Differential cannabinoid‐like effects and pharmacokinetics of ADB‐BICA, ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA in mice: A comparative study

Abstract

Despite synthetic cannabinoids' (SCs) prevalent use among humans, these substances often lack comprehensive pharmacological data, primarily due to their rapid emergence in the market. This study aimed to discern differences and causal factors among four SCs (ADB‐BICA, ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA), with respect to locomotor activity, body temperature and nociception threshold. Adult male C57BL/6 mice received intraperitoneal injections of varying doses (0.5, 0.1 and 0.02 mg/kg) of these compounds. Three substances (including ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA) demonstrated dose‐ and time‐dependent hypolocomotive and hypothermic effects. Notably, 0.1 mg/kg MDMB‐4en‐PINACA exhibited analgesic properties. However, ADB‐BICA did not cause any effects. MDMB‐4en‐PINACA manifested the most potent and sustained effects, followed by ADB‐4en‐PINACA, ADB‐BINACA and ADB‐BICA. Additionally, the cannabinoid receptor 1 (CB1R) antagonist AM251 suppressed the effects induced by acute administration of the substances. Analysis of molecular binding configurations revealed that the four SCs adopted a congruent C‐shaped geometry, with shared linker binding pockets conducive to robust steric interaction with CB1R. Essential residues PHE²⁶⁸, PHE²⁰⁰ and SER¹⁷³ within CB1R were identified as pivotal contributors to enhancing receptor–ligand associations. During LC‐MS/MS analysis, 0.5 mg/kg MDMB‐4en‐PINACA exhibited the highest plasma concentration and most prolonged detection window post‐administration. The study of SCs' pharmacological and pharmacokinetic profiles is crucial for better understanding the main mechanisms of cannabinoid‐like effects induced by SCs, interpreting clinical findings related to SC uses and enhancing SCs risk awareness.

ADB‐BINACA, ADB‐4en‐PINACA, and MDMB‐4en‐PINACA, but ADB‐BICA not, demonstrated the cannabinoid‐like effects in mice. CB1R mediated the cannabinoid‐like effects. The differences of each compound's effect are related to its pharmacokinetic and pharmacological characteristics.

1. INTRODUCTION

Synthetic cannabinoids (SCs) have gained popularity in many countries over the past decade as an alternative to natural cannabis. ¹ , ² As of 31 December 2021, the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) has been tracking 224 SCs that have surfaced on the drug market since their initial identification in 2008. ³ Importantly, the widespread misuse of SCs worldwide has precipitated a global public health crisis.

It is now understood that SCs are synthetic analogues of delta‐9‐tetrahydrocannabinol (∆9‐THC), the primary psychotropic compound and partial cannabinoid receptor (CBR) agonist found in cannabis. ⁴ Despite being structurally distinct from Δ9‐THC, SCs demonstrate higher affinity and efficacy in binding to and activating cannabinoid receptor 1 (CB1R) compared to Δ9‐THC. ⁵ , ⁶ Beyond mimicking the typical effects of THC, SCs often provoke more severe adversities and health risks. ⁷ , ⁸ The pronounced potential for abuse and the deleterious effects underscore the grave concerns surrounding SCs.

Many new SCs with chemical structures akin to ‘conventional’ SCs have emerged in response to consumer demand. ADB‐BICA (1‐benzyl‐N‐(1‐carbamoyl‐2,2‐dimethylpropan‐1‐yl)‐1H‐indole‐3‐carboxamide) and ADB‐BINACA (N‐[1‐(aminocarbonyl)‐2,2‐dimethylpropyl]‐1‐(phenylmethyl)‐1H‐indazole‐3‐carboxamide) were initially detected from a clandestine laboratory in China in 2016. ⁹ ADB‐BICA belonged to the indole‐3‐carboxamide family. After being first reported, six metabolites of ADB‐BICA were identified using human liver microsomes in 2018. ¹⁰ Unlike ADB‐BICA, ADB‐BINACA belonged to the indazole‐3‐carboxamide family. The potency and efficacy of ADB‐BINACA for CB1R activation were evaluated using a live cell‐based nanoluciferase complementation reporter assay. ¹¹ ADB‐4en‐PINACA (N‐[1‐amino‐3,3‐dimethyl‐1‐oxobutan‐2‐yl]‐1‐[pent‐4‐en‐1‐yl]‐1H‐indazole‐3‐carboxamide), was first reported in the United States in 2021. ¹² Similar to ADB‐BINACA, ADB‐4en‐PINACA belonged to the indazole‐3‐carboxamide family. The metabolism, quantitative analysis, binding ability, the potency and efficacy for CB1R have also been reported. ¹² , ¹³ , ¹⁴ But no information about their in vivo pharmacological and toxicological information are available for now. MDMB‐4en‐PINACA (methyl 3,3‐dimethyl‐2‐[1‐(pent‐4‐en‐1‐yl)‐1H‐indazole‐3‐carboxamido]‐butanoate) first was available on the European drug market in 2017 and then was formally notified as a new psychoactive substance by the EMCDDA on 23 August 2018. ¹⁵ According to the latest data from the United Nations Office on Drugs and Crime (UNODC), MDMB‐4en‐PINACA, the predominant SCs, accounted for nearly 42% of 339 reported SCs cases in 2021 and up until October 2022. ¹⁶ , ¹⁷ As a widely popular potent agonist, in addition to its synthesis, metabolism, binding and receptor activation characteristics being documented, intraperitoneal injection of MDMB‐4en‐PINACA could impair the movement, coordination and balance even a decline in spatial learning and memory in rats. ¹⁸

