam zures and life-threatening (Anderson and Kjeligren, 2017; tablets (Arens et al., 2016). Phenazepam, together with synthetic can- Corkery et al., 2012; Gupta and Garg, 2014; Nishi et al., 2014; WHO, nabinomimetics, was identified in smokable products called “Pineapple 2015, 2017). Analysis of anonymous self-reports published on a Express” and “Purple Haze”, marketed in New Zealand as containing Swedish drug forum revealed that flubromazolam withdrawal

11 J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16 symptoms are worse and longer than those produced by any benzo- were found (Shearer et al., 2015). Another report presents tissue dis- diazepine, and include muscle aches, sleeping disorders, severe anxiety tribution and concentration of phenazepam and its metabolite, 3-hy- and panic attacks, symptoms, perceptual distortions, droxyphenazepam, in biological fluids and tissues from 29 post-mortem cramping, chills, seizures, and vomiting (Anderson and Kjeligren, cases from Scotland, investigated between January 2011 and April 2017). 2013 (Crichton et al., 2015). In 27 cases, where the cause of death was not directly related to phenazepam, its median concentration in fe- 6. Acute intoxication cases involving designer benzodiazepines moral, cardiac and subclavian blood was 0.097 mg/L (0.007–0.360 mg/ L), 0.086 mg/L (0.014–0.310 mg/L) and 0.039 mg/L (0.016–0.270 mg/ Bäckberg et al. (2018) published data on acute intoxication cases L), respectively. Apart from blood, phenazepam was also detected in presenting for emergency care in Sweden during 2012–2016 and col- vitreous humour: 0.013 mg/L (0.007–0.054 mg/L), urine: 0.016 mg/L lected within the STRIDA program. DBZD were detected in 217 (11.3%) (0.007–0.049 mg/L), thalamus: 0.325 mg/kg (0.065–1.013 mg/kg), out of 1913 urine samples analyzed. The mean age of patients was 28 : 0.584 mg/kg (0.099–2.125 mg/kg) and psoas muscle: 0.166 mg/ years (range 15–66; median 26), and 81% were men. The first analy- kg (0.034–0.469 mg/kg). In the remaining two cases phenazepam was tically confirmed intoxication involving DBZD (etizolam) was found in either the certified cause of death or a contributing factor. In the former January 2012. The frequency of samples tested positive for DBZD in- one, concentrations of phenazepam and 3-hydroxyphenazepam in fe- creased from 4% in 2012, 8% in 2013, 11% in 2014, to 19% in 2015, moral blood were 1.64 mg/L and 0.43 mg/L, and in urine 0.08 mg/L and then declined to 6% in 2016. Altogether 14 DBZD were found: and 0.19 mg/L. In the latter case (multidrug fatal intoxication) con- flubromazolam (92 cases), pyrazolam (33), flubromazepam (33), me- centrations of phenazepam and 3-hydroxyphenazepam in femoral clonazepam (26), etizolam (20), clonazolam (16), 3-hydro- blood were 0.97 mg/L and 0.17 mg/L, and in urine 0.55 mg/L and xyphenazepam (eight), nifoxipam (five), diclazepam (four), metizolam 3.52 mg/L (Crichton et al., 2015). The authors of this report suggest (four), bromazepam (one), deschloroetizolam (one), (one), that phenazepam may undergo post-mortem redistribution (Crichton and phenazepam (one). Twenty four patients (11%) tested positive only et al., 2015). Whether post-mortem distribution might occur in the case for DBZD. In the remaining cases other psychoactive drugs were de- of other DBZD remains to be elucidated. tected, including standard prescription benzodiazepines (49%), other Maskell et al. (2011) reported nine cases of death in the United such as opioids, ethanol and (29%), stimulants Kingdom where phenazepam was detected in post-mortem toxicology including phenethylamines and synthetic cathinones (11%); in half of but not implicated in the cause of death. Death was attributed to the samples a mixture of drugs was identified. Medical records and opioids in seven cases, but other drugs in two cases. The deceased were clinical data obtained for 23 patients who were intoxicated only with men and women aged 31–45 years. Phenazepam was found in 17 au- DBZD show that the most prominent clinical signs were CNS depression topsy cases in Finland between July 1, 2010 and June 30, 2011. Most of with a sedative- toxidrome, , dilated pupils, and the deaths were accidental overdoses. All