# Unveiling the potential abuse liability of α-D2PV: A novel α-carbon phenyl-substituted synthetic cathinone

Núria Nadal-Gratacós, Sandra Mata, Pol Puigseslloses, Morgane de Macedo, Virginie Lardeux, Stephanie Pain, Fu-Hua Wang, Liselott Källsten, David Pubill, Xavier Berzosa, et al.

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## To cite this version:

Núria Nadal-Gratacós, Sandra Mata, Pol Puigseslloses, Morgane de Macedo, Virginie Lardeux, et al. Unveiling the potential abuse liability of α-D2PV: A novel α-carbon phenyl-substituted synthetic cathinone. Neuropharmacology, 2025, 272, pp.110425. 10.1016/j.neuropharm.2025.110425. inserm-05348280

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# Unveiling the potential abuse liability of α-D2PV: A novel α-carbon phenyl-substituted synthetic cathinone

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Núria Nadal-Gratacós<sup>a,●</sup>, Sandra Mata<sup>b</sup>, Pol Puigseslloses<sup>a,●</sup>, Morgane De Macedo<sup>c</sup>, Virginie Lardeux<sup>c</sup>, Stephanie Pain<sup>c</sup>, Fu-Hua Wang<sup>d</sup>, Liselott Källsten<sup>d</sup>, David Pubill<sup>a,●</sup>, Xavier Berzosa<sup>b,●</sup>, Jan Kehr<sup>d</sup>, Marcello Solinas<sup>c</sup>, Jordi Camarasa<sup>a</sup>, Elena Escubedo<sup>a</sup>, Raul López-Arnau<sup>a,∗</sup>

## ARTICLE INFO

Handling Editor: Thomas Gould

**Keywords:**

Synthetic cathinones
Addiction
α-D2PV
Dopamine
Self-administration

## ABSTRACT

Synthetic cathinones are emerging psychoactive substances designed to mimic the effects of classical psychostimulants. Among them, α-D2PV, a novel pyrrolidine-containing cathinone characterized by a phenyl group on the α-carbon atom, has gained significant attention. This study investigates the *in vitro* and *in silico* mechanism of action as well as the abuse liability of α-D2PV using rodent models. Dopamine (DA), noradrenaline (NE), and serotonin (5-HT) uptake inhibition assays were conducted in HEK293 cells expressing the corresponding human monoamine transporter, complemented by molecular docking studies at the DA transporter (DAT). Behavioral studies in male Swiss CD-1 mice assessed locomotor activity and conditioned place preference, while microdialysis and self-administration experiments were performed in male Sprague-Dawley rats. The findings reveal that α-D2PV is a potent DA and NE uptake inhibitor, with minimal activity at the 5-HT transporter (SERT). Docking studies showed that the benzene rings of α-PVP and α-D2PV align precisely in their most stable conformations at DAT. In vivo, α-D2PV elicited dose-dependent hyperlocomotion, thigmotaxis, and rewarding effects in mice, alongside increased extracellular DA levels in the nucleus accumbens of awake rats. Self-administration experiments confirmed α-D2PV's high reinforcing efficacy, indicating a significant risk of abuse in humans. Finally, these results underscore the necessity for continued surveillance of α-D2PV within the illicit drug market. Furthermore, novel synthetic cathinones incorporating a phenyl ring at the α-carbon side chain warrant proactive monitoring due to their potential to retain dopaminergic activity and evade initial legal controls.

## 1. Introduction

New psychoactive substances (NPS) are a diverse group of psychoactive compounds initially sold as legal alternatives of classical and scheduled drugs of abuse. Although some efforts have been made by authorities to reduce its widespread impact, the drug market is continuously evolving with an ever-increasing number of NPS that substitute the already scheduled ones. Synthetic cathinones are a class of NPS derived from cathinone, an active compound of the khat plant, which mimic the effects of classical psychostimulants, such as amphetamine,

methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA) or cocaine (for a review see (Riley et al., 2020)). Among synthetic cathinones, pyrrolidine-containing cathinones, such as α-pyrrolidinopentiophenone (α-PVP, also known as “flakka”) and 3, 4-methylenedioxypyrovalerone (MDPV) (Fig. 1), have gained widespread popularity due to numerous intoxications, fatalities and their high potential for abuse (Beck et al., 2015, 2018; Gannon et al., 2018a; Karila et al., 2018; Kesha et al., 2013; Murray et al., 2012; Patocka et al., 2020; Ross et al., 2012; Wright and Harris, 2016), sparking significant concern among public health authorities. In this sense,

<footnote><sup>a</sup> Department of Pharmacology, Toxicology and Therapeutic Chemistry, Pharmacology Section and Institute of Biomedicine (IBUB), Faculty of Pharmacy, Universitat de Barcelona, 08028, Barcelona, Spain</footnote>

<footnote><sup>b</sup> Chemical Reactions for Innovative Solutions (CRISOL), IQS School of Engineering, Universitat Ramon Llull, 08017, Barcelona, Spain</footnote>

<footnote><sup>c</sup> Laboratoire de Neurosciences Expérimentales et Cliniques, Université de Poitiers, INSERM, U-1084, Poitiers, France</footnote>

<footnote><sup>d</sup> Pronexus Analytical AB, Grindstuvägen 44, 167 33, Bromma, Sweden</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: raullopezarnau@ub.edu (R. López-Arnau).</footnote>
α-pyrrolidino-2-phenylacetophenone (α-D2PV), a novel pyrrolidine-containing cathinone defined by the presence of a phenyl group on the α-carbon atom (Fig. 1), has been recently detected and identified in the illicit drug market (Bade et al., 2024; EMCDDA, 2022; Puljević et al., 2024; Wood et al., 2024). Given the rapid development of new designer drugs, it is crucial to provide data about the addictive potential of this class of novel compounds to assist law enforcement and public health services, as well as to raise awareness among the general population through scientific-based evidence.

Synthetic cathinones are known to interact with monoaminergic transporters such as dopamine, noradrenaline and serotonin transporters (DAT, NET and SERT, respectively), among other targets, leading to increased extracellular levels of dopamine (DA), noradrenaline (NE) and/or serotonin (5-HT) in the brain through monoamine reuptake inhibition. In vivo studies using rodent models have demonstrated that many synthetic cathinones can induce rewarding and reinforcing effects (Aarde et al., 2015a,b; Dolan et al., 2018; Gannon et al., 2018a,b; King and Riley, 2017; Riley et al., 2020; Watterson and Olive, 2014). In fact, DA increases in the nucleus accumbens (NAcc), are thought to underlie such reinforcing properties (Di Chiara and Imperato, 1988; Koob et al., 1998; Nestler, 2005). Furthermore, some authors have demonstrated that high DAT/SERT ratio of interaction with cathinones is associated with an increased abuse liability (Bauer et al., 2013; Baumann et al., 2011; Dolan et al., 2018; Glennon and Young, 2016; Negus and Banks, 2017; Schindler et al., 2016). Particularly, Gannon and colleagues demonstrated a correlation between the reinforcing effectiveness of pyrrolidine-containing cathinones and their selectivity for DAT vs SERT (Gannon et al., 2018b).

