14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Received: 7 January 2021 Revised: 19 July 2021 Accepted: 23 July 2021 DOI: 10.1111/bph.15645 THEMED ISSUE ARTICLE Remote local photoactivation of morphine produces analgesia without opioid-related adverse effects  pez-Cano1,2 | Marc Lo Joan Font3,4 | Ester Aso1,2 | Kristoffer Sahlholm1,2,5,6 | 7 8,9,10 11,12 Gisela Cabré | Jesús Giraldo | Yves De Koninck | Jordi Hernando7 | Amadeu Llebaria3 | Víctor Fernández-Dueñas1,2 | Francisco Ciruela1,2 1 Pharmacology Unit, Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, Institute of Neurosciences, University of Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain 2 Neuropharmacology & Pain Group, Neuroscience Program, Bellvitge Institute for Biomedical Research, IDIBELL, L'Hospitalet de Llobregat, Barcelona, Spain 3 MCS, Laboratory of Medicinal Chemistry, Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain 4 Institut de Génomique Fonctionnelle (IGF), University of Montpellier, CNRS, INSERM, Montpellier, France 5 Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 6 Department of Integrative Medical Biology, Umeå University, Umeå, Sweden 7 Departament de Química, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, Spain 8 Laboratory of Molecular Neuropharmacology and Bioinformatics, Unitat de Bioestadística and Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain 9  i Innovacio Unitat de Neurociència Traslacional, Parc Taulí Hospital Universitari, Institut d'Investigacio  Parc Taulí (I3PT), Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain 10 n Biomédica en Red de Salud Mental, CIBERSAM, Barcelona, Spain Instituto de Salud Carlos III, Centro de Investigacio 11 Institut Universitaire en Santé Mentale de Québec, Québec, Quebec, Canada 12 Department of Psychiatry and Neuroscience, Université Laval, Québec, Quebec, Canada Correspondence Francisco Ciruela and Víctor Fernández- Background and Purpose: Opioid-based drugs are the gold standard medicines for Dueñas, Pharmacology Unit, Department of pain relief. However, tolerance and several side effects (i.e. constipation and depen- Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, dence) may occur upon chronic opioid administration. Photopharmacology is a prom- Institute of Neurosciences, University of ising approach to improve the benefit/risk profiles of these drugs. Thus, opioids can Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain. be locally activated with high spatiotemporal resolution, potentially minimizing Email: fciruela@ub.edu; vfernandez@ub.edu systemic-mediated adverse effects. Here, we aimed at developing a morphine photo- Amadeu Llebaria, MCS, Laboratory of Medicinal Chemistry, Institute for Advanced derivative (photocaged morphine), which can be activated upon light irradiation both Chemistry of Catalonia (IQAC-CSIC), in vitro and in vivo. Barcelona, Spain. Email: amadeu.llebaria@iqac.csic.es Experimental Approach: Light-dependent activity of pc-morphine was assessed in cell-based assays (intracellular calcium accumulation and electrophysiology) and in mice (formalin animal model of pain). In addition, tolerance, constipation and depen- dence were investigated in vivo using experimental paradigms. Key results: In mice, pc-morphine was able to elicit antinociceptive effects, both using external light-irradiation (hind paw) and spinal cord implanted fibre-optics. In Abbreviations: CPP, conditioned place preference; DRG, dorsal rood ganglion; GIRK, G protein-coupled inwardly rectifying potassium channel; PC-morphine, photocaged-morphine. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. Br J Pharmacol. 2021;1–17. wileyonlinelibrary.com/journal/bph 1 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2 LÓPEZ-CANO ET AL. Funding information Instituto de Salud Carlos III, Grant/Award addition, remote morphine photoactivation was devoid of common systemic opioid- Number: PIE14/00034; Catalan government, related undesired effects, namely, constipation, tolerance to the analgesic effects, Grant/Award Numbers: 2017 SGR 465, 2017 SGR 1604; FEDER/Ministerio de Ciencia, rewarding effects and naloxone-induced withdrawal. Innovacion y Universidades-Agencia Estatal de n, Grant/Award Numbers: Conclusion and Implications: Light-dependent opioid-based drugs may allow effec- Investigacio CTQ2017-89222-R, SAF2017-87199-R, tive analgesia without the occurrence of tolerance or the associated and severe SAF2017-87349-R opioid-related undesired effects. KEYWORDS dependence, morphine, pain, photopharmacology, tolerance 1 | I N T RO DU CT I O N What is already known Pain is a multidimensional pathological condition that diminishes the quality of life of patients, interfering in their daily family and work • Prescribing opioid medications for pain relief exposes activities. Different epidemiological studies conclude that, at pre- people to the risk of opioid use disorders. sent, the prevalence of chronic pain is very high, with at least 20% • Safe, effective, and non-addictive strategies to manage of the population being affected (Blyth et al., 2004; Cousins & pain urge to be explored. Lynch, 2011). This is the reason why analgesics are among the most-consumed drugs (Urquhart, 2018). Importantly, the choice for What does this study add analgesic medications is currently based on the World Health Organization (WHO) analgesic three-step ladder (Vargas-Schaffer & • We apply a novel pharmacological approach based on the Cogan, 2014). This ladder classifies drugs according to their use of light controlling opioid activity, photocaged analgesic potency and suggests a progressive use (from less to more morphine. potent drugs) depending on the needs. On the top step of the • Photocaged morphine has high spatiotemporal resolution WHO ladder, as the most effective drugs for pain relief, we mainly and reduced side effects. find the so-called major opioids (morphine, methadone, hydromorphone, oxycodone, oxycodone/naloxone, fentanyl, What is the clinical significance buprenorphine and tapentadol) (Law & Loh, 2013; McDonald & Lambert, 2016). • This novel light-based approach may provide an optimal Major opioids exhibit important and severe side effects benefit/risk ratio for opioid-based therapies. (i.e. respiratory depression, constipation, dependence, hyperalgesia and neurotoxicity) and patients might develop tolerance to their anal- gesic effects, which can limit their use (Law & Loh, 2013; McNicol et al., 2003; Volkow & Thomas McLellan, 2016). These drugs have been classically held in reserve for a limited number of conditions Photopharmacology is a new discipline that allows for the temporal (i.e. cancer-related pain). In recent years, however, opioid use has and spatial remote control of ligand activity specifically in the target increased exponentially (Cheung et al., 2014; Nuckols et al., 2014). tissues of select pharmacological compounds (Lerch et al., 2016; Epidemiological data indicate that a more careful approach should be Velema et al., 2014). Recently, we developed a photosensitive nega- taken when using opioids regularly (McNicol et al., 2003). In tive allosteric modulator of the metabotropic glutamate type the ongoing opioid epidemic in the United States, more than 5 receptor (mGlu5 receptor), which elicited analgesic effects upon 100 people die every day from opioid overdosing (Kolodny peripheral and central irradiation (Font et al., 2017). Based on this et al., 2015). In line with this, 30% of patients prescribed with experience, here we aimed to design, synthesize and characterize a opioids do not use them properly, and 10% of patients develop photosensitive morphine derivative allowing the local light-dependent dependence (Kolodny et al., 2015). release of morphine to minimize the appearance of analgesic toler- Altogether, it seems clear that finding novel strategies to reduce ance and opioid-related side-effects. Indeed, we managed to photo- tolerance, which counteracts analgesic efficacy and drives dose esca- deliver morphine to the spinal cord by means of flexible fibre-optics lation, and opioid-related side effects should be a major goal in anal- (Bonin et al., 2016), which could represent a highly translational strat- gesic research. One of the most exciting approaches to reduce egy. Overall, the novel light-based approach presented here may rep- off-target effects mediated by systemic drug administration resent a proof of concept model for the safe use of opioid drugs in is photopharmacology (Lerch et al., 2016; Velema et al., 2014). clinical settings. 