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Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1

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Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1 Research Articles: Cellular/Molecular Citric acid in drug formulations causes pain by potentiating acid-sensing ion channel 1 https://doi.org/10.1523/JNEUROSCI.2087-20.2021 Cite as: J. Neurosci 2021; 10.1523/JNEUROSCI.2087-20.2021 Received: 8 August 2020 Revised: 8 December 2020 Accepted: 10 April 2021 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2021 Yang and Lai This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. 1 Citric acid in drug formulations causes pain by potentiating 2 acid-sensing ion channel 1 3 4 Abbreviated title: ASIC1 mediates pain caused by citric acid 5 6 Ya Lan Yang1, Ted Weita Lai1,2,3* 7 8 1Graduate Institute of Biomedical Sciences, China Medical University, Taichung, Taiwan. 9 2Drug Development Center, China Medical University, Taichung, Taiwan. 10 3Translational Medicine Research Center, China Medical University Hospital, Taichung, 11 Taiwan. 12 13 *Correspondence should be addressed to Ted Weita Lai at [email protected] 14 15 Number of pages: 24 16 17 Number of figures: 9 18 19 Number of words: Abstract, 250; Introduction, 565; Discussion, 1500. 20 21 Conflict of interest statement: The authors declare no competing financial interests. 22 23 Acknowledgements: This study was supported by research grants from the China Medical 24 University (CMU109-MF-28), Ministry of Science and Technology (MOST109-2320-B-039- 25 010), the National Health Research Institutes (NHRI-EX110-10803NI) and the ‘Drug 26 Development Center, China Medical University’ from The Featured Areas Research Center 27 Program within the framework of the Higher Education Sprout Project by the Ministry of 28 Education (MOE) in Taiwan. Y.L.Y. received the Excellent Senior Research Assistant Award 29 of NHRI IRG in Health and Medical Sciences. 1 30 Abstract 31 Pain at the injection site is a common complaint of patients receiving therapeutic 32 formulations containing citric acid. Despite the widely acknowledged role of acid-sensing ion 33 channels (ASICs) in acid-related perception, the specific ASIC subtype mediating pain 34 caused by subcutaneous acid injection and the mechanism by which citrate affects this 35 process are less clear. Here, male mice subjected to intraplantar acid injection responded by 36 executing a withdrawal reflex, and this response was abolished by ASIC1 but not ASIC2 37 knockout. Although intraplantar injection of neutral citrate solution did not produce this 38 response, intraplantar injection of acidic citrate solution produced a withdrawal reflex greater 39 than that produced by acidity alone. Consistent with the behavioral data, neutral citrate failed 40 to produce an electrophysiological response in HEK293 cells, which express ASIC1, but 41 acidic citrate produced a whole-cell inward current greater than that produced by acidity 42 alone. Saturating the intracellular solution with citrate had no effect on the potentiating effect 43 of extracellular citrate, suggesting that citrate acted extracellularly to potentiate ASIC1. 44 Moreover, exposure to citrate immediately before acid stimulation failed to potentiate ASIC1 45 currents, which ruled out the involvement of a metabotropic receptor gated by a citrate 46 metabolite. Finally, removal of calcium ions from the extracellular solution mimicked the 47 potentiating effect of citrate and prevented citrate from further potentiating ASIC1. Our data 48 demonstrate that ASIC1 is necessary for the nociceptive response caused by subcutaneous 49 acid infusion and that neutral citrate, despite not inducing ASIC1 currents or nociceptive 50 behavior on its own, potentiates acid nociception by removing the inhibitory effect of 51 extracellular calcium ions on ASIC1. 52 53 Significance Statement 54 Citric acid is a common ingredient used in pharmaceutical formulations. Despite the 55 widespread clinical use of these formulations, it remains unclear how citric acid causes pain 56 when injected into patients. We identified ASIC1 as the key receptor used to detect injection- 57 site pain caused by acid, and we showed that neutral citrate does not stimulate ASIC1; 58 instead, citrate substantially potentiates ASIC1 activation when injected simultaneously with 59 acid. In addition, we demonstrated that citrate potentiates ASIC1 by removing the inhibitory 60 action of calcium on the extracellular side of the receptor. Given that injection-site pain is the 61 primary complaint of patients receiving citrate-containing medical products, our data provide 62 mechanistic insight into a common medical complaint and suggest a means of avoiding 63 injection pain. 2 64 Introduction 65 Acid causes pain when injected subcutaneously or intramuscularly into human subjects 66 (Steen and Reeh, 1993; Steen et