Among pyrrolidine-containing cathinones, α-PVP became available after the scheduling of MDPV, a first generation synthetic cathinone with a similar structure and mechanism of action (Drug Enforcement Administration, 2011; Karila et al., 2018). In spite of the schedule of α-PVP in 2017 (Drug Enforcement Administration, 2017), its use and abuse as well as intoxication and fatality cases continue to be reported

globally (Heinonen et al., 2021; Kriikku and Ojanperä, 2024; Lunetta et al., 2020; Patocka et al., 2020; Pierluigi et al., 2018; Richman et al., 2018). Like MDPV, α-PVP is a potent DA and NE uptake inhibitor with little to no action at SERT and is capable of increasing synaptic DA levels in the NAcc (Gannon et al., 2018a; Marusich et al., 2014; Schindler et al., 2020; Wojcieszak et al., 2018). Furthermore, it has been demonstrated that α-PVP induces rewarding and reinforcing effects in rodent models (Aarde et al., 2015a; Gannon et al., 2018b; Javadi-Paydar et al., 2018; Marusich et al., 2019; Nelson et al., 2019).

α-D2PV, a novel synthetic cathinone with a singular structural substitution, – the presence of a phenyl ring instead of the alkyl chain that distinguishes from other synthetic cathinones – has garnered considerable attention on drug user's forums. In fact, some users mention that this NPS possess strong effects, highlighting euphoria, stimulation or increased libido as the most frequent symptoms. According to such drug user's forums, a common dose of α-D2PV is 20–50 mg administered by inhalation, nasal insufflation or intravenous injection (Kuropka et al., 2022; Reddit.com, 2021a, 2021b). However, despite its emergence and detection in the illicit drug market, no published pharmacological or scientific studies are available about the mechanism of action and the abuse potential of α-D2PV. Given the obvious limitations of controlled human trials, the use of preclinical models of drug use and abuse is crucial. Therefore, the aim of the present work was to provide data regarding the mechanism of action and the potential abuse liability of α-D2PV in rodent models. Particularly, we investigated its potency in inhibiting DA, NE and 5-HT uptake, its interaction mechanism at hDAT, as well as its dose-dependent induction of hyperlocomotion and rewarding effects in mice. Additionally, we investigated its ability to alter extracellular DA levels (and metabolites) in the NAcc and its reinforcing effects in rats.

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Chemical structure of MDPV, α-PVP, methylphenidate and α-D2PV.</figcaption></figure>
## 2. Materials and methods

### 2.1. Drugs and reagents

α-D2PV was synthetized in racemic form as hydrochloride salt and identified as described in Supplementary Information (Figs. S1 and S2, respectively). [³H]1-Methyl-4-phenylpyridinium ([³H]MPP⁺) and [³H] 5-HT were purchased from PerkinElmer Inc. (Boston, MA, USA). Cocaine was generously provided by the Spanish National Institute of Toxiciology. α-PVP was synthetized in racemic form as hydrochloride salt as described by (Duart-Castells et al., 2021). Cell culture media [Dulbecco's modified Eagle's medium (DMEM) high-glucose] was purchased from Merck/Sigma-Aldrich (St. Louis, MO, USA). Solutions for injection were prepared daily in isotonic saline solution (0.9 % NaCl, pH 7.4). Dopamine hydrochloride (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenylacetic acid (HVA) and all other chemicals were purchased from Sigma-Aldrich, except of acetonitrile (ACN, LC-MS grade) and methanol (MeOH, LC-MS grade), which were purchased from Thermo Fisher Scientific (Gothenburg, Sweden). The stable isotope deuterium labeled DA-d4, DOPAC-d5, and HVA-d3 were purchased from Toronto Research Chemicals (Toronto, ON, Canada) and were used as the internal standards. Ultrapure water was generated by a Direct-Q-3 UV water purification system (Merck Millipore, Darmsatadt, Germany). All other reagents were of analytical grade and purchased from several commercial sources.

### 2.2. Subjects

Male Swiss CD-1 mice (Janvier, Le Genest, France) weighing 30–35 g (6–8 weeks old), were used for horizontal locomotor activity (HLA) and conditioned place preference (CPP) experiments. Male Sprague-Dawley rats (Janvier, Le Genest, France) weighing 300–400 g (8–12 weeks old), were used for microdialysis and self-administration (SA) experiments. All animals were housed in temperature-controlled conditions (22 ± 1 °C) under a 12 h light/dark cycle and with free access to food and drinking water and were randomly assigned to an experimental group. All animal care and experimental protocols are approved by the Animal Ethics Committee of the University of Barcelona under the supervision of the Autonomous Government of Catalonia, by the regional ethical committee at Stockholm county court (Stockholms Norra djurförsökstiska nämnd) following the directives of the Swedish Animal Welfare Act 1988:534 or the local ethics committee (COMETHEA), and are in accordance and comply with the guidelines of the European Community Council (2010/63/EU), as amended by Regulation(EU) 2019/1010, the ARRIVE guidelines for reporting experiments involving animals (McGrath et al., 2010). All efforts were made to minimize animal use and suffering.

### 2.3. Cell culture

Human embryonic kidney cells (HEK293) stably transfected with the human isoforms of the transporters DAT, NET and SERT (hDAT, hNET and hSERT) were used for DA, NE and 5-HT uptake inhibition assays. Cells were maintained in DMEM supplemented with heat-inactivated 10 % fetal bovine serum (FBS), 1 µg/ml streptomycin, 100IU/ml penicillin, geneticin (G418; 50 µg/ml) and cultured in a humidified atmosphere (5 % CO2, 37 °C) up to a subconfluent state. For uptake inhibition, cells were seeded at a density of 0.36 million cells/well onto 96-well plates previously coated (24h) with poly-D-lysine (PDL) 24 h prior to the experiment.

### 2.4. DA, NE and 5-HT uptake inhibition assays

On the day of the experiment, the culture medium was removed and immediately replaced with 200µl/well of Krebs-HEPES-Buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 20 mM

D-glucose; pH 7.3). A preincubation step, in which cells were incubated with different concentration of the drug in KHB at a final volume of 50 µL/well for 5 min, was performed to ensure equilibrated conditions. The preincubation solution was then removed and cells were incubated with 0.02 µM [³H]MPP⁺ during 3 min for DAT and NET or 0.1 µM [³H]5-HT for 1 min for SERT, along with different concentrations of the drug in KHB. The tritiated substrate was then removed, cells were washed with ice-cold KHB and lysed with 1 % sodium dodecyl sulfate (SDS). The lysate was added to scintillation fluid and the radioactivity was quantified with a beta-scintillation counter (PerkinElmer, Waltham, MA, USA). Non-specific uptake was determined in the presence of cocaine (100 µM) for DAT and NET or paroxetine (3 µM) for SERT and subtracted throughout. Non-specific uptake represented <10 % of total uptake. Uptake in absence of the drug was normalized to 100 %, and uptake in the presence of different concentrations of drugs was expressed as a percentage thereof.