Together, while recent reports have detailed the synthesis, metabolism and CB1R affinity of these compounds, their toxicity and effects remain insufficiently explored and largely uncharted. ¹¹ , ¹² , ¹⁹ , ²⁰ , ²¹ Considering widespread misuse of SCs worldwide, understanding the structure–effect relationship involved in SCs and CB1R is crucial. This study aimed to probe the differences in potency and duration of action of the four drugs in cannabimimetic effects, shedding light from both pharmacokinetic and pharmacodynamic perspectives.

2. RESULTS

2.1. Inhibition of motor function by ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA in mice in a dose‐ and time‐dependent manner

We first examined the impact of various doses (0.5, 0.1 and 0.02 mg/kg) of the four drugs on motor function using the spontaneous locomotor activity test. As illustrated in Figure 1A, the results demonstrated a significant reduction in the total distance travelled by mice for 1 h after administering there indazole drugs including ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA at a 0.5 mg/kg dose compared to the control group. MDMB‐4en‐PINACA maintained its hypolocomotive effect at 0.1 mg/kg, whereas ADB‐4en‐PINACA and ADB‐BINACA did not exhibit such an effect. However, no hypolocomotive effect was observed for the three indazole drugs at a 0.02 mg/kg dose and ADB‐BICA at three doses (ADB‐BICA, H = 1.099, P = 0.7772, n = 8; ADB‐BINACA, H = 13.67, P = 0.0034, n = 8; ADB‐4en‐PINACA, H = 22.89, P < 0.0001, n = 8; MDMB‐4en‐PINACA, H = 24.2, P < 0.0001, n = 8). Notably, the total distance travelled by mice following administration of 0.5 mg/kg MDMB‐4en‐PINACA was significantly lower (29.25‐fold) than ADB‐BICA, (17.6‐fold) ADB‐BINACA and (10.01‐fold) ADB‐4en‐PINACA, which indicated that MDMB‐4en‐PINACA induced the most potent hypolocomotive effect and ADB‐BICA yielded the weakest effect.

FIGURE 1.

Hypolocomotion effects resulting from systemic administration of four drugs. (A) Impact of the four drugs on total distance travelled 1 h post‐administration in spontaneous locomotor activity at doses of 0.02, 0.1 and 0.5 mg/kg. (B) Visual representation of homecage behaviour for the four drugs 6 h after administration at 0.5 mg/kg. (C) Temporal effects on total distance travelled 6 h after administration in homecage behaviour tests at doses of 0.02, 0.1, and 0.5 mg/kg. Data represent mean ± SEM, *P < 0.05 versus 0.5 mg/kg, ***P < 0.001 versus 0.5 mg/kg, ****P < 0.0001 versus 0.5 mg/kg, ^&& P < 0.01 versus 0.1 mg/kg, ^&&& P < 0.001 versus 0.1 mg/kg, ^&&&& P < 0.0001 versus 0.1 mg/kg.

We conducted homecage behaviour tests to further corroborate these hypolocomotive effects and assess their duration. Visualization data, which encompassed 12 parameters, including total distance travelled, revealed a time‐ and dose‐dependent inhibition (Figure 1B). We recorded and analysed the total distance travelled over 6 h following drug administration using a two‐way repeated measures ANOVA. Consistent with the spontaneous locomotor activity results, indole ADB‐BICA did not yield a reduction in total distance travelled by mice following the drugs administration at three doses (effect of administration (F _3,28 = 0.8542, P = 0.4762), time interval (F _{3.440, 96.31} = 54.78, P < 0.0001), administration × time interval (F _15,140 = 0.9664, P = 0.4937), n = 8). For three indazole drugs, the total distance travelled by mice following the drugs administration of 0.5 mg/kg was significantly reduced. At 0.1 mg/kg, ADB‐4en‐PINACA and MDMB‐4en‐PINACA continued to induce substantial hypolocomotive effects, while ADB‐BINACA did not yield a reduction in total distance. No hypolocomotive effect was observed at 0.02 mg/kg for any of the three drugs (ADB‐BINACA, effect of administration (F _3,28 = 2.831, P = 0.0564), time interval (F _3.693,103.4 = 35.48, P < 0.0001), administration × time interval (F _15,140 = 3.538, P < 0.0001), n = 8; ADB‐4en‐PINACA, effect of administration (F _3,28 = 0.9823, P = 0.4152), time interval (F _3.43,96.04 = 30.82, P < 0.0001), administration × time interval (F _15,140 = 7.167, P < 0.0001), n = 8; MDMB‐4en‐PINACA, effect of administration (F _{3, 28} = 29.93, P < 0.0001), time interval (F _{3.450, 96.61} = 21.39, P < 0.0001), administration × time interval (F _15,140 = 10.53, P < 0.0001), n = 8). Similarly, the order of hypolocomotive potency among the three indazole drugs remained consistent. Furthermore, these hypolocomotive effects exhibited different durations, with MDMB‐4en‐PINACA's effect lasting up to 5 h post‐administration, while ADB‐BINACA and ADB‐4en‐PINACA induced effects lasting only 1 h (Figure 1C). Detailed data on hanging time and rearing time are provided in the Supplementary Materials, Figures S1–S4. Taken together, these findings indicate that the three indazole drugs induced hypolocomotion in mice in a dose‐dependent and time‐dependent manner during homecage behaviour tests, whereas indole ADB‐BICA cannot.

