Synthetic opioids, such as fentanyl and fentanyl analogs (F/FA), are clinically administered to alleviate severe chronic and acute pain, and induce anesthesia (Lee and Ho, 2013; Fujii et al., 2019; Strang et al., 2020). However, their clinical efficacy is complicated by their inclination to induce analgesic tolerance, pleasure, and addictive states (Lee and Ho, 2013). In 2021, opioid use disorder (OUD) and subsequent overdose resulted in an estimated 80,411 deaths in the US, incurring a medical cost of more than 1–1.5 trillion dollars (Serdarevic et al., 2017; Torralva and Janowsky, 2019; CDC, 2020; National Center on Health Statistics, 2021; NIDA, 2023). OUD is a chronically relapsing disorder that is characterized by compulsive drug-seeking behavior that can be enhanced by drug abstinence and is maintained by encounters with drug-associated cues which elicit strong cravings and promote drug-seeking behaviors (Mueller et al., 2002; Zanda et al., 2021; Zhang et al., 2022). A large body of literature shows that sex differences impose clinically relevant changes in the vulnerability to OUD with women being prescribed and escalating to higher doses of opioid faster with greater craving and withdrawal symptoms compared to their male counterparts (Lee and Ho, 2013; Manubay et al., 2015; Serdarevic et al., 2017; Huhn et al., 2019a; Huhn et al., 2019b; Pisanu et al., 2019; Torralva and Janowsky, 2019; CDC, 2020; National Center on Health Statistics, 2021; Wightman et al., 2021). Despite this, sex differences in relation to the experience of opioid reward remain ambiguous with contradictory findings in both human and animal studies (Lee and Ho, 2013; Huhn et al., 2019a; Wightman et al., 2021). Since 1999, deaths resulting from synthetic opioid-induced overdose have increased by 103.2-fold in men, representing 73% of all synthetic opioid deaths in 2020, compared to women who have experienced a smaller 46.2-fold increase (Serdarevic et al., 2017; Barbosa-Leiker et al., 2018; Torralva and Janowsky, 2019; CDC, 2020; National Center on Health Statistics, 2021; NIDA, 2023). This divergence in the vulnerability to OUD and rate of overdose have been attributed to socio-economic pressures and differences in pain manifestation, coping, drug pharmacokinetics, and sensitivity to aversive drug effects (Lee and Ho, 2013; Manubay et al., 2015; Pisanu et al., 2019). However, little attention has been given to sex-dependent effects of fentanyl on opioid-induced reward with current literature largely focused on outcomes related to opioid-induced analgesia and respiratory depression (Manubay et al., 2015; Huhn et al., 2019a).

In the U.S., overdose from drugs of abuse is now the leading cause of accidental death in adults aged 18–50, resulting in ∼1,000 emergency visits and ∼91 deaths per day with more than 67% involving F/FA (Serdarevic et al., 2017; Torralva and Janowsky, 2019; CDC, 2020; National Center on Health Statistics, 2021). Overdose from opioids results in opioid-induced respiratory depression (OIRD) and cardiac toxicity which culminate in respiratory and cardiovascular failure. Opioid-induced respiratory conditions are reversed by administration of competitive opioid receptor antagonists such as naloxone and naltrexone (Barbosa-Leiker et al., 2018; Torralva and Janowsky, 2019). However, these treatments terminate opioid-induced analgesia, precipitate severe withdrawal symptoms, and are not effective against wooden chest syndrome (WCS, respiratory muscle rigidity and laryngospasm), uniquely resulting from the use of F/FA (Barbosa-Leiker et al., 2018; Torralva and Janowsky, 2019). In fact, despite the increased clinical and public availability of naloxone over the last decade, there has been no significant reduction in F/FA-related deaths (Torralva et al., 2020). As of 2022, an estimated 3 million people in the US and 16 million worldwide suffer from OUD (Azadfard et al., 2022). Medication-assisted treatments for OUD (i.e., methadone, buprenorphine, and ER naltrexone), which operate through direct antagonistic or agonistic action on the mu-opioid receptor, prevent subsequent clinical use of opioids in analgesia or anesthesia (Huhn et al., 2019a). These reactive intervention-based strategies for OUD are not preventative and burden patients with self-admittance and retention of treatment which is marginally effective with only 11% of people receiving treatment and less than 20.7% of those sustaining abstinence for at least 5 years (Zhu et al., 2018; Davis et al., 2021; Volkow, 2022). Accordingly, there is an urgent unmet medical need to develop drugs which can 1) reverse OIRD/WCS resulting from highly potent synthetic opioids without compromising analgesic efficacy or inducing aversive withdrawal states, and 2) prevent the development of and/or aid in the arrest of OUD.

