Propacetamol-Induced Injection Pain Is Associated with Activation of Transient Receptor Potential Vanilloid 1 Channels S

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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/07/25/jpet.116.233452.DC1

1521-0103/359/1/18–25$25.00 http://dx.doi.org/10.1124/jpet.116.233452 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:18–25, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

Propacetamol-Induced Injection Pain Is Associated with Activation of Transient Receptor Potential Vanilloid 1 Channels s

Florian Schillers, Esther Eberhardt, Andreas Leffler, and Mirjam Eberhardt Department of Anaesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany (F.S., A.L., M.E.); and Department of Anaesthesiology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany (E.E.) Received March 8, 2016; accepted July 22, 2016

ABSTRACT Downloaded from Propacetamol (PPCM) is a prodrug of paracetamol (PCM), were expressed in human embryonic kidney 293 cells and which was generated to increase water solubility of PCM for investigated by means of whole-cell patch clamp and ratio- intravenous delivery. PPCM is rapidly hydrolyzed by plasma metric calcium imaging. PPCM (but not PCM) activated esterases to PCM and diethylglycine and shares some struc- TRPV1, sensitized heat-induced currents, and caused an tural and metabolic properties with lidocaine. Although PPCM increase in intracellular calcium. In TRPA1-expressing cells is considered to be comparable to PCM regarding its analgesic however, both PPCM and PCM evoked calcium responses but

properties, injection pain is a common side effect described for failed to induce inward currents. Intracutaneous injection of jpet.aspetjournals.org PPCM but not PCM. Injection pain is a frequent and unpleasant PPCM, but not of PCM, in human volunteers induced an intense side effect of numerous drugs in clinical use, and previous and short-lasting pain and an increase in superficial blood flow, reports have indicated that the ligand gated ion channels indicating activation of nociceptive C fibers and subsequent transient receptor potential ankyrin 1 (TRPA1) and transient neuropeptide release. In conclusion, activation of human TRPV1 receptor potential vanilloid 1 (TRPV1) can mediate this effect by PPCM seems to be a relevant mechanism for induc- on sensory neurons. This study aimed to investigate molecular tion of pain upon intracutaneous injection and thus also for mechanisms by which PPCM, in contrast to PCM, causes pain reported as an adverse side effect upon intravenous injection pain. Therefore, human TRPV1 and TRPA1 receptors administration. at ASPET Journals on September 24, 2021

Introduction intravenous application of PPCM (Depré et al., 1992). In addition to pain and local irritation at the injection site, health Paracetamol (PCM; acetaminophen) is a popular analgesic care–associated cases of allergic reactions have been reported drug for light to medium pain. However, because of its poor for PPCM (Barbaud et al., 1995). solubility and stability in water-based solutions at physiologic Injection pain is a common problem causing discomfort and pH values, it is less suitable for intravenous application. distress during induction of general anesthesia with propofol Therefore, propacetamol (PPCM), a prodrug of PCM, was (Doenicke et al., 1996; Tan and Onsiong, 1998) or etomidate generated by esterification of PCM and the carboxylic acid and even after local infiltration with local anesthetics such as diethylglycine to improve solubility (see Fig. 1 for a compar- lidocaine. Many of these proalgesic substances were described ison of molecular structures) (Barbaud et al., 1995). PPCM to activate transient receptor potential (TRP) channels was effectively used to treat fever and pain in several expressed in nociceptive nerve endings, and transient receptor European countries when oral application was not appropri- potential vanilloid 1 (TRPV1) and transient receptor potential ate. Intravenous infusion leads to rapid hydrolysis of PPCM ankyrin 1 (TRPA1) currently seem to be the most relevant within 7 minutes by plasma esterases in PCM and diethylgly- candidates mediating activation and/or sensitization of sen- cine (Bannwarth et al., 1992). Although analgesic properties of sory neurons by both propofol and local anesthetics (Leffler PPCM are proven to be superior to placebo and comparable to et al., 2008, 2011; Matta et al., 2008). Both TRPV1 and TRPA1 the effects of PCM (McNicol et al., 2011), it has become evident nonselective cation channels are expressed in peripheral that injection pain occurs quite frequently (up to 39%) upon nerve endings of nociceptive C and Ad fibers and also along the peripheral axon and in central nerve terminals in the spinal cord. As polymodal receptors, TRPA1 and TRPV1 can This research was supported by intramural grants from Hannover Medical be activated by a multitude of different agonists related to School. dx.doi.org/10.1124/jpet.116.233452. pain, inflammation, and oxidative stress. Among several s This article has supplemental material available at jpet.aspetjournals.org. mechanisms resulting in activation, the modification of

ABBREVIATIONS: ANOVA, analysis of variance; BCTC, 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide; GSK1016790A, N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene- 2-carboxamide; HC030031, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; HEK, human embryonic kidney; HSD, honestly significant difference; hTRP, human transient receptor potential; PCM, paracetamol; PPCM, propacetamol; TRP, transient receptor potential; TRPA, transient receptor potential ankyrin; TRPV, transient receptor potential vanilloid.