### 2.5. Molecular docking

The structural model of DAT was obtained from the Protein Data Bank (PDB ID: 9EO4), which corresponds to the cryo-EM reconstruction of the outward-open structure of the hDAT bound to cocaine, with a resolution of 2.66 Å. First, the SMILES strings of the molecules were submitted to SwissParam, which generates information on alternative tautomers and protonation states of the query molecule. The resulting mol2 files were used as input ligands for docking experiments. The docking protocol was validated by reproducing the interaction mechanism described for cocaine (See Supplementary Information/Fig. S3). Both cathinones were built as (S)-enantiomer in protonated form, and they were docked into the hDAT model using the SwissDock web server (Bugnon et al., 2024; Grosdidier et al., 2011), which provides a free, user-friendly, yet state-of-the-art protein-small molecule docking tool, leveraging the Attracting Cavities algorithm (Röhrig et al., 2023; Zoete et al., 2016). To ensure the accuracy of the procedure, docking experiments were perfomed with R-cocaine as reference. Moreover, due to structural similarities with α-D2PV (a phenyl ring at the α-carbon side chain), methylphenidate was also included (see Fig. 1). All figures were prepared using Open-Source PyMOL 2.5.0.

### 2.6. Horizontal locomotor activity

In the habituation phase, which lasted for two consecutive days, mice received an intraperitoneal (i.p.) saline injection and were immediately placed into a black Plexiglass open field arena (25 x 25 × 40 cm) under low-light conditions and white noise for 30 min. On the test day, the HLA was measured as described (Nadal-Gratacos et al., 2021). In short, independent group of mice received an i.p. Injection of saline (5 ml/kg) or different doses (3, 10 or 30 mg/kg) of α-D2PV and were immediately placed in the open field arena with the same conditions of light and noise. HLA was video-monitored for 1 h and a tracking software (Smart 3.0 Panlab, Barcelona, Spain) was used to measure their total travelled distance (in cm). All doses and route of administration were chosen according to previous published studies on synthetic cathinones performed in our laboratory (Duart-Castells et al., 2021; Nadal-Gratacos et al., 2021, 2023). Moreover, locomotor activity was also recorded in a separate cohort of rats in order to confirm that the selected dose for microdialysis experiments was effective in evoking a robust locomotor activation. The behavioral test was performed in a quiet and dimmed (80 lux) experimental room. Initially, each rat was placed into a transparent polycarbonate cage (48 × 38 cm, height 40 cm) and allowed to habituate for 60 min. Thereafter, the rats were administered s.c. With α-D2PV (3 mg/kg) or vehicle and placed back to the cages. Locomotor activity was video-recorded by use of a computerized video-system Smart 3.0 (Panlab, Harvard Apparatus, USA). The computerized system was set to calculate the distance travelled in 5-min bins (cm/5 min).
## 2.7. Conditioned place preference (CPP) test

The potential of the studied compounds to induce rewarding effects was determined using a place conditioning paradigm, as described (Duart-Castells et al., 2021), using the same doses and route of administration as in the locomotor activity test. For this experiment, an apparatus with two distinct compartments, with differences in tactile and visual cues, connected by a central corridor, was used. Briefly, conditioned place preference was performed in three phases: pre-conditioning test, conditioning and post-conditioning test. During the preconditioning phase (Day 0), mice were placed in the middle of the corridor and had free access to both compartments for 15 min. The time spent in each compartment was recorded and monitored using a specific tracking software (Smart 3.0 Panlab, Barcelona, Spain). During the conditioning phase (Day 1- Day 4; two sessions per day separated by a 5-h period), the access to the corridor was closed, and mice received an i. p. Injection of α-D2PV (3, 10 or 30 mg/kg) and were immediately placed into one of the compartments for 20 min. On alternate sessions, mice received a saline i.p. Injection and were placed for 20 min in the other compartment. Mice in the control group received a saline injection in every session. The compartment and session in which mice received the drug was counterbalanced. On the test day (post-conditioning phase), the same conditions as in the pre-conditioning phase were applied. A preference score was calculated as the difference between the time spent in the drug-paired compartment in the post-conditioning test minus the time spent in the preconditioning phase.

## 2.8. Microdialysis experiments

The microdialysis experiments were carried out on awake rats following the protocol described elsewhere (Kehr and Yoshitake, 2006, 2017).

### 2.8.1. Surgery

The stereotaxic surgeries were carried out under aseptic conditions following current recommendations for rodent surgery and analgesia (Flecknell, 2018; Hoogstraten-Miller and Brown, 2008). About 30-60 min before the surgery, each animal received buprenorphine (Temgesic, 0.05 mg/kg s.c.) and carprofen ('Rimadyl', Pfizer) 5 mg/kg subcutaneously (s.c.). The rats were anaesthetized with isoflurane using a Univentor 400 anesthesia unit (AgnThos, Lidingo, Sweden) and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) using a flat skull position with incisor bar set to -3.2 mm. An ocular lubricant (Viscotears, Novartis) was applied to both eyes to prevent drying of the cornea during surgical procedure. The body temperature of the animal was controlled by a thermometer and a heating pad maintained at 37 °C during the operation by use of a CMA/105 temperature controller (CMA Microdialysis, Stockholm, Sweden). Hair was clipped from the appropriate surgical site and disinfected with chlorhexidine solution (1 %) and the local anesthetic Marcaine (bupivacaine) was injected s.c. at the site of the skin incision. A middle scalp incision of 1.5-2 cm was made, and the flaps were kept aside using the homeostatic forceps. After exposing the skull, one small hole was drilled on the right side and two small holes were drilled for the anchor screws using a fine trephine drill. Two microscrews were placed into the skull. A guide cannula (CMA Microdialysis) was implanted into the NAcc at the following coordinates: AP +2.2 mm; L -1.2 mm; DV -5.6 mm, providing the final DV -7.6 mm for the tip of the microdialysis probe. The coordinates were specified from bregma and the dural brain surface according to the atlas of (Paxinos and Watson, 2006). The guide cannula was fixed firmly to the skull surface using dental cement (Dentalon Plus, Heraeus, Germany). After completing the surgery, the animals were kept under a warm (IR) lamp in their home cages and allowed to recover from anesthesia. The operated rats were kept individually in their home cages (Eurostandard III H, Tecniplast, Italy) until the day of microdialysis experiment. During this period, the general health status of the animals was monitored on a

regular basis. On the following day after the surgery, the rats were additionally treated with carprofen (5 mg/kg s.c.) and twice with buprenorphine (0.05 mg/kg s.c.) with 8 h in between the injections. The animals were kept under close supervision and were allowed to recover during the following 7 days.

