Novel N-normetazocine Derivatives with Opioid Agonist/Sigma-1 Receptor Antagonist Profile as Potential Analgesics in Inflammatory Pain

Although opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are the most common drugs used in persistent pain treatment; they have shown many side effects. The development of new analgesics endowed with mu opioid receptor/delta opioid receptor (MOR/DOR) activity represents a promising alternative to MOR-selective compounds. Moreover, new mechanisms, such as sigma-1 receptor (σ1R) antagonism, could be an opioid adjuvant strategy. The in vitro σ1R and σ2R profiles of previous synthesized MOR/DOR agonists (−)-2R/S-LP2 (1), (−)-2R-LP2 (2), and (−)-2S-LP2 (3) were assayed. To investigate the pivotal role of N-normetazocine stereochemistry, we also synthesized the (+)-2R/S-LP2 (7), (+)-2R-LP2 (8), and (+)-2S-LP2 (9) compounds. (−)-2R/S-LP2 (1), (−)-2R-LP2 (2), and (−)-2S-LP2 (3) compounds have Ki values for σ1R ranging between 112.72 and 182.81 nM, showing a multitarget opioid/σ1R profile. Instead, (+)-2R/S-LP2 (7), (+)-2R-LP2 (8), and (+)-2S-LP2 (9) isomers displayed a nanomolar affinity for σ1R, with significative selectivity vs. σ2R and opioid receptors. All isomers were evaluated using an in vivo formalin test. (−)-2S-LP2, at 0.7 mg/kg i.p., showed a significative and naloxone-reversed analgesic effect. The σ1R selective compound (+)-2R/S-LP2 (7), at 5.0 mg/kg i.p., decreased the second phase of the formalin test, showing an antagonist σ1R profile. The multitarget or single target profile of assayed N-normetazocine derivatives could represent a promising pharmacological strategy to enhance opioid potency and/or increase the safety margin.