Preclinical investigations have demonstrated that the thioester drug, D-cysteine ethylester (D-CYSee), delivers rapid and long-lasting recovery of respiratory function from OIRD in male and female rats with minimal effect on analgesic efficacy (Getsy et al., 2022c). These findings strongly indicate that D-CYSee and related thioesters (Mendoza et al., 2013; Gaston et al., 2021; Getsy et al., 2022b; Getsy et al., 2022c) act through an opioid receptor-independent mechanism that is metabolically distinct from those engaged by L-thiol esters which, while effective at recovering respiratory drive, induce maladaptive adverse effects on upper airway function (Gaston et al., 2021; Getsy et al., 2022c; Getsy et al., 2022b). Despite the clear therapeutic potential for D-CYSee in the recovery of respiratory drive following OIRD, the fact that it alters OIRD and that it readily passes the blood brain barrier suggests that indirect interactions with opioid-reward related effects are possible. Further, the potential aversive or rewarding behavioral effects of D-CYSee in the absence of opioids have yet to be fully characterized. While several previous studies have evaluated dose effects on the rewarding characteristics of fentanyl, these evaluations were often limited to male subjects and/or to a narrow dose range (Mucha and Herz, 1985; Vitale et al., 2003; Gaulden et al., 2021). Systematic analysis of the sex and dose specific differences in the rewarding properties of fentanyl could greatly aid in the development of studies evaluating treatment effects on fentanyl use disorder. Thus, experiments in the present study aimed to evaluate 1) the dose and sex dependent effects of fentanyl to induce rewarding states, and 2) the extent to which D-CYSee alters affective state and the acquisition of fentanyl-induced reward seeking.

In the present study, we utilized a rat model of CPP to evaluate 1) the dose and sex dependent effects of fentanyl on reward-seeking behavior, and 2) the extent to which D-CYSee alters affective state and the acquisition of fentanyl-induced reward. Our results demonstrate significant differences in the fentanyl dose response between male and female Long-Evans rats. Fentanyl CPP in males followed a range-bound dose response centered at a dose of 5 μg/kg, with only doses of 5 μg/kg and 10 μg/kg eliciting a significant change in preference that also trended with doses of 0.5 μg/kg—1 μg/kg. Fentanyl doses above 10 μg/kg or below 0.5 μg/kg failed to induce any effect on preference in male rats. Conversely, fentanyl preference in female rats demonstrated a multifaceted dose response that was comprised of two sub-populations which responded in a divergent manner at higher fentanyl doses. While not significant, a linear trend toward preference was observed at doses from 1 μg/kg to 10 μg/kg. However, at a dose of 50 μg/kg a dual modality was observed wherein 25% of animals showed a significant CPP with the remaining 75% showing no difference in preference from saline control animals. No consistent changes or trends in preference were observed across the testing period of 5 days in males or females under any treatment condition, although test day one showed greater variability than later test days. Pretreatment with D-CYSee was effective at preventing the acquisition of fentanyl seeking in both male and female animals, and in the absence of fentanyl, D-CYSee induced no change in affective state. Critically, in males, but not females, D-CYSee exhibited a dose dependency wherein 100 mg/kg but not 10 mg/kg prevented the acquisition of fentanyl CPP. A partial non-significant reduction in preference compared to fentanyl controls was observed under the 10 mg/kg dose, whereas 100 mg/kg D-CYSee prevented fentanyl CPP. Critically, a dose dependency for D-CYSee was not observed in females as both the low and high dose were equally effective at preventing the acquisition of fentanyl CPP. These data suggest that males are more at risk for the development of OUD from lower dose fentanyl than females, and that females exhibit a dual modality in opioid reward experience. That is, a greater percentage of females are resistant to the rewarding effects of fentanyl at higher doses while a smaller percentage find these doses tremendously rewarding.