of the deceased were male slurred speech (Bäckberg et al., 2018). Carpenter et al. (2018) analyzed with a mean age of 35 ± 7 years. The median phenazepam blood cases of intoxication related to DBZD and reported by 40 states to the concentration was 0.048 mg/L (range 0.007–1.600 mg/L). One or sev- U.S. National Poison Data System during January 1, 2014 – December eral opioids were detected in blood/urine in 12 of these cases, alcohol 31, 2017. Among 474 cases, 234 were classified as single compound in nine and amphetamine in four (Krrikku et al., 2012). exposures, and their number steadily increased from 26 in 2014, 30 in In Scotland, etizolam was the third most frequently detected sub- 2015, 66 in 2016 to 122 in 2017. Most of the cases (77%) represented stance reported among all drug-related deaths in 2016, after heroin/ acute intoxication. The median age of patients was 25 years (range and methadone. The number of deaths related to etizolam 13–63), and 86% were men. The most common benzodiazepine found increased from 58 in 2015 to 270 in 2016, with more women than men in these single-agent exposures was etizolam (162 cases; 69%), fol- being among the deceased (UNODC, 2018). In addition, 134 deaths due lowed by clonazolam (50 cases; 21%). Other identified compounds to intoxication with diclazepam were reported in Scotland in 2016 include flubromazolam (13 cases), diclazepam (three), flubromazepam (UNODC, 2018). (one), meclonazepam (one), and norflurazepam (one). The most A case of a 42-year old Caucasian male who died as the result of common clinical signs of intoxications were drowsiness/lethargy multidrug intoxication was described by Bailey et al. (2010). Near the (65%), slurred speech (17%), and confusion (14%). Less frequently body, a vial of white powder labeled "Phenazepam, Purity 99%, CAS observed symptoms were agitation/irritability, ataxia and tachycardia No. 51753-57-2, Research Sample", was found. Phenazepam, morphine, (each 9%), hypotension (8%), coma (4%), and bradycardia (4%). There , and were present in his femoral blood at concentra- was one death among the cluster with a reportedly intentional ingestion tions of 386, 116, 85 and 72 ng/mL, respectively. Elsewhere, a number of etizolam. In 85% of the subjects, the duration of clinical symptoms of substances were detected in femoral blood of a 22-year old man, was shorter than 24 h. In one case, intoxication with etizolam, the whose death was determined to be the aspiration of chyme, possibly symptoms persisted for longer than one month (Carpenter et al., 2018). due to a loss of consciousness: mitragynine (790 μg/L), etizolam (280 μg/L), pregabaline (3 mg/L), pipamperon (7.4 μg/L), 7. Fatal intoxications related to the use of designer (6.9 μg/L), (1.1 μg/L), fluoxetine (89 μg/L), benzodiazepines (18 μg/L), (5.9 μg/L), and (likely) 2-MMC (∼5.2 μg/L) (Domingo et al., 2017). Another report describes the case of a 42-year By analogy to pharmaceutical benzodiazepines, DBZD are involved old man who died in the course of myocardial infarction due to a mostly in polydrug deaths. In South Scotland during 2010–2014, there multidrug intoxication. Toxicological analysis revealed the presence of were 228 cases where phenazepam was detected in post-mortem fe- etizolam (0.3 mg/L) in his blood, as well as the synthetic cathinones moral blood (Shearer et al., 2015). In two of these cases, the cause of 3,4-methylenedioxypyrovalerone (0.046 mg/L) and pentedrone death was solely attributed to phenazepam (blood concentration of (0.16 mg/L), together with ephedrine (0.068 mg/L), olanzapine 1.2 mg/L and 1.6 mg/L). In 54 cases, the cause of death was related to (4.2 mg/L) and mitrazapine (0.57 mg/L) (Liveri et al., 2016). Other phenazepam along with one or more other drugs, mainly methadone, fatal poisoning cases were described by Tanaka and coworkers (Tanaka morphine and alcohol. The blood concentration of phenazepam in these et al., 2011a, 2011b). In one, toxicological analysis revealed the pre- cases ranged from < 0.005 to 0.9 mg/L (median 0.10 mg/L). Of the 83 sence of etizolam, , and in cases where phenazepam was present but not included in the cause of femoral blood at concentrations of 86 ng/mL, 5082 μg/mL, 0.107 μg/ death, its concentration in blood ranged from 0.005 to 0.46 mg/L mL and 0.144 μg/mL, respectively (Tanaka et al., 2011a). In another (median 0.05 mg/L). In these cases methadone, morphine and ethanol case, , 7-aminoflunitrazepam (a metabolite of flunitrazepam)