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 3 2 | METHODS as mobile phase. The fluorescent-yellow oil was solved in 7 ml of ether, and HCl in dioxane (4N) was added dropwise. The precipitate 2.1 | Drug synthesis was collected by filtration and washed several times with ether, to afford the title compound (215 mg, 47%) as a yellow solid. This com- All the chemicals and solvents were provided from commercial sup- pound is light sensitive and should be maintained and manipulated pliers and used without purification, except the anhydrous solvents, protected from illumination. Mp: 301–303 C. 1H-NMR (400 MHz, which were treated previously through a system of solvent purifica- DMSO-d6) δ 7.57 (d, J = 9.0 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.71 tion (PureSolv), degasified with inert gases and dried over alumina or (dd, J = 9.1, 2.5 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 6.55 (d, J = 2.5 Hz, molecular sieves (dimethyl formamide). 1H), 6.14 (s, 1H), 5.73–5.66 (m, 1H), 5.33 (s, 2H), 5.30–5.26 (m, 1H), 4.18 (dq, J = 5.7, 2.7 Hz, 1H), 4.12 (d, J = 6.9 Hz, 1H), 3.57 (s, 3H), 3.44 (q, 4H), 3.30–3.17 (m, 2H), 3.08 (s, 1H), 3.02 (d, J = 5.0 Hz, 1H), 2.1.1 | Synthesis of 4-(bromomethyl)- 2.85 (d, J = 4.7 Hz, 2H), 2.81–2.72 (m, 1H), 2.36–2.26 (m, 1H), 1.19 7-(diethylamino)-2H-chromen-2-one (t, J = 7.1 Hz, 6H). 13C-NMR (101 MHz, DMSO-d6) δ 160.76, 155.78, 151.57, 147.67, 139.92, 134.89, 129.81, 125.78, 125.38, 125.09, A solution of 7-(diethylamino)-4-(hydroxymethyl)coumarin (DEACM) 119.34, 116.79, 105.63, 91.08, 66.73, 65.84, 59.33, 46.14, 44.10, (358 mg, 1.45 mmol) and triethylamine (0.40 ml, 2.9 mmol) in DCM 42.47, 41.36, 40.40, 37.52, 32.45, 21.05, 12.29. HPLC-PDA-MS (12 ml) was cooled to 0 C, methanesulfonyl chloride (0.17 ml, (using method A) RT: 2.59 min, λmax = 214, 246, 317, 389 nm; purity 2.1 mmol) was added dropwise, and the reaction mixture was stirred > 98% (254 nm). HRMS (m/z): [M + H]+ calcd. For C31H34N2O5, 2 h at 0 C. The mixture was quenched with cold saturated NaHCO3 515.2546; found, 515.2537. (50 ml), and DCM (20 ml) was added. The organic layer was sepa- rated, washed with brine (2  50 ml), dried over Na2SO4, filtered off and concentrated under reduced pressure. Afterwards, the crude 2.2 | Photochemical characterization was solved in THF (12 ml), and LiBr (503 mg, 5.8 mmol) was added. The reaction mixture was stirred for 2.5 h. The mixture was concen- To investigate the uncaging process of photocaged-morphine a 7 μM trated, DCM (30 ml) was added, washed with brine (2  30 ml), solution of this compound in phosphate-buffered saline (PBS):DMSO dried over Na2SO4 and the solvent removed under reduced pres- 99:1 was irradiated at 405 nm and 0.56 Wcm2 using a cw laser sure. The crude was purified through flash silica column chromatog- (MDL-E-405, Scitec Instruments Ltd., Wiltshire, UK). The changes in raphy using DCM/AcOEt 99:1 as mobile phase, affording the title UV–vis absorption were monitored in time using an HP 8453 spec- compound (140) (200 mg, 44%) as a yellow solid (Seven trophotometer (Agilent Technologies, Inc., Colorado Springs, CO, et al., 2014). 1 H-NMR (400 MHz, Chloroform-d) δ 7.49 (d, USA). An aliquot of the irradiated sample after 120 min was analysed J = 9.0 Hz, 1H), 6.62 (dd, J = 9.0, 2.6 Hz, 1H), 6.51 (d, J = 2.6 Hz, by MS (ESIMS) in a micrOTOF-Q spectrometer (Bruker Corporation, 1H), 6.13 (s, 1H), 4.40 (d, J = 0.7 Hz, 2H), 3.42 (q, J = 7.1 Hz, 4H), Billerica, MA, USA) (Figure S1). Control experiments were conducted 1.21 (t, J = 7.1 Hz, 6H). by irradiating morphine and DEACM (Indofine Chemical Co., Hillsborough, NJ) solutions at the same conditions. From the UV–vis absorption measurements, the photouncaging quantum yield of 2.1.2 | Synthesis of 7-(diethylamino)- photocaged-morphine was determined, as previously described 4-((((4R,7S,12bS)-7-hydroxy-3-methyl-2,3,4,4a,7,7a- (Lees, 1996), with the photoisomerization process of the closed hexahydro-1H-4,12-methanobenzofuro[3,2-e] state of 1,2-bis(5-chloro-2-methyl-3-thienyl)perfluorocyclopentene isoquinolin-9-yl)oxy)methyl)-2H-chromen-2-one in hexane at 405 nm as a reference (Φiso = 0.13) (Higashiguchi hydrochloride (photocaged-morphine). et al., 2005). To a solution of (4R,7S,12bS)-3-methyl-2,3,4,4a,7,7a-hexahydro- 1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7,9-diolhydrochloride 2.3 | Cell culture (morphine hydrochloride) (250 mg, 0.87 mmol) in DMF (7 ml) was added K2CO3 (266 mg, 1.93 mmol), and the mixture was HEK-293T cells (RRID:CVCL_0063), permanently expressing μ-opioid stirred 15 min. After this time, a solution of 4-(bromomethyl)- receptors were used (kindly provided by Laboratorios Dr. Esteve, Bar- 7-(diethylamino)-2H-chromen-2-one (299 mg, 0.96 mmol) in DMF celona, Spain). Cells were grown by seeding them in a 96-well plate (2 ml) was added to the reaction mixture and stirred 4 days at room (10,000 cells per well) using DMEM (Sigma-Aldrich, St. Louis, MO, temperature in the dark. Afterwards, AcOEt (50 ml) was added. Then, USA) supplemented with 100 Uml1 penicillin (Biowest, Nuaillé, the mixture was washed with a saturated solution of NaHCO3 France), 100 mgml1 streptomycin (Biowest), 10% v/v FBS (3  50 ml), brine (3  50 ml), dried over Na2SO4, filtered off and the (Invitrogen, Carlsbad, CA, USA), 1 mM pyruvic acid (Biowest), non- solvent removed under vacuum. The brown crude solid was purified essential amino acids (Biowest) and 2 mM L-glutamine (Biowest) in the through flash silica column chromatography using DCM/MeOH 94:6 presence of 0.1 mgm1 geneticin (InvivoGen, Tolouse, France). Cells 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 LÓPEZ-CANO ET AL. were kept at 5% CO2, 37 C, and 95% humidity conditions. Cells were 2.6 | Electrophysiology tested for mycoplasma content; thus, only mycoplasma-free cells were used. HEK-293T cells stably expressing μ receptors were cultured as described above. Cells were seeded into six-well plates (300,000 cells per well) and transiently transfected the following day with cDNA 2.4 | Dorsal rood ganglion (DRG) isolation and (in μg per well) encoding G protein-coupled inwardly rectifying primary culture potassium channel type 1 (Kir3.1;GIRK1)-YFP (1.5) and Kir3.2 (GIRK2) (1.5) using polyethylenimine (PEI, linear, 25 kDa; Polysciences Europe DRG from adult male and female CD-1 mice was extracted as previ- GmbH, Hirschberg an der Bergstrasse, Germany) (Longo et al., 2013). ously described (Tulleuda et al., 2011). In brief, animals were killed by Twenty-four hours after transfection, cells were lifted off using cervical dislocation preceded by anaesthesia before the thoracic cav- Versene (Gibco), diluted 1/5 and re-seeded onto 18-mm glass cover- ity was exposed, and the viscera removed to access to the vertebras slips (VWR International Eurolab, Llinars del Vallès, Spain) coated with before performing a laminectomy. Sensory ganglia (containing DRGs) poly-L-ornithine (Sigma–Aldrich), and kept in culture for additional 2– were dissected, cleaned and maintained in ice-cold Mg2+/Ca2+ free- 4 days prior to experiments. Subsequently, coverslips were mounted PBS (Sigma-Aldrich) supplemented with glucose (10 mM), HEPES in an Attofluor holder and placed at an inverted Axio Observer micro- (10 mM), penicillin (100 UIml1) and streptomycin (100 μgml1) until scope (Zeiss, Oberkochen, Germany) equipped with a 63 dissociation. Subsequently, sensory ganglia were chemically dissoci- oil-immersion objective. A Polychrome V (Till Photonics GmbH, ated in 2 ml of HAM's F-12 (Biowest) containing collagenase type IA Gräfelfing, Germany) was used as light source for fluorescence excita- 1 1 (1 mgml ; Sigma-Aldrich) and BSA (1 mgml ; Sigma-Aldrich) for tion. The extracellular high-potassium buffer contained (in mM): 1 h and 45 min at 37 C. Afterwards, ganglia were centrifuged at 113 NaCl, 0.34 Na2HPO4, 25 KCl, 0.44 KH2PO4, 0.5 MgCl2, 0.4 100 g for 5 min. The pellet was