Over the last few decades, several studies have utilized the CPP paradigm to evaluate the impacts of mu-opioid receptor agonists such as fentanyl on contextual preference and reward (Finlay et al., 1988; Vitale et al., 2003; Rech et al., 2011; Gaulden et al., 2021). The earliest study assessed the motivational properties of fentanyl at doses of 0.25, 1, 4, and 16 μg/kg in male Sprague Dawley rats and found a nearly identical range-bound fentanyl dose response centered at 4 μg/kg (Figure 7A; Mucha and Herz, 1985). Studies since have assumed equivalence between the male and female dose response curves employing only a few doses of fentanyl between 0.25 μg/kg and 16 ug/kg (Finlay et al., 1988; Vitale et al., 2003; Rech et al., 2011; Gaulden et al., 2021). Recent work found that significant fentanyl CPP could be elicited in females with a dose of 16 μg/kg but not 4 μg/kg (Gaulden et al., 2021). Interestingly, the data at the 16 μg/kg but not 4 μg/kg dose showed greater variability in which 25% (n = 4) expressed a preference above the standard error of the mean (SEM), ≈31% (n = 5) expressed a preference below the SEM, and 37% (n = 7) exhibited increased preference within the SEM. Critically, the data centered around the mean closely aligns with our linear dose response at lower fentanyl doses while those above and below the standard error are similar to, with smaller effect size, our findings at 50 μg/kg. Taken together these findings support a model of a transitional fentanyl dose response curve in female rats (Figure 7B) that exhibit a low-grade linear relationship with induced CPP at low doses (≈1 µg/kg-10 μg/kg). Doses above 10 μg/kg provoke a divergent response wherein animals progressively fall away from the linear model splitting into high preference or low-zero preference fentanyl response groups. This dual modality in fentanyl responding is consistent with findings showing that women with OUD are more motivated to obtain opioids and that female rats will self-administer more fentanyl than males and express higher levels of drug seeking (Lee and Ho, 2013; Serdarevic et al., 2017; Huhn et al., 2019a; Huhn et al., 2019b; Townsend et al., 2019; Bakhti-Suroosh et al., 2021; Towers et al., 2021; Wightman et al., 2021; NIDA, 2022).

Current literature on differences between female-female and female-male reward experience to drugs of abuse including opioids have largely centered on estrous cycling. Supporting this, recent work showed that the estrous cycle phase during the first exposure to fentanyl was predictive of elicited preference (Gaulden et al., 2021). However, the response pattern previously discussed within their 16 μg/kg treatment group was maintained within both estrus and non-estrus groups. Thus, while the estrous cycling may impact the magnitude of the elicited preference at lower doses, it does not appear to underlie the divergent responding seen in females in response to high-dose fentanyl. Recent findings have identified sex differences in the behavioral inhibition system centered in the prefrontal cortex, and in hippocampal plasticity and cognitive processing which are critical for opioid-reward processing and the formation of a CPP (Giacchino and Henriksen, 1998; Euston et al., 2012; Gonzalez et al., 2014; Baldo, 2016; Otis et al., 2018; Nam et al., 2019; Yagi and Galea, 2019; Jung et al., 2022). These functional and morphological discrepancies between males and females in these regions may underlie the observed sex differences in opioid-reward processing and the efficacy of D-CYSee (Yagi and Galea, 2019; Jung et al., 2022). Further evaluation of the impacts of these sex differences on reward processing are needed to better inform the clinical application of opioids and the development of treatment(s) for OUD, OIRD and WCS. Further, the successful translation of these models to humans could greatly inform the clinical application of opioids in pain management and clarify the divergence between the predicted vulnerability to OUD, and the number of women who advance to OUD and overdose.

Current non-preventative treatment strategies for OUD and OIRD are less effective in females (Barbosa-Leiker et al., 2018; Huhn et al., 2019a; Hoopsick et al., 2020; Davis et al., 2021). Our data demonstrate that D-CYSee, while effective at preventing fentanyl reward in both sexes, is more effective in female rats. D-CYSee prevents the acquisition of fentanyl-induced CPP in both males and females suggesting that D-CYSee could be an effective co-treatment with prescribed opioids to reduce the development of OUD. D-CYSee has been shown to effectively and persistently reverse OIRD in rat and mouse models (Getsy et al., 2022c). As a cysteine derivative, D-CYSee shares properties with N-acetyl-L-cysteine (NAC) which has also been shown to reduce OIRD (Getsy et al., 2022a) and cocaine-seeking behavior (Madayag et al., 2007; Amen et al., 2011; Reichel et al., 2011; Schmaal et al., 2012; Corbit et al., 2014; Reissner et al., 2015). Thus, D-CYSee may act in a similar fashion to NAC through normalization of glutamate and restoration of glutamate uptake by the glutamate transporter 1 (GLT-1) (Schmaal et al., 2012; Reissner et al., 2015). Although limited data are available on the effects of NAC on opioid seeking, NAC has been shown to reduce reinstatement of heroin self-administration (Zhou and Kalivas, 2008). D-CYSee may share these same mechanisms, and the ethylester addition likely facilitates its effects through its cell/blood brain barrier permeability (Mendoza et al., 2013; Gaston et al., 2021; Jenkins et al., 2021; Getsy et al., 2022c). Thus, D-CYSee may be a more effective cysteine derivative than NAC at preventing OUD in addition to reversing OIRD in both males and females (Getsy et al., 2022c). Determining the molecular mechanism(s) by which D-CYSee affords its promising outcomes and whether it could be effective in the treatment of OUD will be the subject of future studies.