12 J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16 and ethanol were found to be present in femoral blood at concentra- with a median of 0.17 mg/L. In five cases phenazepam was the only tions of 0.025 μg/mL, 0.094 μg/L and 0.29 mg/mL, respectively drug detected. The observed effects where symptomatic of CNS de- (Tanaka et al., 2011b). The drugs were also detected in the stomach pression, with slurred speech, lack of balance, slow reactions, drowsi- contents of these two deceased (Tanaka et al., 2011a, 2011b). ness and confusion (Stephenson et al., 2013). There are four case reports of deaths related to intoxication with DBZD in combination with synthetic opioids (MT-45, U-47700, AH- 9. Metabolism 7921) used as NPS. Papsun et al. (2016) describe the case of a 35-year old man with a known history of drug abuse who was found dead. MT- Unlike pharmaceutical benzodiazepines, the vast majority of DBZD 45 and etizolam were detected in his femoral blood at concentrations of have not undergone clinical trials, and our knowledge of their phar- 520 ng/mL and 35 ng/mL, respectively. Similarly, Partridge et al. macokinetic properties and biotransformation is very limited. Assuming (2018) present a case of a 28-year-old man with a history of illicit drug that these substances could be extensively metabolized, there is a high use. U-47700 (330 μg/L), diclazepam (70 μg/L), flubromazepam need for effective analytical methods to detect their biotransformation (10 μg/L), methylamphetamine (290 μg/L) and amphetamine (150 μg/ products in biological samples, particularly urine. L) were found in post-mortem peripheral blood. Three main strategies are used to examine the metabolism of DBZD: In another study, mixed intoxication by U-47700 and flu- (1) incubation of substances with human liver microsomes (HLM) fol- bromazepam was found to have a lethal outcome in a 24‐year‐old man lowed by analysis of the produced metabolites, (2) analysis of urine admitted to a hospital after suffering apnoea associated with their samples of a large cohort of NPS users, and in rare cases (3) analysis of consumption. The concentrations of U‐47700 and flubromazepam in a urine samples in controlled self-administration studies. blood sample collected after admission were 370 μg/L and 830 μg/L, The two main biotransformation pathways for benzodiazepines are respectively. The man died six days later (Koch et al., 2018). Finally, generally oxidation and glucuronidation; for an excellent review see intoxication with AH-7921 in combination with other psychoactive Moosmann and Auwäter (2018). Phase I metabolism of DBZD involves drugs, including DBZD, was the cause of death of a young woman. The , N-dealkylation, and in the case of nitrobenzodiazepines concentrations of drugs in her blood were as follows: AH-7921 – – nitro reduction to the 7-amino compound; compounds containing 0.33 mg/L, methoxetamine – 0.064 mg/L, etizolam – 0.27 mg/L, phe- atom in their structure can also undergo debromination. The nazepam – 1.33 mg/L, 7-aminonitrazepam – 0.043 mg/L, diazepam – phase II reaction involves glucuronidation of the parent compound 0.046 mg/L, nordiazepam – 0.073 mg/L, and – 0.018 mg/L and/or its phase I metabolites, and acetylation of 7-amino metabolites (Karinen et al., 2014). of nitrobenzodiazepines (see Table 2). It should emphasized that not only some active metabolites of registered benzodiazepines are sold as 8. Designer benzodiazepines and impaired driving NPS, but also some metabolites of DBZD (i.e., 3-hydroxyphenazepam, deschloroetizolam and desmethyletizolam; , lorazepam Over the last decade, an increasing number of apprehended drivers and formed from diclazepam; clonazepam produced have been found to test positive for DBZD. The subjects commonly from cloniprazepam) can be responsible for the pharmacological ac- exhibited slurred speech, lack of balance, slow reactions, drowsiness tivity and toxic effects of the