Introduction
Inflammatory pain is a pathological condition related to an increased sensitivity caused by tissue damage. The increasing incidence and impact of inflammatory diseases have encouraged the search for new pharmacological strategies to face them. The conventional therapies for inflammation, including steroidal (SAID), nonsteroidal anti-inflammatory drugs (NSAID), and disease-modifying antirheumatic drugs (DMARDs) [1,2], have shown many side effects and deficiencies. Moreover, opioid therapy for inflammatory pain for the treatment of chronic diseases is often associated with several side effects. Therefore, to date, the treatment of chronic inflammatory pain is one of the unmet clinical needs for patients. σ1R is a 24 kDa protein of 223 aminoacids anchored to the endoplasmic reticulum and plasma membranes that acts as a molecular chaperone, modulating the functionality of different receptors and ion channels [13,14]. It is expressed in peripheral organs and many areas of the central nervous system (CNS), including key areas for pain modulation, such as dorsal root ganglia, superficial layers of dorsal horn, locus coeruleus, and periaqueductal grey matter [15]. Literature data demonstrate that most of the endogenous ligands for σ1R are steroids synthesized in the nervous system (neurosteroids), such as pregnenolone-sulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEA-S), that have a role as σ1R agonists [16]. On the other hand, progesterone is an endogenous antagonist of this receptor, and it induces down-regulation of TRPV1 expression in the plasma membrane of sensory neurons, causing a decrease in capsaicin-induced nociceptive responses [17].
The σ1R modulation of analgesia was first proposed by Chien and Pasternak, who identified σ1R as a potent anti-opioid endogenous system [18]. It has been demonstrated that σ1R agonists are able to strongly counteract spinal and supraspinal opioid-induced analgesia, while σ1R antagonists potentiate opioid-induced analgesia [19]. Furthermore, selective σ1R antagonists have demonstrated analgesic efficacy in acute and chronic inflammatory pain models [20,21].
Several hypotheses explain the involvement of σ1R in inflammatory pain modulation. One possible mechanism is that σ1R activation enhances the bradykinin induced Ca 2+ release and nitric oxide (NO) signalling, thereby strengthening the inflammatory process [22,23]. Moreover, a peripheral σ1R-related mechanism, proposed by Tejada et al. [24], might be even more important in the modulation of inflammatory pain sustained by a conspicuous enhancement of peripheral sensitization [25]. Additionally, pain sensitization after peripheral inflammation involves plastic changes characterized by an increase in spinal excitatory neurotransmission [26]. It has been described as a modulatory role of σ1Rs in spinal sensitization and point to N-methyl-D- σ 1 R is a 24 kDa protein of 223 aminoacids anchored to the endoplasmic reticulum and plasma membranes that acts as a molecular chaperone, modulating the functionality of different receptors and ion channels [13,14]. It is expressed in peripheral organs and many areas of the central nervous system (CNS), including key areas for pain modulation, such as dorsal root ganglia, superficial layers of dorsal horn, locus coeruleus, and periaqueductal grey matter [15]. Literature data demonstrate that most of the endogenous ligands for σ 1 R are steroids synthesized in the nervous system (neurosteroids), such as pregnenolonesulfate (PREG-S) and dehydroepiandrosterone sulfate (DHEA-S), that have a role as σ 1 R agonists [16]. On the other hand, progesterone is an endogenous antagonist of this receptor, and it induces down-regulation of TRPV1 expression in the plasma membrane of sensory neurons, causing a decrease in capsaicin-induced nociceptive responses [17].
The σ1R modulation of analgesia was first proposed by Chien and Pasternak, who identified σ1R as a potent anti-opioid endogenous system [18]. It has been demonstrated that σ1R agonists are able to strongly counteract spinal and supraspinal opioid-induced analgesia, while σ1R antagonists potentiate opioid-induced analgesia [19]. Furthermore, selective σ1R antagonists have demonstrated analgesic efficacy in acute and chronic inflammatory pain models [20,21].
Several hypotheses explain the involvement of σ1R in inflammatory pain modulation. One possible mechanism is that σ1R activation enhances the bradykinin induced Ca 2+ release and nitric oxide (NO) signalling, thereby strengthening the inflammatory process [22,23]. Moreover, a peripheral σ1R-related mechanism, proposed by Tejada et al. [24], might be even more important in the modulation of inflammatory pain sustained by a conspicuous enhancement of peripheral sensitization [25]. Additionally, pain sensitization after peripheral inflammation involves plastic changes characterized by an increase in spinal excitatory neurotransmission [26]. It has been described as a modulatory role of σ1Rs in spinal sensitization and point to N-methyl-D-aspartate (NMDA) receptors and Ca 2+ -dependent intracellular cascades as underlying mechanisms. The overall effect of activating σ1Rs is to increase intracellular Ca 2+ concentration by potentiating Ca 2+ entry at the plasma membrane level (NMDA-induced Ca 2+ influx) and Ca 2+ mobilization from endoplasmic stores (IP3-induced Ca 2+ mobilization) [27]. The modulatory role of σ1R in pain transmission was identified using σ1R knockout (KO) mice that showed attenuated pain responses in both phases of the formalin test [28,29], confirmed also by the administration of σ1R antagonists [24].
First, by competition binding assays, we evaluated the affinity for opioid receptors and σ1R, σ2R of all (−)-LP2 and (+)-LP2 isomers. Moreover, based on the role of simultaneous MOR-DOR activation and σ1R antagonism in inflammatory pain modulation, we tested all (−)-LP2 and (+)-LP2 isomers in the mouse formalin test. To highlight the contribution of opioid receptors and σ1R in the observed effects, coadministration of the opioid antagonist naloxone or selective σ1R agonist PRE-084, respectively, were evaluated.
The N-normetazocine nucleus has mainly been employed for the design of opioid analgesics, although it is a versatile structure. In fact, its stereochemistry plays a crucial role in directing N-normetazocine-based compounds to different targets [30]. The literature data reports that the (−)-(1R,5R,9R) configuration mainly interacts with opioid receptors [32], while, the (+)-(1S,5S,9S) antipode is able to bind σ1R. Alazocine ((+)-SKF-10,047) was the first compound discovered that highlighted a remarkable σ1R affinity. Conversely its (−)isomers are more tightly bound to MOR and KOR [33]. Furthermore, Carrol et al. [34] synthesized (+)-and (−)-N-substituted analogues of (+)-SKF-10,047, proving that σ1R affinity and selectivity of all compounds were influenced by the nature of the N-substituent and the N-normetazocine stereochemistry. Subsequently, regardless of their stereochemistry, the (+)-and (−)-phenazocines, characterized by an N-2-phenylethyl substituent, showed opioid agonist/σ1R antagonist profiles and a significant in vivo analgesia [35]. Differently, the analgesic effect of (+)-LP1, with an N-phenylpropanamide substituent, was not reversed by naloxone, suggesting a σ1R antagonist profile [36]. Thus, the stereochemistry of the N-normetazocine scaffold as well as the size, electronic, and steric properties of its Nsubstituents could shift the functional profile from selective σ1R to mixed-target compounds.