18 Propacetamol Activates TRPV1 to Evoke Pain 19

(PAA, Pasching, Austria). Cells were cultured at 37°C and 5% CO2 for 24–48 hours before use for patch clamp or calcium imaging. Patch Clamp Experiments. Cells were examined by whole-cell patch clamp with an EPC10 USB HEKA amplifier (HEKA Elektronik, Lamprecht, Germany), low-passed at 1 kHz, and sampled at 2 kHz. Pipettes were pulled from borosilicate glass tubes (TW150F- 3; World Precision Instruments, Berlin, Germany) to give a resistance of 2–5MV. The external calcium-free solution included 140 mM NaCl,

5 mM KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, and 10 mM glucose (pH 7.4 was adjusted with tetramethylammonium hydroxide).

The internal solution contained 140 mM KCl, 2 mM MgCl2,5mM EGTA, and 10 mM HEPES (pH was adjusted with KOH). Cells were either held at 260 mV or currents evoked by 500-millisecond voltage ramps from 2100 to 1100 mV were measured. All experiments were performed at room temperature. Na1 currents were recorded from the ND7/23-hybrid cell line derived from neonatal rat spinal sensory neurons fused with mouse neuroblastoma cells (Wood et al., 1990). For these experiments, data

were sampled at 20 kHz and filtered at 5 kHz. The series resistance Downloaded from Fig. 1. Molecular structure of PCM and PPCM. Ester hydrolysis of the was compensated by 60%–70% and the capacitance artifacts were water-soluble prodrug PPCM yields PCM and diethylglycine. cancelled using Patchmaster software (HEKA Elektronik). Pipettes were pulled with a resistance of 1 to 2 MV and filled with internal

