Tryptamines are monoamine alkaloids with hallucinogenic properties and are widely abused worldwide. To hasten the regulations of novel substances and predict their abuse potential, we designed and synthesized four novel synthetic tryptamine analogs: Pyrrolidino tryptamine hydrochloride (PYT HCl), Piperidino tryptamine hydrochloride (PIT HCl), N,N-dibutyl tryptamine hydrochloride (DBT HCl), and 2-Methyl tryptamine hydrochloride (2-MT HCl). Then, we evaluated their rewarding and reinforcing effects using the conditioned place preference (CPP) and self-administration (SA) paradigms. We conducted an open field test (OFT) to determine the effects of the novel compounds on locomotor activity. A head-twitch response (HTR) was also performed to characterize their hallucinogenic properties. Lastly, we examined the effects of the compounds on 5-HTR1a and 5-HTR2a in the prefrontal cortex using a quantitative real-time polymerase chain reaction (qRT-PCR) assay. None of the compounds induced CPP in mice or initiated SA in rats. PYT HCl and PIT HCl reduced the locomotor activity and elevated the 5-HTR1a mRNA levels in mice. Acute and repeated treatment with the novel tryptamines elicited HTR in mice. Furthermore, a drug challenge involving a 7-day abstinence from drug use produced higher HTR than acute and repeated treatments. Both the acute treatment and drug challenge increased the 5-HTR2a mRNA levels. Ketanserin blocked the induced HTR. Taken together, the findings suggest that PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl produce hallucinogenic effects via 5-HTR2a stimulation, but may have low abuse potential.
Tryptamines are a group of monoamine alkaloids (Fig. 1) that are synthesized through decarboxylation of the amino acid tryptophan (Tittarelli
Previous studies show that tryptamines have an affinity for 5-HT1a, 5-HT2a, and 5-HT2c receptors (Nagai
As part of the continuing efforts of the Drug Abuse Research Institute of Korea (DARC) to hasten the regulations of novel synthetic tryptamines and to predict the abuse potential of future synthetic tryptamine compounds with similar modifications, we synthesized four novel synthetic tryptamine analogs, (C) pyrrolidino tryptamine hydrochloride (PYT HCl), (D) piperidino tryptamine hydrochloride (PIT HCl), (E) N,N-dibutyl tryptamine hydrochloride (DBT HCl), and (F) 2-methyl tryptamine hydrochloride (2-MT HCl) (Fig. 1), and examined their abuse potential. We performed the conditioned place preference (CPP) in mice and self-administration (SA) test in rats to assess their rewarding and reinforcing effects. We also examined the effects of the new tryptamines on locomotor activity and 5-HTR1a mRNA expression in the prefrontal cortex (PFC). In addition, we investigated the effects of the acute and repeated treatment as well as the challenge in the HTR paradigm on the expression of 5-HTR2a in the PFC. We chose the PFC because it has been implicated in motor activity and the HTR (Fuster, 1991; Willins and Meltzer, 1997; Erberk and Rezaki, 2007). Another cohort of mice was pretreated with KS during the HTR test to determine the involvement of 5-HTR2a receptors in the hallucinogenic effects of the compounds.
All animals were purchased from Hanlim Animal Laboratory Co. (Hwasung, Korea) and were housed in a temperature- and humidity-controlled (22 ± 2°C, 55 ± 5%) room with a 12-h light/dark (07:00–19:00 h light) schedule. Male C57BL/6 mice, aged 6 weeks and weighing 20–25 g, were maintained at 6 per cage, and Sprague Dawley rats, aged 8 weeks weighing 250–300 g, were maintained at 4 per cage. The mice were used for the open field, CPP, and HTR assays. The rats were used for the SA test. Before the experiments, all animals were habituated to the laboratory settings for five days. They had free access to food and water during habituation and experiments; however, this was not the case for rats during the lever training and actual SA sessions. Animal care and maintenance were carried out in compliance with the Principles of Laboratory Animal Care (NIH publication No. 85–23 revised 1985) and the Animal Care and Use Guidelines of Sahmyook University, Korea. All efforts were made to minimize the number of animals used and their suffering.
PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl powders were synthesized and generously provided by the Laboratory of Medicinal Chemistry of Kyunghee University (Seoul, Korea), whereas KS and methamphetamine (METH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-MT HCl was diluted with 5% dimethyl sulfoxide, 5% polysorbate 80, and normal saline (0.9% w/v NaCl). PYT HCl, PIT HCl, DBT HCl, KS, and METH were diluted with normal saline. All drugs were administered either intraperitoneally (open field test [OFT], CPP and HTR) or intravenously (SA), and the doses were based on prior experiments that addressed abuse potential and tryptamine induced-HTR (Darmani
The place preference apparatus that was used consisted of two Plexiglas compartments (17.4×12.7×12.7 cm3) made of polyvinylchloride and separated by a guillotine door. To provide a visual and tactile difference between the compartments, one compartment was white with a 6.352-mm stainless steel mesh floor, while the other was black with a stainless-steel grid floor (3.2-mm diameter rods placed 7.9 mm apart); the compartments were covered and illuminated. Behavior was recorded and analyzed using a computer-based video tracking system (Ethovision, Noldus, Netherlands).
The CPP test consisted of three phases: habituation and pre-conditioning, conditioning, and post-conditioning. During habituation, the mice were allowed to explore both compartments for 20 min once a day for two days. On the day after habituation, the time spent on each side was recorded (pre-conditioning). To eliminate side-preference bias, the data from the pre-conditioning phase were used to group the animals that spent similar amounts of time in each compartment. Mice that spent over 840 s in one compartment were excluded from the test (n=5). In the conditioning phase, mice (n=10 per group) received PYT HCl, PIT HCl, DBT HCl, 2-MT HCl (0.3, 1, or 3 mg/kg), METH (1 mg/kg), or vehicle (veh) and were confined to a randomly designated compartment for 45 min. Parallel experiments were also performed with METH because this drug has been demonstrated to induce conditioned place preference (Berry
For the drug SA test, rats were placed in standard operant conditioning chambers (Coulbourn Instruments, Allen-town, PA, USA) with ventilation fans to mask the external noise. Each operant chamber had a food pellet dispenser, two 4.5-cm-wide response levers (left and right), a stimulus light located 6 cm above the left lever, and a centrally positioned house light (2.5 W, 24 V) at the top of the chamber. Downward pressure (approximately equivalent to 25 g) on the levers resulted in a programmed response (described below). Located beside the operant chamber was a motor-driven syringe pump that delivered solutions at 0.01 mL/s. Solutions flowed through polytetrafluoroethylene tubing connected to a swivel, which was mounted on a counterbalanced arm at the top of the chamber, allowing free movement of the animals within the chamber. The tubing was connected to an intravenous catheter implanted in the animal. Graphic State Notation software (Coulbourn Instruments) was used to control experimental parameters and collect data.
Initially, rats were trained to press the active (drug-paired) lever (30 min/day for 3 days) for a contingent food pellet reward on a continuous schedule of reinforcement. During training, rats were food-restricted such that no rat dropped below 90% of its starting body weight. Only those rats that acquired at least 80 pellets during the last session of training were selected and prepared for surgery. Intrajugular surgeries were performed according to a previously described procedure (De La Peña
The OFT was performed according to a previous study (Botanas
The HTR is a distinct twitching behavior of the head of rodents. The HTR procedure followed that described by our previous study (Abiero
To assess the acute and repeated effects, mice (n=6 per group) were treated i.p. with vehicle, PYT HCl, PIT HCl, DBT HCl and 2-MT HCl (3 mg/kg) once a day for 7 days and challenged with the same drug and dose following 7 days of drug abstinence. A dose of 3mg/kg was selected for the present study because in a previous study it had submaximal effects and reliably increased the number of head twitches (Abiero
Mice (n=6 per group) treated with PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl (3 mg/kg) or veh for 1-day, 7-day and challenge day were sacrificed by cervical dislocation and decapitation 30 min after the last drug dose. Brains were rapidly and carefully removed as described previously (Spijker, 2011) and placed in ice-cold saline to prevent damage to the brain. The PFC was dissected out and immediately frozen at −70°C until further use. The PFC was used because this brain region has been implicated in motor activity and in the induction of the HTR (Fuster, 1991; Willins and Meltzer, 1997; Erberk and Rezaki, 2007). Total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The RNA was further purified using the Hybrid-RTM kit (Geneall Biotechnology, Seoul, Korea). The concentration of total RNA was determined using a Colibri Microvolume Spectrometer (Titertek-Berthold, Pforzheim, Germany).