### 2.8.2. Microdialysis

On the day of microdialysis, the awake rats were implanted with the microdialysis probes and connected to the microdialysis perfusion system. Briefly, two microdialysis probes (CMA/12: 0.5 mm o.d., 2 mm membrane length with cut-off 20 kDa) were inserted into the respective guide cannula in the NAcc of the awake rat. The artificial CSF (aCSF) solution contained 148 mM NaCl, 4 mM KCl, 0.8 mM MgCl2, 1.4 CaCl2, 1.2 mM Na2HPO4, 0.3 mM NaH2PO4, pH 7.2 and was filtered through a sterile 0.2 µm disc filter and aspirated into a 1 ml glass, gas-tight syringe (CMA Microdialysis). Each rat in its home cage was placed into the frame of a Rotating animal cage system, RACS (Microbiotech/se, Årsta, Sweden). The RACS allows a swivel-free connection of tubing to the syringe pump and to the fraction collector. Following 120-min stabilization period, the first 3 samples were collected for quantification of basal extracellular levels of neurotransmitters. Thereafter, saline solution (5 ml/kg, s.c.) or α-D2PV (3 mg/kg, s.c.) were administered, and the samples were collected for additional 4 h. The dose and route of administration were chosen according to a previous published microdialysis study performed on synthetic cathinones in our laboratory (Kehr et al., 2011).

### 2.8.3. Quantification of neurotransmitters and metabolites by UHPLC-MS/MS

Concentrations of DA and the metabolites DOPAC and HVA in the microdialysates were measured by ultra high-performance liquid chromatography with electrospray tandem mass spectroscopy (UHPLC-MS/MS) following derivatization with benzoyl chloride as described by (Song et al., 2012) following some minor modifications. Briefly, the UHPLC-MS/MS system included an Elute HTG binary gradient UHPLC pump, an Elute column oven and an EVOQ Elite triple quadrupole mass spectrometer (all from Bruker Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization (ESI) source operating in a positive mode. The samples were injected by use of a PAL autosampler (CTC Analytics, Switzerland) operating at +6 °C and equipped with a 10-µl sample loop. The system was controlled by a Compass 2.0/HyStar 4.0 software (Bruker), the compound screening and quantitation was performed by a TASQ 2.2 data acquisition and processing software (Bruker). The mass spectrometer was supplied by nitrogen and air generated by a Genius 3045 nitrogen/air generator (Peak Scientific Instruments, Inchinnan, Scotland, UK). The source parameters were as follows: probe gas flow 30, nebulizer gas flow 45, probe temperature +400 °C, cone gas flow 15, cone temperature +350 °C, CID gas Ar 1.5 mTorr. The mobile phase A included 10 mM ammonium formate and 0.15 % (v/v) formic acid in water, the mobile phase B was ACN. The mobile phase gradient and flow rate are as follows: initial, 0 % B, 150 µL/min; 0.1 min, 10 % B, 150 µL/min; 1.5 min, 30 % B, 175 µL/min; 1.6 min, 30 % B, 200 µL/min; 3 min, 50 % B, 200 µL/min; 3.5 min, 55 % B, 200 µL/min; 3.9 min, 100 % B, 200 µL/min; 4.9 min, 100 % B, 200 µL/min; 5.0 min, 0 % B, 200 µL/min; 6.4 min, 0 % B, 200 µL/min; 6.5 min, 0 % B, 150 µL/min. The separation was achieved on a Cortecs UHPLC C18 column (100 × 2.1 mm, 1.6 µm, Waters, USA). The derivatization procedure was performed at room temperature. Briefly, to a 10 µl of the microdialysate or to a calibration standard, 6 µl of the internal standard mixture (1 µM of each deuterated analyte standards in ACN), followed by 6 µl of 0.1 M sodium carbonate buffer and 6 µl benzoyl chloride (1 % v/v in ACN) were pipetted. The resulting solution was mixed for 5 min, thereafter 26 µl of 1 % FA in ACN was be added. The samples were shaken and centrifuged, thereafter 40 µl of the supernatant were aspirated and dried in a miniVac Duo concentrator (Genevac, Ipswich, UK). The volumes of 10 µl were injected onto the column. The
calibration curves were constructed in the range of 0,1–1000 nM.

## 2.9. Self-administration experiments

### 2.9.1. Catheterization surgery

Rats were prepared for α-D2PV self-administration by surgical catheterization of the right jugular vein. Briefly, rats were anaesthetized with isoflurane (5 % induction, 2.5 % maintenance) in O₂ and administered with the nonsteroidal anti-inflammatory ketoprofen (2.5 mg/kg, s.c.). Animals were placed on a heating pad from the induction of the anesthesia to the end of the surgery. A handmade silastic catheter was inserted into the jugular vein and the distal end was led to the back between the scapulae. Rats were allowed to recover for 5–7 days and flushed daily with 0.1 ml sterile saline (0.9 %), and heparin (100 UI/mL) in sterile saline.

### 2.9.2. Self-administration apparatus and design

Self-administration experiments were performed in Imetronic experimental chambers equipped with nose-pokes as operand (Imetronic, Pessac, France). The initial dose for α-D2PV self-administration was chosen based on pilot substitution experiments in rats trained to self-administer methamphetamine. Rats were initially allowed to nose-poke to self-administer α-D2PV with this dose (20 µg/kg/inj) for nine 2h sessions under a fixed-ratio 1 (FR1) schedule and on the 10th session we investigated the reinforcing efficacy of this dose in a progressive ratio (PR) schedule with the exponential progression based on the equation: response ratio = $(5e^{(0.2 \times \text{reinforcer number})} - 5$, rounded to the nearest integer. Thus, the values of the steps were 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, and 737 (Richardson and Roberts, 1996). Sessions under the progressive-ratio schedule ended if a rat did not complete a step within 1 h or after 3h. Then, the dose was changed, and rats were allowed to self-administer α-D2PV for 3–5 days under FR1 before being tested again in the PR at the new dose in a single session. A total of five unit-doses were tested: 20, 40, 80, 160 and 320 µg/kg/inj. Dose-responses were established using the average of the last 3 days of FR1 and the value obtained in the corresponding PR session.

## 2.10. Data and statistical analysis

Data were expressed as Mean ± SEM. Non-linear regression was used to fit the plotted competition curves, and data were best fitted to a sigmoidal dose-response curve to obtain an IC₅₀ value. One-way or two-way ANOVA of repeated measures and subsequent Tukey's post hoc test, conducted only if F was significant, was used to determine the effects of α-D2PV on locomotor activity, thigmotaxis and conditioned place preference experiments. The mean basal levels (DA, DOPAC and HVA) in the control and treated group were compared by use of two-way ANOVA mixed-effects model followed by Bonferroni's multiple comparison test. Differences in the relative AUC (0–240 min) values between the groups and treatments were analysed by unpaired student's t-test. HLA in rats were analysed by two-way ANOVA of repeated measures, followed by Bonferroni's multiple comparison test.