solution containing 140 mM CsF, 2 mM MgCl2, 5 mM EGTA, and N-terminal cysteines has been shown as the underlying 10 mM HEPES (pH was adjusted to 7.4 by KOH). The external mechanism of how reactive agents sensitize and activate these solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.2 mM ion channels (Hinman et al., 2006; Chuang and Lin, 2009). We CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4 was adjusted by jpet.aspetjournals.org noted that PPCM indeed shows some structural similarities tetramethylammonium hydroxide). Sodium currents were elicited by to lidocaine. Furthermore, PPCM is metabolized by cleavage depolarizing pulses to 210 mV at 0.1 or 10 Hz using 2120 mV as the of diethylglycine which is similar to ethylglycin generated holding potential. Fitmaster (HEKA Elektronik) and Origin 8.5.1 (OriginLab, Northampton, MA) software were used for data analysis. by hydrolysis of lidocaine. Therefore, we hypothesized that 21 activation of TRPV1 and/or TRPA1 may account for the Ratiometric [Ca ]i Measurements. Cells were stained by 3 mM Fura-2-AM and 0.01% pluronic for about 45 minutes. After injection pain caused by PPCM as well. We thus aimed to wash out to allow Fura-2-AM deesterification, coverslips were investigate whether activation of human (h)TRPV1 and/or mounted on an inverse microscope with a 20Â objective (Axio observer at ASPET Journals on September 24, 2021 hTRPA1 channels could account for injection pain caused by D1; Carl Zeiss, Jena, Germany). Cells were constantly superfused with intravenous PPCM application. We combined cellular meth- extracellular solution at room temperature containing 145 mM NaCl, ods including whole-cell patch clamp and calcium imaging. 5 mM KCl, 1.25 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM Furthermore, we wanted to indirectly explore activation of HEPES using a software-controlled seven-channel, gravity-driven, epidermal C fibers by PPCM and PCM in human volunteers by common-outlet superfusion system. Fura-2 was excited using a quantifying injection pain and flare reaction measured by microscope light source and an LEP filter wheel (Ludl Electronic laser Doppler imaging. Products Ltd., Hawthorne, NY) to switch between 340-nm and 380-nm wavelengths. Images were exposed for 40 and 20 milliseconds, respectively, and were acquired at a rate of 1 Hz with a charge-coupled device camera (Cool SNAP EZ; Photometrics, Tucson, AZ). Data were Materials and Methods recorded using VisiView 2.1.1 software (Visitron Systems GmbH, Chemicals. Chemicals were purchased and solved as follows. Puchheim, Germany). Background fluorescence was subtracted before Acetaminophen/PCM, GSK1016790A [N-((1S)-1-{[4-((2S)-2-{[(2,4- calculation of ratios. Either 0.3 mM capsaicin or 250 mM carvacrol was dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]- used to identify hTRPV1- or hTRPA1-expressing cells on a functional carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide], and level and 5 mM ionomycin was applied as a control at the end of each probenecid were from Sigma-Aldrich (Taufkirchen, Germany). TRPV1 experiment. Experiments were repeated at least five times and were blocker BCTC [4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)- performed on different days. Averaged results are reported as means 6 phenyl]-1-piperazinecarboxamide; 100 mM stock solution in ethanol] S.E.M. of area under the curve (D ratio F340/380 nm) for regions of and TRPA1 blocker HC030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6- interest adapted to the cells. Calculation of the area under the curve tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; 100 mM included 60 seconds from the beginning of PPCM/PCM application to stock solution in dimethylsulfoxide] were from Tocris (Bio-Techne, the maximum of calcium responses, and baseline calcium levels were Wiesbaden-Nordenstadt, Germany) and Propacetamol MolPort subtracted. (Riga, Latvia), respectively. PCM and PPCM solutions were prepared Psychophysics Studies. For studies with human volunteers, in external solution before each experiment. experimental procedures met the requirements of the Declaration of Cell Culture. Stable cell lines were used for experiments on Helsinki and were approved by the ethics committee of Hannover human TRPV1 and TRPA1 channels. The hTRPV1-expressing human Medical School (Hannover, Germany). The recommended dose to treat embryonic kidney (HEK) 293 cell line was a kind gift from Dr. Peter postoperative pain in adults is an intravenous infusion of 1 g PCM Zygmunt (Clinical Chemistry and Pharmacology, Department of usually applied in 100 ml isotonic saline solution, resulting in a Laboratory Medicine, Lund University Hospital, Lund, Sweden) and concentration of 66.2 mM (Sinatra et al., 2005). As an equivalent dose, the hTRPA1-expressing cell line was established in our laboratory (de 2 g PPCM is clinically used, resulting in 66.5 mM if applied in 100 ml la Roche et al., 2013). For some experiments, HEK 293T cells were isotonic saline infusion. Therefore, either 50 mM PCM or PPCM was transfected with plasmids of hTRPV1, hTRPV2, hTRPV4, and intradermally injected in a 75-ml volume into the volar forearm of the hTRPA1 (1 mg each) and cotransfected with enhanced green fluores- volunteer (n 5 7 each; three men and four women) in a double-blinded cent protein (0.25 mg) for patch clamp experiments using Nanofectin manner. Pain and itching were assessed on a numerical rating scale, 20 Schillers et al. ranging from 0 (no sensation) to 10 (maximum pain/itch imaginable) in responding to 0.3 mM capsaicin (n 5 475; Fig. 2, F and H, upper 15-second intervals for a period of 10 minutes. For quantitative panel and blue bar, respectively). Again, this PPCM-induced sensory testing, numerical ratings (0–10) to a 10-second heat stimulus effect could be abolished by the TRPV1-blocker BCTC (100 nM; of 47°C or to a 10-second cold stimulus of 0°C, applied by metal rods Fig. 2, F and H, lower panel and white bar, respectively) and (0.8 cm in diameter) heated by a water bath or cooled with ice, were 20 mM PCM did not evoke any increase in intracellular calcium compared before and after application of substances. The skin was in hRPV1-expressing cells (n 5 427, ANOVA F 5 457.5, tested for dynamic mechanical allodynia by stroking with a fine brush. [2, 1157] , , A laser Doppler imager (moorLDI2-IR; Moor, London, UK) was used to P 0.001; followed by HSD post hoc test, P 0.001each;Fig.2,G record changes in superficial blood flow. Two baseline scans of 0.6-mm and H, cyan bar). spatial resolution were taken, followed by seven scans every 2 minutes Both PPCM and PCM Induce Small Membrane 21 starting right after the injections and a final one after 20 minutes. The Currents and an Increase in [Ca ]i through TRPA1. In areas of superficial vasodilation were analyzed with moorLDI soft- hTRPA1-expressing cells responding to the TRPA1-agonist ware (version 5.3) and defined as pixels in which intensity exceeded carvacrol (250 mM), outwardly rectifying ramp currents were the mean of basal values plus two standard deviations. evoked by high concentrations ($10 mM) of PPCM (n 5 8; Fig. Statistical Analysis. For analysis of patch clamp data and intra- 3A). By contrast, 20 mM PPCM completely failed to induce individual quantitative sensory testing data from human volunteers, membrane currents in hTRPV2- and hTRPV4-expressing two groups were compared by a nonparametric Wilcoxon matched- HEK 293 cells (n 5 5 each; Supplemental Fig. 2, A and B). pairs test for n , 10. A comparison of more than two groups for calcium PPCM and PCM applied at a concentration of 20 mM to

imaging and psychophysics data was performed by analysis of Downloaded from variance (ANOVA) after honestly significant difference (HSD) post hTRPA1-expressing cells constantly held at 260 mV did not hoc tests as stated using Statistica 7 software (StatSoft, Tulsa, OK). lead to inward currents (n 5 5 and 6; Supplemental Fig. 2, C Differences were considered significant at P , 0.05. All data are and D). However, 50 mM PPCM evoked small inward currents displayed as means 6 S.E.M. through activation of hTRPA1 and these inward currents could reliably be inhibited by 50 mM of the TRPA1-blocker HC030031 (n 5 6, P , 0.05, Wilcoxon matched-pairs test; Fig.