resuspended with 1 ml of trypLE Mg2SO4, 1.26 CaCl2, 10 HEPES, 2 D-glucose and 1 ascorbic acid; express (Gibco, Carlsbad, CA, USA) in supplemented PBS for 15 min pH 7.4 with NaOH. at 37 C. Ganglia were collected by centrifugation at 100 g for 5 min Single cells were selected for recording based on their plasma and resuspended in 2 ml of supplemented DMEM. Finally, ganglia membrane expression of the tagged GIRK1-YFP protein, as judged were mechanically disaggregated by forcing them to pass through a by their fluorescence (Figure 3c). Cells selected for recording were fire-polished Pasteur pipette until no cell aggregates were observed. voltage-clamped in the whole-cell configuration using an Axopatch DRG neurons were plated onto poly-L-lysine and laminin-coated 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). A 96-well plates with supplemented Dulbecco's Modified Eagle Medium Digidata 1440 analogue/digital converter (Molecular Devices) was (DMEM). used for interfacing the amplifier with a personal computer. Patch micropipettes were pulled from borosilicate glass capillaries (GC120F-10, Harvard Apparatus, Edenbridge, UK) to have a resis- 2.5 | Intracellular calcium determinations tance of 4–10 MΩ when filled with intracellular solution [in mM; 10 NaCl, 120 KCl, 5 MgCl2, 1 EGTA, 5 HEPES, 5 ATP, 0.2 GTP Intracellular calcium determinations were performed using Fluo-4 NW (pH 7.2 with NaOH)]. Cells were voltage-clamped at 80 mV, and Calcium Assay Kit (ThermoFisher Scientific, Waltham, MA, USA) as test compounds were applied using a pressure-driven, computer- previously described (Font et al., 2017). In brief, HEK-293T cells per- controlled perfusion system (Octaflow; ALA Scientific Instruments manently expressing μ receptors or DRGs neurons grown in 96-well Inc., Westbury, NY, USA). Increases in inward current were used as black plates were incubated with Fluo4 NW following manufacturer's readout of μ receptor activation. During each recording, cells were indications. Subsequently, cells were treated with vehicle (HBSS), first exposed to a saturating concentration (10 μM) of morphine, in morphine or photocaged-morphine maintained in dark or previously order to evoke a control response against which the subsequent irradiated for 15 min at 405 nm (1 Hz frequency, 500 ms pulses and responses could be normalized. Increasing concentrations (10 nM 2000 mA intensity LED and 23 mW output power). Fluo4 NW emis- to 10 μM) of morphine, or the uncaged (1 Hz frequency, 500 ms sion (i.e. 535 nm) was recorded in real-time upon 485 nm excitation in pulses and 2000 mA intensity LED and 23 mW output power) or a POLARstar Omega multi-mode microplate reader (BMG Labtech caged derivative (pc-Mor), were then applied consecutively, each GmbH, Ortenberg, Germany). The results were expressed as the per- application lasting 20 s. Experiments were carried out at room centage of μ receptor activation induced by treatments following the temperature. equation: h  i Ca2þ accumulation ð%Þ ¼ AUCveh  AUCdrug =AUCveh  100 2.7 | Animals Adult male and female CD-1 mice (RRID:MGI:2686808; animal facil- where AUCveh and AUCdrug represent the AUC value in the vehicle- ity of University of Barcelona) weighing 20–25 g were used and and drug-treated conditions, respectively. randomly assigned to each experimental group. The University of 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 5 Barcelona Committee on Animal Use and Care approved the proto- 2.9 | Formalin test col (number: 10034). Following the approved experimental protocol, all animals were supervised daily to assess signs of adverse effects The formalin animal model of pain was performed as previously during treatment. A retrospective analysis of the protocol demon- pez-Cano et al., 2019). In brief, mice (n = 5–6 per group) described (Lo strated that no corrective measures (i.e. use of analgesics) were were administered intraperitoneally (i.p.) with vehicle, morphine or needed. Animals were housed and tested in compliance with the photocaged-morphine 35 min before a diluted formalin solution (20 μl guidelines provided by the Guide for the Care and Use of Labora- of 2.5% formalin/0.92% formaldehyde; Sigma-Aldrich) was tory Animals (Clark et al., 1997) and following the European Union intraplantarly (i.pl.) injected in the mid-plantar surface of the right hind directives (2010/63/EU). Mice were housed in groups of five in paw of the mouse. The formalin-induced nociceptive behaviour in standard cages with ad libitum access to food and water and light or mock manipulated (dark) conditions was quantified as the time maintained under a non-reversed 12 h dark/light cycle (starting light spent licking or biting the injected paw during the 35 min after the period at 7:30 AM), 22 C temperature, and 66% humidity (standard injection of formalin. The initial acute phase (0–5 min; phase I), was conditions). All animal experimentation was carried out in a period followed by a relatively short quiescent period (15 min), which was comprehended between 9:00 AM to 6:00 PM by a researcher blind then followed by a prolonged response (20–35 min; phase II). For the to drug treatments. Animal studies are reported in compliance with peripheral photocaged-morphineuncaging, the hind paw was directly the ARRIVE guidelines (Percie du Sert et al., 2020) and with the rec- irradiated with a LED-based fibre-optic system (Doric Lenses Inc.) at ommendations made by the British Journal of Pharmacology (Lilley 405 nm light (or dark) for 15 min before the recording of each phase. et al., 2020). The present interval was selected according to preliminary pilot experiments (Figure S2) showing non-optimal photo-uncaging reliable processes for periods less than 15 min. We did not test larger irradia- 2.8 | Epidural fibre-optic implantation tion times (i.e. more than 15 min), since phase II licking/biting determi- nations in the formalin injected paw would have been interfered Both epidural fibre-optics production and implantation surgery were (Figure 5a). Nevertheless, a 15-min irradiation of photocaged- performed as previously described (Bonin et al., 2016). In brief, a morphine produced a significant antinociceptive effect in both forma- length of multimode plastic fibre (240 μm core, 250 μm diameter with lin test phases (Figure S2). To uncage photocaged-morphine at the cladding, 0.63 NA) modified with a diffusive tip to enable mul- lumbar segment (L4–L6) of the spinal cord, mice were irradiated tidirectional diffusion of light from the fibre was used to manufacture through the epidural implanted flexible fibre-optic (Doric Lenses Inc.) the epidural fibre-optic implant (MMF_POF_240/250-0.63_8cm_DLF; with 405 nm light (or dark) for 15 min before the recording of each Doric Lenses, Quebec, Canada). The fibre was fitted with a stainless phase. The 405 nm light pulses lasted 500 ms each and were adminis- ferrule (2.5 mm diameter, 270 μm bore; Thor Labs, Bergkirchen, tered at 1 Hz frequency with 23 mW output power and 2000 mA Germany) using epoxy blue-dye (Fosco, Pleasanton, CA, USA). The intensity. Antinociception induced by the different treatments was length of the fibre from the end of the ferrule to the diffusive tip was calculated with the equation: 40 mm for 20–25 g adult CD-1 mice. A base of dental cement (Agnthos, Lidingö, Sweden) was added to the ferrule prior to implanta- Antinociceptive effect ð%Þ ¼ ½ðLTV – LTDÞ=LTV  100 tion to facilitate fixation on the skull of the mouse. Mice were anaesthetized with a combination of ketamine/xylacine (dose of where LTV and LTD represent the licking/biting time in the vehicle- 100 mgkg1 body weight for ketamine and 10 mgkg1 for xylacine) and drug-treated animals, respectively. animals, spontaneously breathing, were keep on a heated blanket before the epidural fibre implantation surgery was performed as described (Bonin et al., 2016). Thus, the fibre-optic was carefully 2.10 | Gastrointestinal transit inserted through the atlanto-occipital membrane and under the first vertebrae (C1), immediately rostral to C1, with the head held at an Gastrointestinal transit (GIT) studies were performed to assess the acute, downward angle. The correct positioning of the fibre tip near constipation level caused by