Formalin Test
Mice injected with 5% formalin into the mid-plantar surface of the right hind paw developed the characteristic formalin-induced pain behaviour, evidenced by a biphasic flinching and lifting/licking response. The time course of formalin-induced behaviours evidenced an immediate and acute nociceptive activity (0-5 min, phase I) that, after a short quiescent period (5-10 min, interphase), was followed by a more prolonged response (10-60 min, phase II).
as the size, electronic, and steric properties of its N-substituents could shift the fu profile from selective σ1R to mixed-target compounds.

Formalin Test
Mice injected with 5% formalin into the mid-plantar surface of the right hi developed the characteristic formalin-induced pain behaviour, evidenced by a b flinching and lifting/licking response. The time course of formalin-induced beh evidenced an immediate and acute nociceptive activity (0-5 min, phase I) that, afte quiescent period (5-10 min, interphase), was followed by a more prolonged respo 60 min, phase II).
To evaluate the systemic antinociceptive effect in both phases of the forma vehicle, (−)-2R-LP2 (2) (2mg/kg), (−)-2S-LP2 (3) (0.7 mg/kg), (+)-2R/S-LP2 (7) (5 and (+)-2R-LP2 (8) (5 mg/kg) were administrated intraperitoneally (i.p.) 20 min formalin injection, at doses chosen in accordance to previous studies [28]. As sh Figure 2, the administration of compound (−)-2S-LP2 showed a significant analges reducing the flinching/licking time in both phases of the formalin test. The (−) isomer showed the same profile with a minor potency, proving that the stere influences pharmacological characteristics such as affinity, as demonstra radioligand binding assays. To prove that the systemic antinociceptive effect was mainly mediated by the system, we administrated a subcutaneous (s.c.) solution of naloxone hydrochl mg/kg), a non-selective opioid receptor antagonist, 20 min before the injection o LP2 (the more representative of the two compounds under study). As shown in F To prove that the systemic antinociceptive effect was mainly mediated by the opioid system, we administrated a subcutaneous (s.c.) solution of naloxone hydrochloride (3 mg/kg), a non-selective opioid receptor antagonist, 20 min before the injection of (−)-2S-LP2 (the more representative of the two compounds under study). As shown in Figure 3, naloxone antagonized the antinociceptive effect of (−)-2S-LP2, confirming its main effect through the opioid system. Moreover, given the affinity for σ1R of the compounds, a contribution of this receptor to the opioid-mediated antinociception effect should be considered. naloxone antagonized the antinociceptive effect of (−)-2S-LP2, confirming its ma through the opioid system. Moreover, given the affinity for σ1R of the compo contribution of this receptor to the opioid-mediated antinociception effect sh considered. It is well established that σ1R is involved in nociception, and the effects repor σ1R ligands are consistent with a role of this receptor in pain hypersensitivity [37 considering the results obtained in the radioligand binding assay with a (+)-2R/S value two times lower than (+)-2R-LP2 and (+)-2S-LP2 and with a significant σ2R/ opioid receptors/σ1R selectivity ratio, we then moved to test the analgesic eff compounds 7, 8, and 9 in the mouse formalin test.
As shown in Figure 4, administration of (+)-2R/S-LP2 (7) at a dose of 5 mg/kg before formalin injection by the i.p. route did not alter the severity of the first p pain. The second phase, instead, was significantly decreased, as evidenced by a re in both the number of flinches and the lifting/licking time as compared with treated mice. (+)-2R-LP2 (8) effect was not significative. The same result was obtain the isomer (+)-2S-LP2 (9). It is well established that σ1R is involved in nociception, and the effects reported with σ1R ligands are consistent with a role of this receptor in pain hypersensitivity [37]. Thus, considering the results obtained in the radioligand binding assay with a (+)-2R/S-LP2 K i value two times lower than (+)-2R-LP2 and (+)-2S-LP2 and with a significant σ2R/σ1R and opioid receptors/σ1R selectivity ratio, we then moved to test the analgesic efficacy of compounds 7, 8, and 9 in the mouse formalin test.
As shown in Figure 4, administration of (+)-2R/S-LP2 (7)   It is well established that σ1R is involved in nociception, and the effects σ1R ligands are consistent with a role of this receptor in pain hypersensitiv considering the results obtained in the radioligand binding assay with a (+ value two times lower than (+)-2R-LP2 and (+)-2S-LP2 and with a significan opioid receptors/σ1R selectivity ratio, we then moved to test the analge compounds 7, 8, and 9 in the mouse formalin test.
As shown in Figure 4, administration of (+)-2R/S-LP2 (7)   In inflammatory pain, spinal cord neuronal sensitization occurs duri phase of the formalin test. As NMDA receptor activation plays an imp (+)-2R/S-LP2 reduced nociceptive behaviour in the mouse formalin test. Intraperitoneal administration of (+)-2R/S-LP2 (5 mg/kg) was able to decrease nociceptive behaviour only in the second phase of the mouse formalin test, while intraperitoneal administration of (+)-2R-LP2 (5 mg/kg) and (+)-2S-LP2 (5 mg/kg) did not differ significantly from the vehicle. Results are expressed as mean ± SEM (n = 6-8 mice per group) and are represented as the percentage of inhibition of pain responses of the respective vehicle group (100%). * p < 0.05 vs. Vehicle, two-way ANOVA followed by Bonferroni post-hoc test.
In inflammatory pain, spinal cord neuronal sensitization occurs during the second phase of the formalin test. As NMDA receptor activation plays an important role in formalin-induced pain the obtained results apparently support the hypothesis that σ1R facilitates the expression of formalin-induced pain, through their known ability to modulate NMDA-mediated responses [38].
Molecules 2022, 27, x FOR PEER REVIEW formalin-induced pain the obtained results apparently support the hypothesis t facilitates the expression of formalin-induced pain, through their known ab modulate NMDA-mediated responses [38].