Results jpet.aspetjournals.org 3B). Furthermore, 50 mM PCM evoked comparably small PPCM Activates Membrane Currents and Induces inward currents in hTRPA1-expressing cells (n 5 6; Fig. 3C). 21 an Increase in [Ca ]i through TRPV1. To investigate Using ratiometric calcium imaging, 20 mM PPCM induced whether PPCM exhibits hTRPV1-activating properties, HEK calcium responses in 86% of all cells also responding to 250 mM 293 cells expressing wild-type hTRPV1 were examined by carvacrol (n 5 218; Fig. 3, D and F, upper panel and blue bar, standard whole-cell voltage clamp experiments. Measuring respectively). According to the notion that this effect is ramp currents by 500-millisecond depolarizations from 2100 mediated by hTRPA1, these PPCM-induced responses were to 1100 mV, we applied PPCM at increasing concentrations completely blocked by HC030031 (50 mM, ANOVA F[1, 381] 5 at ASPET Journals on September 24, 2021 (1 mM to 10 mM). In hTRPV1-expressing cells, concentrations 408.0, P , 0.001; followed by HSD post hoc test, P , 0.001; Fig. of $100 mM activated membrane currents with a strong 3, D and F, lower panel and white bar, respectively). After outward rectification in all cells that also generated capsaicin washout of HC030031, we observed an immediate recovery of (1 mM)–induced currents (n 5 9; Fig. 2A). calcium signals in 82% of PPCM-responsive cells, indicating a PPCM applied at a 20-mM concentration to hTRPV1- sustained activation of hTRPA1 by PPCM. Similar to PPCM, expressing cells constantly held at 260 mV induced large 20 mM PCM induced increases in intracellular calcium in 92% inward currents that could be completely blocked by 100 nM of of hTRPA1-expressing HEK cells (n 5 299, Fig. 3, E and G, the TRPV1-blocker BCTC (n 5 8, P 5 0.01, Wilcoxon matched- upper panel and cyan bar, respectively). Again, this effect pairs test; Fig. 2B). The same concentration of PCM (20 mM) could be inhibited by HC030031 (ANOVA F[1, 465] 5 590.6, P , did not evoke any currents in hTRPV1-expressing HEK cells 0.001; followed by HSD post hoc test, P , 0.001; Fig. 3, E and (n 5 8; Fig. 2C). G, lower panel and white bar). Sensitization of TRPV1-mediated heat responses has been High Concentrations of PPCM Induce Tonic Block of described for many TRPV1 agonists such as capsaicin or Neuronal Voltage-Gated Sodium Channels. To test the lidocaine (Tominaga et al., 1998; Leffler et al., 2008). We hypothesis that PPCM might block voltage-gated sodium investigated heat-induced currents by repetitively applying channels because of structural similarities to lidocaine (Sup- 5-second ramps from 25°C to 45°C. Indeed, PPCM at 10 mM plemental Fig. 1), we investigated whether PPCM or PCM enhanced heat-induced currents from 3392 6 795 pA to 7313 6 inhibits Na1 currents in neuroblastoma ND7/23 cells known 661 pA (P 5 0.028, n 5 6; Fig. 2D). By contrast, PCM had no to functionally express tetrodotoxin-sensitive Na1 channels. effect on heat-induced inward currents (4999 6 1353 pA Cells were held at 2120 mV to examine the block of resting versus 4657 6 1138 pA, P 5 0.48, n 5 8; Fig. 2E). Conse- channels and currents were elicited by depolarizing pulses quently, the threshold for heat-induced currents was shifted to 210 mV. Tonic block by PPCM was determined by from 42 6 1°C to 37 6 2°C by 10 mM PPCM (P 5 0.028, n 5 6; application of 10 mM, 100 mM, 1 mM, and 10 mM PPCM Fig. 2D), whereas the temperature threshold of hTRPV1 or PCM, respectively. Only high concentrations of PPCM remained unchanged by the application of PCM (42 6 1°C (.10 mM) induced a substantial block of Na1 currents. Although versus 41 6 1°C, n 5 8, P 5 0.12, all Wilcoxon matched-pairs 10 mM PPCM reduced current amplitude to 66.4% 6 1.4% tests; Fig. 2E). (P 5 0.0002, n 5 6; Fig. 4A), 10 mM PCM decreased current To confirm the results obtained by patch clamp recordings, amplitude only to 93.3% 6 1.7% (P 5 0.002, n 5 7; Fig. 4B). we applied ratiometric calcium imaging to Fura-2–stained These data indicate that PPCM induces a stronger tonic block hTRPV1-expressing HEK 293 cells. PPCM was applied at a of Na1 currents compared with PPCM (P 5 0.0002, all 20-mM concentration for 40 seconds and evoked a robust increase ANOVA F[9, 55] 5 81.37; followed by HSD post hoc test, P , in intracellular calcium in 68% of hTRPV1-expressing cells also 0.0001; Fig. 4C). No use-dependent block of Na1 channels Propacetamol Activates TRPV1 to Evoke Pain 21 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