Acquisition of α-D2PV self-administration was analysed by two-way ANOVA of repeated measures with session and active/inactive nose-poke as factors, followed by Tukey's multiple comparison test. Dose Responses (number of responses and total intake) under FR1 schedule were analysed by one-way ANOVA for repeated measures followed by Tukey's multiple comparison test. Dose responses (number of responses and injections) under PR Schedule were analysed by Friedman test (non-parametric one-way ANOVA of repeated measures), followed by Dunn's multiple comparison test.

The α error probability was set at 0.05 (p < 0.05). The size group for the behavioral experiments is shown in the corresponding figure legends. All statistic calculations were carried out using GraphPad Prism (GraphPad software, San Diego, CA, USA).

# 3. Results

## 3.1. Monoamine uptake inhibition induced by α-D2PV in transfected HEK293 cells

The corresponding concentration-response curves and IC₅₀ values are depicted in Fig. 2 and Table 1, respectively. Data on uptake inhibition at DAT and SERT for α-PVP and cocaine from a previous study performed in our lab and at the same experimental conditions have been added for comparison (Nadal-Gratacos et al., 2023). α-D2PV and α-PVP displayed similar and low IC₅₀ values for hDAT, 2-fold more potent at hDAT than cocaine as well as little to no activity at hSERT. Moreover, α-D2PV was 4-fold less potent at hNET than α-PVP. In addition, both pyrrolidine-containing cathinones showed higher hDAT/hSERT inhibition ratios than cocaine (Table 1).

## 3.2. Molecular docking of α-D2PV at hDAT

As mentioned in the methods section, R-cocaine was included to validate docking protocol. The top-ranked pose closely aligned with the crystallized cocaine structure in PDB ID: 9EO4 (Nielsen et al., 2024). When assayed, methylphenidate resides within the same binding site as cocaine, with its phenyl group nestled between TM3 and TM8 and its piperidine group directed towards TM1b and TM6a. Both, cocaine' and methylphenidate's benzene ring, established an edge-to-face π-π interaction with mainly Tyr156. Moreover, the amino group of both compounds also formed an ionic bond with the highly conserved Asp79 residue (Fig. S3). On the other hand, the most stable conformations of α-PVP and α-D2PV were located within the binding site, with their benzene rings precisely overlapping. This arrangement also leads to an edge-to-face π-π interaction with Tyr156, Phe76 and Phe326. Moreover, the amino group of the pyrrolidine ring also forms an ionic bond with Asp79 while the ketone group can also form hydrogen bond with Tyr156 (Fig. 3A). Conversely, similar interactions with the ketone group of methylphenidate were not found since the methyl acetate group of methylphenidate points outward (Fig. 3B). Finally, the alkyl and benzene ring side chain of α-PVP and α-D2PV, respectively, are also oriented towards the extracellular side. (Fig. 3A).

## 3.3. α-D2PV induces dose-dependent hyperlocomotion and thigmotaxis in mice

As shown in Fig. 4A and B, acute administration of α-D2PV induced a dose-dependent increase in the locomotor activity of mice. One-way ANOVA of HLA (cumulative distance travelled in 60 min; Fig. 4A) yielded a significant effect of the variable *Dose* (F(3, 52) = 68.54; p < 0.001). Subsequent post-hoc Tukey's test revealed a significant (p < 0.001) increase in the distance travelled after injection of 10 and 30 mg/ kg of α-D2PV compared to the saline group. Regarding HLA time course profile (Fig. 3B), two-way ANOVA of repeated measures also yielded a significant effect of the variable *Dose* (F(3, 52) = 68,01; p < 0.001), Time (F(11, 572) = 67,49; p < 0.001) and the interaction between both variables (F(33, 572) = 4889; p < 0.001). Additionally, one-way ANOVA of the time spent in the center of the arena and subsequent post-hoc Tukey's test (F(3, 52) = 21.90; p < 0.001) revealed a dose-dependent thigmotactic effect after α-D2PV administration (Fig. 4C).

## 3.4. α-D2PV induces dose-dependent conditioned place preference in mice

The conditioning effects of α-D2PV were studied using the conditioned place preference paradigm. During the preconditioning phase, no statistical difference was found between compartments, indicating a total lack of preference for either compartment (data not shown). After conditioning, on the test day, one-way ANOVA of the results yielded a significant effect of the variable *Dose* (F(3, 52) = 10.89; p < 0.001). Tukey's multiple comparison test revealed a significant increase in the
<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Effects of α-D2PV, α-PVP and cocaine on [³H]MPP⁺ uptake inhibition at hDAT (A) and hNET (B), and [³H]5-HT uptake inhibition at hSERT (C) in HEK293 cells. Data are expressed as a percentage of control uptake (mean ± SEM) of at least 3 independent experiments carried out on triplicates.</figcaption></figure>

<caption>**Table 1**</caption>

Monoamine uptake inhibition at hDAT, hNET and hSERT of α-D2PV, α-PVP and cocaine. Values are IC₅₀ given as µM (mean ± SEM). hDAT/hSERT ratio = 1/ DAT IC₅₀: 1/SERT IC₅₀. hDAT/hSERT ratio for α-D2PV and α-PVP were calculated using the mean IC₅₀ experimental values of the 5-HT uptake; IC₅₀ (α-D2PV): 202,3 ± 21,31 and IC₅₀ (α-PVP): 398,7 ± 49,90.

<table><thead><tr><th rowspan="3">Compound</th><th colspan="4">Transfected HEK293 cells</th></tr><tr><th colspan="4">Monoamine uptake inhibition (IC<sub>50</sub> (µM) ± SEM)</th></tr><tr><th>[³H]MPP<sup>+</sup><br>uptake at hDAT</th><th>[³H]MPP<sup>+</sup><br>uptake at hNET</th><th>[³H]5-<br>HT<br>uptake<br>at hSERT</th><th>hDAT/<br>hSERT<br>inhibition<br>ratio</th></tr></thead><tbody><tr><td>α-D2PV</td><td>0.113 (±0.008)</td><td>0.223 (±0.022)</td><td>>100</td><td>1788</td></tr><tr><td>α-PVP</td><td>0.124 (±0.006)<sup>a</sup></td><td>0.051 (±0.003)</td><td>>100<sup>a</sup></td><td>3222<sup>a</sup></td></tr><tr><td>Cocaine</td><td>0.238 (±0.016)<sup>a</sup></td><td>0.347 (±0.037)</td><td>2.01<br>(±0.28)<sup>a</sup></td><td>8<sup>a</sup></td></tr></tbody></table>

preference score for all doses tested (Fig. 4D).

