Zhoumou Chen

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

Endogenous adenosine A3 receptor activation selectively alleviates persistent pain states

Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR, lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically, engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.

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.

Sphingosine 1-Phosphate Mediates Hyperalgesia via a Neutrophil-Dependent Mechanism

Novel classes of pain-relieving molecules are needed to fill the void between non-steroidal anti-inflammatory agents and narcotics. We have recently shown that intraplantar administration of sphingosine 1-phosphate (S1P) in rats causes peripheral sensitization and hyperalgesia through the S1P1 receptor subtype (S1PR1): the mechanism(s) involved are largely unknown and were thus explored in the present study. Intraplantar injection of carrageenan in rats led to a time-dependent development of thermal hyperalgesia that was associated with pronounced edema and infiltration of neutrophils in paw tissues. Inhibition of 1) S1P formation with SK-I, a sphingosine kinase inhibitor, 2) S1P bioavailability with the S1P blocking antibody Sphingomab, LT1002 (but not its negative control, LT1017) or 3) S1P actions through S1PR1 with the selective S1PR1 antagonist, W146 (but not its inactive enantiomer, W140) blocked thermal hyperalgesia and infiltration of neutrophils. Taken together, these findings identify S1P as an important contributor to inflammatory pain acting through S1PR1 to elicit hyperalgesia in a neutrophil-dependant manner. In addition and in further support, we demonstrate that the development of thermal hyperalgesia following intraplantar injection of S1P or SEW2871 (an S1PR1 agonist) was also associated with neutrophilic infiltration in paw tissues as these events were attenuated by fucoidan, an inhibitor of neutrophilic infiltration. Importantly, FTY720, an FDA-approved S1P receptor modulator known to block S1P-S1PR1 signaling, attenuated carrageenan-induced thermal hyperalgesia and associated neutrophil infiltration. Targeting the S1P/S1PR1 axis opens a therapeutic strategy for the development of novel non-narcotic anti-hyperalgesic agents.

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.