2.2. Hypothermia induced by ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA in mice in a dose and time‐dependent manner

The present study assessed hypothermic responses in mice following intraperitoneal administration of four drugs. Figure 2 illustrates the results at different doses, measured in 30‐min intervals. For indole ADB‐BICA, it did not change the body temperature following administration at three doses (Figure 2A; administration effect (F _3,28 = 4.262, P = 0.0134), time interval effect (F _4.504,126.1 = 1.285, P = 0.2771), administration × time interval interaction (F _18,168 = 0.2759, P = 0.9987), n = 8). For three indazole drugs, at 0.5 mg/kg ADB‐BINACA, hypothermia was observed within 30 min, leading to a peak temperature reduction of 2.14°C. The body temperature returned to baseline within 1 h. However, doses of 0. and 0.02 mg/kg did not induce hypothermic effects (Figure 2B; administration effect (F _3,28 = 7.195, P = 0.001), time interval effect (F _4.584,128.4 = 7.078, P < 0.0001), administration × time interval interaction (F _18,168 = 8.223, P < 0.0001), n = 8). For ADB‐4en‐PINACA, the hypothermic effect lasted 2 h at the 0.5 mg/kg dose. The most significant reduction occurred at 30 min, with a peak temperature drop of 3.94°C. At the 0.1 mg/kg dose, a similar effect was observed at 30 min, followed by a return to baseline. The 0.02 mg/kg dose did not induce hypothermia (Figure 2C; administration effect (F _3,28 = 44.33, P < 0.0001), time interval effect (F _3.31,92.67 = 27.93, P < 0.0001), administration × time interval interaction (F _18,168 = 13.27, P < 0.0001), n = 8). MDMB‐4en‐PINACA produced a pronounced and sustained hypothermic response at the 0.5 mg/kg dose, lasting approximately 11.0 h. The temperature reduction peaked at 8.64°C after 1 h and gradually returned to baseline levels. At the 0.1 mg/kg dose, hypothermia persisted for about 1.5 h, with a maximum temperature reduction of 3.98°C at 30 min. The 0.02 mg/kg dose did not induce hypothermia (Figure 2D; administration effect (F _3,28 = 161.3, P < 0.0001), time interval effect (F _3.229,90.42 = 79.57, P < 0.0001), administration × time interval interaction (F _72,672 = 49.93, P < 0.0001), n = 8). These findings indicate that the three indazole drugs induced hypothermia in a dose‐ and time‐dependent manner in mice, whereas indole ADB‐BICA can not. Among them, MDMB‐4en‐PINACA exhibited the most potent and longest‐lasting hypothermic effect.

FIGURE 2.

Time course effects on body temperature and response latency due to systemic administration of four drugs. (A–D) Influence of the four drugs on body temperature after administration at doses of 0.02 mg/kg, 0.1 mg/kg, and 0.5 mg/kg. (E‐H) Effect of the four drugs on response latency in the hot plate test 30 min after administration at doses of 0.02 and 0.1 mg/kg. Data represent mean ± SEM, *P < 0.05 versus 0.5 mg/kg, **P < 0.01 versus 0.5 mg/kg, ***P < 0.001 versus 0.5 mg/kg, ****P < 0.0001 versus 0.5 mg/kg, ^& P < 0.001 versus 0.1 mg/kg, ^&& P < 0.001 versus 0.1 mg/kg, ^&&&& P < 0.0001 versus 0.1 mg/kg. The green dashed line represents the period from 0.5 to 1 h, the blue dashed line signifies the interval from 0.5 to 5.5 h, the red dashed line corresponds to the time from 6.5 to 7.5 h, and the purple dashed line depicts the duration from 8 to 11 h in (D).

2.3. Analgesia induced by 0.1 mg/kg MDMB‐4en‐PINACA in mice

A hot plate test was conducted 30 ± 5 min after injection to evaluate the analgesic effects of the four drugs. Since 0.5 mg/kg of three indazole drugs significantly inhibited locomotor activity, resulting in response latencies exceeding 60 seconds, the focus shifted to assessing the impact of the 0.02 and 0.1 mg/kg doses. Figure 2E–H presents the data. None of the drugs affected response latency at 0.02 mg/kg. However, only MDMB‐4en‐PINACA induced a significant increase in response latency at 0.1 mg/kg compared to the control group (ADB‐BICA, H = 1.633, P = 0.442, n = 8; ADB‐BINACA, H = 1.198, P = 0.5494, n = 8; ADB‐4en‐PINACA, H = 0.9928, P = 0.6087, n = 8; MDMB‐4en‐PINACA, H = 13.33, P = 0.0013, n = 8).

