Paclitaxel-induced Mechanical Allodynia Research Articles

DNMT3a-triggered downregulation of K2p 1.1 gene in primary sensory neurons contributes to paclitaxel-induced neuropathic pain.

Antineoplastic drugs induce dramatic transcriptional changes in dorsal root ganglion (DRG) neurons, which may contribute to chemotherapy-induced neuropathic pain. K2p 1.1 controls neuronal excitability by setting the resting membrane potential. Here, we report that systemic injection of the chemotherapy agent paclitaxel time-dependently downregulates the expression of K 2p 1.1 mRNA and its coding K2p 1.1 protein in the DRG neurons. Rescuing this downregulation mitigates the development and maintenance of paclitaxel-induced mechanical allodynia and heat hyperalgesia. Conversely, in the absence of paclitaxel administration, mimicking this downregulation decreases outward potassium current and increases excitability in the DRG neurons, leading to the enhanced responses to mechanical and heat stimuli. Mechanically, the downregulation of DRG K 2p 1.1 mRNA is attributed to paclitaxel-induced increase in DRG DNMT3a, as blocking this increase reverses the paclitaxel-induced the decrease of DRG K2p 1.1 and mimicking this increase reduces DRG K2p 1.1 expression. In addition, paclitaxel injection increases the binding of DNMT3a to the K 2p 1.1 gene promoter region and elevates the level of DNA methylation within this region in the DRG. These findings suggest that DNMT3a-triggered downregulation of DRG K2p 1.1 may contribute to chemotherapy-induced neuropathic pain.

Cisplatin educates CD8+ T cells to prevent and resolve chemotherapy-induced peripheral neuropathy in mice.

The mechanisms responsible for the persistence of chemotherapy-induced peripheral neuropathy (CIPN) in a significant proportion of cancer survivors are still unknown. Our previous findings show that CD8 T cells are necessary for the resolution of paclitaxel-induced mechanical allodynia in male mice. In this study, we demonstrate that CD8 T cells are not only essential for resolving cisplatin-induced mechanical allodynia, but also to normalize spontaneous pain, numbness, and the reduction in intraepidermal nerve fiber density in male and female mice. Resolution of CIPN was not observed in Rag2 mice that lack T and B cells. Reconstitution of Rag2 mice with CD8 T cells before cisplatin treatment normalized the resolution of CIPN. In vivo education of CD8 T cells by cisplatin was necessary to induce resolution of CIPN in Rag2 mice because adoptive transfer of CD8 T cells from naive wild-type mice to Rag2 mice after completion of chemotherapy did not promote resolution of established CIPN. The CD8 T-cell-dependent resolution of CIPN does not require epitope recognition by the T-cell receptor. Moreover, adoptive transfer of cisplatin-educated CD8 T cells to Rag2 mice prevented CIPN development induced by either cisplatin or paclitaxel, indicating that the activity of the educated CD8 T is not cisplatin specific. In conclusion, resolution of CIPN requires in vivo education of CD8 T cells by exposure to cisplatin. Future studies should examine whether ex vivo CD8 T cell education could be applied as a therapeutic strategy for treating or preventing CIPN in patients.

Monoclonal Antibody Targeting the Matrix Metalloproteinase 9 Prevents and Reverses Paclitaxel-Induced Peripheral Neuropathy in Mice

Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling condition accompanying several cancer drugs, including the front-line chemotherapeutic agent paclitaxel. Although CIPN can force dose reduction or even discontinuation of chemotherapy, affecting survival in cancer patients, there is no US Food and Drug Administration–approved treatment for CIPN. CIPN in mice is characterized by neuropathic pain (eg, mechanical allodynia) in association with oxidative stress and neuroinflammation in dorsal root ganglia (DRGs), as well as retraction of intraepidermal nerve fibers. Here, we report that paclitaxel-induced mechanical allodynia is associated with transcriptional increase in matrix metalloproteinase (MMP) 2 and 9 and decrease in metallopeptidase inhibitor 1 (TIMP1), a strong endogenous MMP9 inhibitor. Consistently, MMP9 protein levels are increased in DRG neurons in vivo and in vitro after paclitaxel treatment, and it is demonstrated, for the first time, that intrathecal injections of exogenous TIMP1 or a monoclonal antibody targeting MMP9 (MMP9 mAb) significantly prevented and reversed paclitaxel-induced mechanical allodynia in male and female mice. Analyses of DRG tissues showed that MMP9 mAb significantly decreased oxidative stress and neuroinflammatory mediators interleukin-6 and tumor necrosis factor α, as well as prevented paclitaxel-induced loss of intraepidermal nerve fibers. These findings suggest that MMP signaling plays a key role in paclitaxel-induced peripheral neuropathy, and MMP9 mAb may offer new therapeutic approaches for the treatment of CIPN. PerspectiveChemotherapy-induced peripheral neuropathy (CIPN) remains ineffectively managed in cancer patients, potentially leading to the discontinuation of an otherwise life-saving treatment. Here, we demonstrate that a monoclonal antibody targeting MMP9 alleviates neuropathic pain and several mechanisms linked to CIPN. This study is particularly relevant, because a humanized MMP9 antibody is already in advanced clinical trials for the treatment of colitis and cancer, and it may be straightforwardly repurposed for the relief of CIPN.

