Leesa Bryant

Targeting the Overproduction of Peroxynitrite for the Prevention and Reversal of Paclitaxel-Induced Neuropathic Pain

Chemotherapy-induced peripheral neuropathy (CIPN) accompanied by chronic neuropathic pain is a major dose-limiting side effect of a large number of antitumoral agents including paclitaxel (Taxol). Thus, CIPN is one of most common causes of dose reduction and discontinuation of what is otherwise a life-saving therapy. Neuropathological changes in spinal cord are linked to CIPN, but the causative mediators and mechanisms remain poorly understood. We report that formation of peroxynitrite (PN) in response to activation of nitric oxide synthases and NADPH oxidase in spinal cord contributes to neuropathological changes through two mechanisms. The first involves modulation of neuroexcitatory and proinflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10 and IL-4) cytokines in favor of the former. The second involves post-translational nitration and modification of glia-derived proteins known to be involved in glutamatergic neurotransmission (astrocyte-restricted glutamate transporters and glutamine synthetase). Targeting PN with PN decomposition catalysts (PNDCs) not only blocked the development of paclitaxel-induced neuropathic pain without interfering with antitumor effects, but also reversed it once established. Herein, we describe our mechanistic study on the role(s) of PN and the prevention of neuropathic pain in rats using known PNDCs (FeTMPyP5+ and MnTE-2-PyP5+). We also demonstrate the prevention of CIPN with our two new orally active PNDCs, SRI6 and SRI110. The improved chemical design of SRI6 and SRI110 also affords selectivity for PN over other reactive oxygen species (such as superoxide). Our findings identify PN as a critical determinant of CIPN, while providing the rationale toward development of superoxide-sparing and “PN-targeted” therapeutics.

Counter-Regulation of Opioid Analgesia by Glial-Derived Bioactive Sphingolipids

The clinical efficacy of opiates for pain control is severely limited by analgesic tolerance and hyperalgesia. Herein we show that chronic morphine upregulates both the sphingolipid ceramide in spinal astrocytes and microglia, but not neurons, and spinal sphingosine-1-phosphate (S1P), the end-product of ceramide metabolism. Coadministering morphine with intrathecal administration of pharmacological inhibitors of ceramide and S1P blocked formation of spinal S1P and development of hyperalgesia and tolerance in rats. Our results show that spinally formed S1P signals at least in part by (1) modulating glial function because inhibiting S1P formation blocked increased formation of glial-related proinflammatory cytokines, in particular tumor necrosis factor-α, interleukin-1βα, and interleukin-6, which are known modulators of neuronal excitability, and (2) peroxynitrite-mediated posttranslational nitration and inactivation of glial-related enzymes (glutamine synthetase and the glutamate transporter) known to play critical roles in glutamate neurotransmission. Inhibitors of the ceramide metabolic pathway may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

Manganese(III) complexes of bis(hydroxyphenyl)dipyrromethenes are potent orally active peroxynitrite scavengers.

We report a new series of biscyclohexano-fused Mn(III) complexes of bis(hydroxyphenyl)dipyrromethenes, 4a-c, as potent and orally active peroxynitrite scavengers. Complexes 4a-c are shown to reduce peroxynitrite through a two-electron mechanism, thereby forming the corresponding Mn(V)O species, which were characterized by UV, NMR, and LC-MS methods. Mn(III) complex 4b and its strained BODIPY analogue 9b were analyzed by X-ray crystallography. Finally, complex 4a is shown to be an orally active and potent analgesic in a model carrageenan-induced hyperalgesia known to be driven by the overproduction of peroxynitrite.

