The Journal of Toxicological Sciences (J. Toxicol. Sci.) 329 Vol.41, No.3, 329-337, 2016

Original Article Novel psychoactive benzofurans strongly increase extracellular level in mouse corpus striatum

Tatsu Fuwa1, Jin Suzuki1, Toyohito Tanaka1, Akiko Inomata1, Yoshiko Honda2 and Tohru Kodama2

1Department of Pharmaceutical and Environmental Sciences, Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunincyou, Shinjuku-ku, Tokyo 169-0073, Japan 2Laboratory of Physiological Psychology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506, Japan

(Received November 24, 2015; Accepted February 19, 2016)

ABSTRACT — We examined the effects of three benzofurans [1-(Benzofuran-5-yl)-N-methylpropan-2 - (5-MAPB), 1-(Benzofuran-2-yl)-N-methylpropan-2-amine (2-MAPB), and 1-(Benzofuran-5-yl)- N-ethylpropan-2-amine (5-EAPB)] on the extracellular monoamine level in mouse corpus striatum by the microdialysis method and compared them with the effects of psychoactive 3,4-Methylenedioxymeth- (MDMA). The effects of benzofurans on the extracellular monoamine level were qualita- tively analogous to that of MDMA, with an increase in serotonin (5-HT) level exceeding (DA) level. The effects of 2-MAPB and 5-EAPB were almost the same as the effect of MDMA. However, 5-MAPB strongly increased extracellular monoamine level than MDMA. These differences in the poten- cy appear to have a structure-activity relationship. The administration of 5-MAPB (1.6 × 10-4 mol/kg B.W.) resulted in the death of two-thirds of the mice. The same dose of MDMA did not cause any deaths. The administration of 5-MAPB (1.6 × 10-4 mol/kg B.W.) produced a 3.41°C ± 0.28°C rise in rectal tem- perature after 1 hr, whereas the administration of MDMA (1.6 × 10-4 mol/kg B.W.) produced an approxi- mate 1.85°C ± 0.26°C rise. These results suggest that benzofurans have 5-HT toxicity similar to MDMA, and 5-MAPB has a higher risk of lethal intoxication than MDMA. Furthermore, 5-APB, the metabol- ic product of 5-MAPB demethylation, may be involved in the acute 5-HT toxicity and may cause lethal intoxication in mice.

Key words: Benzofuran, 3, 4-Methylenedioxymethamphetamine, Microdialysis, Serotonin, Lethal intoxication

INTRODUCTION firmed chemical constitution, we have been examining the effect on the extracellular monoamine levels in the mouse Novel psychoactive substances (NPSs) have been central nervous system (CNS) following an oral admin- increasingly emerging in the recent years. These prod- istration. We found that most of these substances cause a ucts mimic the euphoric effect of other well-known illicit significant increase in monoamine levels in mouse CNS. drugs but are advertised as “legal highs” and are sold via We offer these scientific data to the Governor of Tokyo illegal drug outlets, such as head shops, delivery shops, Advisory Committee so the drugs can be declared illegal. and Internet shops. Because there is very little informa- In 2014, we examined the effect of 20 NPSs on the tion regarding the components, chemical formulas, and extracellular monoamine level. The compounds were clas- the activity of these products, NPS use has a high risk, sified in two chemical groups. The first group comprised and drug abuse remains a major public health issue world- synthetic (13 substances). The second group wide. comprised phenylethylamines (seven substances), except In Tokyo, we started analyzing the components and synthetic cathinones. This second group included three chemical formulas of NPSs and conducting marketing benzofurans, 1-(Benzofuran-5-yl)-N-methylpropan-2- research in 1996. Since 2007, for the substances with con- amine (5-MAPB), 1-(Benzofuran-2-yl)-N-methylpropan- Correspondence: Tatsu Fuwa (E-mail: [email protected])