Abstract 602: The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity

Abstract While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our laboratories have previously shown that nicotine, the prototypical nicotinic acetylcholine receptor (nAChR) agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy without increasing lung tumor growth in mice. Our current studies have demonstrated that the α7 nAChR silent agonist R-47 may be a promising preventative and therapeutic treatment for CIPN as well. R-47 (10 mg/kg, p.o.) was administered to male C57BL/6J mice treated with paclitaxel (8 mg/kg, i.p., every other day for a total of four injections). Von Frey filament testing revealed that R-47 can both prevent and reverse paclitaxel-induced mechanical allodynia; the latter effect can be inhibited by the α7 nAChR antagonist methyllycaconitine. In support of these findings, R-47 also prevents the reduction of intra-epidermal nerve fibers in the hind paws of paclitaxel-treated mice. In addition, α7 nAChR knockout mice exhibit a greater and sustained increase in mechanical allodynia after paclitaxel administration when compared to wild-type mice, suggesting the involvement of this receptor subtype in hindering CIPN development and/or maintenance. With regard to cancer, R-47 fails to increase A549 and H460 non-small cell lung cancer cell viability, colony formation, or proliferation alone, and does not interfere with paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, R-47 does not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that the use of an α7 nAChR silent agonist may be a safe and efficacious approach for the prevention and treatment of CIPN. Citation Format: S Lauren Kyte, Wisam Toma, Ganeshsingh Thakur, M Imad Damaj, David A. Gewirtz. The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 602.

Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a painful and debilitating side effect of cancer chemotherapy with an unclear pathogenesis. Consequently, the available therapies for this neuropathic pain syndrome are inadequate, leading to a significantly reduced quality of life in many patients. Complement, a key component of the innate immune system, has been associated with neuroinflammation, a potentially important trigger of some types of neuropathic pain. However, the role of complement in CIPN remains unclear. To address this issue, we developed a C3 knockout (KO) rat model and induced CIPN in these KO rats and wild-type littermates via the i.p. administration of paclitaxel, a chemotherapeutic agent associated with CIPN. We then compared the severity of mechanical allodynia, complement activation, and intradermal nerve fiber loss between the groups. We found that 1) i.p. paclitaxel administration activated complement in wild-type rats, 2) paclitaxel-induced mechanical allodynia was significantly reduced in C3 KO rats, and 3) the paclitaxel-induced loss of intradermal nerve fibers was markedly attenuated in C3 KO rats. In in vitro studies, we found that paclitaxel-treated rat neuronal cells activated complement, leading to cellular injury. Our findings demonstrate a previously unknown but pivotal role of complement in CIPN and suggest that complement may be a new target for the development of novel therapeutics to manage this painful disease.

Monoacylglycerol Lipase Inhibitors Reverse Paclitaxel-Induced Nociceptive Behavior and Proinflammatory Markers in a Mouse Model of Chemotherapy-Induced Neuropathy.