2.4. Blockade of hypolocomotion, hypothermia and analgesia effects by CB1R antagonist AM251 in mice

Given that the biological effects of SCs primarily hinge on CB1R activation, the study explored whether the CB1R antagonist AM251 or the CB2 receptor antagonist AM630 could counter the effects induced by the drugs. Notably, administration of AM251 or AM630 alone had no impact on locomotor activity (H = 0.7550, P = 0.6856, n = 8), body temperature (H = 7.423, P = 0.0244, n = 8) or thermal nociceptive threshold (H = 2.403, P = 0.3007, n = 8) in mice. However, pretreatment with AM251 effectively reversed hypolocomotion (ADB‐BINACA, H = 7.94, P = 0.0189, n = 8; ADB‐4en‐PINACA, H = 15.16, P = 0.0005, n = 8; MDMB‐4en‐PINACA, H = 15.16, P = 0.0004, n = 8) and hypothermia (ADB‐BINACA, H = 12.40, P = 0.002, n = 8; ADB‐4en‐PINACA, H = 15.71, P = 0.0004, n = 8; MDMB‐4en‐PINACA, H = 16.67, P = 0.0002, n = 8) effects induced by the three indazole drugs at the 0.5 mg/kg dose. Similarly, AM251 reversed the analgesic effect induced by MDMB‐4en‐PINACA at 0.1 mg/kg (H = 15.42, P = 0.0004, n = 8). Notably, AM630 did not impede the observed effects of the drugs. These findings underscore the pivotal role of CB1R, as opposed to CB2 receptors, in mediating the effects induced by the three indazole drugs in mice (Figure 3).

FIGURE 3.

Blockage effects of AM251 and AM630 on hypolocomotion, hypothermia, and analgesia effects induced by the three indazole drugs in mice. (A) Influence of AM251 and AM630 on mice locomotor activity. (B–D) Blockage effects of AM251 and AM630 on hypolocomotion effects induced by the three indazole drugs in mice at a dose of 0.5 mg/kg. (E) Influence of AM251 and AM630 on mice body temperature. (F–H) Blockage effects of AM251 and AM630 on hypothermia effects induced by the three indazole drugs in mice at a dose of 0.5 mg/kg. (I) Influence of AM251 and AM630 on mice thermal nociceptive threshold. (F) Blockage effects of AM251 and AM630 on analgesia effects induced by MDMB‐4en‐PINACA in mice at a dose of 0.1 mg/kg. **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control.

2.5. Molecular docking analysis

A comprehensive molecular docking analysis was undertaken to elucidate the structure–activity mechanisms underlying the interactions between the four drugs and CB1R. As shown in Figure 4A, the key constituents of the binding pocket for the four drugs are situated within the residues of TM2–TM3 and TM5–TM7. Despite variations in their chemical compositions, all four drugs and the initial ligand in the crystal structure adopt a similar C‐shaped conformation with overlapping ligand binding pockets. Upon docking ADB‐BICA, ADB‐BINACA and ADB‐4en‐PINACA, hydrogen bonds formed between the carboxamide and the residues of SER¹⁷³ of TM5, alongside π–π interactions between the indazole core of ADB‐4en‐PINACA or the phenylmethyl tail of ADB‐BICA, ADB‐BINACA and the residues of PHE²⁶⁸ within ECL2 (Figure 4B–D). For MDMB‐4en‐PINACA, a π–π interaction takes shape between the indazole core of CB1R and the residues of PHE²⁰⁰ (Figure 4E).

FIGURE 4.

Molecular docking of four drugs. (A) Binding pockets of the four drugs and comparison of binding conformations with the active conformation of CB1R (grey) using ADB‐BICA (pink), ADB‐BINACA (yellow), ADB‐4en‐PINACA (blue), MDMB‐4en‐PINACA (green) and initial cocrystal ligand (brown) (PDB: 6N4B). (B–E) Four‐dimensional ligand interaction diagrams of CB1R, with blue lines indicating π–π interactions and yellow lines representing hydrogen bonds.

2.6. Blood concentration analysis

A comprehensive analysis of blood concentration was performed employing LC‐MS/MS techniques to investigate the structure–activity mechanisms of the four drugs. As depicted in Figure 5A1–D1, no traces of the drugs were observed within the control group samples. Four drugs and SKF525A, an internal standard, were recorded in blood sample after administration, respectively (Figure 5A2–D2,E). Notably, the plasma concentration of four drugs administered at 0.5 mg/kg peaked at the 30‐min mark. Subsequently the plasma concentration of ADB‐BICA, ADB‐BINACA and ADB‐4en‐PINACA demonstrated a rapid decline and nearly reaching undetectable levels within 2 h, in contrast, MDMB‐4en‐PINACA followed by a gradual decrease, persisting for more than 6 h until it was almost undetectable at 7 h. Moreover, MDMB‐4en‐PINACA exhibited the most elevated plasma concentration, followed by ADB‐BICA, ADB‐4en‐PINACA and ADB‐BINACA at the corresponding time points, as illustrated in Figure 5F.

FIGURE 5.