General Remarks
All commercial chemicals were purchased from Merck (Darmstadt, Germa were used without further purification. (±)-cis-N-normetazocine was obtaine Fabbrica Italiana Sintetici. Melting points were determined in open capillary tube Büchi 530 apparatus and are uncorrected. Analytical TLC was performed on silic F254 aluminium sheets (Merck) with a fluorescent indicator. Components were vis by UV light (λ = 254 nm) and iodine vapour. Flash column chromatography was out on Merck silica gel 60 (230-400 mesh). Optical rotations were determined in solution with a Perkin-Elmer 241 polarimeter. 1 H and 13 C NMR spectra were ro recorded on a Varian Inova-200 spectrometer in CDCl3 solution; chemical shif expressed in ppm with reference to tetramethylsilane as an internal standard. El analyses (C, H, and N) were performed on a Carlo Erba 1106 analyzer and the results were within ± 0.4% of the theoretical values. All reported compounds had of at least 95%.

General Remarks
All commercial chemicals were purchased from Merck (Darmstadt, Germany) and were used without further purification. (±)-cis-N-normetazocine was obtained from Fabbrica Italiana Sintetici. Melting points were determined in open capillary tubes with a Büchi 530 apparatus and are uncorrected. Analytical TLC was performed on silica gel 60 F 254 aluminium sheets (Merck) with a fluorescent indicator. Components were visualized by UV light (λ = 254 nm) and iodine vapour. Flash column chromatography was carried out on Merck silica gel 60 (230-400 mesh). Optical rotations were determined in MeOH solution with a Perkin-Elmer 241 polarimeter. 1 H and 13 C NMR spectra were routinely recorded on a Varian Inova-200 spectrometer in CDCl 3 solution; chemical shifts δ are expressed in ppm with reference to tetramethylsilane as an internal standard. Elemental analyses (C, H, and N) were performed on a Carlo Erba 1106 analyzer and the analysis results were within ± 0.4% of the theoretical values. All reported compounds had a purity of at least 95%.