Although paclitaxel effectively treats various cancers, its debilitating peripheral neuropathic pain side effects often persist long after treatment has ended. Therefore, a compelling need exists for the identification of novel pharmacologic strategies to mitigate this condition. As inhibitors of monoacylglycerol lipase (MAGL), the primary hydrolytic enzyme of the endogenous cannabinoid, 2-arachidonyolglycerol, produces antinociceptive effects in numerous rodent models of pain, we investigated whether inhibitors of this enzyme (i.e., JZL184 and MJN110) would reverse paclitaxel-induced mechanical allodynia in mice. These drugs dose dependently reversed allodynia with respective ED50 values (95% confidence limit) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Complementary genetic and pharmacologic approaches revealed that the antiallodynic effects of each drug require both cannabinoid receptors, CB1 and CB2 MJN110 reduced paclitaxel-mediated increased expression of monocyte chemoattractant protein-1 (MCP-1, CCL2) and phospho-p38 MAPK in dorsal root ganglia as well as MCP-1 in spinal dorsal horn. Whereas the antinociceptive effects of high dose JZL184 (40 mg/kg) underwent tolerance following 6 days of repeated dosing, repeated administration of a threshold dose (i.e., 4 mg/kg) completely reversed paclitaxel-induced allodynia. In addition, we found that the administration of MJN110 to control mice lacked intrinsic rewarding effects in the conditioned place preference (CPP) paradigm. However, it produced a CPP in paclitaxel-treated animals, suggesting a reduced paclitaxel-induced aversive state. Importantly, JZL184 did not alter the antiproliferative and apoptotic effects of paclitaxel in A549 and H460 non-small cell lung cancer cells. Taken together, these data indicate that MAGL inhibitors reverse paclitaxel-induced neuropathic pain without interfering with chemotherapeutic efficacy.

Orally active Epac inhibitor reverses mechanical allodynia and loss of intraepidermal nerve fibers in a mouse model of chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of cancer treatment that significantly compromises quality of life of cancer patients and survivors. Identification of targets for pharmacological intervention to prevent or reverse CIPN is needed. We investigated exchange protein regulated by cAMP (Epac) as a potential target. Epacs are cAMP-binding proteins known to play a pivotal role in mechanical allodynia induced by nerve injury and inflammation. We demonstrate that global Epac1-knockout (Epac1-/-) male and female mice are protected against paclitaxel-induced mechanical allodynia. In addition, spinal cord astrocyte activation and intraepidermal nerve fiber (IENF) loss are significantly reduced in Epac1-/- mice as compared to wild-type mice. Moreover, Epac1-/- mice do not develop the paclitaxel-induced deficits in mitochondrial bioenergetics in the sciatic nerve that are a hallmark of CIPN. Notably, mice with cell-specific deletion of Epac1 in Nav1.8-positive neurons (N-Epac1-/-) also show reduced paclitaxel-induced mechanical allodynia, astrocyte activation, and IENF loss, indicating that CIPN develops downstream of Epac1 activation in nociceptors. The Epac-inhibitor ESI-09 reversed established paclitaxel-induced mechanical allodynia in wild-type mice even when dosing started 10 days after completion of paclitaxel treatment. In addition, oral administration of ESI-09 suppressed spinal cord astrocyte activation in the spinal cord and protected against IENF loss. Ex vivo, ESI-09 blocked paclitaxel-induced abnormal spontaneous discharges in dorsal root ganglion neurons. Collectively, these findings implicate Epac1 in nociceptors as a novel target for treatment of CIPN. This is clinically relevant because ESI-09 has the potential to reverse a debilitating and long-lasting side effect of cancer treatment.

Differential Inhibition of Nav1.7 and Neuropathic Pain by Hybridoma-Produced and Recombinant Monoclonal Antibodies that Target Nav1.7 : Differential activities of Nav1.7-targeting monoclonal antibodies.

The voltage-gated Na+ channel subtype Nav1.7 is important for pain and itch in rodents and humans. We previously showed that a Nav1.7-targeting monoclonal antibody (SVmab) reduces Na+ currents and pain and itch responses in mice. Here, we investigated whether recombinant SVmab (rSVmab) binds to and blocks Nav1.7 similar to SVmab. ELISA tests revealed that SVmab was capable of binding to Nav1.7-expressing HEK293 cells, mouse DRG neurons, human nerve tissue, and the voltage-sensor domain II of Nav1.7. In contrast, rSVmab showed no or weak binding to Nav1.7 in these tests. Patch-clamp recordings showed that SVmab, but not rSVmab, markedly inhibited Na+ currents in Nav1.7-expressing HEK293 cells. Notably, electrical field stimulation increased the blocking activity of SVmab and rSVmab in Nav1.7-expressing HEK293 cells. SVmab was more effective than rSVmab in inhibiting paclitaxel-induced mechanical allodynia. SVmab also bound to human DRG neurons and inhibited their Na+ currents. Finally, potential reasons for the differential efficacy of SVmab and rSVmab and future directions are discussed.

Icariin, a flavonoid with anti-cancer effects, alleviated paclitaxel-induced neuropathic pain in a SIRT1-dependent manner.

