Abuse liability of isopropylphenidate via modulation of D1 receptor signalling pathway

Central nervous system (CNS) stimulants, including methamphetamine (METH) and methylphenidate (MPH), are used to treat attention-deficit/hyperactivity disorder (ADHD). In the United States, the number of dispensed prescriptions for CNS stimulants increased by approximately 60%, from 50.4 million in 2012 to 80.8 million in 2023 (IQVIA Government Solutions, 2024). These agents produce psychostimulant and rewarding effects that contribute to their abuse potential. Chronic exposure is also associated with physical and psychological adverse effects, including headaches, cardiovascular complications, and psychotic symptoms, and severe cases may be fatal. Consistent with these risks, overdose deaths involving stimulants have risen substantially in the United States over the same period (National Institute on Drug Abuse, 2024). Accordingly, elucidating the addictive properties and underlying mechanisms of these stimulants remains an important public health priority. Isopropylphenidate (IPH) is a structural analogue of MPH and has recently been regulated as a controlled substance due to its pharmacological similarity to METH and reports of adverse effects. However, the addictive potential of IPH and its underlying mechanisms remain incompletely understood. Here, we assessed the abuse liability of IPH in mice using conditioned place preference (CPP) and behavioral sensitization (BS) paradigms. To examine the involvement of dopaminergic signaling, we administered dopamine receptor antagonists during CPP and BS and, in separate CPP cohorts, selectively inhibited dopamine D1 receptor (D1R)-expressing medium spiny neurons (MSNs) in the nucleus accumbens (NAc) using a chemogenetic approach. In addition, we measured dopamine concentrations and the expression of dopamine-related proteins in the NAc and ventral tegmental area (VTA) using enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence. IPH administration significantly increased CPP scores in both male and female mice, and acute IPH exposure induced hyperlocomotion. In the BS paradigm, repeated IPH exposure produced robust behavioral sensitization and elicited IPH-primed expression of locomotor sensitization following a withdrawal period. Notably, SCH23390, a dopamine D1 receptor antagonist, blocked both IPH-induced CPP and the expression of behavioral sensitization, whereas raclopride, a dopamine D2 receptor antagonist, had no effect. Moreover, chemogenetic inhibition of D1R-expressing MSNs using Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) prevented the expression of IPH-induced CPP. At the molecular level, IPH increased dopamine concentrations in both the NAc and the VTA and activated downstream D1R signaling in the NAc. These changes were accompanied by increased numbers of c-Fos–positive neurons and elevated dopamine transporter (DAT) levels in the NAc, along with reduced tyrosine hydroxylase (TH) levels in the VTA. Importantly, D1R blockade attenuated these molecular alterations in both the NAc and the VTA. Collectively, these findings indicate that IPH induces addiction-related behaviors by modulating the dopaminergic system, primarily through D1R-dependent signaling. Furthermore, these results provide mechanistic insight into IPH addiction and offer behavioral and molecular support for the recent regulation of IPH as a controlled substance. This research was supported by the National Research Foundation of Korea (2022R1A6A1A03054419, RS-2025-16067597) and the Ministry of Food and Drug Safety (23212MFDS218) of Korea.

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