General Procedure for the Synthesis of Compounds 7−9
A mixture of (+)-cis-N-normetazocine (1.63 mmol, 1 eq), the appropriate tosylate intermediates (1.63 mmol, 1 eq), NaHCO 3 (2.45 mmol, 1,5 eq), and a catalytic amount of KI was stirred in DMF at 65 • C for 24 h. After cooling, the reaction mixture was filtered and concentrated under vacuum to remove DMF. The resulting residue was purified by flash chromatography on a silica gel column using CH 2 Cl 2 /C 2 H 5 OH (97:3 v/v) as an eluent to yield the final compounds (7)(8)(9). The free bases were converted into hydrochloride salts by dissolution in a minimum amount of THF and adding 1N HCl in Et 2 O to the solution.

Radioligand Binding Assays Animals
Brain and liver homogenates for σ1R and σ2R binding assays were prepared from male Dunkin-Hartley guinea pigs and Sprague-Dawley rats, respectively (ENVIGO RMS S.R.L., Udine, Italy). Animals (200-250 g) were euthanized with CO 2 in a euthanasia chamber and sacrificed by decapitation. Guinea pig brains without cerebellum (~2.5 g each) and rat livers (~7 g each) were kept on dry ice and stored at −80 • C. Brains for MOR, DOR, and KOR binding assays were explanted from male Sprague-Dawley rats and Dunkin-Hartley guinea pigs (Italian Minister of Health project code 335/1984F.N.JLT; ENVIGO RMS S.R.L., Udine, Italy). Animals (200-250 g guinea pigs) were euthanized with CO 2 in a euthanasia chamber and sacrificed by decapitation.  35.5 Ci/mmol) were purchased from PerkinElmer (Zaventem, Belgium). Ultima Gold MV Scintillation cocktail was from PerkinElmer (Milan, Italy). All the other materials were obtained from Merck Life Science S.r.l. (Milan, Italy). The test compound solutions were prepared by dissolving approximately 10 µmol of the test compound in DMSO so that a 10 mM stock solution was obtained. The required test concentrations for the assay (from 10 −5 to 10 −11 M) have been prepared by diluting the DMSO stock solution with the respective assay buffer. All experiments were performed using ultrapure water obtained with a Millipore Milli-Q Reference Ultrapure Water Purification System. All the laboratory glassware was first washed with a 6 M HCl water solution and then rinsed with ultrapure water.

Preparation of Membrane Homogenates from Pig Brain
Fresh guinea pig brain cortices (~25 g) were homogenized in two portions with 10 volumes of ice-cold Tris (50 mM, pH 7.4) containing 0.32 M sucrose with a Potter-Elvehjem glass homogenizer. The suspension was centrifuged at 1030× g for 10 min at 4 • C. The supernatant was separated and centrifuged at 41,200× g for 20 min at 4 • C. The obtained pellet was suspended with 3 volumes of ice-cold Tris (50 mM, pH 7.4), incubated at RT for 15 min, and centrifuged at 41,200× g for 15 min at 4 • C. The final pellet was resuspended with~2 volumes of ice-cold Tris buffer, and frozen at −80 • C in~1 mL portions containing about 5 mg protein/mL [40,41].

Preparation of Membrane Homogenates from Rat Liver
Rat livers (~21 g) were cut into small pieces with a scalpel and homogenized in two portions with 6 volumes of cold 0.32 M sucrose with a Potter-Elvehjem glass homogenizer. The suspension was centrifuged at 1030× g for 10 min at 4 • C. The supernatant was separated and centrifuged at 31,100× g for 20 min at 4 • C. The pellet was resuspended with 6 volumes of ice-cold Tris buffer (50 mM, pH 8) and incubated at rt for 30 min. Then, the suspension was centrifuged at 31,100× g for 20 min at 4 • C. The final pellet was resuspended with 6 volumes of ice-cold Tris buffer and stored at −80 • C in~1 mL portions containing about 6 mg of protein/mL [40,41].