BackgroundOne of the most common side effects of paclitaxel was dosage-dependently painful neuropathy. Various reports indicated that spinal neuroinflammation was involved in paclitaxel-induced neuropathic pain. This study investigated the effect of icariin on paclitaxel-induced neuroinflammation and peripheral neuropathy in rats.MethodsTwo parts were included in this study. In part one, the effect of icariin on paclitaxel-induced neuropathic pain was investigated. Mechanical thresholds were measured as primary outcomes. Production of proinflammatory factors (tumor necrosis factor-α, interleukin-1 β, and interleukin-6), activation of nuclear factor-κB (NF-κB(p65)) signal, and activation of astrocytes were detected as secondary outcomes. Spinal Sirtuin 1 (SIRT1) expression, H4 acetylation, and NAD+ content were measured to investigate the effect of icariin on spinal SIRT1 signal pathway. In part two, the role of SIRT1 signal on icariin-induced effect in rats was investigated, and EX527, a SIRT1 inhibitor, was employed.ResultsThe results showed paclitaxel treatment induced significant decrease in mechanical thresholds. Paclitaxel treatment also induced NF-κB(p65) activation and upregulation of proinflammatory factors (TNF-α, IL-1β, and IL-6). Paclitaxel also induced astrocyte activation in the spinal cord. However, 100 mg/kg icariin treatment significantly alleviated paclitaxel-induced mechanical allodynia and spinal neuroinflammation. Furthermore, icariin treatment dosage-dependently reversed paclitaxel-induced SIRT1 downregulation and H4 acetylation. EX527, a selective SIRT1 inhibitor, completely reversed icariin-induced anti-neuroinflammation and anti-allodynia effects in paclitaxel-induced neuropathic pain rats.ConclusionsThis meant that spinal SIRT1 activation was involved in icariin-induced effects in paclitaxel-induced neuropathic pain rats. Icariin could be a potential agent for the treatment of paclitaxel-induced neuropathic pain.

Modulating the endocannabinoid pathway as treatment for peripheral neuropathic pain: a selected review of preclinical studies.

Chemotherapy-induced neuropathic pain is a distressing and commonly occurring side effect of many commonly used chemotherapeutic agents, which in some cases may prevent cancer patients from being able to complete their treatment. Cannabinoid based therapies have the potential to manage or even prevent pain associated with this syndrome. Pre-clinical animal studies that investigate the modulation of the endocannabinoid system (endogenous cannabinoid pathway) are being conducted to better understand the mechanisms behind this phenomenon. Five recent pre-clinical studies identified from Medline published between 2013 and 2016 were selected for review. All studies evaluated the effect of small-molecule agonists or antagonists on components of the endocannabinoid system in rats or mice, using cisplatin or paclitax-el-induced allodynia as a model of chemotherapy-induced neuropathic pain. Activation of the cannabinoid receptor-2 (CB-2) receptor by AM1710 blocked paclitaxel-induced mechanical and cold allodynia in one study. Four studies investigating the activation of both cannabinoid receptor-1 (CB-1) and CB-2 receptors by dual-agonists (WIN55,21 and CP55,940), or by the introduction of inhibitors of endocannabinoid metabolisers (URB597, URB937, JZL184, and SA-57) showed reduction of chemotherapy-induced al-lodynia. In addition, their results suggest that anti-allodynic effects may also be mediated by additional receptors, including TRPV1 and 5-hydroxytryptamine (5-HT1A). Pre-clinical studies demon-strate that the activation of endocannabinoid CB-1 or CB-2 receptors produces physiological effects in animal models, namely the reduction of chemotherapy-induced allodynia. These studies also provide in-sight into the biological mechanism behind the therapeutic utility of cannabis compounds in managing chemotherapy-induced neuropathic pain, and provide a basis for the conduct of future clinical studies in patients of this population.

Nicotine Prevents and Reverses Paclitaxel-Induced Mechanical Allodynia in a Mouse Model of CIPN.

Chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration, continues to be a dose-limiting and debilitating side effect during and/or after cancer chemotherapy. Paclitaxel, a taxane commonly used to treat breast, lung, and ovarian cancers, causes CIPN in 59-78% of cancer patients. Novel interventions are needed due to the current lack of effective CIPN treatments. Our studies were designed to investigate whether nicotine can prevent and/or reverse paclitaxel-induced peripheral neuropathy in a mouse model of CIPN, while ensuring that nicotine will not stimulate lung tumor cell proliferation or interfere with the antitumor properties of paclitaxel. Male C57BL/6J mice received paclitaxel every other day for a total of four injections (8 mg/kg, i.p.). Acute (0.3-0.9 mg/kg, i.p.) and chronic (24 mg/kg per day, s.c.) administration of nicotine respectively reversed and prevented paclitaxel-induced mechanical allodynia. Blockade of the antinociceptive effect of nicotine with mecamylamine and methyllycaconitine suggests that the reversal of paclitaxel-induced mechanical allodynia is primarily mediated by the α7 nicotinic acetylcholine receptor subtype. Chronic nicotine treatment also prevented paclitaxel-induced intraepidermal nerve fiber loss. Notably, nicotine neither promoted proliferation of A549 and H460 non-small cell lung cancer cells nor interfered with paclitaxel-induced antitumor effects, including apoptosis. Most importantly, chronic nicotine administration did not enhance Lewis lung carcinoma tumor growth in C57BL/6J mice. These data suggest that the nicotinic acetylcholine receptor-mediated pathways may be promising drug targets for the prevention and treatment of CIPN.