Controlling murine and rat chronic pain through A3 adenosine receptor activation

Clinical management of chronic neuropathic pain is limited by marginal effectiveness and unacceptable side effects of current drugs. We demonstrate A3 adenosine receptor (A3AR) agonism as a new target‐based therapeutic strategy. The development of mechanoallodynia in a well‐characterized mouse model of neuropathic pain following chronic constriction injury of the sciatic nerve was rapidly and dose‐depend‐ently reversed by the A3AR agonists: IB‐MECA, its 2‐chlorinated analog (Cl‐IB‐MECA), and the structurally distinct MRS1898. These effects were naloxone insensitive and thus are not opioid receptor mediated. IB‐MECA was ≥1.6‐fold more efficacious than morphine and >5‐fold more potent. In addition, IB‐MECA was equally efficacious as gabapentin (Neurontin) or amitriptyline, but respectively >350‐ and >75‐fold more potent. Besides its potent standalone ability to reverse established mechanoallodynia, IB‐MECA significantly increased the antiallodynic effects of all 3 analgesics. Moreover, neuropathic pain development in rats caused by widely used chemotherapeutics in the taxane (paclitaxel), platinum‐complex (oxaliplatin), and proteasome‐inhibitor (bortezomib) classes was blocked by IB‐MECA without antagonizing their antitumor effect. A3AR agonist effects were blocked with A3AR antagonist MRS1523, but not with A1AR (DPCPX) or A2AAR (SCH‐442416) antagonists. Our findings provide the scientific rationale and pharmacological basis for therapeutic development of A3AR agonists for chronic pain.—Chen, Z., Janes, K., Chen, C., Doyle, T., Bryant, L., Tosh, D.K., Jacobson, K.A., Salvemini, D. Controlling murine and rat chronic pain through A3 adenosine receptor activation. FASEB J. 26, 1855‐1865 (2012). www.fasebj.org

Bioenergetic deficits in peripheral nerve sensory axons during chemotherapy-induced neuropathic pain resulting from peroxynitrite-mediated post-translational nitration of mitochondrial superoxide dismutase

Summary Peroxynitrite contributes to chemotherapy‐induced neuropathic pain via post‐translational nitration and inactivation of mitochondrial superoxide dismutase ultimately leading to ATP depletion within peripheral sensory nerve axons. Abstract Many of the widely used anticancer drugs induce dose‐limiting peripheral neuropathies that undermine their therapeutic efficacy. Animal models of chemotherapy‐induced painful peripheral neuropathy (CIPN) evoked by a variety of drug classes, including taxanes, vinca alkaloids, platinum‐complexes, and proteasome‐inhibitors, suggest that the common underlying mechanism in the development of these neuropathies is mitotoxicity in primary nerve sensory axons (PNSAs) arising from reduced mitochondrial bioenergetics [eg adenosine triphosphate (ATP) production deficits due to compromised respiratory complex I and II activity]. The causative mechanisms of this mitotoxicity remain poorly defined. However, peroxynitrite, an important pro‐nociceptive agent, has been linked to mitotoxicity in several disease states and may also drive the mitotoxicity associated with CIPN. Our findings reveal that the development of mechano‐hypersensitivity induced by paclitaxel, oxaliplatin, and bortezomib was prevented by administration of the peroxynitrite decomposition catalyst Mn(III) 5,10,15,20‐tetrakis(N‐n‐hexylpyridinium‐2‐yl)porphyrin (MnTE‐2‐PyP5+) without interfering with their anti‐tumor effects. Peak CIPN was associated with the nitration and inactivation of superoxide dismutase in the mitochondria, but not in the cytosol, as well as a significant decrease in ATP production within the PNSAs; all of these events were attenuated by MnTE‐2‐PyP5+. Our results provide continued support for the role of mitotoxicity in the development of CIPN across chemotherapeutic drug classes, and identify peroxynitrite as a key mediator in these processes, thereby providing the rationale towards development of “peroxynitrite‐targeted” therapeutics for CIPN.

Supraspinal inactivation of mitochondrial superoxide dismutase is a source of peroxynitrite in the development of morphine antinociceptive tolerance

