Biol. Chem. 2015; 396(6-7): 783–794
Dong Dong Zhang, Bona Linke, Jing Suo, Aleksandra Zivkovic, Yannick Schreiber, Nerea Ferreirós, Marina Henke, Gerd Geisslinger, Holger Stark and Klaus Scholich*
Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors Abstract: FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive
*Corresponding author: Klaus Scholich, Institut für Klinische Pharmakologie, pharmazentrum frankfurt/ZAF, E.S., Klinikum der Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany, e-mail: [email protected] Dong Dong Zhang, Bona Linke, Jing Suo, Yannick Schreiber, Nerea Ferreirós and Gerd Geisslinger: Institut für Klinische Pharmakologie, pharmazentrum frankfurt/ZAF, E.S., Klinikum der Goethe-Universitä/t Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany Aleksandra Zivkovic and Holger Stark: Institute of Pharmaceutical and Medical Chemistry, Heinrich Heine University, Düsseldorf, Germany Marina Henke: Institut für Klinische Pharmakologie, pharmazentrum frankfurt/ZAF, E.S., Klinikum der Goethe-Universitä/t Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany; and Fraunhofer Institute of Molecular Biology and Applied Ecology, Project Group Translational Medicine and Pharmacology (IME-TMP), Frankfurt/Main, Germany
effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes. Keywords: FTY720; neuropathic pain; sphingosine-1-phosphate; S1P receptor; spinal cord. DOI 10.1515/hsz-2014-0276 Received November 27, 2014; accepted January 16, 2015; previously published online January 23, 2015
Introduction The bioactive sphingolipid metabolite sphingosine1-phosphate (S1P) is synthesized by phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2) in a variety of cell types. Both kinases are activated in response to a variety of extracellular stimuli (Karliner, 2009; Pyne et al., 2009) and are involved in the regulation of a multiplicity of cellular functions, such as apoptosis, cell differentiation, and migration. S1P can mediate its actions either through the activation of a family of five G-protein coupled receptors (S1P1–5) or by acting as an intracellular second messenger (Spiegel and Milstien, 2003; Anliker and Chun, 2004; Saba and Hla, 2004). Throughout the nervous system, S1P promotes survival of neurons and astrocytes, induces proliferation of neural progenitor cells and astrocytes, and differentially modulates synaptic transmission depending on the neuronal subtypes (Spiegel and Milstien, 2003; Anliker and Chun, 2004; Colombaioni and Garcia-Gil, 2004; Coste et al., 2008a). In the peripheral nerve system, S1P increases neuronal excitability. For example, intra- as well as extracellular-applied S1P facilitates NGF-induced excitability of sensory neurons from dorsal root ganglia (DRG) (Zhang et al., 2006a,b) and induces thermal hyperalgesia (Mair et al., 2011). Accordingly, injection
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784 D.D. Zhang et al.: Spinal antinociceptive mechanisms of FTY720 of S1P in peripheral tissue elicits hyperalgesic responses either directly through neuronal expressed S1P receptors S1P1 and S1P3 (Mair et al., 2011; Camprubi-Robles et al., 2013) or indirectly through immune cells such as neutrophil granulocytes (Finley et al., 2013). In the central nervous system, peripheral inflammation reduces the S1P-release in the spinal cord, causing a disinhibition of cAMP-mediated sensitization and, as a consequence, increased pain-related (nociceptive) behavior (Coste et al., 2008a,b). Accordingly, intrathecal application of S1P, sphinganine-1-phosphate (dhS1P) or the non-selective S1P receptor agonist FTY720 reduces effectively nociceptive behavior in animal models for inflammatory and trauma-induced neuropathic pain (Coste et al., 2008a,b). More recently, it was shown that activation of S1P1 receptors on microglia in the spinal cord induces the release of pro-inflammatory mediators (i.e. TNFα, IL1β) and decreases the release of the anti-inflammatory mediators IL10 during chemotherapy-induced neuropathies (Janes et al., 2014). Fittingly, downregulation of S1P1 on spinal microglia through FTY720 reduced the nociceptive behavior in one model of chemotherapy-induced neuropathic pain. FTY720 (fingolimod) is phosphorylated by SPHK2 and is in its phosphorylated form (FTY720-P) a potent agonist at all known S1P receptor subtypes (S1P1,3,4,5), except S1P2 (Brinkmann et al., 2002; Billich et al., 2003; Brinkmann, 2007). It is an approved immunosuppressive drug for the treatment of multiple sclerosis that acts as functional antagonist on S1P1. After S1P1 activation FTY720-P induces an irreversible internalization and downregulation of S1P1. By downregulating S1P1, FTY720 traps lymphocytes in lymph nodes, causing the depletion of circulating lymphocytes (Tedesco-Silva et al., 2005; Mulgaonkar et al., 2006; Zhang et al., 2007). Previously, FTY720 has been shown to reduce the nociceptive behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain to a similar extent as gabapentin, a commonly used drug for the treatment of neuropathic pain (Coste et al., 2008b). Here, we investigated the mechanisms of the antinociceptive effect of FTY720 in a model for trauma-induced neuropathic pain and showed that the antinociceptive effect is based on the activation of spinal S1P1 and S1P3 receptors. These findings differ from the antinociceptive mechanisms of FTY720 described for chemotherapyinduced neuropathic pain, highlighting the different mechanisms that underlie the development of chemotherapy- and trauma-induced neuropathic pain (Sisignano et al., 2014).