A quantitative real-time polymerase chain reaction (qRT-PCR) was performed to determine the levels of 5-HTR1a and 5-HTR2a mRNA in the PFC. The method was similar to that used in our previous study (Botanas
The results from CPP (Fig. 2), open field tests (Fig. 4A–4D) and qRT-PCR for 5-HTR1a (Fig. 4E) experiments were analyzed by one-way analysis of variance (ANOVA), followed by Dunnett’s test to compare the effects of PYT HCl, PIT HCl, DBT HCl, 2-MT HCl, and METH to the veh. Data from the active lever responses and the number of infusions (Fig. 3) during the 2-h/d, 7-day SA test, the HTR (Fig. 5A) and qRT-PCR for 5-HTR2a (Fig. 5C) experiments were analyzed using a two-way repeated measures analysis of variance (ANOVA) with treatment as the between-subject factor and SA days/treatment days as the within-subject factor. Bonferroni’s test was utilized to further compare the effects of PYT HCl, PIT HCl, DBT HCl, 2-MT HCl, and METH to the veh and each SA day/treatment day. The mean infusion during stable days (days 4–7) was also presented and analyzed with one-way ANOVA for PYT HCl, PIT HCl, DBT HCl, 2-MT HCl, and the unpaired, two tailed
Fig. 2 displays the CPP scores of mice treated with the test drugs (i.e., PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl), METH, and veh. A one-way ANOVA revealed that METH significantly increased the CPP score [F (13,105)=16.12,
Fig. 3 shows the number of active lever responses and infusion of veh, PYT HCl, PIT HCl, DBT HCl-and 2-MT HCl-treated rats during the 2-h/day, 7-day SA sessions under the FR1 schedule. Rats self-administering PYT HCl displayed a significant difference between treatments [F (3,168)=3.829,
Fig. 4A–4D illustrate the locomotor activity of the mice treated with PYT HCl, PIT HCl, DBT HCl, 2-MT HCl (1, 3, 10 mg/kg), METH (1 mg/kg), or veh. It was revealed that PYT and PIT dose-dependently decreased the locomotor activity of the mice. One-way ANOVA showed a significant locomotor alteration [PYT HCl [F (4,48)=25.90,
Fig. 5A demonstrates the acute and repeated effects of mice treated with PYT HCl, PIT HCl, DBT HCl, 2-MT HCl, or veh in the HTR assay. A two-way repeated measures ANOVA revealed a significant difference between treatments [F (4,25)=83.90,
In the present study, we found that the novel synthetic tryptamine analogs PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl did not induce CPP in mice and were not reliably self-administered by rats, suggesting that these drugs have no or minimal rewarding and reinforcing effects. This result is similar to other tryptamines that fail to produce CPP and SA in animals (Fantegrossi
Consistent with the results of other tryptamines, PYT HCl and PIT HCl dose-dependently decreased the locomotor activity of the mice (Krebs-Thomson
Similar to other tryptamines, acute treatment with PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl induced the HTR in mice (Fig. 5A) (Fantegrossi
In conclusion, PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl do not induce rewarding and reinforcing effects; therefore these drugs are likely to have a low potential for abuse. Furthermore, these novel tryptamines induce hallucinogenic effects, as evidenced in the HTR assay, which is probably mediated by the activation of 5-HTR2a in the PFC. However, additional studies (e.g. structure-activity relationship) are necessary to clarify the differential effects of each analog in locomotor activity and HTR assays. Nonetheless, the present study provides valuable information that may be useful in predicting the abuse potential and psychopharmacological effects of potential synthetic tryptamines.
This work was supported by the Ministry of Food and Drug Safety (MFDS) of Korea (14182MFDS979, 19182MFDS410). The authors have no conflicts of interest to declare.