Opioid overdose fatalities have sharply accelerated in the US in the 21st century, and the specific substances involved in these overdose deaths have shifted from prescription opioids, to heroin, to non-pharmaceutical fentanyl and its highly potent analogs (Jalal et al., 2018). In 2019, it was estimated that 1.2 % of the world population misused opioids, and opioids were present in over 70 % of overdose deaths worldwide (United Nations Office on Drugs and Crime World Drug Report, 2021). The exponential rise in opioid overdose fatalities has been linked to concurrent use of fentanyl and synthetic drugs from multiple classes, including stimulants like methamphetamine (Han et al., 2021, Hoopsick and Yockey, 2023, Strickland et al., 2021), prescription benzodiazepines and novel abused analogs (Beletsky and Davis, 2017, Ciccarone, 2017, Laing et al., 2021), and depressants such as gabapentin and xylazine (Friedman et al., 2022, Johnson et al., 2021, Nunez et al., 2021, Thangada, 2021). Concurrent use of fentanyl with other drugs may at least partially explain the growing phenomenon of naloxone-resistant overdose (Britch and Walsh, 2022, Moss and Carlo, 2019).

Synthetic cannabinoid receptor agonists (SCRAs) were originally developed to probe cannabinoid receptor type 1 (CB1) function (Howlett et al., 2021, Wiley et al., 1998). SCRAs bind to CB1 and elicit psychoactive effects similar to – but more extreme than – those of Δ⁹-tetrahydrocannabinol (THC) from plant cannabis, due to greater affinity for and efficacy at CB1 (Ford et al., 2017, Tai and Fantegrossi, 2017). SCRAs have become a new class of illicit drugs, and over 200 distinct SCRAs have been detected in abuse-ready preparations in the US and abroad since 2008 (Uchiyama et al., 2009, Vardakou et al., 2010, Auwarter et al., 2009). Regulation of specific SCRAs fuels clandestine efforts to design newer drugs which evade detection while maintaining CB1 activity, yet to this day, all abused SCRAs are characterized by structures containing a tail, core, linker and linked group (Ford et al., 2017) (see Fig. 1). Conservation of this basic SCRA structure maintains binding affinity for CB1, but substitutions at each moiety alter agonist interactions with the receptor and affinity for drug-metabolizing Phase I enzymes. SCRAs elicit frequent and severe adverse effects in human users, including persistent psychosis, tachycardia, arrhythmias, myocardial infarction, seizures, convulsions, and even death (Ford et al., 2017, Tai and Fantegrossi, 2017).

In mice, SCRAs elicit acute cannabimimetic effects, including all four of the classical “cannabinoid tetrad” measures of antinociception, locomotor suppression, catalepsy and hypothermia. Despite qualitative similarities in drug effects following acute administration, quantitative differences in potency, maximal effects, onset and duration of action are observed among SCRAs (Howlett et al., 2021, Wiley et al., 1998). Further differences among individual SCRAs are demonstrated after repeated administration, as for example progressive and complete tolerance to hypothermic effects of the aminoaklylindole JWH-018 is observed, but only slow and incomplete tolerance to hypothermic effects of the indazole carboxamide 5F-ADB-PINACA is achieved following the same injection regimen (Wilson et al., 2022). Because these cannabimimetic effects of JWH-018 and 5F-ADB-PINACA are attenuated by the CB1 antagonist / inverse agonist rimonabant, these and other (Grafinger et al., 2021b, Pike et al., 2021, Grafinger et al., 2021a) data strongly imply that individual SCRAs may differentially impact CB1 signaling. SCRAs may therefore elicit diverse profiles of respiratory depressant effects, and very different interactions with fentanyl may be observed among SCRAs.