Vol. 41 No. 3 330

T. Fuwa et al.

2-amine (2-MAPB), and 1-(Benzofuran-5-yl)-N-ethyl- propan-2-amine (5-EAPB). In Europe, these compounds were first mentioned in the early warning in 2013 and 2014 by the European Monitoring Centre for Drugs and Drug (EMCDDA, 2013, 2014). In Japan, these benzofurans were discovered in the Tokyo illegal drug market in 2014 (Suzuki et al., 2014). The chemical structures of 5-MAPB, 2-MAPB, and 5-EAPB are illustrated in Fig. 1, along with other sub- stances associated with these compounds. The benzofurans are molecules combining a benzene ring and an attached heterocyclic furan ring. 5-MAPB, and 5-EAPB are deriv- atives of ring-substituted , particular- ly 5-MAPB, which condensed dioxolane of methamphet- amine structure [3,4-Methylenedioxymethamphetamine (MDMA, “Ecstasy”)] is modified to furan. 2-MAPB and 5-MAPB are structural isomers of 1-(n-benzofuranyl)-N- methylpropan-2-amine. 5-(2-Aminopropyl) benzofuran (5-APB) and 6-(2-Aminopropyl) benzofuran (6-APB) are derivatives of ring-. 5-APB and 6-APB were first mentioned in the early warnings in 2010 and 2011 by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA, 2010, 2011). Green et al. (2013) summarized user reports regarding psychoac- tive effects of 5-APB and 6-APB; however, there is lit- tle information regarding 2-MAPB and 5-EAPB. Advi- sory Council on the Misuse of Drugs (ACMD, 2013) Fig. 1. Structures and chemical properties. Left column: di- has reported that “the N-methyl analogue of 5-APB was oxolane (IUPAC: 1, 3-dioxolane), furan (IUPAC: offered for sale as “5-MAPB,” and structurally this mate- 1-Oxa-2,4-cyclopentadiene), Benzofuran (IUPAC: 1-Benzofuran), (IUPAC: N-methyl- rial is closer to Ecstasy than APBs.” 1-phenylpropan-2-amine), and Amphetamine (IUPAC: 5-APB, 6-APB, and 5-EAPB inhibit monoamine re- 1-Phenylpropan-2-amine). Middle column: Tested uptake in in vitro assay (Iversen et al., 2013; Rickli et substances MDMA (IUPAC: 1-(Benzo[d][1,3]di- al., 2015). However, the effect of 5-MAPB and 2-MAPB oxol-5-yl)-N-methylpropan-2-amine, purity: 98%), has not been determined in vitro. Monte et al. (1993) had 5-MAPB (IUPAC: 1-(Benzofuran-5-yl)-N-methyl- propan-2-amine, purity: 98%), 2-MAPB (IUPAC: postulated that because of their structural similarity to 1-(Benzofuran-2-yl)-N-methylpropan-2-amine, purity: , benzofurans are likely to act as catecho- 98%), and 5-EAPB (IUPAC: 1-(Benzofuran-5-yl)-N- lamine-releasing or re-uptake-inhibiting agents. However, ethylpropan-2-amine, purity: 98%). Right column: The the effect of benzofurans on the extracellular monoamine related substances 5-APB [IUPAC: 5-(2-Aminopropyl) level has not been verified in animal studies. benzofuran, purity: 98%] and 6-APB [IUPAC: 6-(2- Aminopropyl) benzofuran]. In this study, we compared the effect of three benzo- furans, and the psychoactive MDMA on the level of extra- cellular monoamines in the mouse corpus striatum using Japan) one week before use. They were housed under the microdialysis method. conditions of constant room temperature (24°C ± 1°C) and humidity of 50% ± 5%, with a regular 12 hr/12 hr MATERIALS AND METHODS light/dark cycle. All experiments were approved by the Animal Experiment Committee of Tokyo Metropolitan Animal and experimental design Institute of Public Health. Eighty male Crlj:CD1 (IGS) mice (28-33 g, 6-7 weeks Extracellular levels of serotonin (5-HT), dopamine old) were used in this experiment. To habituate the ani- (DA), and (NE) were assayed in corpus mals to the experimental laboratory, the mice were pur- striatum of freely moving mouse using the microdialy- chased from Charles River Laboratories (Kanagawa, sis method and high-performance liquid chromatography