Detection of four drugs by LC‐MS/MS. (A1–D1) Chromatograms depicting four drugs in mice blank plasma. (A2–D2) Chromatograms depicting four drugs in mice plasma 30 min post‐drugs administration at 0.5 mg/kg. (E) Chromatograms of internal standard in mice plasma. (F) Mean blood concentration–time curve of the four drugs in mice 30 min post‐drugs administration at 0.5 mg/kg, n = 4.

3. DISCUSSION

The assessment of classical tetrad effects encompassing analgesia, hypothermia, catalepsy and hypolocomotion remains the conventional approach to discern the cannabimimetic nature of novel compounds. ²² In the context of this study, we scrutinized the acute effects of four drugs on motor function, body temperature and antinociceptive thresholds across varying doses in mice. The evaluation of motor function involved the assessment of both spontaneous locomotor activity over 1 h and homecage behaviour tests spanning 6 h. Our findings unveiled that three indazole drugs yielded hypolocomotive effects in a dose‐dependent and time‐dependent manner in mice. The temporal window of efficacy for hypolocomotion persisted for an hour following the administration of ADB‐BINACA and ADB‐4en‐PINACA, while MDMB‐4en‐PINACA extended this window to 5 h. Notably, three indazole drugs exhibited varying degrees of hypolocomotion, ascending in potency from ADB‐BINACA to ADB‐4en‐PINACA and peaking with MDMB‐4en‐PINACA, both in the context of spontaneous locomotor activity and homecage behaviour tests. Although the majority of studies have highlighted the hypolocomotion effects triggered by SCs, ²³ , ²⁴ preclinical investigations have reported a biphasic modulation of rodent spontaneous locomotion by SCs, inducing facilitation at lower doses and inhibition at higher doses. ²³ , ²⁵ Nonetheless, in the present study, the administration of all three indazole drugs elicited an immediate hypolocomotive response, precluding the biphasic pattern, attributed to the rapid penetration of highly lipophilic SCs across the blood–brain barrier into the central nervous system. ²⁶ In terms of body temperature, the three indazole drugs evoked hypothermia in a dose‐dependent and time‐dependent manner in mice, with MDMB‐4en‐PINACA yielding the most potent and enduring hypothermic effect. The hot plate test was employed to assess analgesic effects, with low doses of the drugs chosen to avoid potential interference from their hypolocomotion effects. At 0.1 mg/kg, ADB‐4en‐PINACA and ADB‐BINACA exhibited no impact on response latency, whereas MDMB‐4en‐PINACA significantly prolonged response latency. It is highly conceivable that the analgesic effect of 0.1 mg/kg MDMB‐4en‐PINACA could be confounded by its hypolocomotive impact. Previous studies have reported the absence of antinociceptive effects for certain SCs, such as AB‐FUBINACA and AB‐CHMINACA, in hot plate assays. ²⁷ Consequently, the present results underscore the disparity in above effects among various SCs, with MDMB‐4en‐PINACA demonstrating the strongest impact, ADB‐BICA the weakest and ADB‐4en‐PINACA and ADB‐BINACA positioned in between.

Two major cannabinoid receptors have been identified, namely, CB1R and CB2R, both of which exhibit high binding affinities for SCs. ²² CB1R, predominantly expressed in the brain, is integral to behavioural functions and chiefly responsible for the psychoactive outcomes of SCs consumption. ²³ , ²⁸ , ²⁹ CB2R was highly expressed in peripheral tissues. ²² , ²⁹ While past research has attributed cannabinoid tetrad effects induced by Δ9‐THC and SCs to CB1R, ²² , ³⁰ , ³¹ our study sought to elucidate the receptor mechanisms governing the hypolocomotion, hypothermia and analgesia effects of the three indazole drugs. We found that the observed effects were solely contingent on CB1R activity, as AM251 reversed the effects, while AM630 failed. Notably, the dissimilarities in the effects of the four drugs, all binding to the same receptor, remain enigmatic.