opioid treatment. Briefly, 6 h prior to L1 (corresponding to spinal segments L4-L6) was confirmed by con- the gastrointestinal transit experimental procedure, mice were individ- necting the fibre to the LED-based light source and observing the illu- ually housed and fasted. Food was restricted but animal had free minated region on the back. Finally, once the fibre was correctly access to water for the entire study. Afterwards, mice were positioned, the dental cement base of the fibre was fixed to the skull intragastrically administered with 0.25 ml of a suspension of 10% with Loctite 454 Prism instant adhesive and the incision sutured. The vegetable charcoal Norit A® (Sigma-Aldrich) in 5% gum acacia fibre-optic implanted mice were let to recover from anaesthesia and (Sigma-Aldrich) plus vehicle, morphine (10 mgkg1, i.p.) or supervised to ensure lack of paralysis for 1 week. After recovery, ani- photocaged-morphine(10 mgkg1, i.p.). Immediately, the hind paw mals looked healthy. However, mice presenting any movement abnor- was directly irradiated with a LED-based fibre-optic system (Doric mality (less than 10% of total number surgeries performed) were Lenses Inc.) at 405 nm light or mock manipulated (dark). The 405 nm automatically excluded. light regime consisted of pulses that lasted 500 ms each and were 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 LÓPEZ-CANO ET AL. administered at 1 Hz frequency with 23 mW output power and confined into the drug-paired conditioning compartment for 30 min. 2000 mA intensity for 15 min. Twenty minutes later, mice were killed, During the evening sessions of the conditioning phase (days 2 to 4) and the stomach and small intestine dissected to collect the intestinal (Figure 8a), separated at least 6 h from the morning sessions, mice segment between the stomach and the ileocecal junction. The received vehicle followed by direct light-irradiation of the hind paw distance from the pyloric sphincter to the ileocecal junction was con- with a LED-based fibre-optic system at 405 nm light (500 ms pulses, sidered to reflect the whole length of the small intestine. The distance 1 Hz frequency, 2000 mA intensity and 23 mW output power) or from the pylorus to the frontier of activated charcoal was measured mock manipulated (dark) for 15 min and then were immediately con- as the migration distance. The gastrointestinal transit (GIT) rate was fined into the vehicle-paired conditioning compartment for 30 min. calculated using the following formula: Thus, a total of three pairings were carried out with vehicle, morphine or photocaged-morphine(drug-paired compartment) and three GIT rate ð%Þ ¼ ½MAC=LSI  100 pairings with vehicle (vehicle-paired compartment). Treatments were counterbalanced as closely as possible between compartments. where MAC and LSI represent the migration distance of activated Finally, the post-conditioning phase (day 5, Figure 8a) was conducted charcoal and the whole length of small intestine, respectively. exactly as the preconditioning phase, allowing mice to freely explore both compartments for 18 min. The time spent in each compartment was recorded. The CPP score was calculated as follows:- CPP score 2.11 | Opioid tolerance (s) = (Time spent in the drug-paired compartment during post-condi- tioning phase)  (Time spent in the drug-paired compartment during Opioid tolerance was evaluated by performing the formalin test after pre-conditioning phase). chronic morphine treatment. Briefly, animals were administered with vehicle, morphine (10 mgkg1, i.p.) or photocaged-morphine (10 mgkg1, i.p.) twice a day (within a 12 h interval) for 5 days. After 2.13 | Naloxone-precipitated morphine withdrawal each administration, the hind paw was directly irradiated for 15 min with a LED-based fibre-optic system (Doric Lenses Inc.) at 405 nm The naloxone-precipitated withdrawal syndrome in morphine- light or mock manipulated (dark). The 405 nm light regime consisted dependent mice was evaluated based on a modified protocol previ- of pulses that lasted 500 ms each and were administered at 1 Hz fre- ously described (Maldonado et al., 1997). Briefly, animals were quency with 23 mW output power and 2000 mA intensity. After administered with vehicle, morphine (10 mgkg1, i.p.) or photocaged- chronic treatment, the formalin animal model of pain was performed morphine(10 mgkg1, i.p.) twice a day (within a 12 h interval) for as described above. 5 days, except for the last day when only the morning dose was administered. Immediately after each drug administration, the hind paw was directly irradiated with a LED-based fibre-optic system 2.12 | Morphine-induced conditioned place (Doric Lenses Inc.) at 405 nm light or mock manipulated (dark) for preference 15 min. The 405 nm light regime consisted of pulses that lasted 500 ms each and were administered at 1 Hz frequency with 23 mW The rewarding effects of morphine were evaluated using the condi- output power and 2000 mA intensity. Afterwards, withdrawal was tioned place preference (CPP) paradigm, which was performed based precipitated by injecting naloxone (1 mgkg1, s.c.) 2 h after the last on a modified protocol previously described (Maldonado et al., 1997). morphine administration. Animals were placed individually into obser- The apparatus consisted of two main square conditioning compart- vational test chambers to evaluate the behavioural signs of with- ments (15  15  15 cm), with differences in texture ground surface drawal 10 min before naloxone administration and for 30 min (i.e. rough and smooth) and different black and white pattern walls immediately after naloxone injection. The chambers consisted of (i.e. squared and zebra stripes patterns), separated by a triangular cen- transparent round glass containers (20 cm in diameter) above a dark tral area with sliding doors. The light intensity within the conditioning surface. Wet dog shakes, jumping, paw tremor and sniffing were chambers was 30 lux. The CPP test consisted of three phases, as indi- counted over 5-min periods. Teeth chattering, piloerection, ptosis, cated in Figure 8a. During the preconditioning phase (day 1), drug- diarrhoea and tremor were scored 1 for appearance or 0 for naive mice were placed in the middle of the central area with free nonappearance within each 5-min period. Locomotor activity over access to both compartments for 18 min (Figure 8a). The time spent 5-min periods was rated 0 (normal activity), 1 (low activity) or 2 (inac- in each compartment was recorded. During the morning sessions of tivity). Body weight was determined the first day and the last day the conditioning phase (days 2 to 4), mice received vehicle (saline), (before naloxone injection). Considering all the individual signs, a morphine (10 mgkg1, i.p.) or photocaged-morphine(10 mgkg1, i.p.) global withdrawal score was calculated for each animal, as previously followed by direct light-irradiation of the hind paw with a LED-based reported (Maldonado et al., 1992), by giving each individual sign a rel- fibre-optic system (Doric Lenses Inc.) at 405 nm light (500 ms pulses, ative weight: 0.5 for each episode of wet dog shake, jumping, paw 1 Hz frequency, 2000 mA intensity and 23 mW output power) or tremor or sniffing; and 1 for the presence of teeth chattering, mock manipulated (dark) for 15 min and then were immediately piloerection, ptosis, diarrhoea and tremor during each observation 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 7 period of 5 min. Locomotor activity score for each period was added PHARMACOLOGY http://www.guidetopharmacology.org and are to the global withdrawal score. permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019). 