Fig. 2. PPCM activates hTRPV1. (A) PPCM evokes concentration-dependent ramp currents in HEK 293 cells expressing hTRPV1. (B and C) In representative experiments at a holding potential of 260 mV, 20 mM PPCM evokes large inward currents in hTRPV1-expressing cells that can be inhibited by BCTC (B) but 20 mM PCM does not induce inward currents (C). (D and E) PPCM (D) but not PCM (E) increases heat ramp–induced (25–45°C) inward currents and shifts temperature thresholds of evoked currents to lower temperatures (see red dotted lines). Bar diagrams show maximum of heat-induced inward currents (means 6 S.E.M.) and temperature needed to evoke those currents before and after treatment with PPCM (D) and PCM (E). *P = 0.028 (PPCM) and P . 0.05 (PCM), all Wilcoxon matched-pairs test (n = 6 and 8). (F) PPCM (20 mM) applied for 40 seconds evokes an increase in intracellular calcium in hTRPV1-expressing Fura-2–stained HEK 293 cells (n = 475, 68% of 0.3 mM capsaicin–responsive cells; upper panel). Responses are blocked by the TRPV1 antagonist BCTC (100 nM, n = 198; lower panel). (G) PCM (20 mM) does not induce increases in intracellular calcium in hTRPV1- expressing cells (n = 427). Responses are shown as means 6 S.E.M. (bold black trace and dashed lines) and calcium measurements from representative cells (thin gray traces). (H) Area under the curve of evoked calcium signals. PPCM responses (blue bar) are significantly reduced by the TRPV1 channel blocker BCTC (white bar) and no responses can be evoked by 20 mM PCM (cyan bar, ANOVA F[2, 1157] = 457.5, P , 0.001; followed by HSD post hoc test, P , 0.001 each). ***P , 0.001. AUC, area under the curve. could be observed at 10 mM PPCM or 10 mM PCM when widespread and sometimes patchy reddening of the skin. currents were activated at 10 Hz (Supplemental Fig. 3). Because infusion of PPCM (but not PCM) causes pain and Injection of PPCM Evokes Pain and an Increase in skin irritations in some patients, we hypothesized that in- Cutaneous Blood Flow in Humans. Activation of TRPV1 tracutaneous injection of PPCM might lead to pain and axon and TRPA1 in endings of nociceptive nerve fibers has been reflex flares in human skin. This was tested in human proposed to be responsible for the injection pain caused by volunteers (n 5 7; three men and four women) by a double- many clinically used drugs such as lidocaine and propofol. blinded intracutaneous injection of 75 ml PPCM or PCM at Depolarization of the membrane after TRP channel activation 50 mM (e.g., concentrations slightly below the concentrations gives rise to action potentials, which are propagated to the of infusions of PPCM and PCM used in clinical settings). central nervous system where they lead to the experience of Indeed, the injection of PPCM caused an immediate pain with pain. However, vasoactive neuropeptides, such as the potent a mean maximum pain rating of 6.3 6 0.7 (on a numerical vasodilator calcitonin gene-related peptide, are also released rating scale of 0–10). This intense pain rapidly declined over from the activated nerve endings and from collaterals of wide- approximately 5 minutes (Fig. 5A) and was described as branching peptidergic C fibers by antidromic action potential burning or stinging. Apart from the initial pain caused by propagation. This so-called “axon-reflex” flare leads to a the injection needle, application of PCM was not Furthermore, 22 Schillers et al. Downloaded from jpet.aspetjournals.org

Fig. 3. PPCM and PCM induce an increase in intracellular calcium by activating hTRPA1 but hardly evoke inward currents. (A) PPCM sensitizes ramp currents in hTRPA1-expressing HEK 293 cells but only at concentrations above 1 mM. (B) Representative experiments of 50 mM PPCM to evoke small inward currents in hTRPA1-expressing cells which can be inhibited by the TRPA1-blocker HC030331. Cells were held at 260 mV. (C) PCM (50 mM) similarly induces small inward currents. (D) In hTRPA1-expressing HEK 293 cells, 20 mM PPCM induces an increase in intracellular calcium in 86% of cells that also respond to carvacrol (250 mM, upper panel, n = 178). Responses are prevented by the TRPA1 blocker HC030031 (50 mM, n = 123) for the duration of application but reoccurr after washout of HC030031 (lower panel). (E) Similar to PPCM, 20 mM PCM evokes increases in intracellular