Effective treatment of chronic pain with morphine is limited by decreases in the drugs analgesic action with chronic administration (antinociceptive tolerance). Because opioids are mainstays of pain management, restoring their efficacy has great clinical importance. We have recently reported that formation of peroxynitrite (ONOO(-), PN) in the dorsal horn of the spinal cord plays a critical role in the development of morphine antinociceptive tolerance and have further documented that nitration and enzymatic inactivation of mitochondrial superoxide dismutase (MnSOD) at that site provides a source for this nitroxidative species. We now report for the first time that antinociceptive tolerance in mice is also associated with the inactivation of MnSOD at supraspinal sites. Inactivation of MnSOD led to nitroxidative stress as evidenced by increased levels of products of oxidative DNA damage and activation of the nuclear factor poly (ADP-ribose) polymerase in whole brain homogenates. Co-administration of morphine with potent Mn porphyrin-based peroxynitrite scavengers, Mn(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+) and Mn(III) 5,10,15,20-tetrakis(N-n-hexylpyridinium-2-yl)porphyrin (MnTnHex-2-PyP5+) (1) restored the enzymatic activity of MnSOD, (2) attenuated PN-derived nitroxidative stress, and (3) blocked the development of morphine-induced antinociceptive tolerance. The more lipophilic analogue, MnTnHex-2-PyP5+ was able to cross the blood-brain barrier at higher levels than its lipophylic counterpart MnTE-2-PyP5+ and was about 30-fold more efficacious. Collectively, these data suggest that PN-mediated enzymatic inactivation of supraspinal MnSOD provides a source of nitroxidative stress, which in turn contributes to central sensitization associated with the development of morphine antinociceptive tolerance. These results support our general contention that PN-targeted therapeutics may have potential as adjuncts to opiates in pain management.

NMDA-receptor activation and nitroxidative regulation of the glutamatergic pathway during nociceptive processing.

&NA; The role of peroxynitrite (PN) as a mediator of nociceptive signaling is emerging. We recently reported that the development of central sensitization that follows the intraplantar injection of carrageenan in rats is associated with spinal PN synthesis. We now demonstrate that a significant pathway through which spinal PN modulates central sensitization is post‐translational tyrosine nitration of key proteins involved in the glutamatergic pathway, namely glutamate transporter GLT‐1 and glutamine synthetase (GS). We also reveal that spinal activation of the N‐methyl‐d‐aspartate (NMDA) receptor provides a source of PN in this setting. Intraplantar injection of carrageenan led to the development of thermal hyperalgesia as well as nitration of GLT‐1 and GS in dorsal horn tissues. Pretreatment with the PN decomposition catalyst FeTM‐4‐PyP5+ [Fe(III)5,10,15,20‐tetrakis(N‐methylpyridinium‐4‐yl)porphyrin] or the NMDA receptor antagonist MK‐801 blocked the development of hyperalgesia. Carrageenan‐induced hyperalgesia was also associated with nitration and inactivation of spinal mitochondrial superoxide dismutase (MnSOD) known to provide a critical source of PN during central sensitization. Nitration of GLT‐1 and GS contributes to central sensitization by enhancing glutamatergic neurotransmission. Our results support the critical role of nitroxidative stress in the development of hyperalgesia and suggest that post‐translational nitration of enzymes and transporters linked to glutamatergic neurotransmission represent a novel mechanism of central sensitization.

Spinal NADPH oxidase is a source of superoxide in the development of morphine-induced hyperalgesia and antinociceptive tolerance

The role of superoxide and its active byproduct peroxynitrite as mediators of nociceptive signaling is emerging. We have recently reported that nitration and inactivation of spinal mitochondrial superoxide dismutase (MnSOD) provides a critical source of these reactive oxygen and nitrogen species during central sensitization associated with the development of morphine-induced hyperalgesia and antinociceptive tolerance. In this study, we demonstrate that activation of spinal NADPH oxidase is another critical source for superoxide generation. Indeed, the development of morphine-induced hyperalgesia and antinociceptive tolerance was associated with increased activation of NADPH oxidase and superoxide release. Co-administration of morphine with systemic delivery of two structurally unrelated NADPH oxidase inhibitors namely apocynin or diphenyleneiodonium (DPI), blocked NADPH oxidase activation and the development of hyperalgesia and antinociceptive tolerance at doses devoid of behavioral side effects. These results suggest that activation of spinal NADPH oxidase contributes to the development of morphine-induced hyperalgesia and antinociceptive tolerance. The role of spinal NADPH oxidase was confirmed by showing that intrathecal delivery of apocynin blocked these events. Our results are the first to implicate the contribution of NADPH oxidase as an enzymatic source of superoxide and thus peroxynitrite in the development of central sensitization associated with morphine-induced hyperalgesia and antinociceptive tolerance. These results continue to support the critical role of these reactive oxygen and nitrogen species in pain while advancing our knowledge of their biomolecular sources.