substance. and confusion (Kerrigan et al., 2013; Stephenson et al., 2013). Phena- DBZD vary significantly in terms of effective doses and duration of zepam was detected in 0.5% of 166 individuals driving under the in- action (Table 3), from short-acting drugs, e.g., etizolam and adinazolam fluence of drugs (DUID) that were killed in road traffic accidents in (elimination half-life of approximately three hours) to long-acting ones, Norway during 2006–2008 (Gjerde et al., 2011). Also in Norway, DBZD e.g., diclazepam (elimination half-life of around 42 h), phenazepam were detected in 77 out of 22,022 samples collected during the period (elimination half-lives of around 15 h after intravenous injection and July 1, 2013 – May 31, 2016. Sixty-nine were found to be under the 103 h after oral administration), and flubromazepam (elimination half- influence of drugs, and 14 of them were involved in a traffic accident life of around 106 h) (Lomas and Maskell, 2015; Moosmann et al., 2013, (Høiseth et al., 2016). The following median (range) blood concentra- 2014). The delayed onset of action may lead users to re-dose the drug tions of DBZD were recorded: flubromazolam – 0.012 mg/L before the effects of the first dose have been experienced, thus poten- (0.00048–0.10; n = 25), flubromazepam – 0.055 mg/L (0.0047–1.2; tially leading to overdose (Corkery et al., 2012). Drugs with a long n = 24), diclazepam – 0.013 mg/L (0.0021–0.057; n = 15), etizolam – elimination half-life are likely to accumulate in an organism, increasing 0.050 mg/L (0.019–0.17; n = 14), clonazolam – 0.0053 mg/L the risk of intoxication. (0.0019–0.011; n = 7), and pyrazolam – 0.074 mg/L (n = 1). In six cases, DBZD were the only drugs detected in blood samples. The other 10. Analytical detection of designer benzodiazepines and their most frequently detected drugs were Δ9-THC (51% of the cases), am- metabolites phetamine (44% of the cases) and ethanol (14% of the cases). In three cases, the DBZD were detected together with other NPS: cannabino- The identification and quantification of DBZD, both parent com- mimetics - 5F-APINACA (n = 2) and APINACA (n = 1); psychostimu- pounds and their metabolites, is an important task in the field of clinical lants - methiopropamine (n = 1) and α-PVP (n = 1) (Høiseth et al., and forensic toxicology. A liquid-liquid extraction and solid-phase ex- 2016). traction are used for sample clean-up and extraction of DBZD. Urine In Finland, 141 out of 4007 blood samples collected from appre- samples should be hydrolyzed using for example β-glucoronidase (Kintz hended drivers during the period between July 1, 2010 and June 30, et al., 2017a; Pettersson Bergstrand et al., 2016). Some DBZD have been 2011 tested positive for phenazepam. The majority of the drivers, 88%, detected in blood and urine using immunochemical assays with high were male. The mean age of the men was 34 ± 2 years and of women cross-reactivity, such as enzyme multiplied immunoassay technique 27 ± 5 years. The median phenazepam concentration in blood was (EMIT), cloned enzyme donor immunoassay (CEDIA), enzyme-linked 0.061 mg/L (range 0.004–3.600 mg/L). In seven cases phenazepam was immunosorbent assay (ELISA), and kinetic interaction of microparticles the only psychoactive substance present (median concentration in solution (KIMS) technique (O’Connor et al., 2016; Pettersson 0.270 mg/L). Other substances identified together with phenazepam Bergstrand et al., 2017). However, immunochemical screening of bio- were amphetamine and various pharmaceutical benzodiazepines logical specimens for DBZD has two important drawbacks: (1) the (Kriikku et al., 2012). drugs, especially the newest ones, may be missed when screened by In the state of Georgia (U.S.) phenazepam was found in blood immunoassay if they are not in the scope of the confirmation panel of samples taken from 11 impaired drivers between March, 2010 and benzodiazepines; (2) blood/serum levels of DBZD can be too low to be August, 2011. Concentrations of the drug ranged from 0.04 to 3.2 mg/L, detected by immunoassays (see Moosmann and Auwäter, 2018;

13 J.B. Zawilska and J. Wojcieszak Neurotoxicology 73 (2019) 8–16

Table 2 Metabolism of designer benzodiazepines.