Protein Determination
The protein concentration was determined by Bradford's method. The Bradford solution was prepared by dissolving 10 mg of Coomassie Brilliant Blue G 250 in 5 mL of 95% ethanol. To this solution, 10 mL of 85% phosphoric acid were added, and the mixture was stirred and filled to a total volume of 100 mL with ultrapure water. The calibration was carried out with bovine serum albumin as a standard at different concentrations. In a 96well plate, 30 µL of the calibration solution or 30 µL of the membrane receptor preparation were mixed with 240 µL of the Bradford solution, respectively. After 5 min of incubation at rt, the UV absorbance was measured at λ = 595 nm using a microplate spectrophotometer reader (Synergy HT, BioTek).

σ1. R Ligand Binding Assays
In vitro σ 1 R ligand binding assays were carried out in a Tris buffer (50 mM, pH 7.4) for 150 min at 37 • C. The thawed membrane preparation of guinea pig brain cortex (250 µg/sample) was incubated with increasing concentrations of test compounds and [ 3 H] (+)-pentazocine (2 nM) in a final volume of 0.5 mL. The K d value of [ 3 H] (+)-pentazocine was 2.9 nM. Unlabelled (+)-pentazocine (10 µM) was used to measure non-specific binding. Bound and free radioligand were separated by fast filtration under reduced pressure using a Millipore filter apparatus through Whatman GF/6 glass fiber filters, which were pre-soaked in a 0.5% poly (ethyleneimine) water solution for 120 min. Each filter paper was rinsed three times with 3 mL of ice-cold Tris buffer (50 mM, pH 7.4), dried at rt, and incubated overnight with 3 mL of scintillation cocktail into pony vials. The bound radioactivity has been determined using a liquid scintillation counter (Beckman LS 6500).
3.4.6. σ2. R Ligand Binding Assays In vitro σ 2 R ligand binding assays were carried out in Tris buffer (50 mM, pH 8.0) for 120 min at rt. The thawed membrane preparation of rat liver (250 µg/sample) was incubated with increasing concentrations of test compounds and [ 3 H]DTG (2 nM) in the presence of (+)-pentazocine (5 µM) as σ 1 R masking agent in a final volume of 0.5 mL. The K d value of [ 3 H]DTG was 17.9 nM. Non-specific binding was evaluated with unlabeled DTG (10 µM). Bound and free radioligand were separated by fast filtration under reduced pressure using a Millipore filter apparatus through Whatman GF/6 glass fiber filters, which were presoaked in a 0.5% poly(ethyleneimine) water solution for 120 min. Each filter paper was rinsed three times with 3 mL of ice-cold Tris buffer (10 mM, pH 8), dried at rt, and incubated overnight with 3 mL of scintillation cocktail into pony vials. The bound radioactivity has been determined using a liquid scintillation counter (Beckman LS 6500).

Data Analysis
The K i -values were calculated with the program GraphPad Prism ® 5.0 (GraphPad Software, San Diego, CA, USA). The K i -values are given as the mean value ± SD from at least two independent experiments performed in duplicate. The bound radioactivity has been determined using a Beckman LS 6500 liquid scintillation counter (Beckman Coulter, Brea, CA, USA).

Preparation of Membrane Homogenates from Sprague-Dawley Rat Brains for MOR and DOR Binding Assays or Guinea Pig Brains for KOR Binding Assays
Sprague-Dawley rat brains (for MOR and DOR binding assays) or guinea pig brains (for KOR binding assays) were homogenized in ice-cold Tris buffer (50 mM, pH 7.4) by using a Dounce glass homogenizer (Wheaton, Millville, NJ, USA) with a loose inner tolerance pestle first and a tight inner tolerance pestle later in a cylindrical glass tube of 40 mL volume. The suspension was centrifuged at 40,000× g for 20 min at 4 • C (Beckmann J2-20 centrifuge and a JA-21 rotor). The pellet was resuspended in ice-cold Tris buffer and then incubated at 37 • C for 30 min to remove endogenous ligands. After incubation, the suspension was centrifuged at 40,000× g for 20 min at 4 • C and the final pellet was resuspended in ice-cold Tris buffer and frozen at −80 • C in~1 mL portions containing about 10 mg protein/mL.