Vol. 41 No. 3 331

Benzofurans increase extracellular serotonin level with electrochemical detection (HPLC-ECD). The test compounds were dissolved in distilled water (D.W.), and the solution was orally administered at 0.2 mL/10 g body weight. This main study included nine groups. They were as follows: D.W.-administered mice (n = 6), mice administered with 0.8 × 10-4 mol/kg B.W. of MDMA (n = 9), 5-MAPB (n = 9), 2-MAPB (n = 9), 5-EAPB (n = 9), mice administered 0.4 × 10-4 mol/kg B.W. of MDMA or 5-MAPB (n = 6 for each), and mice administered 1.6 ×10-4 mol/kg B.W. of MDMA or 5-MAPB (n = 6 for each). Furthermore, we examined the effect of 5-APB (0.8 × 10-4 mol/kg B.W.; n = 6). Sepa- rately from microdialysis experiments, we measured the rectal temperature of all four mice administered MDMA (1.6 × 10-4 mol/kg B.W.) or 5-MAPB (1.6 × 10-4 mol/kg B.W.) before administration and 1 hr after administration, using a device for the measurement of rectal temperature (Terumo Finer CTM-303; Terumo, Tokyo, Japan) and a thermistor probe (ME-PDK061; Terumo). The thermis- tor probe head was inserted at approximately 1.5 cm from the anus, and temperature was recorded after temperature Fig. 2. Experimental setup for monoamine chromatographic stabilization (after approximately 3 min). analysis using a free-moving mouse. The connection of microdialysis lines adopted the free-moving can- nula system using a liquid swivel. Dialyzate samples Stereotaxic surgery and microdialysis probe were collected using an online sampling system with Mice were anesthetized with 2% (Mylan, auto-injector. The chromatogram data processing was Tokyo, Japan), and then transferred to a stereotaxic frame performed by Data Processor (EPC-500; Eicom). (SR-5M; Narishige, Tokyo, Japan) with a mouse anesthe- sia mask (GM-4; Narishige). A microdialysis probe was cm, height: 30 cm). The connection of microdialysis lines stereotaxically implanted in the corpus striatum [0.7 mm used the free-moving cannula system (with liquid swiv- anterior and 2.2 mm lateral from bregma at a depth of 3.4 el). Inlet tubing of the microdialysis probe with a liquid mm from the brain dorsal surface (Franklin and Paxinos, swivel (TCS2-23; Eicom) was connected to a 2.5-mL 2008)]. A concentric style microdialysis probe (D-I-06-02; gas-tight syringe (81417; Hamilton Company, NV, USA) Eicom, Kyoto, Japan) with an outer diameter of 0.24 mm filled with artificial cerebrospinal fluid (NaCl: 148.8 mM, and an active length of 6 mm was attached to a 2-mm cel- CaCl2. 2H2O: 1.8 mM, and KCl: 4.02 mM with pH 7.4). lulose semipermeable membrane. The probe was secured The microdialysis probe was perfused using a syringe to the cranial bone of the mouse using three stainless steel pump (ESP-64; Eicom) at a perfusion rate of 1.5 μL/min. screws (1-mm diameter, 3-mm length) and dental acrylic Outlet tubing of the microdialysis probe was connected to resin (Toughron Rebase; Miki Chemical Product Co. Ltd., an auto-injector (EAS-20S; Eicom). The analysis of dia- Kyoto, Japan). The surgical operation included maintain- lyzate samples was performed using a sampling online ing the mouse temperature at approximately 37°C on a system with the auto-injection time set at 20-min inter- thermal insulation mat (BWT-100A; Bio Research Center vals. We started measuring monoamine levels 180 min Co., Ltd., Tokyo, Japan) and was completed 40 min later after initiating dialysis when these levels stabilized. The after anesthesia. Microdialysis was performed a day after dialyzate samples were analyzed until 180 min after com- the stereotaxic probe implantation surgery, using the mice pound administration. that were sufficiently recovered from the procedure. Data analysis of monoamines level and statistics Microdialysis procedure The dialyzate samples were analyzed for monoam- Figure 2 illustrates the experimental setup for ines using high-performance liquid chromatography and monoamine chromatographic analysis using free mov- electrochemical detection (HPLC-ECD). The HPLC- ing mouse. Microdialysis experiments were performed ECD (HTEC-500; Eicom) comprised an ion-exchange in Semitransparent Testing Cage (bottom: 19 cm, top: 24 column (CAX; Eicom) and Pure Graphite Electrode