Compound Metabolites Reference

Adinazolam In vitro (HLM): N‐desmethyladinazolam, N, N‐didesmethyladinazolam Moosmann et al., 2016 Diclazepam In vitro (HLM): monohydroxylation → delorazepam, desmetylation → lormetazepam El Balkhi et al., 2017 Human urine: delorazepam, lorazepam, lormetazepam El Balkhi et al., 2017; Moosmann et al., 2014 Human serum: delorazepam Etizolam In vitro (HLM): three monohydroxylated metabolites, keto-metabolite, etizolam glucuronide El Balkhi et al., 2017; Pettersson Bergstrand Post-mortem blood: α-hydroxyetizolam, 8-hydroxyetizolam et al., 2019 Nakame et al., 2008 Deschloroetizolam In vitro (HLM): hydroxydeschloroetizolam, dihydroxydeschloroetizolam, El Balkhi et al., 2017; Pettersson Bergstrand deschloroetizolam glucuronide et al., 2019 Flubromazolam In vitro (HLM, human hepatocytes): 4-hydroxyflubromazolam, α-hydroxyflubromazolam, El Balkhi et al., 2017; Noble et al., 2017; dihydroxyflubromazolam, flubromazolam glucuronide Pettersson Bergstrand et al., 2019; Wohlfarth Mice urine: α-hydroxyflubromazolam,4‐hydroxyflubromazolam, α‐hydroxyflubromazolam et al., 2017 glucuronide, α,4‐dihydroxyflubromazolam, flubromazolam glucuronide Wohlfarth et al., 2017 Human urine: α-hydroxyflubromazolam,4‐hydroxyflubromazolam, flubromazolam Huppertz et al., 2018; Noble et al., 2017; glucuronide, α‐hydroxyflubromazolam glucuronide, 4‐hydroxyflubromazolam glucuronide, Wohlfarth et al., 2017; Pettersson Bergstrand dihydroxyflubromazolam et al., 2018 Metizolam In vitro (HLM): 2-hydroxymetizolam, N-hydroxymetizolam, metizolam glucuronide Kintz et al., 2017a; Moosmann et al., 2016; Human urine: 2-hydroxymetizolam, N-hydroxymetizolam, 2-hydroxymetizolam glucuronide Pettersson Bergstrand et al., 2019 Kintz et al., 2017a Norflurazepam In vitro (HLM): hydroxynorflurazepam, dihydroxynorflurazepam. Moosmann et al., 2018 Phenazepam Human urine: 3-hydroxyphenazepam, 5-bromo-(2-chlorophenyl)-2-aminobenzophenone WHO, 2015 (ABPH), 6-bromo-(2-chlorophenyl) quinazoline-2-one (QNZ) Pyrazolam Human urine: pyrazolam glucuronide Pettersson Bergstrand et al., 2018 COMPOUNDS WITH NITRO GROUP Clonazolam In vitro (HLM): aminoclonazolam, desmethylclonazolam, hydroxyclonazolam El Balkhi et al., 2017 Human urine: 7-aminoclonazolam, 7-acetaminoclonazolam, hydroxyclonazolam, 7- Meyer et al., 2016 aminoclonazolam glucuronide, 7-acetaminoclonazolam glucuronide, hydroxyclonazolam glucuronide Cloniprazepam In vitro (HLM): 7-aminocloniprazepam, hydroxycloniprazepam, dihydroxycloniprazepam, 3- Moosmann et al., 2016; Mortelé et al., 2018 ketocloniprazepam, clonazepam, 7‐aminoclonazepam, hydroxyclonazepam, 3-hydroxy-7- aminoclonazepam, hydroxycloniprazepam glucuronide In vitro (HLM): hydroxyflunitrazolam, dihydroxyflunitrazolam, aminoflunitrazolam, Moosmann et al., 2018; Petterson Bergstrand flunitrazolam glucuronide et al., 2019 Human urine: desnitroflunitrazolam, 7-aminoflunitrazolam, 7-acetamidoflunitrazolam, Ameline et al., 2018 hydroxyflunitrazolam Fonazepam (norflunitrazepam) In vitro (HLM): 7-aminofonazepam (7-aminonorflunitrazepam), 3-hydroxyfonazepam (3- Moosmann et al., 2016 