calcium in hTRPA1-expressing HEK cells (92%, n = 299; upper panel). These responses are completely inhibited by HC030031 (n = 168; lower panel). at ASPET Journals on September 24, 2021 Responses are shown as means 6 S.E.M. (bold black trace and dashed lines) and calcium measurements from representative cells (thin gray traces). (F and G) Area under the curve of evoked calcium signals. PPCM responses (blue bar) are significantly reduced by the TRPA1 channel blocker HC030031 (white bar) and so are PCM-evoked increases (cyan bar) (for PPCM: ANOVA F[1, 381] = 408.0, P , 0.001; for PCM: F[1, 465] = 590.6, P , 0.001; followed by HSD post hoc test, P , 0.001 each). ***P , 0.001. AUC, area under the curve. neither PPCM nor PCM caused itching within 15 minutes C fibers by means of pronounced PPCM-evoked pain and axon after the injection. No allodynia was reported by any subject, reflex erythema in healthy volunteers. and responses to noxious heat (10 seconds, 47°C) or cold Injection pain as a result of activation of TRP channels has (10 seconds, 0°C) did not change significantly after injection of been shown for propofol (Fischer et al., 2010), etomidate PPCM or PCM (n 5 7 each, P . 0.05, Wilcoxon matched-pairs (Matta et al., 2008), and lidocaine as well as for other local test; data not shown). In addition to injection pain, PPCM anesthetics (Leffler et al., 2008, 2011). Although TRPA1 is the induced a large axon-reflex erythema visualized by laser channel that seems to be predominantly activated by etomi- Doppler scanning of superficial blood flow of the forearm date and propofol (Matta et al., 2008), the latter at least also (Fig. 5B). By contrast, apart from the irritation caused by the activates TRPV1 after receptor sensitization in mouse dorsal injection needle, we could not detect any erythema after root ganglion neurons (Fischer et al., 2010). Local anesthetics 5 , injection of PCM (ANOVA F[9, 4] 10.37, P 0.02; followed activate TRPV1, and lidocaine induces a strong sensitization by HSD post hoc test, P # 0.04; Fig. 5, B and C; Supplemental to heat and capsaicin. The EC50 value for lidocaine to activate Fig. 3). rat TRPV1 was determined to be approximately 12 mM

(Leffler et al., 2008), and an EC50 value of 24 mM was subsequently found for the activation of rat TRPA1 (Leffler Discussion et al., 2011). Although TRPV2 and TRPV4 are expressed in TRPV1 and TRPA1 are coexpressed in a large population of sensory neurons, we could not detect a sensitizing or activat- nociceptive sensory neurons (Story et al., 2003). Our data ing effect by PPCM on these channels. Although activation of suggest that PPCM activates hTRPV1 more than hTRPA1 in TRP channels on nociceptive nerve endings induces pain, cellular models by means of patch clamp and calcium imaging. sensitization and activation of spinal TRPA1 receptors was PPCM (but not PCM) also sensitized heat-induced currents proposed to contribute to the analgesic effects of PCM and lowered the temperature threshold for heat-induced (Andersson et al., 2011). To induce this effect, however, PCM currents through hTRPV1. Therefore, it seems plausible that must be metabolized to the reactive metabolites N-acetyl-p- TRPV1 predominantly mediates activation of nociceptive C benzoquinoneimine and parabenzoquinone in the spinal cord. fibers likely to cause injection pain after PPCM administra- Several reactive molecules have been shown to modify N-terminal tion. Accordingly, we showed activation of human nociceptive cysteines of both TRPA1 and TRPV1, leading to channel Propacetamol Activates TRPV1 to Evoke Pain 23

Fig. 4. At high concentrations, PPCM blocks Na+ channels more potently than PCM. (A) Representative current traces displaying tonic block of Na+ currents by PPCM. Cells were held at 2120 mV and currents were activated by test pulses to 210 mV at 0.1 Hz. Increasing concentrations of PPCM were applied inducing block only at 10 mM. (B) Representative recording of PCM applied in increasing concentrations, barely affecting Na+ currents evoked by the same protocol in ND7 cells. (C) PPCM (10 mM) reduces current amplitude more than 10 mM PCM (66% versus 93%, n = 6to7,P , 0.001, ANOVA followed by HSD post hoc test). ***P , 0.001.