Results Antinociceptive effects of FTY720 are mediated by spinal S1P receptors To test whether or not orally (p.o.) administered FTY720 decreases the nociceptive behavior in the SNI model in adult mice, we administered FTY720 (p.o.) once daily, starting at 7 days after the SNI operation when a stable pain behavior has been developed. The antinociceptive efficacy of 0.01 mg/kg FTY720 was comparable to the effect observed for gabapentin (2 × daily, 2.5 mg/kg), which serves as gold standard for the treatment of neuropathic pain (Figure 1A). While 0.001 mg/kg did not have an antinociceptive effect, 0.01 mg/kg had the strong antinociceptive effect that lasted for up to 8 h (Figure 1B). A further increase of the dose to 0.1 mg/kg resulted in a small, short-lasting antinociceptive effect (Figure 1B). This narrow therapeutic window has been described earlier for the antinociceptive effects of FTY720 (Coste et al., 2008a,b) and seems to be based on the prevailing activation of S1P1 at low FTY720 doses in contrast to a dominating inhibitory effect of higher doses of FTY720. Repeated daily single administration of FTY720 over 11 days showed that the antinociceptive effect was stable over this time period and disappeared within 24 h after the last treatment (Figure 1C). To study whether or not FTY720 inhibits neuropathic pain through central or peripheral mechanisms, FTY720P was administered 14 days after inducing the nerve lesion either intraplantarly (i.pl.) or intrathecally (i.th.) (Figure 2A,B). While a single injection of FTY720-P (0.1 and 1 μg/kg) in the paw did not evoke antinociceptive effects in the SNI model (Figure 2A), we found that a single intrathecal (i.th.) administration of FTY720-P (1 μg/kg) significantly increased the mechanical pain thresholds for up to 5 h after application (Figure 2B). To determine whether FTY720 or FTY720-P mediates the antinociceptive effects, we used SPHK2-knockout mice, which are unable to convert FTY720 to FTY720-P. While no antinociceptive effect of systemically administered FTY720 mice was observed in SPHK2-knockout (Figure 2C), intrathecal administration of FTY720-P increased mechanical pain thresholds in these mice (Figure 2C). Likewise, intrathecal injection of dhS1P (1 μg/kg) 14 days after nerve injury significantly increased paw withdrawal latency times (Figure 2D). Notably, dhS1P binds to S1P receptors with similar affinities as S1P without mimicking its intracellular second messenger properties or sharing the ability to irreversibly downregulate S1P1 receptors with FTY720-P
Brought to you by | EP Ipswich Authenticated Download Date | 5/5/15 8:17 AM
D.D. Zhang et al.: Spinal antinociceptive mechanisms of FTY720 785
(Van Brocklyn et al., 1998). Thus, so far the data suggest that the activation of spinal S1P receptors is necessary for the antinociceptive effects of FTY720.