Current evidence suggests that the biological effects of SCs are influenced by various factors, including pharmacology and pharmacokinetics. ²⁶ , ³² , ³³ When evaluating pharmacology characteristics, researchers often rely on parameters such as affinity for CB1R, potency and efficacy in vitro. ³⁴ , ³⁵ At present, research on ADB‐BICA mainly focuses on identification, analysis and metabolism, and research on its pharmacology characteristics and toxicity has not been reported yet. ¹⁰ , ³⁶ , ³⁷ For three indazole drugs, previous studies have shown that MDMB‐4en‐PINACA (Ki = 0.28 nM) and ADB‐4en‐PINACA (Ki = 0.17 nM) exhibit greater affinity for CB1R binding compared to the traditional SC JWH‐018 (Ki = 2.6 nM). ²⁹ This suggests that ADB‐4en‐PINACA has a slightly higher affinity than MDMB‐4en‐PINACA. However, our in vitro animal experiments yielded inconsistent results. Notably, our docking findings revealed favourable binding capabilities of all four drugs with CB1R, akin to potent SC agonists. Yet, these results did not significantly differentiate between the four, failing to fully elucidate the observed variations in biological effects. To gain further insights, a β‐Arrestin 2 recruitment assay was conducted, indicating that MDMB‐4en‐PINACA (EC50 = 2.33 nM, Emax = 299%), ADB‐4en‐PINACA (EC50 = 3.43 nM, Emax = 261%) and ADB‐BINACA (EC50 = 6.36 nM, Emax = 290%) exhibited heightened potency and efficacy compared to SC JWH‐018 (EC50 = 21.4 nM, Emax = 100%) ¹¹ , ¹⁹ (Table 1). These data suggested a correlation between the potency measure (EC50) and biological effects, while the efficacy (Emax) was relatively less consistent. This prompts speculation that the molecular signalling post‐CB1R activation contributes to the differing effects of the drugs, making EC50 a reliable gauge for evaluating their biological impact. However, molecular pharmacology could not entirely account for the differences in the duration of drug effects. Specifically, MDMB‐4en‐PINACA exhibited a more prolonged duration of hypolocomotive and hypothermia effects than ADB‐4en‐PINACA and ADB‐BINACA. Consequently, an in‐depth evaluation of SCs' impact on biological effects necessitated the examination of plasma concentrations for the four drugs. Notably, for ADB‐BICA, ADB‐4en‐PINACA and ADB‐BINACA, peak plasma concentrations were achieved at 30 min, rapidly declining to minimal levels within an hour. Conversely, MDMB‐4en‐PINACA reached its peak plasma concentration 30 min after administration, but remaining detectable for approximately 6 h. Remarkably, the blood concentration–time profiles correlated well with the biological effects' durations for the three indazole drugs. Literature suggests that tert‐leucinate indole and indazole‐3‐carboxamide SCs tend to bind extensively to proteins in vivo and accumulate in lipid‐rich tissues, with subsequent redistribution into the circulatory system. This intricate process could account for the prolonged detection windows of these substances. ²⁶ It is worth noting that although ADB‐BICA showed higher plasma concentrations than ADB‐BINACA and ADB‐4en‐PINACA, it did not cause any observed effects in mice. These findings strongly indicate that both pharmacodynamics and pharmacokinetics of the drugs significantly contribute to their respective biological effects.

TABLE 1.

Ki, EC50 and Emax of three drugs.

SCs	Ki (nM)	EC50 (nM) (95% CI)	Emax (%) (95% CI) relative to JWH‐018	References [11, 29]
MDMB‐4en‐PINACA	0.28	2.33	299%	19, 29
		1.88	221	11
ADB‐BINACA		6.36	290%	11
ADB‐4en‐PINACA	0.17	3.43	261%	19, 29

The EMCDDA's generic structure model of SCs comprises four essential structural components: ‘the core’, ‘the link’, ‘the head’, and ‘the tail’. ³ By manipulating these foundational elements through substitutions, additions or modifications, a multitude of novel SCs can be generated. However, even slight structural alterations can lead to changes in biochemical attributes such as dependence liability and neurotoxicity. ²⁶ In this study, it was observed that ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA possess the indazole core, while ADB‐BICA exhibits the indole core. ADB‐4en‐PINACA incorporates the L‐tert‐leucinamide head group and a 4‐pentenyl (4en‐P) tail, while MDMB‐4en‐PINACA substitutes the head group with methyl L‐tert‐leucinate, and ADB‐BINACA replaces the tail with phenylmethyl. The presence of the L‐tert‐leucinamide head group led to more potent and sustained effects than the L‐tert‐leucinamide head group. Similarly, the 4‐pentenyl (4en‐P) tail induced more potent effects than the phenylmethyl tail. And indazole head induced more potent effects than the indole head. These results will provide a new perspective for a better and more comprehensive understanding of the relationship between the structure and effects of SCs.

In this study, we compared the differences in cannabinoid‐like effects induced by four synthetic cannabinoids in mice. For four drugs, MDMB‐4en‐PINACA manifested the most potent and sustained effects, followed by ADB‐4en‐PINACA, ADB‐BINACA and ADB‐BICA. Although we explicated the causes for the differences from the perspectives of pharmacology, molecular interaction and pharmacokinetics, the mechanism for the differences have not been thoroughly explored. Therefore, a more comprehensive exploration including downstream molecular signal, in vivo distribution of drugs and key interacting residues is warranted in future research to elucidate the nuances in both pharmacology and pharmacokinetics.

4. MATERIALS AND METHODS

4.1. Animals

Male C57BL/6 mice (6–8 weeks old, weighing 20–22 g) were procured from Beijing Vital River Laboratory Animal Technology Co., Ltd., China. The mice were accommodated in groups of four under controlled conditions: a 12‐h light/dark cycle (lights on from 07:00 to 19:00 h) with unrestricted access to food and water. The experimental protocol was approved by the Ethics Committee of Hebei Medical University and adhered to the guidelines for animal experimentation outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (registration number: IACUC‐Hebmu‐P 2022118). Prior to testing, the animals were acclimatized in the testing room for 1 h to familiarize them with the experimental environment. Measures were taken to minimize pain and discomfort.