### 3.5. Administration of α-D2PV increases extracellular levels of DA in the NAcc of awake rats

As shown in Fig. 5A–sc Injection of α-D2PV (3 mg/kg) induced a rapid increase in extracellular DA levels in NAcc. Two-way ANOVA mixed-model statistical analysis yielded a significant effect of the variable *Time* (F(14, 111) = 10,93; p < 0.001), *Treatment* (F(1, 8) = 12,58; p < 0.01) and interaction of both variables (F(14, 111) = 11,27; p < 0.001). Subsequent Bonferroni's multiple comparison test revealed significant difference in the extracellular levels of accumbal DA in α-D2PV treated animals in comparison with saline group 20, 40, 60, 80 and 100 min

after administration (p < 0.001) (Fig. 5A). In addition, when analyzing the AUC relative to control group, Student's *t*-test revealed a significant increase of extracellular accumbal DA levels in α-D2PV-treated animals (t = 3768; p < 0.01) (Fig. 5A inlet). On the other hand, α-D2PV administration caused a decrease in extracellular accumbal DOPAC and HVA levels, as seen in Fig. 5B and C. Particularly, two-way ANOVA mixed-effects model statistical results demonstrated significant effect of the variable *Time* (DOPAC: F(14, 139) = 4572; p < 0.001, HVA: F(14, 139) = 6998; p < 0.001), *Treatment* (DOPAC: F(1, 10) = 14,83; p < 0.01, HVA: F(1, 10) = 11,35; p < 0.01) and interaction of both variables (DOPAC: F(14, 139) = 3355; p < 0.001, HVA: F(14, 139) = 2982; p < 0.001). In a separate cohorts of rats, the effects of α-D2PV on locomotor activity were recorded (Fig. 5B). Two-way ANOVA of repeated measures revealed significant differences for the treatment (F(1, 10) = 38,34; p < 0.001), time (F(59, 590) = 14,30; p < 0.001) and the interaction time x treatment (F(59, 590) = 14,02; p < 0.001). Regarding DA metabolite levels, Bonferroni's multiple comparison test revealed a decrease in extracellular accumbal DOPAC levels 60, 80, 100, 120, 140, 160 and 200 min as well as in extracellular accumbal HVA levels 80, 120, 140, 160 and 200 min after α-D2PV injection in comparison with the saline group (Fig. 5C and D). Furthermore, Student's *t*-test also revealed a significant decrease in the AUC of accumbal DOPAC and HVA levels of α-D2PV-treated rats in comparison with the saline group (Fig. 5C and D inlet) (DOPAC: t = 4.123; p < 0.01, HVA: t = 3.547; p < 0.01).

### 3.6. α-D2PV is reinforcing in rats

Fig. 6A shows the acquisition of self-administration behavior of α-D2PV (20 µg/kg/inj) under an FR1 schedule. Two of the ten rats tested did not acquire self-administration at this dose (less than 10 injections) and were excluded from the analysis. The eight remaining rats quickly learned to self-administer α-D2PV and showed significant

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Illustrative example of the interaction mechanism predicted by molecular docking for α-D2PV (yellow) at hDAT in comparison with α-PVP (neon blue; Panel A) and methylphenidate (green; Panel B). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<footnote>a Data from Nadal-Gratacos et al. (2023).</footnote>
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Effect of α-D2PV on cumulative HLA (A), its time course profile (B), time spent in the center of the arena (C) and CPP (D) in mice. For HLA, Bars represent mean ± SEM of the total distance (cm) travelled in 60 min. For HLA time course profile, each time point represent mean ± SEM of the distance (cm) travelled in 5 min blocks. Statistical results (Tukey's multiple comparison test) are included in Supplementary Information/Table S1, for clarity purposes. For the thigmotaxis experiment, bars represent mean ± SEM of the time spent in the center of the arena in 60 min. For CPP, bars represent the mean ± SEM of the preference score (difference between the time spent in the drug-paired compartment on the test day and the preconditioning day). Tukey's multiple-comparison test: \*p < 0.05; \*\*p < 0.01 and \*\*\*p < 0.001 vs Saline group. \#p < 0.05, \#\#p < 0.01 and \#\#\#p < 0.001 vs 3 mg/kg group, \$$p < 0.001 vs 10 mg/kg group. N = 14 animals/group.</figcaption></figure>

discrimination of active vs inactive nose-pokes by the fifth day. Statistical analysis revealed a significant effect of session active/inactive nose-poke (F(1,14) = 13.05; p < 0.01) but no significant effect of time and no significant session x active/inactive nose-poke interaction.

When different doses of α-D2PV were tested under the FR1 schedule, statistical analysis of number of injections and α-D2PV total intake revealed a significant effect of dose (F(4,32) = 14.77, p < 0.001 and F(4,32) = 33.09, p < 0.001, respectively). Moreover, Tukey's multiple comparison test revealed that the number of injections/responses was maximum at the lowest doses tested and lowest at the highest dose tested (Fig. 6B); conversely, the α-D2PV total intake was lowest at the lowest dose tested (20 µg/kg/inj) and maximal at the highest doses tested (80, 160 and 320 µg/kg/inj) (Fig. 6C).

When different doses of α-D2PV were tested under the PR schedule, statistical analysis of number of injections and responses revealed a significant effect of dose (Friedman statistic = 22.47; p < 0.001 and Friedman statistic = 15.29; p < 0.01, respectively). Moreover, the dose response curves for both number of injections (Fig. 6D) and number of responses (Fig. 6E) showed an inverted U shape with intermediate doses maintaining the highest levels of self-administration. It should be noticed that rats emitted an average of 1000 responses at intermediate doses of α-D2PV, showing very high reinforcing efficacy of this synthetic cathinone.

## 4. Discussion

The illicit drug market for synthetic cathinones is characterized by continually increasing and uncontrolled growth in the number of newly detected substances, often featuring only minor modifications of the chemical structure of previously scheduled (or not yet scheduled) synthetic cathinones. In this sense, MDPV and α-PVP (also known as “flakka”) are first and second-generation of synthetic cathinones,

respectively, already subjected to control measures (Council of the European Union, 2015, Council of the European Union, 2016, Drug Enforcement Administration, 2011, 2017); for a review on legal status see (Bonson et al., 2019). Unlike these well-characterized pyrrolidine-containing cathinones (for reviews see (Baumann et al., 2018; Kolesnikova et al., 2019; Riley et al., 2020)), both possessing a propyl group at the α-carbon side chain, the recently emerged α-D2PV remains largely unstudied, particularly regarding its mechanism of action and addictive properties. This novel compound is distinguished by a phenyl group at the α-carbon side chain, representing a unique and unprecedented substitution within the field of synthetic cathinone's drug market. Thus, the present study unveils the addiction profile and mechanism of action of α-D2PV, focusing on its interaction with monoamine transporters and subsequent effects on dopamine and related metabolite levels *in vivo*, as well as its ability to induce psychostimulant, rewarding and reinforcing effects in rodents. This study used only male rodents, consistent with prior research on synthetic cathinones to establish baseline pharmacological effects. This approach ensures comparability with existing literature and facilitates clearer interpretation of results. However, future studies should examine potential sex differences in α-D2PV's effects to provide a more comprehensive understanding of its abuse liability.