2.14 | Data and statistical analysis 3 | RE SU LT S The data and statistical analysis in this study comply with the rec- ommendations of the British Journal of Pharmacology on experi- 3.1 | Design and synthesis of photocaged mental design and analysis in pharmacology (Curtis et al., 2018). morphine Data are represented as mean ± standard error of mean (SEM) with statistical significance set at P < 0.05. The number of samples/ To generate a morphine-based photosensitive compound (photo- animals (n) in each experimental condition is indicated in the caged-morphine), we applied a caging strategy based on the chemical corresponding figure legend. Statistical analysis was undertaken binding of morphine to a photoremovable coumarine (i.e. coumarinyl only for data sets where each group size was at least n = 5. Out- phototrigger, Figure 1). Thus, we synthesized photocaged-morphine liers were assessed by the Iterative Grubbs' test; no animals were by tethering one of the hydroxyl groups of morphine to a excluded. Comparisons among experimental groups were performed bromoderivative of the violet-light absorbing coumarin DEACM in by two- or three-way factor ANOVA followed by Tukey's multiple a one-pot procedure. comparisons post hoc test using GraphPad Prism 9 (RRID: Next, we evaluated the UV–visible absorption spectrum of the SCR_002798; San Diego, CA, USA), as indicated. newly synthesized compound. As shown in Figure 2a, photocaged- morphine showed a maximum absorption peak at 394 nm, resem- bling that of the coumarin unit, thus in agreement with the lack of 2.15 | Materials absorption in the visible region of morphine. Subsequently, we assessed the photocaged-morphine photochemical behaviour upon The commercial suppliers for chemical were Acefe, Gavà, Spain; 405 nm light irradiation. Under these experimental conditions, spec- Panreac Química, Castellar del Vallès, Spain; and Fisher Scientific, tral changes consistent with the photolysis of the coumarin ben- Madrid, Spain. All the other suppliers are alreaded provided. zylic bond and release of the appended morphine fragment were observed (Figure 2b) (Wong et al., 2017). This was further ascertained by MS analysis after photocaged-morphine photolysis 2.16 | Nomenclature of targets and ligands (Figure S1). A quantum yield of Φchem = 0.004 was determined for the uncaging process of photocaged-morphine, which is in Key protein targets and ligands in this article are reasonable agreement with the photochemical behaviour hyperlinked to corresponding entries in the IUPHAR/BPS Guide to reported for other biologically-relevant hydroxyl groups caged with F I G U R E 1 Design and synthesis of photocaged morphine. The synthesis of photocaged-morphine (pc-Mor) from morphine involves a one-pot procedure using morphine and 7-(diethylamino)-4-(hydroxymethyl)coumarin (DEACM, 128b). Conditions: (a) NEt3, MsCl, LiBr, DCM, THF, r.t., 3 h, 44%. (b) K2CO3, DMF, r.t., 4 days, 47% (see Section 2) 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 LÓPEZ-CANO ET AL. F I G U R E 2 Photochemical properties of photocaged-morphine (pc-MOR). (a) Absorption spectra of pc-Mor and its separate morphine and 7-(diethylamino)-4-(hydroxymethyl)coumarin (DEACM; coumarin)) units in PBS:DMSO 99:1 (c = 7.0 μM). For comparison purposes, the maximum of the spectra is normalized to unity. (b) Variation of the absorption spectrum of pc-Mor in PBS:DMSO 99:1 (c = 7.0 μM) upon continuous excitation at 405 nm (0.56 Wcm2; irradiation time = 0, 1, 2, 3, 5, 7, 10.5, 15, 20, 30, 40, 60, 90 and 120 min) 7-diethylamino-4-hydroxymethylcoumarin through an ether linkage rectifying current (pEC50 = 6.90 ± 0.12) (Figure 3e), as expected (Wong et al., 2017). (Johnson et al., 2006). Again, photocaged-morphine was able to induce a concentration-dependent GIRK-mediated current only following irradiation with 405 nm light (pEC50 = 6.53 ± 0.11) 3.2 | Optical control of μ-opioid receptor in (Figure 3c), thus validating the caging approach to photocontrol the cultured cells and primary neurons intrinsic activity of this μ receptor caged agonist. Finally, we performed intracellular calcium accumulation experi- To assess photocaged-morphine-mediated photocontrol of μ recep- ments in cultured neurons from mouse dorsal root ganglion (DRG; tors, we first evaluated intracellular calcium accumulation (Hauser Figure 4a), to assess the light-dependent activity of photocaged- et al., 1996) in stable MOR-expressing HEK cells. While morphine morphine in a native system. Similar to that observed in stable μ (100 nM) induced a robust intracellular calcium rise both in dark receptor-expressing HEK cells, while morphine was able to induce a and under 405 nm illumination, photocaged-morphine (100 nM) significant increase in calcium levels both in dark and under 405 nm only produced intracellular calcium accumulation upon irradiation illumination (Figure 4b,c), photocaged-morphine only elicited intracel- with 405 nm light (Figure 3a). In line with this, morphine lular calcium accumulation upon irradiation with 405 nm light concentration-dependently increased intracellular calcium levels, (Figure 4b,c). Analysis (Treatment  Illumination) revealed a significant both in dark and light conditions (pEC50 = 8.21 ± 0.18 and main effect of treatment, illumination and the interaction between pEC50 = 8.01 ± 0.21, respectively) (Figure 3b). Conversely, both factors was also highly significant between dark and 405 nm photocaged-morphine only elicited a concentration-dependent illumination conditions in photocaged-morphine treated DRGs increase in calcium accumulation upon irradiation with 405 nm light (Figure 4c). Of note, we also aimed at recording electrophysiology (Figure 3b), with a similar potency to that observed for morphine responses to morphine and photocaged-morphinein cultured DRGs (pEC50 = 7.90 ± 0.25). Next, we assessed light-dependent μ but were unable to register consistent electrophysiological responses receptor-mediated GIRK activation. We co-transfected GIRK-1 and (Figure S3). These results could be due to the fact that DRGs are GIRK-2 subunits in stable μ receptor-expressing HEK cells and per- known to form a rather heterogeneous population, with only small formed voltage-clamp experiments. Cells were selected for record- subsets expressing GIRK channel subunits and μ receptors, respec- ing based on their plasma membrane expression of GIRK1-YFP tively, at the mRNA level (Usoskin et al., 2015). Nevertheless, calcium (Figure 3c), voltage-clamped at 80 mV and the morphine and recordings, which allowed to assess μ receptor-dependent activity in photocaged-morphine were applied while the inward current was the whole cell population, confirmed the ability of photocontrolling monitored (Figure 3d). Under these experimental conditions, mor- the intrinsic activity of photocaged-morphine both in heterologous phine evoked a concentration-dependent GIRK-mediated inwardly and endogenous systems. 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 9 F I G U R E 3 Optical modulation of μ receptor activity in living cells. (a) Determination of morphine (MOR)-mediated intracellular calcium mobilization in HEK-293T cells stably expressing MOR. Representative time course of intracellular calcium mobilization in cells incubated with vehicle (Veh), morphine (MOR, 100 nM) or photocaged-morphine (pc-MOR, 100 nM) while monitoring intracellular calcium accumulation in dark (left panel) or upon irradiation at 405 nm (right panel). (b) Concentration-response photo-modulation of MOR-mediated calcium accumulation. The AUC (AUC) of intracellular calcium accumulation in HEK-293T cells stably expressing MOR treated with morphine (circle) or pc-MOR (square) in dark (black) or irradiated at 405 nm (violet) was quantified. All the values are expressed as percentage of the corresponding ionomycin signal (mean ± SEM; n = 5). (c) Representative image of GIRK-YFP expression in HEK-293T cells stably expressing MOR and transiently transfected with GIRK1-YFP and GIRK2. Cells were analysed by fluorescence microscopy. Scale bar: 10 μm. (d) Representative GIRK current recording from HEK-293T cells stably expressing MOR and transiently transfected with GIRK1-YFP and GIRK2. Cells were voltage-clamped at 80 mV, superfused with high-potassium buffer, and exposed to 1 μM of morphine (MOR), pc-MOR or pc-MOR irradiated at 405 nm. (e) Concentration- response curves of evoked GIRK currents. Increasing concentrations of morphine (circle) or pc-MOR (square) in dark (black) or irradiated at 405 nm (violet) were superfused to cells and the increase in inward current was taken as a measure of MOR activation. Results were plotted as a fraction of the maximal response to 10 μM morphine in each cell (mean ± SEM; n = 5) 3.3 | Photocontrolling morphine-mediated model