conformation changes and sometimes to sustained receptor Downloaded from activation (Hinman et al., 2006; Chuang and Lin, 2009; Eberhardt et al., 2014). Activation of TRP channels in the periphery can account for PPCM-induced injection pain. As a result of the rapid hydrolysis of PPCM, an additional analgesic effect caused by the activation of spinal TRP channels seems unlikely. jpet.aspetjournals.org However, once PPCM is metabolized to PCM and further to N-acetyl-p-benzoquinoneimine and parabenzoquinone, TRP channel–mediated effects might again account for the analgesic properties of PPCM. Although PCM induced only minimal membrane currents in TRPA1-expressing cells, it induced a robust increase in intracellular calcium. We can only specu- late on why this calcium influx does not translate into inward at ASPET Journals on September 24, 2021 currents, but a higher sensitivity of TRPA1 to agonists in calcium imaging compared with patch clamp has been observed repeatedly (Eberhardt et al., 2012; Andersson et al., 2015). One reason might be calcium itself, which has been Fig. 5. Injection of PPCM in human skin induces pain and axon-reflex described to sensitize and desensitize TRPA1 (Zurborg et al., erythema. (A) Magnitude and time course of 50 mM PPCM-evoked pain in 2007; Wang et al., 2008). Another issue could be sensitization human volunteers after intracutaneous injection to the volar forearm. Pain was rated on a numerical rating scale from 0 to 10. PCM (50 mM) was of TRPA1 by UV light, which might be the case using Fura-2 used as a control and did not induce pain (n = 7). (B) Laser Doppler (Hill and Schaefer, 2009). Nevertheless, the injection of PCM scanning to measure superficial blood flow reveals increased flow after , did not cause pain or a significant increase in cutaneous blood PPCM injection compared with PCM (ANOVA F[9, 4] = 10.37, P 0.02; followed by HSD post hoc test, P # 0.04). (C) Representative pseudocolor flow. Therefore, the observed activation of TRPV1 by PPCM image series of PPCM and PCM injections in one volunteer. NRS, seems to be the more relevant mechanism for injection pain. numerical rating scale; QST, quantitative sensory testing. In addition to PCM-induced analgesic effects, TRP channel activation has also been discussed to account for adverse effects of PCM. In several epidemiologic studies, exposure to intracellular calcium after TRP channel activation and a therapeutic doses of PCM was reported to predispose children concomitant release of calcitonin gene-related peptide could to asthma and eczema (Eneli et al., 2005; Beasley et al., 2008). play an important role in inducing and modulating sensitizing An important role for TRPA1 in airway hyper-reactivity was immune responses (Shiba et al., 2009). Furthermore, the demonstrated in animal models of asthma (Caceres et al., deletion of TRPA1 in mice revealed a crucial role for TRPA1 2009), and the application of therapeutic dosages of PCM in hapten-induced contact hypersensitivity. In this model, in mice induced TRPA1-dependent airway inflammation TRPA1 in the skin controls levels of inflammatory cytokines (Nassini et al., 2010). (interleukin-4, interleukin-6) and nerve growth factor as well PPCM has been shown to induce contact dermatitis in as substance P and serotonin, which induces pruritogenic health care personnel with occupational exposure to the drug responses (Liu et al., 2013). (Barbaud et al., 1995; Berl et al., 1998). Importantly, activa- Berl et al. (1998) proposed a mechanism of how PPCM tion of TRPA1 and TRPV1 is reported to be involved in skin induces occupational contact dermatitis in sensitized nurses sensitization in contact hypersensitivity. Dermatitis associ- who had positive patch tests to PPCM but negative patch tests ated with direct handling of capsaicin-containing chili peppers to PCM. It remains to be investigated whether TRPA1 or is one of the most obvious examples (Williams et al., 1995). TRPV1 is directly involved in this sensitization, or whether Widely used phthalate esters that are thought to promote these channels indirectly aggravate skin responses by medi- cutaneous allergic responses also exhibit TRPA1- and TRPV1- ating neurogenic inflammation and itching (Bessac and Jordt, activating properties (Shiba et al., 2009). The increase in 2008). 24 Schillers et al.

In our cellular models, PPCM (but not PCM) induced (Department of Anaesthesiology and Intensive Care Medicine, Hann- a substantial sensitization of heat-evoked inward currents over Medical School) for continuous support. through TRPV1. In contrast, only four of seven volunteers in our psychophysical study showed a small increase in pain Authorship Contributions rating to noxious heat. As we expected, PPCM was much less Participated in research design: Leffler, M. Eberhardt. potent than capsaicin to activate TRPV1 in vitro. However, Conducted experiments: Schillers, E. Eberhardt, Leffler, M. pain ratings of our volunteers to PPCM injections were also Eberhardt. moderate compared with capsaicin injections frequently used Performed data analysis: Schillers, E. Eberhardt, Leffler, M. Eberhardt. to cause sensitization in human pain models (Geber et al., Wrote or contributed to the writing of the manuscript: Schillers, 2007). However, heat and mechanical hyperalgesia after Leffler, M. Eberhardt. capsaicin injections have been shown to be concentration dependent and therefore to depend on the extent of TRPV1 References activation (Simone et al., 1987, 1989). Thus, no observed heat Andersson DA, Filipovic MR, Gentry C, Eberhardt M, Vastani N, Leffler A, Reeh P, and Bevan S (2015) Streptozotocin stimulates the ion channel TRPA1 directly: sensitization in our psychophysics studies does not contradict involvement of peroxynitrite. J Biol Chem 290:15185–15196. involvement of TRPV1 in PPCM-induced injection pain. Andersson DA, Gentry C, Alenmyr L, Killander D, Lewis SE, Andersson A, Bucher B, Galzi JL, Sterner O, Bevan S, et al. (2011) TRPA1 mediates spinal antinociception PPCM is rapidly hydrolyzed to PCM and diethylglycine. induced by acetaminophen and the cannabinoid D(9)-tetrahydrocannabiorcol. Nat Besides ethylglycine, diethylglycine is also generated in the Commun 2:551. metabolism of lidocaine (Nelson et al., 1977). Both were Bannwarth B, Netter P, Lapicque F, Gillet P, Péré P, Boccard E, Royer RJ, Downloaded from and Gaucher A (1992) Plasma and cerebrospinal fluid concentrations of para- considered as inert compounds. However, recent studies have cetamol after a single intravenous dose of propacetamol. Br J Clin Pharmacol 34: proposed that ethylglycine induces antinociceptive effects of 79–81. Barbaud A, Trechot P, Bertrand O, and Schmutz JL (1995) Occupational allergy to intravenous lidocaine by being a cosubstrate for the spinal propacetamol. Lancet 346:902. glycine transporter GLY T1 (Werdehausen et al., 2012). Thus, Beasley R, Clayton T, Crane J, von Mutius E, Lai CKW, Montefort S, and Stewart A; ISAAC Phase Three Study Group (2008) Association between paracetamol use in ethylglycine was suggested to hamper spinal glycine uptake infancy and childhood, and risk of asthma, rhinoconjunctivitis, and eczema in and consecutively to enhance spinal synaptic inhibition. In children aged 6-7 years: analysis from Phase Three of the ISAAC programme. jpet.aspetjournals.org Lancet 372:1039–1048. animal models of inflammatory and neuropathic pain, Berl V, Barbaud A, and Lepoittevin JP (1998) Mechanism of allergic contact der- Werdehausen et al. (2015) demonstrated that systemic treat- matitis from propacetamol: sensitization to activated N,N-diethylglycine. Contact Dermat 38:185–188. ment with ethylglycine was sufficient to reduce hyperalgesia. Bessac BF and Jordt SE (2008) Breathtaking TRP channels: TRPA1 and TRPV1 in Whether diethylglycine is further metabolized to ethylglycine airway chemosensation and reflex control. Physiology (Bethesda) 23:360–370. Caceres AI, Brackmann M, Elia MD, Bessac BF, del Camino D, D’Amours M, Witek or whether diethylglycine may also exhibit effects on glycine JS, Fanger CM, Chong JA, Hayward NJ, et al. (2009) A sensory neuronal ion transporters remains to be investigated. However, studies channel essential for airway inflammation and hyperreactivity in asthma. Proc –