al., 1995; Issberner et al., 1996; Ugawa et al., 2002; Jones et 67 al., 2004), and pharmaceutical formulations containing citrate (10-25 mM), widely used for 68 its pH buffering, calcium chelating, and antioxidant properties, cause more pain than citrate- 69 free formulations carrying the same therapeutic agents (Frenken et al., 1993; Veys et al., 70 1998; Yu et al., 1998; Laursen et al., 2006). As a result, pain at the injection site is a major 71 complaint of patients receiving injections of the current best-selling pharmaceutical product 72 adalimumab (Humira, AbbVie), which has a citrate-buffered acidic formulation (pH 5.2), for 73 the treatment of rheumatoid arthritis and Crohn’s disease (Furst et al., 2003; Keystone et al., 74 2004; van de Putte et al., 2004; Nash et al., 2016). To address this unfavorable property, a 75 citrate-free formulation (Humira Citrate-free) recently became commercially available. 76 Moreover, tissue acidosis has been thought to contribute to pain sensation caused by ischemia, 77 inflammation, and surgical incision (Issberner et al., 1996; Benson et al., 1999; Pan et al., 78 1999; Woo et al., 2004), and citrate levels have been associated with episodes of angina 79 pectoris (Bagger et al., 1981); therefore, understanding the mechanism by which acid or 80 citrate causes pain can have broad clinical implications for understanding pain beyond 81 therapeutic injections. Mammalian cells sense tissue acidification primarily through proton- 82 gated ion channels, including the acid-sensing ion channel (ASIC) family (Price et al., 1996; 83 Waldmann et al., 1996; Garcia-Anoveros et al., 1997; Waldmann et al., 1997b; Waldmann et 84 al., 1997a) and transient receptor potential cation channel subfamily V member 1 (TrpV1) 85 (Caterina et al., 1997; Tominaga et al., 1998). ASIC is further categorized into subtypes 86 encoded by four separate genes that encode ASIC1a (Garcia-Anoveros et al., 1997; 87 Waldmann et al., 1997a) and its splice variant ASIC1b (Chen et al., 1998; Bassler et al., 88 2001), ASIC2a (Price et al., 1996; Waldmann et al., 1996; Garcia-Anoveros et al., 1997) and 89 its splice variant ASIC2b (Lingueglia et al., 1997), ASIC3 (Waldmann et al., 1997b), and 90 ASIC4 (Akopian et al., 2000; Grunder et al., 2000); among these ASIC subtypes, all but 91 ASIC2b and ASIC4 form proton-gated ion channels on their own (Lingueglia et al., 1997; 92 Akopian et al., 2000; Grunder et al., 2000). Although it is widely believed that these proton- 93 gated ion channels, which are expressed on the neurite terminals of nociceptors, are likely 94 initiators of acid-related sensory transduction, the receptor subtype that mediates acute pain 95 caused by subcutaneous acid injection remains unclear, as does the contribution of citrate to 96 this painful sensation. 3 97 In this study, we asked whether neutral citrate at a concentration resembling 98 pharmaceutical products causes acid-like pain when injected subcutaneously in mice and 99 whether it affects pain caused by intraplantar injection of acid at a pH resembling 100 pharmaceutical products. We found that intraplantar acid injection causes a rapid withdrawal 101 reflex in mice, a response that can be attenuated by coinjection of the ASIC inhibitor 102 amiloride. Although neutral citrate failed to produce such a response, it substantially 103 augmented the nociceptive response caused by the acid injection. To investigate this 104 phenomenon further, we identified ASIC1 as the receptor subtype required for this type of 105 pain perception in vivo, and, in a cell line natively expressing human ASIC1, we further 106 investigated the mechanism by which citrate contributes to acid-induced nociception by 107 studying its pharmacological effect on ASIC1 electrophysiology in vitro. 108 109 Materials and Methods 110 Mice 111 Male C57BL/6 mice (6-7 weeks old; 20-30 g) were used in most experiments. Mice lacking 112 ASIC1 (B6.129-Asic1tm1Wsh/J; stock no.: 013733) (Wemmie et al., 2002), ASIC2 (B6.129- 113 Asic2 tm1Wsh/J; stock no.: 013126) (Price et al., 2000), and TrpV1 (B6.129X1-Trpv1tm1Jul/J; 114 stock no.: 003770) (Caterina et al., 2000) were purchased from Jackson Laboratory. The mice 115 were provided food and water ad libitum prior to the experiment, and all experiments 116 involving animals followed the Institutional Guidelines of the China Medical University for 117 the Care and Use of Experimental Animals (IGCMU-CUEA) and were approved by its 118 Institutional Animal Care and Use Committee (IACUC) (Protocol No. 2016-213-2 and 2020- 119 401). 120 121 Cell culture 122 HEK 293 cells purchased from American Type Culture Collection (cat no. CRL-1573) were 123 used in the electrophysiological experiments. The cells were cultured in Dulbecco’s Modified 124 Eagle’s Medium supplemented with 10% fetal bovine serum in a CO2 incubator (Thermo 125 Scientific; cat no.
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