Similar to other classical psychostimulant drugs, synthetic cathinones exert their effects by interacting with monoamine transporters (DAT, SERT and NET), inhibiting the reuptake of the corresponding neurotransmitter (Baumann et al., 2012; Cozzi et al., 1999; López-Arnau et al., 2012; Nagai et al., 2007). In this sense, it has been demonstrated that pyrrolidine-containing cathinones are potent DA and NE uptake inhibitors with little to no action at SERT (Baumann et al., 2013; Davies et al., 2023; Kolanos et al., 2015; Nadal-Gratacos et al., 2022), resulting in a high DAT/SERT ratio which is associated with an increased potential for abuse (Glennon and Young, 2016; Negus and Banks, 2017; Schindler et al., 2016). Our results demonstrated that α-D2PV is as
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Effect of a single s.c. administration of α-D2PV (3 mg/kg) on extracellular levels of DA (A), DOPAC (C) and HVA (D) in the NAcc of awake rats. Effects of α-D2PV (3 mg/kg, s.c.) on locomotor activity (distance travelled) recorded in 5 min bins (B). The arrow indicates the time of drug or saline injection. Data are represented as mean ± SEM. Bonferroni's multiple comparison test; \*p < 0.05, \*\*p < 0.01 and \*\*\*p < 0.001 vs saline group. Inlets: Bars represent the mean AUC (0-240min) ± SEM calculated as the difference in relative changes (%) in DA (A inlet), DOPAC (C inlet) and HVA (D inlet) levels over a 240 min period between α-D2PV- and saline-treated animals. Student's t-test; \*\*p < 0.01 vs saline group. N = 4-6 animals/group.</figcaption></figure>

potent as its congener α-PVP and 2-fold more potent than cocaine at inhibiting DA uptake. Given the structural similarity of methylpheni-date's phenyl ring in the α-carbon side chain, we hypothesized that the α-D2PV substitution might adopt a comparable orientation within the binding pocket of hDAT. However, computational analyses revealed that α-D2PV aligns more closely with its analog, α-PVP. Notably, the alkyl side chain of α-PVP and the phenyl ring side chain of α-D2PV overlap and are both oriented toward the extracellular binding side. As ex-pected, α-D2PV has little to no action at inhibiting 5-HT reuptake, as it occurs with α-PVP, which leads to high DAT/SERT inhibition ratios for this novel compound, pointing to a high abuse liability. On the other hand, notable differences were found on NE uptake inhibition. α-D2PV is 1.5-fold more potent than cocaine but 4-fold less potent than α-PVP at inhibiting NE uptake. Altogether, these results suggest that the presence of a phenyl ring at the α-carbon side chain in pyrrolidine-containing cathinones may slightly decrease the potency at NET while still retain-ing a potent DA reuptake inhibition. Further studies are needed with other synthetic cathinones featuring a phenyl ring side chain to corroborate such structure-activity relationship.

Most of the synthetic cathinones found in the recreational drug market are reported to increase the locomotor activity in rodents (Aarde et al., 2013; Baumann et al., 2012; Fantegrossi et al., 2013; Gatch et al., 2013; Huang et al., 2012; López-Arnau et al., 2012; Marusich et al., 2012; Shortall et al., 2013), indicative of their psychostimulant effect. This is likely linked to the ability of psychostimulants to enhance dopaminergic signaling in the mesolimbic pathway (Beaulieu and Gainetdinov, 2011; Ikemoto, 2002; Missale et al., 1998). Particularly,

previous studies have demonstrated that pyrrolidine-containing cath-inones, such as MDPV and its unsubstituted phenyl ring analogs (α-PVP, α-pyrrolidinobutiophenone or α-PBP, and α-pyrrolidinopropiophenone or α-PPP), are able to induce hyperlocomotion in mice (Marusich et al., 2014). Accordingly, our results revealed that this novel pyrrolidine-containing cathinone, α-D2PV, dose-dependently increases the locomotor activity in mice. Moreover, the same assay also demon-strated a thigmotactic effect since mice spent less time in the center than in the periphery of the arena. Although some authors correlate thig-motaxis with anxiety-like behavior (Seibenhener and Wooten, 2015), it must be pointed out that such thigmotactic effect observed in our ex-periments is significant at the same doses able to induce hyper-locomotion, so a dependent-relationship between both effects cannot be discarded. However, at the lowest dose tested, at which no significant effect on locomotion was detected, an almost significant thigmotactic effect was observed, pointing to an anxiogenic-like effect. In fact, it is widely known that psychostimulants are able to acutely induce anxiety-like behaviors at certain doses and experimental conditions (Biala and Kruk, 2007; Ortman et al., 2021; Yang et al., 1992).

Place conditioning paradigm is used to evaluate the conditioned motivational effects, which are typically defined as rewarding effects (Bardo and Bevins, 2000; Tzschentke, 2007). Previous studies have re-ported the ability of synthetic cathinones to support place preference conditioning (Chen et al., 2022; Danda et al., 2022; Espinosa-Velasco et al., 2024; Jones et al., 2023; Karlsson et al., 2014; Manke et al., 2023; Oh et al., 2018; Xu et al., 2016), including pyrrolidine-containing cathinones (Botanas et al., 2017; Cheong et al., 2017; Gatch et al.,
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** (A) Acquisition of self-administration rat behavior of α-D2PV (20 µg/kg/inj) under the FR1 schedule. Tukey's multiple comparison test \*p < 0.05 and \*\*p < 0.01 vs inactive nosepoke. Dose-response of the number of injections (B) and α-D2PV total intake (C) under FR1 schedule. Tukey's multiple comparison test \*\*p < 0.01 and \*\*\*p < 0.001 vs 20 µg/kg/inj; \#\#p < 0.01 and \#\#\#p < 0.001 vs 40 µg/kg/inj; \$\$p < 0.01 vs 80 µg/kg/inj. Dose-response of the number of injections (D) and responses (E) under PR schedule. Dunn's multiple comparison test \*p < 0.05 and \*\*p < 0.01 vs 20 µg/kg/inj; \#p < 0.05 vs 40 µg/kg/inj; \$p < 0.01 vs 80 µg/kg/inj. All data is represented as mean ± SEM.</figcaption></figure>

2015; Hataoka et al., 2017; Kim et al., 2024; King et al., 2015a, 2015b; Nadal-Gratacos et al., 2022; Nelson et al., 2017). Our results demonstrate that α-D2PV is able to induce rewarding effects in mice at all doses tested (3, 10 and 30 mg/kg), showing a typical inverted U-shaped response. Particularly, the rewarding effect was maximum at the medium dose tested (10 mg/kg), a dose that also induced place preference conditioning for α-PVP in previous studies from our research group under the same experimental conditions (Duart-Castells et al., 2021; Nadal-Gratacos et al., 2023). Interestingly, although α-D2PV did not elicit a significant psychostimulant effect at the lowest dose tested (3 mg/kg), it still induced rewarding effects in mice.