Vol. 41 No. 3 332

T. Fuwa et al.

(WE-PG; Eicom) and was maintained at 35°C. Mobile phase 40 min, and in the NE level with a peak value of 804% ± (0.1 M acetic acid with pH 6.0, 30% methanol, 0.05 M 97% at 40 min. 5-MAPB caused an increase in the 5-HT sodium sulfate, and 0.134 mM EDTA-2Na) was deliv- level with a peak value of 2194% ± 415% at 40 min, ered at a flow rate of 250 μL/min. Monoamine concentra- in the DA level with a peak value of 824% ± 168% at tions were calculated each integral calculus level of chro- 40 min, and in the NE level with a peak value of 1147% matograms from calibration of 10 pg. The mean values of ± 148% at 40 min. The effects of 5-MAPB were stronger five dialyzate samples (basal concentrations) of individu- than the effects of MDMA. 2-MAPB caused an increase al mice before compound administration (during 100 min in the 5-HT level with a peak value of 1091% ± 89% at before administration) were used as a baseline and defined 20 min, in the DA level with a peak value of 220% ± 17% as 100%. All values were re-calculated as a percentage at 20 min, and in the NE level with a peak value of 499% of these baselines, and then expressed as mean ± S.E. ± 29% at 40 min. 5-EAPB caused an increase in the 5-HT (n = 6-9 mice for each group). The overall effect of the level with a peak value of 1356% ± 117% at 40 min, in compounds on extracellular monoamine was expressed as the DA level with a peak value of 264% ± 13% at 40 min, the area under the curve (AUC). This gave the sum of rel- and in the NE level with 547% ± 31% at 40 min. ative changes in the extracellular level (increase rate per- Having reached the peak, the elevated monoamine lev- centage) of each monoamine over the 180 min post-treat- els caused by the administration of MDMA, 2-MAPB, or ment period (nine samples). We defined the mean of the 5-EAPB gradually decreased. But the elevated monoam- AUC in the D.W.-administered group as 100%. We recal- ine level was maintained for a long time until the end of culated the relative AUC of each treatment group, using the experiment (after 180 min), whereas D.W. caused a 100% for the D.W. group. The differences between rela- slight increase in monoamine levels for a short time, tive AUC values were analyzed using analysis of variance which rapidly returned to the basal levels. (ANOVA) and post hoc Tukey-Kramer test. In results of The overall effects of MDMA, 5-MAPB, 2-MAPB, dose-related response, Jonckheere test was used to statis- and 5-EAPB on the extracellular levels of 5-HT, DA, tical analyses whether there is dose-related response that and NE were estimated using AUC. The relative AUC dosage of MDMA and 5-MAPB expressed overall effects values are depicted in Fig. 3D. AUCs were examined of monoamine increase. using ANOVA and post hoc Tukey-Kramer test to inves- tigate the differences between the changes in the lev- RESULTS AND DISCUSSION els of monoamines 5HT, DA, and NE. Benzofurans and MDMA affected the levels of these monoamines to a

Our microdialysis analysis of benzofuran analogs rep- different extent (MDMA: F2, 24, = 21.4933, P = 4.47E- resents the first assessment of the neurochemical activity 06; 5-MAPB: F2, 24, = 11.6640, P = 0.000289; 2-MAPB: of these compounds in vivo. F2, 24, = 85.5818, P = 1.2E-11; 5-EAPB: F2, 24, = 58.9304, P = 5.5E-10). Tukey-Kramer test of AUC showed that the Basal extracellular concentrations of DA, 5-HT 5-HT levels significantly increased more than the DA lev- and NE in mouse corpus striatum els (P < 0.001) in all groups and more than the NE levels The basal concentration of DA, 5-HT, and NE in (5-MAPB group: P < 0.01, 2-MAPB group: P < 0.001, mouse corpus striatum was 5.91 × 10-14 ± 3.17 × 10-15; 5-EAPB group: P < 0.001), except for the MDMA group 1.15 × 10-15 ± 8.97 × 10-17; and 1.53 × 10-15 ± 1.81 × 10-16 (P = 0.21). NE level increased significantly more than mol/μL, respectively. the DA level (MDMA group: P < 0.001, 2-MAPB group: P < 0.001, 5-EAPB group: P < 0.05), except for the Effect of MDMA, 5-MAPB, 2-MAPB, and 5-MAPB group (P = 0.43). Benzofurans (5-MAPB, 5-EAPB with 0.8 × 10-4 mol/kg B.W dosage on 2-MAPB, and 5-EAPB) cause the strongest increase of extracellular monoamine levels the 5-HT, and increase the effects of NE more than those Fig. 3 (A, B and C) shows the temporal patterns of the of DA. extracellular monoamine level for 280 min, i.e., between Furthermore, AUCs were analyzed using ANOVA 100 min before and 180 min after oral administration and post hoc Tukey-Kramer test to examine the differ- of D.W. or 0.8 × 10-4 mol/kg B.W. dosage of MDMA, ences between the tested substances. MDMA, 5-MAPB, 5-MAPB, 2-MAPB, or 5-EAPB. 2-MAPB, and 5-EAPB caused significant increases