In this regard, there are no reliable survey data on co-use of opioids and SCRAs, largely because SCRAs are not detected in standard urine toxicology screens. Nevertheless, analytical studies have demonstrated that fentanyl is a common adulterant in SCRA products and that SCRAs are frequently detected as adulterants in street opioids (Di Trana et al., 2022a, Ershad et al., 2020, Minutillo et al., 2019, Solimini et al., 2017, Ti et al., 2021). Similar forensic studies with biological samples from drug users (including overdose victims) have simultaneously detected the presence of fentanyl and various SCRAs (Di Trana et al., 2022b, Giorgetti et al., 2022, Liu et al., 2022, Wiens et al., 2021). Medicinal and recreational cannabis products are also increasingly adulterated with SCRAs (Jones et al., 2025, Monti et al., 2022, Oomen et al., 2022), unknowingly exposing users of otherwise legal cannabis products to these dangerous drugs, and there are reliable survey data indicating sustained high rates of cannabis and opioid co-use (Corroon et al., 2017, Lucas, 2017). This strongly suggests that opioids and SCRAs are used concurrently, either intentionally to enhance drug effects, or inadvertently as a consequence of adulterated drug supplies.

Opioid-induced respiratory depression is well-described (Shook et al., 1990), but the effects of SCRAs on respiratory parameters are understudied, and mechanisms mediating these effects are largely unknown. Nevertheless, several case reports and case series describe respiratory depression following SCRA overdose in humans (Centers for Disease Control and Prevention, 2013, Hermanns-Clausen et al., 2018, Ivanov et al., 2019, Kourouni et al., 2020, Alon and Saint-Fleur, 2017), but these reports are uncontrolled and usually lack analytical confirmation of SCRA exposure. However, one rigorous study (Manini et al., 2022) in Emergency Department patients with confirmed SCRA exposure reported that the incidence of acute respiratory failure following SCRA overdose was 31.3 %, and compared to non-SCRA overdose, confirmed SCRA overdose was significantly associated with respiratory failure. Animal studies in this area are few, but early studies demonstrated CB1-mediated respiratory depressant effects of limited doses of high-efficacy CB1 agonists WIN-55,212–2 and HU-210 in anesthetized rats (Padley et al., 2003, Pfitzer et al., 2004, Schmid et al., 2003). More recently, similar respiratory depressant effects of the first generation SCRA JWH-018 were described in awake, freely-moving mice using pulse oximetry, and these effects were blocked by prior administration of the putative CB1 antagonist AM-251 (Marchetti et al., 2023). However, the antagonist dose used in this study was 6-fold larger than required to block JWH-018-elicited impairment of novel object recognition in mice (Barbieri et al., 2016), and we have previously reported that AM-251 also exhibits antagonist binding to μ-opioid receptors with mid-nanomolar affinity (Seely et al., 2012), which complicates a purely CB1-based interpretation of the apparent respiratory depressant effects of JWH-018.

Thus, in these studies we compared respiratory depressant effects of fentanyl to those of two structurally-distinct SCRAs: the naphthyl indole JWH-018 and the indazole carboxamide 5F-ADB-PINACA, following acute and chronic administration, using whole body plethysmography in adult male NIH Swiss mice. We performed reciprocal antagonism pretreatment studies using the μ-opioid receptor antagonist naloxone and the CB1 antagonist / inverse agonist rimonabant against fentanyl and SCRA doses eliciting similar respiratory depression. Doses of fentanyl and the SCRAs which elicited ~25 % suppression of respiratory rate were co-administered, and antagonist “rescue” studies were conducted using large doses of naloxone, rimonabant, or a combination of both antagonists. In separate groups of mice, fentanyl, JWH-018, 5F-ADB-PINACA, or the combinations of fentanyl +  JWH-018 and fentanyl +  5F-ADB-PINACA were administered to mice, and a single blood sample was drawn at a time of maximal respiratory depression to provide a pharmacokinetic snapshot of blood conentrations of drugs at this overdose-relevant timepoint. The sum of all the data presented here strongly suggests that co-administration of fentanyl and SCRAs – either intentional or inadvertent – will exacerbate respiratory depression and confer resistance to naloxone rescue via pharmacodynamic interactions between μ-opioid and CB1 cannabinoid receptors, and that some SCRAs will also instigate pharmacokinetic drug-drug interactions with fentanyl, resulting in higher blood concentrations which functionally increase the fentanyl dose.