of pain, since it allows for the assessment of both peripheral antinociception and central receptors within the pain neuraxis (Mogil, 2009). We investigated light-dependent photocaged-morphine antinociceptive One of the main goals of developing a morphine-based photo- effects by irradiating peripheral (hind paw) and central (spinal cord) tis- pharmacology strategy was to achieve pain management with sues (Figures 5 and 6, respectively). First, following the protocol indi- high spatiotemporal resolution. We first assessed the ability of cated in Figure 5a, we administered vehicle, morphine or photocaged- photocaged-morphine to elicit light-dependent antinociception in an morphine and assessed its antinociceptive effects in dark and light animal model of pain. We took advantage of the formalin mouse conditions upon peripheral (hind paw) 405 nm irradiation (Figure 5a, 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 10 LÓPEZ-CANO ET AL. F I G U R E 4 Optical modulation of endogenous μ receptor activity in primary DRG neurons. (a) Brightfield image of a primary mouse DRG neuron at 1 day in vitro. Scale bar: 10 μm. (b) Determination of morphine-mediated intracellular calcium accumulation in primary DRG neurons. Representative time course of intracellular calcium mobilization in neurons incubated with vehicle (Veh), morphine (MOR, 100 nM) or photocaged morphine (pc-MOR, 100 nM) while monitoring intracellular calcium accumulation in dark (left panel) or upon irradiation at 405 nm (right panel). (c) Quantification of photo-modulation of MOR-mediated calcium accumulation. The AUC (AUC) of intracellular calcium accumulation in DRG neurons treated with vehicle (Veh), morphine (MOR, 100 nM) or photocaged morphine (pc-MOR, 100 nM) in dark (black) or upon irradiation at 405 nm (violet) was quantified. All the values are expressed as percentage of the corresponding ionomycin signal (mean ± SEM; n = 5). *P < 0.05, two-way ANOVA with Tukey's post hoc test F I G U R E 5 Optical control of peripheral μ receptors in the formalin animal model of pain. (a) Upper part: Scheme of the 405 nm irradiation regime (violet rectangles) and licking recordings (grey rectangles – Phase I and phase II) in the formalin animal model of pain. Thus, animals were intraperitoneally injected with vehicle (Veh, saline), morphine (Mor, 10 mgkg1) or photocaged-morphine (pc-Mor, 10 mgkg1) 20 min before irradiation at 405 nm or mock manipulated (dark) for 15 min. Lower part: Graphical scheme showing the LED-mediated irradiation of the hind paw. (b) Peripheral light-dependent pc-Mor-mediated antinociception in mice was assessed upon irradiation of the hind paw (see Section 2). Total hind paw licking was measured for 0–5 min (phase I) and 15–30 min (phase II) after intraplantar injection of 20 μl of formalin solution (2.5% paraformaldehyde). As a control of peripheral pc-Mor uncaging, the contralateral hind paw (CT) was also irradiated. The antinociceptive effect was calculated as the percentage of the maximum possible effect (see Section 2) and expressed as mean ± SEM (n = 6). *P < 0.05 two-way ANOVA with Tukey's post hoc test  pez-Cano et al., 2019), forma- lower panel). As previously reported (Lo confirmed a significant main effect of treatment on illumination and lin injection induced an innate defensive licking/biting behaviour, the interaction between both factors in phase I and II. Thus, systemic which was not modified upon vehicle injection, neither in dark nor in morphine administration (10 mgkg1, i.p.) produced significant anti- light conditions (Figure 5b). Analysis of treatment  illumination nociceptive effects, both in phases I and II of the formalin test, and 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 11 F I G U R E 6 Optical control of central μ receptors MORs in the formalin animal model of pain. (a) Graphical scheme showing the irradiation of the lumbar region using a LED-based epidural fibre-optic implant. (b) Central light-dependent pc-Mor-mediated antinociception in mice was assessed upon irradiation of the spinal cord (see Section 2). Total hind paw licking was measured for 0–5 min (phase I) and 15–30 min (phase II) after intraplantar injection of 20 μl of formalin solution (2.5% paraformaldehyde). The antinociceptive effect was calculated as the percentage of the maximum possible effect (see Section 2) and expressed as mean ± SEM (n = 6). *P < 0.05, two-way ANOVA with Tukey's post hoc test regardless of light irradiation (Figure 5b). Of note, while morphine was surgery and were submitted to the irradiation protocol either they able to completely abolish formalin pro-nociceptive effects at phase II, were treated with photocaged-morphine or not. There was a signifi- it only partially reduced nociception at phase I (Figure 5b). These cant main effect of treatment in both phase I and II and with illumina- results indicate that morphine was more effective at relieving central tion also with the interaction between both factors. Again, morphine sensitization than acute peripheral pain. On the other hand, when (10 mgkg1, i.p.) was able to exert antinociceptive effects both at administering photocaged-morphine(10 mgkg1, i.p.) in dark condi- phases I and II, regardless of light irradiation (Figure 6b). Interestingly, tions we did not observe a significant antinociceptive effect, com- photocaged-morphine (10 mgkg1, i.p.) was only able to suppress pared with vehicle treated animals (Figure 5b). Importantly, while formalin-induced nocifensive behaviour when light was delivered to photocaged-morphine was unable to suppress nociceptive responses the epidural anatomical space (Figure 6b). Thus, while photocaged- in dark conditions, it did elicit significant antinociceptive effects after morphine did not display antinociceptive effects in dark conditions, it direct hind paw irradiation, both in phase I and II (Figure 5b). This significantly inhibited nociception both in phase I (62.1 ± 15.1%) and result confirmed that, after systemic administration, photocaged- in phase II (72.6 ± 14.2%) upon direct spinal cord illumination morphine was peripherally photo-uncaged and induced anti- and confirmed with a Tukey's post hoc test. Epidural uncaged nociceptive effects. Subsequent analysis revealed that the light- photocaged-morphine had comparable efficacy to systemic morphine dependent photocaged-morphine antinociceptive effects in phase II in phase II (72.6 ± 14.2% vs. 98.2 ± 2.8%). It seems likely that central were significantly lower than that observed for morphine (63.3 μ receptors contribute to a higher extent to block central sensitization ± 13.6% vs. 99.1 ± 1.5%), a difference likely explained by either the (i.e. phase II in the formalin animal model of pain). Overall, these limited local (non-central) effects of photocaged-morphine upon hind- results demonstrated that photocaged-morphine was effective in paw irradiation or the inability to photo-release all the drug systemi- mediating antinociception both upon peripheral and central irradia- cally administered. Importantly, photocaged-morphine did not display tion. In other words, our data support the notion that localized light antinociceptive effects when the contralateral hind paw (CT, non- irradiation targeting peripheral and spinal μ receptors might represent injected with formalin) was irradiated (Figure 5b). a valuable strategy for the treatment of pain-related diseases. Given the higher efficacy of morphine relieving central sensitiza- tion and aiming to develop a potential translational approach to photocontrol opioid-mediated effects, we next evaluated the anti- 3.4 | Reduction of tolerance and opioid-related nociceptive effects of photocaged-morphine after epidural irradiation side effects upon photocaged-morphine treatment (Figure 6a). Although the implantation of a flexible fibre-optic at the lumbar epidural anatomical area did not affect the nociceptive Opioid-based analgesic therapies may lead to tolerance to the analge- responses to formalin injection (Figure 6b), all animals underwent sic effects and a series of side effects (i.e. constipation, dependence 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12 LÓPEZ-CANO ET AL. and addiction) (Grim et al., 2020). Thus, one of