MDMA caused an increase in the 5-HT level with a in dialyzate 5-HT (F4, 37 = 13.8511, P = 5.36E-07), DA peak value of 1207% ± 135% at 20 min after administra- (F4, 37 = 9.7976, P = 1.65E-05), and NE (F4, 37 = 23.1985, tion, in the DA level with a peak value of 302% ± 37% at P = 1.17E-03). AUC of 5-HT significantly increased in

Vol. 41 No. 3 333

Benzofurans increase extracellular serotonin level

Fig. 3. Effects of MDMA, 5-MAPB, 2-MAPB, and 5-EAPB on extracellular monoamine level in mouse striatum. Temporal pat- tern of extracellular levels of 5-HT (A), DA (B), and NE (C) before and after oral administration of D.W. (×), MDMA (♦), 5-MAPB (●), 2-MAPB (○), and 5-EAPB (∆). The dosage was 0.8 × 10-4 mol/kg B.W. respectively. The arrow (↓) indicates the time when substances were administered. The results for each group are shown as means ± S.E. for 6 or 9 mice. D; the overall effects of administered substances as AUC of each monoamine response (from time 0 to 180 min after administra- tion). We used ANOVA followed by Tukey-Kramer test. ***P < 0.001, **P < 0.01, and *P < 0.05 were statistically compared with D.W. group. #P < 0.01 was statistically compared with MDMA group. +P < 0.001 was statistically different between increase ratios for 5-HT and DA. ∆P < 0.01 was statistically different between increase ratios for 5-HT and NE. xx P < 0.001 and xP < 0.05 were statistically different between increase ratios for NE and DA.