S1P1 and S1P3 receptors are necessary to mediate antinociceptive effects of FTY720
Figure 1: FTY720 increases mechanical thresholds in the SNI model for neuropathic pain. (A) Mice were subjected to the SNI operation. Mechanical thresholds were determined at the indicated times and shown as paw withdrawal latencies (PWL). Daily treatment with orally (p.o.) administered FTY720 (1 × daily 0.01 mg/kg) or gabapentin (2 × daily 25 mg/kg) was started at day 7. Mechanical allodynia was always determined 2 h after drug application. Data are shown as mean±S.E.M. of eight, nine or ten mice per group. ANOVA/ Bonferoni *p
Dong Dong Zhang, Bona Linke, Jing Suo, Aleksandra Zivkovic, Yannick Schreiber, Nerea Ferreirós, Marina Henke, Gerd Geisslinger, Holger Stark and Klaus Scholich*
Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors Abstract: FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive
*Corresponding author: Klaus Scholich, Institut für Klinische Pharmakologie, pharmazentrum frankfurt/ZAF, E.S., Klinikum der Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany, e-mail: [email protected] Dong Dong Zhang, Bona Linke, Jing Suo, Yannick Schreiber, Nerea Ferreirós and Gerd Geisslinger: Institut für Klinische Pharmakologie, pharmazentrum frankfurt/ZAF, E.S., Klinikum der Goethe-Universitä/t Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany Aleksandra Zivkovic and Holger Stark: Institute of Pharmaceutical and Medical Chemistry, Heinrich Heine University, Düsseldorf, Germany Marina Henke: Institut für Klinische Pharmakologie, pharmazentrum frankfurt/ZAF, E.S., Klinikum der Goethe-Universitä/t Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt/Main, Germany; and Fraunhofer Institute of Molecular Biology and Applied Ecology, Project Group Translational Medicine and Pharmacology (IME-TMP), Frankfurt/Main, Germany
effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes. Keywords: FTY720; neuropathic pain; sphingosine-1-phosphate; S1P receptor; spinal cord. DOI 10.1515/hsz-2014-0276 Received November 27, 2014; accepted January 16, 2015; previously published online January 23, 2015
Introduction The bioactive sphingolipid metabolite sphingosine1-phosphate (S1P) is synthesized by phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2) in a variety of cell types. Both kinases are activated in response to a variety of extracellular stimuli (Karliner, 2009; Pyne et al., 2009) and are involved in the regulation of a multiplicity of cellular functions, such as apoptosis, cell differentiation, and migration. S1P can mediate its actions either through the activation of a family of five G-protein coupled receptors (S1P1–5) or by acting as an intracellular second messenger (Spiegel and Milstien, 2003; Anliker and Chun, 2004; Saba and Hla, 2004). Throughout the nervous system, S1P promotes survival of neurons and astrocytes, induces proliferation of neural progenitor cells and astrocytes, and differentially modulates synaptic transmission depending on the neuronal subtypes (Spiegel and Milstien, 2003; Anliker and Chun, 2004; Colombaioni and Garcia-Gil, 2004; Coste et al., 2008a). In the peripheral nerve system, S1P increases neuronal excitability. For example, intra- as well as extracellular-applied S1P facilitates NGF-induced excitability of sensory neurons from dorsal root ganglia (DRG) (Zhang et al., 2006a,b) and induces thermal hyperalgesia (Mair et al., 2011). Accordingly, injection
Brought to you by | EP Ipswich Authenticated Download Date | 5/5/15 8:17 AM
784 D.D. Zhang et al.: Spinal antinociceptive mechanisms of FTY720 of S1P in peripheral tissue elicits hyperalgesic responses either directly through neuronal expressed S1P receptors S1P1 and S1P3 (Mair et al., 2011; Camprubi-Robles et al., 2013) or indirectly through immune cells such as neutrophil granulocytes (Finley et al., 2013). In the central nervous system, peripheral inflammation reduces the S1P-release in the spinal cord, causing a disinhibition of cAMP-mediated sensitization and, as a consequence, increased pain-related (nociceptive) behavior (Coste et al., 2008a,b). Accordingly, intrathecal application of S1P, sphinganine-1-phosphate (dhS1P) or the non-selective S1P receptor agonist FTY720 reduces effectively nociceptive behavior in animal models for inflammatory and trauma-induced neuropathic pain (Coste et al., 2008a,b). More recently, it was shown that activation of S1P1 receptors on microglia in the spinal cord induces the release of pro-inflammatory mediators (i.e. TNFα, IL1β) and decreases the release of the anti-inflammatory mediators IL10 during chemotherapy-induced neuropathies (Janes et al., 2014). Fittingly, downregulation of S1P1 on spinal microglia through FTY720 reduced the nociceptive behavior in one model of chemotherapy-induced neuropathic pain. FTY720 (fingolimod) is phosphorylated by SPHK2 and is in its phosphorylated form (FTY720-P) a potent agonist at all known S1P receptor subtypes (S1P1,3,4,5), except S1P2 (Brinkmann et al., 2002; Billich et al., 2003; Brinkmann, 2007). It is an approved immunosuppressive drug for the treatment of multiple sclerosis that acts as functional antagonist on S1P1. After S1P1 activation FTY720-P induces an irreversible internalization and downregulation of S1P1. By downregulating S1P1, FTY720 traps lymphocytes in lymph nodes, causing the depletion of circulating lymphocytes (Tedesco-Silva et al., 2005; Mulgaonkar et al., 2006; Zhang et al., 2007). Previously, FTY720 has been shown to reduce the nociceptive behavior in the spared nerve injury (SNI) model for trauma-induced neuropathic pain to a similar extent as gabapentin, a commonly used drug for the treatment of neuropathic pain (Coste et al., 2008b). Here, we investigated the mechanisms of the antinociceptive effect of FTY720 in a model for trauma-induced neuropathic pain and showed that the antinociceptive effect is based on the activation of spinal S1P1 and S1P3 receptors. These findings differ from the antinociceptive mechanisms of FTY720 described for chemotherapyinduced neuropathic pain, highlighting the different mechanisms that underlie the development of chemotherapy- and trauma-induced neuropathic pain (Sisignano et al., 2014).