hydroxynorflunitrazepam; nifoxipam) Meclonazepam In vitro (HLM): aminomeclonazepam, hydroxymeclonazepam. El Balkhi et al., 2017 Human urine: 7-aminomeclonazepam, 7-acetaminomeclonazepam Meyer et al., 2016; Vikingsson et al., 2017 Nifoxipam In vitro (HLM):7-aminonifoxipam, denitro-nifoxipam, nifoxipam glucuronide El Balkhi et al., 2017; Pettersson Bergstrand Human urine: 7-aminonifoxipam, 7-acetaminonifoxipam, nifoxipam glucuronide et al., 2019 Meyer et al., 2016 Nitrazolam In vitro (HLM): 8‐aminonitrazolam, 4-hydroxynitrazolam/α-hydroxynitrazolam Moosmann et al., 2016

HLM – human liver microsomes. Main metabolites are written in bold.

Pettersson Bergstrand et al., 2017). Thus, results of the initial im- 11. Conclusions munoassay screening need confirmation using chromatographic methods, in particular based on mass spectrometry (MS) with up to date Novel benzodiazepines used as NPS are an example of designer library of references. To fulfill this requirement various methods of drugs consisting of derivatives of prescription medications or their DBZD and their metabolites detection in bodily fluids and tissue sam- metabolites legally used in psychiatry and neurology. Although these ples (blood, serum, urine, vitreous humour, hair, liver, brain, muscle) drugs produce effects similar to registered benzodiazepines, they could have been published. They are based on chromatography (GC), e.g., be very harmful, as they can produce marked cognitive and motor with flame-ionization detection (GC–FID) or coupled to MS (GC–MS), impairment after acute use, which may be a causal factor of traffic e.g., with electron ionization (GC–El–MS) or with negative–ion che- accidents. Secondly, as in the case of medical benzodiazepines, chronic mical ionization (GC–NCI–MS), as well as liquid chromatography (LC), use of DBZD may lead to the development of tolerance and drug de- e.g., with diode-array detection (HPLC–DAD) or coupled to MS (LC–MS pendence. Finally, the pattern of abuse involving the use of DBZDs or LC–MS/MS), e.g., liquid chromatography–triple quadrupole–mass poses a threat of serious interaction with alcohol and other CNS de- spectrometry (LC–QqQ–MS/MS) or liquid chromatography–quadrupole pressants, leading to the hyper-additive accumulation of their effects, time of flight–mass spectrometry (LC–QTOF–MS). (Crichton et al., and a greater risk of death due to respiratory depression. 2015; Kintz et al., 2017b; reviewed by Moosmann and Auwäter, 2018). Recently, an ultra-assisted low-density solvent dispersive liquid-liquid Conflict of interest microextraction (US-LDS-DLLME) plus gas chromatography–triple quadrupole mass spectrometry (GC-QQQ-MS) (Meng et al., 2017), and a The authors declare that they have no conflict of interest. non–aqueous capillary electrophoresis (NACE)–tandem mass spectro- metry (Švidrnoch et al., 2018) have been developed and used for the Acknowledgements identification of DBZD in urine and serum, respectively. Supported by grants from the National Science Centre (NCN, Cracow, Poland (2014/13/B/NZ7/02237)) and Medical University of Łódź, Łódź, Poland (503/3-011-01/503-31-002).