Vol. 41 No. 3 334

T. Fuwa et al. all the tested compound groups in comparison with D.W. istration) and AUC of dose-related responses for MDMA group (MDMA: P < 0.01, 5-MAPB: P < 0.001, 2-MAPB: and 5-MAPB treatments. AUCs were analyzed using P < 0.05 and 5-EAPB: P < 0.01). When we compared ANOVA and post hoc Jonckheere test to examine the benzofurans with MDMA, we found that AUC of 5-HT dose-related response. The statistical result revealed sig- in 5-MAPB group was significantly larger than that in nificant dose-related responses for MDMA and 5-MAPB MDMA group (P < 0.01). However, AUC of 5-HT in administration (P < 0.01 for each monoamine). In all 2-MAPB (P = 1.0) and 5-EAPB (P = 0.35) groups did dosage groups, the magnitude of elevated 5-HT always not significantly differ from that of 5-HT in MDMA exceeded that of DA, and the elevated monoamine lev- group. The temporal patterns of extracellular DA levels els did not return to the basal levels during 180 min after in MDMA, 2-MAPB, and 5-EAPB groups demonstrat- administration. Furthermore, the administration of a ed increases above the basal level after compound admin- 1.6 × 10-4 mol/kg B.W. dose of 5-MAPB conspicuous- istration. However, the Tukey-Kramer test revealed that ly caused a gradual increase in 5-HT levels, which were the relative AUCs of DA in MDMA (P = 0.29), 2-MAPB maintained without decrease until 180 min after dosing. (P = 0.89), and 5-EAPB (P = 0.40) did not differ from Tukey-Kramer test revealed that the relative AUCs of AUC in D.W. group. Only 5-MAPB treatment caused a monoamines in low dosage (0.4 × 10-4 mol/kg B.W.) of significant increase in the DA levels in comparison with 5-MAPB did not differ from the middle dosage (0.8 × the levels in D.W. group (P < 0.001). When we com- 10-4 mol/kg B.W.) of MDMA (5HT; P = 0.99, DA; pared benzofurans with MDMA, AUC of DA in the P = 1.00, NE; P = 0.98). These statistical results show 5-MAPB treated group was significantly larger than that that the titer of 5-MAPB is more than double of MDMA. in the MDMA group (P < 0.01). However, AUC of DA Elevation of the core temperature by administration of in 2-MAPB (P = 0.74) and 5-EAPB (P = 1.0) groups did 5-MAPB (1.6 × 10-4 mol/kg B.W.) was greater than that not significantly differ from that in MDMA group. AUC by MDMA (1.6 × 10-4 mol/kg B.W.). The administration of NE in all treated groups significantly increased in com- of 5-MAPB (1.6 × 10-4 mol/kg B.W.) produced an approx- parison with that in D.W. group (MDMA: P < 0.001, imate 3.41°C ± 0.28°C rise in rectal temperature after 5-MAPB: P < 0.001, 2-MAPB: P < 0.01, and 5-EAPB: 1 hr, whereas the administration of MDMA (1.6 × 10-4 mol/ P < 0.001). The comparison between benzofuran and kg B.W.) produced an approximate 1.85°C ± 0.26°C rise. MDMA treatments demonstrated that AUCs of NE in The administration of 5-MAPB (1.6 × 10-4 mol/kg 5-MAPB (P = 0.29) and 5-EAPB (P = 0.22) groups did B.W) resulted in the death of two thirds of the mice with- not significantly differ from that in MDMA group. AUC in 10 hr, but the same dose of MDMA caused no deaths. of NE in the 2-MAPB group was smaller than that in the These results reveal that 5-MAPB causes lethal intoxica- MDMA group (P < 0.01). tion more often than MDMA. These results suggest a structure-activity relationship. Green (2013) has reported that benzofurans have not The extent of the increase in the extracellular monoam- only clinically positive effects but also toxic effects. ine levels triggered by these compounds depends on the There are some case reports of human lethal intoxi- substitution of methylenedioxy ring by the furan ring cation by 5-APB (Adamowicz et al., 2014; Mclntyre (from dioxolane to furan). The effects of the monoam- et al., 2015). Few studies report such intoxication by ine release were stronger in 5-MAPB (furan ring) than 5-MAPB (personal communication), possibly because in MDMA (dioxolane). 5-MAPB and 5-EAPB are both of its late appearance in the market. There are many case 5′-isomers; however, the extension of the N-alkyl chain in reports of human lethal intoxication caused by MDMA 5-EAPB appears to attenuate its effect on the extracellular (Capela et al., 2009; Hall and Henry, 2006; Mallick and monoamine level. Although both 2-MAPB and 5-MAPB Bodenham, 1997). Hall and Henry give an account of tox- are structural isomers of 1-(n-benzofuranyl)-N-methyl- ic effects of MDMA in their clinical case report: “MDMA propan-2-amine, 5-MAPB (5′-isomer) increases the extra- is one of the many pharmacological triggers of the sero- cellular monoamine levels more strongly than 2-MAPB tonin syndrome.” They also say, “ (2′-isomer). clearly shows great similarity to the acute and multi-organ failure seen with MDMA toxicity and Dose-related responses to MDMA and 5-MAPB also malignant hyperthermia and neuroleptic malignant We analyzed MDMA and 5-MAPB administration in syndrome,” and “Survival with a core temperature great- some more detail. Fig. 4 shows the temporal patterns of er than 42°C is rare.” These reports and the results of our extracellular levels of 5-HT, DA, and NE for 280 min study suggest that benzofurans have 5-HT toxicity, simi- (between 100 min before until 180 min after drug admin- lar to MDMA.

Vol. 41 No. 3 335

Benzofurans increase extracellular serotonin level

Fig. 4. Effects of MDMA and 5-MAPB on dose-related monoamine level in mouse striatum. Temporal pattern of extracellular lev- els of 5-HT (A), DA (B), and NE (C) before and after oral administration of MDMA low dosage (∆); MDMA middle dos- age (□) and MDMA high dosage (○); 5-MAPB low dosage (▲), 5-MAPB middle dosage (■) and 5-MAPB high dosage (●). low dosage; 0.4 × 10-4 mol/kg B.W., middle dosage; 0.8 × 10-4 mol/kg B.W., high dosage; 1.6 × 10-4 mol/kg B.W. The arrow (↓) indicates the time when substances were administered. Each result is the mean ± S.E. for 6-9 mice. D; the overall effects of each dose-response; i.e., AUC of each monoamine (up to 180 min after administration). We used ANOVA followed by Jonckheere test to examine the dose-response. The statistical result revealed significant dose-related responses for MDMA and 5-MAPB administration (P < 0.01 for each monoamine).