Results Antinociceptive effects of FTY720 are mediated by spinal S1P receptors To test whether or not orally (p.o.) administered FTY720 decreases the nociceptive behavior in the SNI model in adult mice, we administered FTY720 (p.o.) once daily, starting at 7 days after the SNI operation when a stable pain behavior has been developed. The antinociceptive efficacy of 0.01 mg/kg FTY720 was comparable to the effect observed for gabapentin (2 × daily, 2.5 mg/kg), which serves as gold standard for the treatment of neuropathic pain (Figure 1A). While 0.001 mg/kg did not have an antinociceptive effect, 0.01 mg/kg had the strong antinociceptive effect that lasted for up to 8 h (Figure 1B). A further increase of the dose to 0.1 mg/kg resulted in a small, short-lasting antinociceptive effect (Figure 1B). This narrow therapeutic window has been described earlier for the antinociceptive effects of FTY720 (Coste et al., 2008a,b) and seems to be based on the prevailing activation of S1P1 at low FTY720 doses in contrast to a dominating inhibitory effect of higher doses of FTY720. Repeated daily single administration of FTY720 over 11 days showed that the antinociceptive effect was stable over this time period and disappeared within 24 h after the last treatment (Figure 1C). To study whether or not FTY720 inhibits neuropathic pain through central or peripheral mechanisms, FTY720P was administered 14 days after inducing the nerve lesion either intraplantarly (i.pl.) or intrathecally (i.th.) (Figure 2A,B). While a single injection of FTY720-P (0.1 and 1 μg/kg) in the paw did not evoke antinociceptive effects in the SNI model (Figure 2A), we found that a single intrathecal (i.th.) administration of FTY720-P (1 μg/kg) significantly increased the mechanical pain thresholds for up to 5 h after application (Figure 2B). To determine whether FTY720 or FTY720-P mediates the antinociceptive effects, we used SPHK2-knockout mice, which are unable to convert FTY720 to FTY720-P. While no antinociceptive effect of systemically administered FTY720 mice was observed in SPHK2-knockout (Figure 2C), intrathecal administration of FTY720-P increased mechanical pain thresholds in these mice (Figure 2C). Likewise, intrathecal injection of dhS1P (1 μg/kg) 14 days after nerve injury significantly increased paw withdrawal latency times (Figure 2D). Notably, dhS1P binds to S1P receptors with similar affinities as S1P without mimicking its intracellular second messenger properties or sharing the ability to irreversibly downregulate S1P1 receptors with FTY720-P
Brought to you by | EP Ipswich Authenticated Download Date | 5/5/15 8:17 AM
D.D. Zhang et al.: Spinal antinociceptive mechanisms of FTY720 785
(Van Brocklyn et al., 1998). Thus, so far the data suggest that the activation of spinal S1P receptors is necessary for the antinociceptive effects of FTY720.
S1P1 and S1P3 receptors are necessary to mediate antinociceptive effects of FTY720
Figure 1: FTY720 increases mechanical thresholds in the SNI model for neuropathic pain. (A) Mice were subjected to the SNI operation. Mechanical thresholds were determined at the indicated times and shown as paw withdrawal latencies (PWL). Daily treatment with orally (p.o.) administered FTY720 (1 × daily 0.01 mg/kg) or gabapentin (2 × daily 25 mg/kg) was started at day 7. Mechanical allodynia was always determined 2 h after drug application. Data are shown as mean±S.E.M. of eight, nine or ten mice per group. ANOVA/ Bonferoni *p