Natl Acad Sci USA 106:9099 9104. at ASPET Journals on September 24, 2021 comparing the analgesic effects of PCM and PPCM apparently Chuang HH and Lin S (2009) Oxidative challenges sensitize the capsaicin receptor did not identify any relevant differences (McNicol et al., 2011). by covalent cysteine modification. Proc Natl Acad Sci USA 106:20097–20102. Despite similarities in molecular structure and metabolism, de la Roche J, Eberhardt MJ, Klinger AB, Stanslowsky N, Wegner F, Koppert W, Reeh PW, Lampert A, Fischer MJM, and Leffler A (2013) The molecular basis for PPCM does not display properties of local anesthetics. Al- species-specific activation of human TRPA1 protein by protons involves poorly though there was a small tonic block of Na1 currents at 10 mM conserved residues within transmembrane domains 5 and 6. J Biol Chem 288: 20280–20292. PPCM, we observed no use-dependent block. 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Florian Schillers, Esther Eberhardt, Andreas Leffler and Mirjam Eberhardt

Journal: THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS (JPET)

Supplemental Figures:

Supplemental Figure 1. Molecular structures of PCM, PPCM, lidocaine and diethylglycine. Ester hydrolysis of the water soluble prodrug propacetamol (PPCM) gives paracetamol (PCM) and diethylglycine

Supplemental Figure 2. 20 mM PPCM does not activate TRPV2 or TRPV4, but induces minimal currents through TRPA1. PPCM did neither sensitize ramp currents in hTRPV2-expressing cells (A, n = 5) nor in TRPV4-expressing cells (B, n = 5). 1 mM probenecid and 100 nM GSK were used to confirm functional expression. Ramp currents were evoked by 500 ms-long voltage ramps from -100 to +100 mV. 20 mM PPCM (C) and PCM (D) did not evoke inward currents in hTRPA1-expressing cells which were held at -60 mV. The TRPA1 blocker HC030031 (50 µM) also reduced basal TRPA1 activity “noise” in current trace (C) for the duration of its application. Representative recording of one cell (n = 5 - 6 each).

Supplemental Figure 3. PPCM and PCM fail to induce use-dependent block of sodium channels in ND7/23 cells. Currents of endogenous sodium channels in ND7/23 cells were activated at 10 Hz by

60 test pulses from the holding potential of -120 mV to -10 mV. Peak current amplitudes were normalized to the amplitude of the first pulse and plotted against the pulse number. Neither 10 mM paracetamol (PCM, cyan, n = 6) nor 10 mM propacetamol (PPCM, blue, n = 8) induced a use- dependent block.

Supplemental Figure 4. PPCM-induced axon reflex erythema . Complete series of laser Doppler scans taken before and every 2 minutes after double-blind intracutaneous injection of PPCM or PCM to the volar forearm of a human volunteer. While there is a strong axon reflex erythema induced by neuropeptide release following antidromic activation of wide branching C-fibers after PPCM injection, there is only a short-lasting weak erythema comparable to physiological saline injection after PCM injection.