Vol. 41 No. 3 336

T. Fuwa et al.

Effect of 5-MAPB and 5-APB on 5-HT release in mouse corpus striatum 5-MAPB, 2-MAPB, and 5-EAPB caused a larger increase of 5-HT than DA, and increased monoamines levels persisted for a considerable time. These two char- acteristics were in accordance with other reports about the effect of MDMA treatment (Baumann et al., 2007, 2008; Kehr et al., 2011). However, the effects of 5-MAPB were conspicuously stronger than the effects of MDMA, and there was an interesting temporal pattern of monoam- ine elevation with the administration of 0.8 × 10-4 mol/ kg B.W. dosage of 5-MAPB. 5-MAPB (0.8 × 10-4 mol/ kg B.W.) caused temporal fluctuation in the pattern of the increasing monoamine; monoamine levels gradually decreased from their peak until 100 min after drug admin- istration, but later increased (Fig. 3). Welter et al. (2015) using GC-MS and LC-(HR)-MS analytical procedures in rats and humans, showed that the main metabolites of 5-MAPB were 5-APB and 3-car- boxymethyl-4-hydroxymethamphetamine. Because we expected that the effects of 5-APB would be involved in effects of 5-MAPB administration, we measured the extracellular monoamine level after the administration of 5-APB. Fig. 5 shows an increase in the 5-HT level after 5-APB (0.8 × 10-4 mol/kg B.W) or 5-MAPB administration (0.8 × 10-4 mol/kg B.W). 5-APB caused gradually increas- ing the 5-HT levels. AUC of 5-HT after 5-APB adminis- tration showed a 23-fold increase over D.W. group, where- as 5-MAPB increased the 5-HT level by 12.5-fold. The increases in 5-HT level in 5-APB and 5-MAPB treatment Fig. 5. Elevation of extracellular 5-HT level in mouse striatum groups differed significantly (P < 0.05). There is a possibil- after 5-MAPB and 5-APB administration. The dosage was 0.8 × 10-4 mol/kg B.W. respectively. A; temporal ity that the increase in 5-HT by 5-MAPB caused the syner- pattern of 5-HT levels after treatment with 5-MAPB gy of the parent drug (5-MAPB) and metabolites (5-APB). (●), 5-APB (○), and D.W. (×). The arrow (↓) indicates However, Welter et al. (2015) described a qualitative and the time when substances were administered. Each not quantitative analysis, and there are no other quantita- result is the mean ± S.E. (5-APB, n = 6; 5-AMPB, tive analytical reports of 5-MAPB. Although it is desir- n = 9). B; the overall effects of 5-MAPB and 5-APB, showing AUC of a 5-HT response (up to 180 min af- able to perform a quantitative analysis of the parent drug ter drug administration). We used ANOVA followed (5-MAPB) and metabolites (5-APB), we speculate that by Tukey–Kramer test. Significant increase versus 5-APB, a demethylated metabolite of 5-MAPB, is involved D.W. group, **P < 0.01, Significant difference between in the acute 5-HT toxicity of 5-MAPB and contributes to 5-MAPB and 5-APB treatment, #P < 0.05 the lethal intoxication in mice. We think that metabolic quantitative analysis could be a topic for future research. This study revealed that the NPSs resembling MDMA Conflict of interest---- The authors declare that there is cause excessive release of 5-HT and might have a greater no conflict of interest. risk of lethal intoxication than MDMA. REFERENCES ACKNOWLEDGMENTS Adamowicz, P., Zuba, D. and Byrska, B. (2014): Fatal intoxication We wish to thank Mr. Yoshikazu Kubo and Mr. with 3-methyl-N-methylcathinone (3-MMC) and 5-(2-aminopro- Katsuhiro Yuzawa for their excellent technical assistance in pyl)benzofuran (5-APB). Forensic Sci. Int., 245, 126-132. ACMD (2013): Benzofurans: A review of the evidence of use and oral drug administration and temperature measurements. harm. https://www.gov.uk/government/publications/benzofurans-