Protein Determination
The protein concentration was determined by the Bradford method. The calibration curve was built with bovine serum albumin as the standard compound at 7 different concentrations, ranging from 60 µg/mL to 210 µg/mL with blank correction. In a 96-well plate, 30 µL of the calibration solution or 30 µL of the membrane receptor preparation were mixed with 240 µL of the Bradford solution (10 mg of Coomassie Brilliant Blue G 250 in 5 mL of 95% ethanol, 10 mL of 85% phosphoric acid, and water up to 100 mL; with ultrapure water). After 5 min of incubation at RT, the UV absorbance was measured at λ = 595 nm using a microplate spectrophotometer reader (Synergy HT, BioTek, Winooski, VT, USA). H]-U69,593 were 1.0, 1.5, and 2.3 nM, respectively. Nonspecific binding was assessed in the presence of 10 µM unlabelled naloxone. The reaction was terminated by filtering the solution under reduced pressure using a Millipore filter apparatus through Whatman glass fiber filters, GF/C for MOR and DOR and GF/B for KOR, presoaked for 1h in a 0.1% poly(ethyleneimine) solution. Filters were washed with 50 mM ice-cold Tris-HCl buffer (2 × 4 mL), dried at rt, soaked overnight in 4 mL of scintillation cocktail into 6 mL pony vials and counted on a liquid scintillation counter.

Data Analysis
Ki values were calculated using nonlinear regression analysis to fit a logistic equation to the competition data using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).
3.6. In Vivo Pharmacology 3.6.1. Animals Experiments were performed on male CD1 mice (Envigo Laboratories, Indianapolis, IN, USA) aged between 8 and 12 weeks. Mice were housed in 5 animals per cage under a 12/12 h light/dark cycle at a constant temperature (23-25 • C) with free access to food and water, and were allowed to acclimate for at least one week upon arrival before starting all experiments, which were conducted between 9:00 a.m. and 3:00 p.m. This study was executed according to the European Communities Council directive and Italian regulation (EEC Council 2010/63/EU and Italian D.Lgs. no. 26/2014) to replace, reduce, and refine the use of laboratory animals. All procedures were approved by the ethical committee of the University of Catania (OPBA) and by the Italian Ministry of Health (authorization n • 385/2021-PR).

Behavioral Experiment
Before starting every experiment, mice were randomly assigned to each experimental group and allowed to acclimate in the room for 20 min. The results were analyzed by a researcher blind to the treatment procedure.

Drugs
Formalin and naloxone hydrochloride were purchased from Merck. PRE-84 synthesis was reported in the supporting information ( Figure S7). All compounds were dissolved in pyrogen-free isotonic saline and dimethyl sulfoxide, (DMSO 0.5% v/v). All LP compounds were administrated i.p. 20 min before formalin injection. Naloxone hydrochloride s.c. and PRE-084 hydrochloride s.c. were administered 20 min before the injection of the LP compounds.

Mouse Formalin Test
Formalin solution (5%, 10 µL) was administered subcutaneously into the plantar surface of the right hind paw (i.pl.), monitoring the nociceptive behavior, such as licking and flicking or shanking the injected paw, for 1 h and recording every 5 min, as previously reported [42].
The formalin injection induces a rapid pain response (phase I), which lasts about 5 min, due to direct nociceptors' activation, characterized by an acute form of pain. After a short quiescent period, another behavioral pain form (phase II) occurs, considered more important clinically, characterized by an inflammatory component and persistent pain due to nociceptive sensitization in the dorsal horn of the spinal cord [43].

Future Directions
Using in vitro and in vivo profiles of (+)-2R/S-LP2 (7), we are investigating its therapeutic potential in neuropathic pain, specifically in a rodent model of unilateral sciatic nerve chronic constriction injury (CCI). Nowadays, neuropathic pain represents a significant burden for patients due to long-term therapeutic regimens and severe side effects. Moreover, a mechanistic study on σ1R involvement in the maintenance of painful states is ongoing.