the main aims of a et al., 1986). There was a significant effect of treatment but no signifi- morphine-based photopharmacology strategy may consist of cant effect of illumination or the interaction between both factors. attempting to minimize these undesired side effects. First, we Importantly, although there was significant inhibition of gastrointesti- assessed the putative tolerance to analgesic effects of chronic nal transit in morphine administered animals both in dark and upon photocaged-morphine treatment, as compared with that developed hind paw irradiation, photocaged-morphine did not produce a change by morphine. Mice were chronically administered with vehicle, mor- in gastrointestinal transit, neither in dark nor in light conditions phine or photocaged-morphine before the formalin-induced behav- (Figure 8a). These results may support the use of photocaged- iour was assessed. There was a significant main effect of treatment in morphine as a potentially effective way to achieve antinociception Phase I and II for illumination, drug regime, the interaction between while avoided the undesirable systemic off-target effects of chronic treatment and illumination and the interaction between treatment opioid use. and drug regime, but not between drug regime and illumination or It seems clear that tolerance and constipation are some of the treatment, illumination and drug regime in both phases of the formalin many factors that contribute to making long term opioid-based thera- test. As expected, chronic morphine administration led to the signifi- pies for pain both difficult and uncomfortable. However, based on epi- cant development of tolerance to its antinociceptive effects, after its demiological data from the last few years, the main issues concerning chronic administration at phase I and II both in light and dark condi- opioid-based treatments relate to the misuse and abuse of these kinds tions (Figure 7). Chronic photocaged-morphine administration did not of drugs (Koob, 2020). We aimed to determine whether systemic lead to tolerance as no significant differences in the antinociceptive administration and photoactivation of photocaged-morphine could effects between acute and chronic of photocaged-morphine treat- circumvent the development of morphine-mediated dependence. ment at phase I and II both in light and dark conditions were found First, we evaluated the rewarding effects of photocaged-morphine by (Figure 7). using a conditioned place preference (CPP) paradigm (Figure 8b), a Next, we evaluated the possibility that photocaged-morphine standard behavioural model used to study the rewarding and aversive induced constipation, one of the most unpleasant side effects of effects of drugs, including opiates, in rodents (Maldonado chronic opioid use and withdrawal cited by patients (Farmer et al., 1997). A significant effect of drug treatment was identified, et al., 2018). We examined gastrointestinal transit in mice receiving whereas there was no significant effect of illumination or the interac- systemic administration of vehicle, morphine, or photocaged-mor- tion between both factors was observed. However, subsequent phine. As shown in Figure 8a, morphine produced a marked light inde- Tukey's post hoc test revealed an expected significant increase in the pendent inhibition of gastrointestinal transit, as expected (Manara time spent in the drug-associated compartment upon repeated F I G U R E 7 Reduced opioid-induced tolerance upon morphine photocontrol. Animals were chronically (twice a day during 5 days) administered with vehicle (Veh, saline, i.p.), morphine (Mor, 10 mgkg1, i.p.) or photocaged morphine (pc-Mor, 10 mgkg1, i.p.) 20 min before hind paw light irradiation (see Section 2 and Figure 5a). Finally, on the last day of treatment, antinociception was determined and the results compared with that obtained upon acute treatment. The antinociceptive effect was calculated as the percentage of the maximum possible effect (see Section 2) and expressed as mean ± SEM (n = 6). *P < 0.05, three-way ANOVA with Tukey's post hoc test 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 13 F I G U R E 8 Pc-morphine induce minimal adverse side effects. (a) Gastrointestinal transit (GIT) assessment. Effect of vehicle (Veh), morphine (Mor, 10 mgkg1, i.p.) and photocaged-morphine (pc-Mor, 10 mgkg1, i.p.) in GIT. Results are represented as percentage of activated charcoal transit according to total length of intestine (see material and Methods) and expressed as mean ± SEM (n = 5). *P < 0.05, two-way ANOVA with Tukey's post hoc test. (b) Experimental schedule to determine morphine induced CPP in mice. The three experimental behavioural phases (i.e. preconditioning, conditioning and postconditioning) with the corresponding pairing treatment (i.e. drug-saline) per session (M, morning and E, evening) and day is indicated. (c) Morphine-induced CPP. Animals were treated with vehicle (Veh, saline), morphine (Mor, 10 mgkg1) or photocaged-morphine (pc-Mor, 10 mgkg1) following the pairing schedule shown in panel (b), as described in materials and Methods section. The CPP score was calculated and expressed as mean ± SEM (n = 5). *P < 0.05,two-way ANOVA with Tukey's post hoc test. (d) Naloxone- precipitated morphine withdrawal. Animals were chronically treated with vehicle (Veh, saline, i.p.), morphine (Mor, 10 mgkg1, i.p.) or photocaged-morphine (pc-Mor, 10 mgkg1, i.p.) 20 min before hind paw irradiation (see Section 2 and Figure 5a). After last drug administration, withdrawal was precipitated by administering naloxone (1 mgkg1, s.c.). the global withdrawal score was calculated considering all the physical signs (i.e. wet dog shakes, jumping, paw tremor, sniffing, teeth chattering, piloerection, ptosis, diarrhoea, tremor and/or decreased locomotor activity) and expressed as mean ± SEM (n = 5). *P < 0.05, two-way ANOVA with Tukey's post hoc test morphine administration, independently of irradiation (Figure 8c). withdrawal. Again, a significant effect of drug treatment was identi- Conversely, photocaged-morphine treatment did not induce drug- fied, whereas there was no significant effect of illumination or the mediated place preference, neither in dark nor in light conditions interaction between both factors was observed. No signs of (Figure 8c). Overall, these results indicated that photocaged-morphine withdrawal were observed in any group of mice during behavioural was devoid of rewarding properties, indicating that locally released observation before the administration of naloxone. Naloxone adminis- morphine would not mediate central effects responsible for opioid tration did not exert any relevant effect on vehicle-injected mice. dependence. Importantly, while in chronic morphine-treated mice naloxone injec- Finally, we evaluated the development of a withdrawal syndrome, tion precipitated a significant withdrawal syndrome manifested by the which is a severe opioid-related side-effect. Mice receiving daily drug presence of several somatic signs (i.e. wet dog shakes, paw tremor, (i.e. vehicle, morphine or photocaged-morphine) injections were sub- jumps, ptosis, decreased locomotor activity and diarrhoea), in mice sequently administered with naloxone (1 mgkg1) to precipitate chronically treated with photocaged-morphine no withdrawal 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 14 LÓPEZ-CANO ET AL. syndrome was observed (Figure 8d). Overall, our data provide promis- periphery and the CNS, thus this technology may allow to address ing indications concerning a potential novel opioid-based treatment, important questions in the field. in which analgesic effects could be achieved with reduced adverse Several issues should be considered when using light as a preci- effects and no analgesic tolerance. sion tool in pharmacology. The first, and more obvious issue, consists of the way to deliver light into the target tissue. As commented above, in some previous works, fibre-optics were implanted into the brain 4 | DISCUSSION (Font et al., 2017). Here, we took advantage of less-invasive proce- dures to administer light. Thus, we used external (hind paw) and inter- The use of opioids for pain relief involves major concerns, the nal (epidural) fibre-optics, previously implemented in pain control possible appearance of tolerance to their analgesic effects and (Bonin et al., 2016; Font et al., 2017). Another important issue when adverse side effects, such as constipation and dependence. In implementing photopharmacology is the wavelength used to achieve recent years, due to high prescription rates, opioid-related side drug photoactivation. Thus, most photoactivable compounds effects have become a major health problem (Volkow & Thomas require UV light irradiation that may readily photodamage biological McLellan, 2016). Here, we provide compelling evidence for a novel tissues (Patton et al., 1999) and display poor tissue penetration pharmacological approach, based on the use of light to control (Frangioni, 2003). Interestingly, a series of coumarin photocleavable drug activity and reach effective pain treatment without these protecting groups, as the one used in this study, which enable the opioid-related undesired effects. To our knowledge, photocaged- conditional release of biologically active ligands at 405 nm, have been morphine is the first caged morphine derivative that has shown developed (Klán et al., 2013) and implemented for in vitro and in vivo light-dependent analgesic effects, while it did not produce consti- use with non-toxic effects (Font et al., 2017; Taura et al., 2018). Fur- pation, tolerance or a naloxone-induced withdrawal, and had no thermore, novel drugs are currently being synthesized aiming to use rewarding effects when activated peripherally by light. The protocol red-shifted wavelengths, which display higher tissue penetration of light administration included the use of external and internal (Li et al., 2014; Wegener et al., 2017). LED-based irradiation, allowing for local photocaged-morphine acti- The use of light might allow for the circumvention of the impor- vation in the hind paw and the spinal cord, respectively. Accord- tant opioid-related drawbacks, mostly related to their abuse and/or ingly, this new photopharmacological approach may provide an misuse. Indeed, prescribed opioid analgesics can be abused by taking optimal benefit/risk ratio for opioid-based therapies, which can the medication at or above the recommended dosage via the intended propel its rapid translation into clinical settings. route of administration (Katz et al., 2011). In addition, opioid drugs Photopharmacology, or the ability of light to control the biological can be manipulated to be administered through alternative routes activity of drugs by changing their pharmacokinetic or pharmacody- (i.e. inhalation), which have been correlated with the increased effi- namic profile (Hüll et al., 2018), has the advantage of providing high cacy of these drugs and an increase likelihood to develop dependency spatial and temporal resolution to drug action and reaction. However, (Samaha & Robinson, 2005). Extended-release opioid formulations the usual need for implantation of fibre-optics in the brain may limit designed for around-the-clock management of severe pain are very its use in clinical settings. Our work is a first-line proof-of-concept attractive to abusers, since they contain a great amount of drug as study that may prompt the transition from preclinical to clinical light- compared with that found in immediate-release formulations (Kirsh based devices. However, it is important to note that further preclinical et al., 2012). To minimize the risk of manipulating prescribed drugs, work is still needed before implementing this photopharmacological novel opioid formulations have been designed (Alexander et al., 2014; approach into a more clinical context. Some of the potential limita- Lourenço et al., 2013). These formulations, termed as abuse-deterrent tions are (i) we used a single photocaged-morphine dose (10 mgkg1), formulations, include physical and chemical barriers to prevent manip- based on previous experience (Font et al., 2017). In addition, it would ulation of the formulation and extraction of the active ingredient, be valuable to assess morphine levels after photocaged-morphine combination with an antagonist, aversion technologies, use of new photo-uncaging, both at plasma and target tissues, to establish the molecular entities or prodrugs, and novel delivery systems exact dosage for each route of administration; (ii) we only used a sin- (Department of Health and Human Services Administration, 2015). gle paradigm of inflammatory pain (i.e. formalin mouse model). It The use of light in opioid-based therapies may not only represent a would be needed to assess the effectiveness of photocaged-morphine novel but also a superior abuse-deterrent formulation. in other animal models of pain and (iii) we determined a number of In conclusion, our results support the notion that light-based opi- undesired effects (i.e. constipation, dependence) but it would be oid drugs allow controlling analgesia in a time- and space-dependent important to assess one of the major concerns when using opioids, manner. In addition, the use of photo-caged compounds may minimize which is respiratory depression (McNicol et al., 2003). In addition, it the severe side effects associated with currently available opioid would be relevant to assess how the use of photo-caged drugs may drugs. influence on emotional aspects of pain, which indeed have limited the transition of a number of pain-related assays from animals to humans. ACKNOWLEDG MENTS On the other hand, the present photopharmacology approach is an n This work was supported by FEDER/Ministerio de Ciencia, Innovacio excellent tool to dissect the mechanism of action of opioids in the  n (SAF2017-87349-R, y Universidades-Agencia Estatal de Investigacio 14765381, 0, Downloaded from https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15645 by Spanish Cochrane National Provision (Ministerio de Sanidad), Wiley Online Library on [02/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LÓPEZ-CANO ET AL. 15 SAF2017-87199-R and CTQ2017-89222-R), ISCIII (PIE14/00034), Blyth, F. M., March, L. M., Brnabic, A. J. M., & Cousins, M. J. (2004). and the Catalan government (2017 SGR 1604 and 2017 SGR 465). Chronic pain and frequent use of health care. Pain, 111, 51–58. https://doi.org/10.1016/j.pain.2004.05.020 We thank Centres de Recerca de Catalunya (CERCA) Programme/ Bonin, R. P., Wang, F., Desrochers-Couture, M., Ga Secka, A., Generalitat de Catalunya for IDIBELL institutional support. We thank Boulanger, M. E., Côté, D. C., & De Koninck, Y. (2016). Epidural n from the Scientific and Techni- Esther Castaño and Benjamín Torrejo optogenetics for controlled analgesia. Mol. Pain, 12, 1–11. cal Services (SCT) group at the Bellvitge Campus of the University of Cheung, C. W., Qiu, Q., Choi, S. W., Moore, B., Goucke, R., & Irwin, M. (2014). Chronic opioid therapy for chronic non-cancer pain: Barcelona for their technical assistance. We thank Lourdes Muñoz, A review and comparison of treatment guidelines. Pain Physician, 17, Carme Serra and Juanlo Catena (SimChem, IQAC-CSIC, Barcelona) for 401–414. support in the synthesis of compounds. Xavier Gasull and Aida Clark, J. D., Gebhart, G. F., Gonder, J. C., Keeling, M. E., & Kohn, D. F. 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Experimental design and analy- A.L. conducted the chemical synthesis and analysed chemical data; sis and their reporting: Updated and simplified guidance for authors G.C. and J.H. performed the photochemical studies; K.S. conducted and peer reviewers. British Journal of Pharmacology, 175(7), 987–993. https://doi.org/10.1111/bph.14153 the electrophysiology experiments; Y.D.-K. designed epidural fibre- Department of Health and Human Services Administration, U.S.A. (2015). optic experiments. M.L.-C., V.F.-D., E.A. and F.C. wrote the paper. All Abuse-deterrent opioids: Evaluation and labeling. Guidance for Industry. listed authors contributed to data collection, analysed data and/or Farmer, A. D., Holt, C. B., Downes, T. J., Ruggeri, E., Del Vecchio, S., & De revised the paper. V.F.-D., E.A. and F.C. supervised the research. Giorgio, R. (2018). Pathophysiology, diagnosis, and management of opioid-induced constipation. 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