Vol. 41 No. 3 337

Benzofurans increase extracellular serotonin level

a-review-of-the-evidence-of-use-and-harm. Hall, A.P. and Henry, J.A. (2006): Acute toxic effects of ‘Ecstasy’ Baumann, M.H., Clark, R.D. and Rothman, R.B. (2008): Locomo- (MDMA) and related compounds: overview of pathophysiology tor stimulation produced by 3,4-methylenedioxymethamphet- and clinical management. Br. J. Anaesth., 96, 678-685. amine (MDMA) is correlated with dialysate levels of seroton- Iversen, L., Gibbons, S., Treble, R., Setola, V., Huang, X.P. and in and dopamine in rat brain. Pharmacol. Biochem. Behav., 90, Roth, B.L. (2013): Neurochemical Profiles of some novel psy- 208-217. choactive substances. Eur. J. Pharmacol., 700, 147-151. Baumann, M.H., Wang, X. and Rothman, R.B. (2007): 3,4-Meth- Kehr, J., Ichinose, F., Yoshitake, S., Goiny, M., Sievertsson, T., ylendioxymethamphetamine (MDMA) neurotoxicity in rats: a Nyberg, F. and Yoshitake, T. (2011): , compared reappraisal of past and present findings. , with MDMA (ecstasy) and amphetamine, rapidly increase both 189, 407-424. dopamine and 5-HT levels in of awake rats. Capela, J.P., Carmo, H., Remião, F., Bastos, M.L., Meisel, A. and Br. J. Pharmacol., 164, 1949-1958. Carvalho, F. (2009): Molecular and cellular mechanisms of Mallick, A. and Bodenham, A.R. (1997): MDMA induced hyper- ecstasy-induced neurotoxicity: an overview. Mol. Neurobiol., thermia: a survivor with an initial body temperature of 42.9°C. J. 39, 210-271. Accid. Emerg. Med., 14, 336-338. EMCDDA (2010): EMCDDA-Europol 2010 Annual Report on the Mclntyre, I.M., Gary, R.D., Trochta, A., Stolberg, S. and Stabley, R. implementation of Council Decision 2005/387/JHA.http://www. (2015): Acute 5-(2-aminopropyl)benzofuran (5-APB) intoxica- emcdda.europa.eu/publications/implementation-reports/2010 tion and fatality: a case report with postmortem concentrations. Accessed August 28, 2015 J. Anal. Toxicol., 39, 156-159. EMCDDA (2011): EMCDDA-Europol 2011 Annual Report on the Monte, A.P., Marona-Lewicka, D., Cozzi, N.V. and Nichols, D.E. implementation of Council Decision 2005/387/JHA. http://www. (1993): Synthesis and pharmacological examination of benzo- emcdda.europa.eu/publications/implementation-reports/2011 furan, indan, and tetralin analogues of 3,4-(methylenedioxy) Accessed August 28, 2015 amphetamine. J. Med. Chem., 36, 3700-3706. EMCDDA (2013): EMCDDA-Europol 2013 Annual Report on the Rickli, A., Kopf, S., Hoener, M.C. and Liechti, M.E. (2015): implementation of Council Decision 2005/387/JHA. http://www. Pharmacological profile of novel psychoactive benzofurans. Br. emcdda.europa.eu/publications/implementation-reports/2013 J. Pharmacol., 172, 3412-3425. Accessed August 28, 2015 Suzuki, J., Ushiyama, K., Nakajima, J., Yoshida, M., Ichikawa, EMCDDA (2014): EMCDDA-Europol 2014 Annual Report on the Y., Takahashi, M., Uemura, N., Shimizu, S., Nagashima, M., implementation of Council Decision 2005/387/JHA. http://www. Shimizu, M., Moriyasu, T. and Nakae, D. (2014): Analysis of emcdda.europa.eu/publications/implementation-reports/2014 illegal drugs in the market in the fiscal year 2013. Ann. Rep. Accessed August 28, 2015 Tokyo Metr. Inst. Pub. Health, 65, 61-75. Franklin, K.B.J. and Paxinos, G. (2008): The Mouse Brain in stere- Welter, J., Kavanagh P., Meyer, M.R. and Maurer, H.H. (2015): otaxic coordinates. Third Edition. Elsevier, New York, USA. Benzofuran analogues of amphetamine and methamphetamine: Green, S.L. (2013): Benzofurans and Benzodifurans. In: Dargan PI, studies on the metabolism and toxicological analysis of 5-APB Wood DM, eds. Novel Psychoactive Substances: Classification, and 5-MAPB in urine and plasma using GC-MS and LC-(HR)- Pharmacology and Toxicology. Elsevier, Amsterdam. MSn techniques. Anal. Bioanal. Chem., 407, 1371-1388.

Vol. 41 No. 3