4.2. Drugs

ADB‐BICA (Figure 6A; CAS No. 2219319‐40‐9, >99% purity), ADB‐BINACA (Figure 6B; CAS No. 2748155‐93‐1, >98% purity), ADB‐4en‐PINACA (Figure 6C; CAS No. 2666932‐44‐9, >98% purity), MDMB‐4en‐PINACA (Figure 6D; CAS No. 2504100‐70‐1, >95% purity), AM251 and AM630 were procured from GLPBIO (Montclair, CA, USA) and APExBIO (Houston, TX, USA). The drugs were initially dissolved in DMSO and then diluted in a solution of absolute DMSO: Tween 80: saline at a 1: 1: 18 ratio. The determination of drugs dosage were adjusted based on literatures. ³⁸ , ³⁹ 0.5, 0.1 and 0.02 mg/kg drugs were administrated by intraperitoneal injections at a dosage of 4 mL per kg of body weight. AM251 or AM630 at a dose of 6 mg/kg was administered 30 min prior to SCs injection. Control mice were injected with an appropriate vehicle solution.

FIGURE 6.

Chemical structures of four drugs copied from the Cayman chemical website (https://www.caymanchem.com). (A) ADB‐BICA. (B) ADB‐BINACA. (C) ADB‐4en‐PINACA. (D) MDMB‐4en‐PINACA.

4.3. Motor function tests

Motor function changes were assessed through spontaneous locomotor activity and homecage behaviour tests. The male C57BL/6J mice were categorized into various groups, including the control group (vehicle, DMSO: Tween 80: saline = 1: 1: 18), drug groups (0.5, 0.1 and 0.02 mg/kg), CB1R antagonist group (6 mg/kg AM251), CB2R antagonist group (6 mg/kg AM630), CB1R antagonist (6 mg/kg AM251) + drugs group (0.5 mg/kg) and CB2R antagonist (6 mg/kg AM630) + drugs group (0.5 mg/kg).

4.3.1. Spontaneous locomotor activity

Eight non‐transparent Plexiglas boxes (24 cm width × 24 cm depth × 50 cm height) were employed to evaluate spontaneous locomotor activity. The mice were assessed for the total distance travelled within the arena for a duration of 1 h post‐drug administration. The Animal Video Analysis System (JLBeh soft‐tech Co. Ltd., Shanghai, China) was utilized for data analysis. The boxes were cleaned with 95% ethanol prior to each test.

4.3.2. Homecage behaviours

An AI homecage system (Shanghai Vanbi Intelligent Technology Co., Ltd.) was employed for homecage behaviour testing. Digital video cameras were mounted perpendicularly to the cages, and video data were analysed using Tracking Master software connected to a computer via a Pelco video processor. Over a 6‐h period during the light phase (10:00 AM to 4:00 PM), active behaviours were monitored under dim light conditions within a sound‐attenuated cage. A total of 12 parameters, including the total distance, were recorded immediately after administration and analysed at hourly intervals for a maximum of 6 h.

4.4. Body temperature measurement

A digital rectal thermometer with a lubricated flexible probe was used to measure body temperature. Temperature measurements were taken at various time points post‐administration, with readings recorded every 30 min for 3 or 12 h after injection. The animal grouping was consistent with the motor function tests.

4.5. Thermal nociceptive threshold measurement

The thermal nociceptive threshold was assessed using the hot plate test. Mice were placed on a 55°C hot plate, and the latency to either a hind‐paw lick or jump response was recorded manually 30 min after the injection of test drugs (0.1 mg/kg). Animals were removed from the hot plate if no response occurred within 60 s to prevent tissue injury, with a latency of 60 s assigned as the response.

4.6. Molecular docking analysis

The 3 Å cryo‐EM crystal structure of MDMB‐FUBINACA‐activated full‐length CB1R in complex with its downstream heterotrimeric Gi protein was obtained from the Protein Data Bank (PDB entry: 6N4B, ⁴⁰ https://www.rcsb.org/). The Gi proteins and cholesterol ligands were excluded, retaining only CB1R and the ligand within the active site. The structure underwent preparation using Protein Preparation Wizard in Maestro v. 9.0 (2009), encompassing protein preprocessing, H‐bond assignment optimization and energy minimization. The Receptor Grid Generation tool defined the docking grid. The three‐dimensional (3D) structures of ligands four drugs were sourced from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Ligands were prepared using the LigPrep tool in Maestro to generate energy‐minimized 3D structures. Standard precision (SP) docking in Glide v.5.5 (2009) was employed to determine docking poses with default parameters. The best conformation structures were saved in PDBQT format, visualized and converted to PDB format using PyMOL software version 2.5.4.

4.7. LC‐MS/MS analysis

Samples were collected from mice into heparin Na⁺‐containing tubes at 0, 0.5, 1, 2, 3, 6, 9 and 12 h after the administration of the four drugs at a dose of 0.5 mg/kg. The blood sample was combined with acetonitrile solution (acetonitrile: ddH2O = 3: 1) containing 2 ng/ml SKF525A as an internal standard (IS). After vortexing for 2 min, the mixture was centrifuged at 12 000 rpm for 10 min at 4°C. The resulting supernatant was then filtered through a 0.2‐μm Millipore filter. A 5‐μl aliquot of the filtered mixture was injected into the LC‐MS/MS system. Separation was carried out on a Kinetex C18 column (50 × 3.0 mm, 2.6 μm; Phenomenex) using a gradient program with a flow rate of 0.4 ml/min. The mobile phase comprised solvent A (20 mmol ammonium acetate containing 0.1% formic acid) and solvent B (acetonitrile). The column thermostat was maintained at 35°C throughout the analysis.