The Development and Maintenance of Paclitaxel-induced Neuropathic Pain Require Activation of the Sphingosine 1-Phosphate Receptor Subtype 1

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a critical dose-limiting side effect of many chemotherapeutic agents, including paclitaxel. Results: Spinal activation of the S1P-to-S1PR1 axis contributes to the development and maintenance of paclitaxel-induced neuropathic pain through enhanced neuroinflammatory processes. Conclusion: Inhibition of S1PR1 blocks and reverses paclitaxel-induced neuropathic pain without interfering with anticancer effects. Significance: Targeting the S1PR1 signaling pathway could be an effective approach for the treatment of CIPN. The ceramide-sphingosine 1-phosphate (S1P) rheostat is important in regulating cell fate. Several chemotherapeutic agents, including paclitaxel (Taxol), involve pro-apoptotic ceramide in their anticancer effects. The ceramide-to-S1P pathway is also implicated in the development of pain, raising the intriguing possibility that these sphingolipids may contribute to chemotherapy-induced painful peripheral neuropathy, which can be a critical dose-limiting side effect of many widely used chemotherapeutic agents. We demonstrate that the development of paclitaxel-induced neuropathic pain was associated with ceramide and S1P formation in the spinal dorsal horn that corresponded with the engagement of S1P receptor subtype 1 (S1PR1)-dependent neuroinflammatory processes as follows: activation of redox-sensitive transcription factors (NFκB) and MAPKs (ERK and p38) as well as enhanced formation of pro-inflammatory and neuroexcitatory cytokines (TNF-α and IL-1β). Intrathecal delivery of the S1PR1 antagonist W146 reduced these neuroinflammatory processes but increased IL-10 and IL-4, potent anti-inflammatory/neuroprotective cytokines. Additionally, spinal W146 reversed established neuropathic pain. Noteworthy, systemic administration of the S1PR1 modulator FTY720 (Food and Drug Administration-approved for multiple sclerosis) attenuated the activation of these neuroinflammatory processes and abrogated neuropathic pain without altering anticancer properties of paclitaxel and with beneficial effects extended to oxaliplatin. Similar effects were observed with other structurally and chemically unrelated S1PR1 modulators (ponesimod and CYM-5442) and S1PR1 antagonists (NIBR-14/15) but not S1PR1 agonists (SEW2871). Our findings identify for the first time the S1P/S1PR1 axis as a promising molecular and therapeutic target in chemotherapy-induced painful peripheral neuropathy, establish a mechanistic insight into the biomolecular signaling pathways, and provide the rationale for the clinical evaluation of FTY720 in chronic pain patients.

Retooling Manganese(III) Porphyrin-Based Peroxynitrite Decomposition Catalysts for Selectivity and Oral Activity: A Potential New Strategy for Treating Chronic Pain

Redox-active metalloporphyrins represent the most well-characterized class of catalysts capable of attenuating oxidative stress in vivo through the direct interception and decomposition of superoxide and peroxynitrite. While many interesting pharmacological probes have emerged from these studies, few catalysts have been developed with pharmaceutical properties in mind. Herein, we describe our efforts to identify new Mn(III)-porphyrin systems with enhanced membrane solubilizing properties. To this end, seven new Mn(III)-tetracyclohexenylporphyin (TCHP) analogues, 7, 10, 12, 15, and 16a-c, have been prepared in which the beta-fused cyclohexenyl rings provide a means to shield the charged metal center from the membrane during passive transport. Compounds 7, 15, and 16a-c have been shown to be orally active and potent analgesics in a model of carrageenan-induced thermal hyperalgesia. In addition, oral administration of compound 7 (10-100 mg/kg, n=5) has been shown to dose dependently reverse mechano-allodynia in the CCI model of chronic neuropathic pain.