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Table 3 publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/ Oral doses and duration of action of designer benzodiazepines (TripSit file/616781/misuse_of_drugs_act_circular_008_2017.pdf. Advisory Council on the Misuse of Drugs, 2011. Phenazepam Advice. Available from: FactSheets). http://www.homeoffice.gov.uk/publications/agencies-public-bodies/acmd1/acmd- Compound Dose (oral) Duration of action advice-phenazepam?view=Binary. Accessed October 12, 2018. Ali, A., Jerry, J.M., Khawam, E.A., 2015. Delirium induced by a new synthetic legal in- toxicating drug: phenazepam. Psychosomatics 56 (4), 414–418. https://doi.org/10. Adinazolam Light 5-15 mg Onset 10-25 min 1016/j.psym.2014.05.018. Common 15-30 mg Duration 2-5 h Ameline, A., Richeval, C., Gaulier, J.-M., Raul, J.-S., Kintz, P., 2018. Detection of the Strong 30-50 mg+ After effects 1-16 h designer benzodiazepine flunitrazolam in urine and preliminary data on its meta- Light 0.5-1 mg Onset 15-45 min bolism. Drug Test. Anal. https://doi.org/10.1002/dta.2480. Common 1-3 mg Duration 5-8 h Anderson, M., Kjeligren, A., 2017. The slippery slope of flubromazolam: experiences of a Strong 3-5 mg+ After effects 1-12 h novel psychoactive benzodiazepine as discussed on a Swedish online forum. Nordic Studies Alc. Drugs 34 (3), 217–229. https://doi.org/10.1177/1455072517706304. Clonazolam Threshold 50-75 μg Onset 10-30 min μ Arens, A.M., van Wijk, X.M., Vo, K.T., Lynch, K.L., Wu, A.H., Smollin, C.G., 2016. Adverse Light 75-200 g Duration 6-10 h effects from counterfeit alprazolam tablets. JAMA Intern. Med. 176 (10), 1554–1555. μ ff Common 200-400 g After e ects 1-12 h https://doi.org/10.1001/jamainternmed.2016.4306. Heavy 500-1000 μg Bäckberg, M., Pettersson Bergstrand, M., Beck, O., Helander, A., 2018. Occurrence and Desmethyl- Light 0.5-1 mg Onset 40-60 min time course of NPS benzodiazepines in Sweden - results from intoxication cases in the flunitrazepam Common 1-2 mg Duration 5-10 h STRIDA project. Clin. Toxicol. Phila. (Phila) https://doi.org/ 10.1080/ Strong 2-4 mg+ After effects 2-12 h 15563650.2018.1506130. Diclazepam Light 0.25-1 mg Onset 15-90 min Bailey, K., Richards-Waugh, L., Clay, D., Gebhardt, M., Mahmoud, H., Kraner, J.C., 2010. Fatality involving the ingestion of phenazepam and poppy seed tea. J. Anal. Toxicol. Common 1-2 mg Duration 8-12 h 34 (8), 527–532. ff Strong >2mg After e ects 1-24 h Benerjee, D., 2018. Etizolam withdrawal catatonia: the first case report. Asian J. Etizolam Light 0.5-1 mg Onset 10-40 min Psychiatr. 37, 32–33. https://doi.org/10.1016/j.ajp.2018.07.019. Common 1-2 mg Duration 5-8 h Benesch, M.G.K., Iqbal, S.J., 2018. 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