Mass spectrometry acquisition was performed using a Waters Micromass Quattro Micro triple quadrupole instrument equipped with an electrospray ionization source operating in positive (ESI+) mode with ionization polarity switching. The instrument was optimized with the following settings: ion source voltage, 5.5 kV; ion source temperature, 600°C. The dwell time for each channel of the sample and IS was set to 20 ms. Data were collected in multiple response monitoring (MRM) mode and processed using MultiQuant 3.0.3 software.

4.8. Statistical analysis

GraphPad Prism version 8.4.3 was used for data analysis. The data analysis of spontaneous locomotor activity, nociceptive threshold and antagonists blockade effect was performed by Kruskal–Wallis H Test. Two‐way ANOVA with Geisser–Greenhouse's epsilon was applied to evaluate the difference of homecage behaviour tests and body temperature. A P‐value less than 0.05 was statistically significant.

AUTHOR CONTRIBUTIONS

Fenghua Zhou: Data curation; resources; formal analysis; writing—original draft preparation. Xiaoli Wang: Data curation; writing—original draft preparation. Sujun Tan: Visualization; investigation. Yan Shi and Ping Xiang: Project administration. Bing Xie: Methodology. Bin Cong: Project administration; supervision. Chunling Ma: Conceptualization; funding acquisition; supervision. Di Wen: Project administration; conceptualization; funding acquisition; supervision; writing—original draft preparation.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest related to this work. Each author has indicated that he/she has met the journal's requirements for authorship.

Supporting information

Figure S1. Effects of systemic administration of ADB‐BICA at doses of 0.02 mg/kg, 0.1 mg/kg, and 0.5 mg/kg. (A) Slow movement time (B) Middle movement time (C) Fast movement time (D) Digging time (E) Grooming time (F) Eating time (G) Squatting time (H) Hanging time (I) Rearing time (J) Drinking time (K) Resting time. Data represent mean ± SEM, *P < 0.05 vs 0.5 mg/kg, **P < 0.01 vs 0.5 mg/kg, ^& P < 0.05 vs 0.1 mg/kg.

Figure S2. Effects of systemic administration of ADB‐BINACA at doses of 0.02 mg/kg, 0.1 mg/kg, and 0.5 mg/kg. (A) Slow movement time (B) Middle movement time (C) Fast movement time (D) Digging time (E) Grooming time (F) Eating time (G) Squatting time (H) Hanging time (I) Rearing time (J) Drinking time (K) Resting time. Data represent mean ± SEM, *P < 0.05 vs 0.5 mg/kg, **P < 0.01 vs 0.5 mg/kg, ***P < 0.001 vs 0.5 mg/kg, ^& P < 0.05 vs 0.1 mg/kg, ^$ P < 0.05 vs 0.5 mg/kg, ^$$ P < 0.05 vs 0.5 mg/kg.

Figure S3. Effects of systemic administration of ADB‐4en‐PINACA at doses of 0.02 mg/kg, 0.1 mg/kg, and 0.5 mg/kg. (A) Slow movement time (B) Middle movement time (C) Fast movement time (D) Digging time (E) Grooming time (F) Eating time (G) Squatting time (H) Hanging time (I) Rearing time (J) Drinking time (K) Resting time. Data represent mean ± SEM, *P < 0.05 vs 0.5 mg/kg, **P < 0.01 vs 0.5 mg/kg, ***P < 0.001 vs 0.5 mg/kg, ****P < 0.0001 vs 0.5 mg/kg, ^& P < 0.05 vs 0.1 mg/kg, ^&& P < 0.01 vs 0.1 mg/kg, ^&&&& P < 0.0001 vs 0.1 mg/kg.

Figure S4. Effects of systemic administration of MDMB‐4en‐PINACA at doses of 0.02 mg/kg, 0.1 mg/kg, and 0.5 mg/kg. (A) Slow movement time (B) Middle movement time (C) Fast movement time (D) Digging time (E) Grooming time (F) Eating time (G) Squatting time (H) Hanging time (I) Rearing time (J) Drinking time (K) Resting time. Data represent mean ± SEM, *P < 0.05 vs 0.5 mg/kg, **P < 0.01 vs 0.5 mg/kg, ***P < 0.001 vs 0.5 mg/kg, ****P < 0.0001 vs 0.5 mg/kg, ^& P < 0.05 vs 0.1 mg/kg, ^&& P < 0.01 vs 0.1 mg/kg, ^&&& P < 0.001 vs 0.1 mg/kg, ^&&&& P < 0.0001 vs 0.1 mg/kg.

Zhou F, Wang X, Tan S, et al. Differential cannabinoid‐like effects and pharmacokinetics of ADB‐BICA, ADB‐BINACA, ADB‐4en‐PINACA and MDMB‐4en‐PINACA in mice: A comparative study. Addiction Biology. 2024;29 (2): e13372. doi: 10.1111/adb.13372.

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding authors.