It is widely known that increased levels of synaptic DA in the NAcc is a key process involved in the reinforcing and rewarding properties of a

drug (Nestler, 2001). Some studies have already demonstrated the increase of extracellular DA concentrations in NAcc of rodents induced by first- and second-generation of pyrrolidine-containing cathinones (Kaizaki et al., 2014; López-Arnau et al., 2017; Saha et al., 2015; Schindler et al., 2016, 2020; Wojcieszak et al., 2018). As expected by the potent uptake inhibition at DAT *in vitro* and the psychostimulant and rewarding effects observed in mice, α-D2PV (3 mg/kg, s.c.) also induced a rapid (within 20–40 min) increase in accumbal DA levels in awake rats (maximum effect around 322 % increase). For instance, Schindler and colleagues showed similar DA levels increases in NAcc of awake rats after i.v. administration of 0.1–0.3 mg/kg of racemic α-PVP (Schindler et al., 2020). Moreover, Wojcieszak and colleagues also demonstrated a similar extracellular DA increase in mouse striatum after s.c.
administration (3 mg/kg) of α-PVP (Wojcieszak et al., 2018). Importantly, the temporal profile of elevated dopamine (DA) levels aligns with the locomotor activity pattern elicited by α-D2PV in rats. α-D2PV also induced a concomitant decrease in DOPAC and HVA levels in awake rats. This decrease in DA metabolite concentrations can be attributed to DA uptake inhibition by α-D2PV. The key enzyme in the metabolism of DA, namely monoaminoxidase (MAO), is localized inside the nerve terminal, so that monoamines have to be taken up to be metabolized. As α-D2PV blocks reuptake, a decrease in metabolites is detected despite the increase in monoamine levels. A similar effect for other psychostimulant drugs has been previously described (Kehr et al., 2011; Kimura et al., 1993; López-Arnau et al., 2018; Sorg and Kalivas, 1991; Zocchi et al., 1997).

Intravenous self-administration is a preclinical models with the high face validity for studying human drug-taking behavior (O'Connor et al., 2011). The number of responses (nosepokes in this study) emitted to obtain injections of the drug is a measure of the reinforcing effects of a drug (Sanchis-Segura and Spanagel, 2006; Zernig et al., 2007). Pyrrolidine-containing cathinones are self-administered by rodents (Aarde et al., 2013, 2015a,b; Cheong et al., 2017; Gannon et al., 2017, 2018b; Kim et al., 2024; Marusich et al., 2019, 2021; Nagy et al., 2020; Schindler et al., 2016, 2020; Sewalia et al., 2018; Watterson et al., 2014; Xu et al., 2021). In the present study, naïve rats readily learned to self-administer α-D2PV (20 µg/kg/injection) and maintained very high levels of self-administration (100 injections in 120-min sessions, i.e. one injections every approximately 72 s). Similarly, Gannon and colleagues observed comparable acquisition patterns for α-PVP, with similar response rates and acquisition times under an FR1 schedule at a dose of 32 µg/kg/injection in male Sprague-Dawley rats (Gannon et al., 2018b). Furthermore, the dose-response curve for α-D2PV under FR1 revealed a decrease in the number of injections as the dose increased, with maximal effects observed at doses between 20 and 80 µg/kg/injection. This pattern aligns with findings by Aarde and colleagues, who reported a similar dose-effect relationship for α-PVP in male Wistar rats (Aarde et al., 2015a).

The progressive ratio (PR) schedule provides a measure of drug motivation by evaluating the effort animals are willing to exert for drug access (Tsibulsky and Norman, 2022). In this study, α-D2PV produced an inverted U-shaped dose-response curve under the PR schedule, consistent with the profiles observed for other psychostimulants (Briscoe et al., 1998; Clemens et al., 2006; Griffiths et al., 1979; Martelle et al., 2008; Roth and Carroll, 2004). Notably, the highest number of injections and responses were observed at doses ranging from 40 to 80 µg/kg/injection, with more than 500 and 1000 responses, respectively. This pattern, similar to that found by (Aarde et al., 2015a) with α-PVP, demonstrates high reinforcing efficacy of α-D2PV that suggests high risks of abuse liability of this compound in humans. These findings are consistent with previous reports correlating the reinforcing effects of synthetic cathinones, particularly pyrrolidine-containing cathinones, with their potent DAT inhibitory properties (Gannon et al., 2018a). In fact, the rewarding and reinforcing effects observed are in accordance with the ability of α-D2PV to induce an increase of synaptic DA levels in NAcc.

In summary, α-D2PV exhibits a pharmacological and behavioral profile indicative of a high abuse liability, similar to other pyrrolidine-containing cathinones. Its potent DAT inhibition and the resulting increase in accumbal DA levels combined with its psychostimulant, rewarding and reinforcing properties, highlight the need for continued monitoring of this compound in the illicit drug market. Finally, greater attention and proactive monitoring are essential for future novel synthetic cathinones featuring a phenyl ring at the α-carbon side chain, as these compounds may retain dopaminergic activity and abuse potential while creates NPS able to evade initial legal controls. Such a unique substitution in various cathinone derivatives may pose significant health risks and contribute to their potential emergence in the illicit drug market.

## CRediT authorship contribution statement

**Núria Nadal-Gratacos:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Data curation.
**Sandra Mata:** Methodology, Investigation. **Pol Puigseszloses:** Writing – review & editing, Methodology, Investigation. **Morgane De Macedo:** Methodology, Investigation, Formal analysis, Data curation. **Virginie Lardeux:** Methodology, Investigation, Formal analysis, Data curation.
**Stephanie Pain:** Methodology, Investigation, Formal analysis, Data curation. **Fu-Hua Wang:** Methodology, Investigation. **Liselott Källsten:** Methodology, Investigation. **David Pubill:** Writing – review & editing, Methodology. **Xavier Berzosa:** Writing – review & editing, Resources, Methodology, Investigation, Funding acquisition. **Jan Kehr:** Writing – review & editing, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Marcello Solinas:** Writing – review & editing, Funding acquisition, Formal analysis, Data curation, Conceptualization.
**Jordi Camarasa:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **Elena Escubedo:** Writing – review & editing, Writing – original draft, Visualization, Software, Methodology, Investigation, Funding acquisition, Conceptualization. **Raul López-Arnau:** Writing – review & editing, Writing – original draft, Visualization, Supervision, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Funding – acknowledgements

This study was supported by European Union (EU) Home Affairs Funds, NextGenPS project (101045825) and Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación/MICIU/AEI/10.13039/501100011033 (PID2022-1375410B-100). The UB group belongs to 2021SGR0090 from Generalitat de Catalunya. PP received a doctoral scholarship grant from Generalitat de Catalunya (AGAUR), 2022 FISDU 00004. This study has benefited from the facilities and expertise of PREBIOS platform (Université de Poitiers) and the technical help of Catherine Le Goff.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuropharm.2025.110425.

## Data availability

Data will be made available on request.

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