Electroacupuncture alleviates chemotherapy-induced pain through inhibiting phosphorylation of spinal CaMKII in rats.

Current medical treatments for chemotherapy-induced pain (CIP) are either ineffective or have adverse side effects. Acupuncture may alleviate CIP, but its effectiveness against this condition has not been studied. Paclitaxel causes neuropathic pain in cancer patients. We evaluated the effects of electroacupuncture (EA) on paclitaxel-induced CIP in a rat model. Paclitaxel (2mg/kg) or vehicle was injected (i.p.) on alternate days of 0-6. The resulting pain was treated with 10Hz/2mA/0.4ms pulse EA for 30min at the equivalent of human acupoint GB30 (Huantiao) once every other day between days 14 and 26. For sham control, EA needles were inserted into GB30 without stimulation. Von Frey filaments with bending forces of 2-8g and 15g were used to assess mechanical allodynia and hyperalgesia, respectively, on day 13 and once every other day between 14-26days and then for 2-3weeks after EA treatment. Compared to sham control, EA significantly alleviated paclitaxel-induced mechanical allodynia and hyperalgesia, as shown by less frequent withdrawal responses to the filaments. The alleviation of allodynia/hyperalgesia lasted up to 3weeks after the EA treatment. EA significantly inhibited phosphorylation of Ca2+ /calmodulin-dependent protein kinase II (CaMKII) in the spinal cord. KN-93, a selective inhibitor of p-CaMKII, inhibited mechanical allodynia/hyperalgesia and p-CaMKII. 5-HT1A receptor antagonist blocked EA inhibition of allodynia/hyperalgesia and p-CaMKII. Electroacupuncture activates 5-HT 1A receptors in the spinal cord and inhibits p-CaMKII to alleviate both allodynia and hyperalgesia. The data support acupuncture/EA as a complementary therapy for CIP. Electroacupuncture (EA) activates spinal 5-HT1A receptors to inhibit p-CaMKII to alleviate paclitaxel-induced pain. Acupuncture/EA may be used as a complementary therapy for CIP.

Abstract 986: Targeting nicotinic acetylcholine receptors for the prevention and reversal of chemotherapy-induced peripheral neuropathy

Abstract While chemotherapy has played a significant role in the survival of cancer patients, exposure to drugs such as the taxanes, platinum-based compounds, and bortezomib often results in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, burning pain, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a taxane that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor or delay treatment. Our studies have established that the prototypical nicotinic acetylcholine receptor (nAChR) agonist, nicotine, is capable of preventing and reversing the development of paclitaxel-induced CIPN, and does not stimulate tumor growth or interfere with the cytotoxic properties of paclitaxel. Male C57BL/6J mice were treated with paclitaxel (8 mg/kg, i.p.) every other day for a total of four injections. Nicotine was either infused via a subcutaneous 7-day osmotic minipump (24 mg/kg/day) starting 48 hours prior to paclitaxel treatment, or acutely injected (0.9 mg/kg, i.p.) following paclitaxel treatment. Von Frey filament testing revealed that nicotine can prevent and reverse paclitaxel-induced mechanical allodynia; the latter effect of nicotine can be inhibited by the α7 nAChR antagonist methyllycaconitine (10 mg/kg, s.c.). Also, α7 nAChR knockout mice exhibit a greater sustained decrease in mechanical threshold after paclitaxel administration than wild-type mice, suggesting the involvement of this receptor in CIPN development and maintenance. This hypothesis was supported by the reversal of paclitaxel-induced mechanical allodynia by the α7 nAChR silent agonist R-47. While nicotine activates its receptors in the tumors cells, based on stimulation of the PI3K/Akt pathway, MTT/MTS colorimetric assays showed that concentrations of nicotine ranging from 0.1 to 10 µM fail to significantly increase the viability of A549 or H460 non-small cell lung cancer cells, murine Lewis lung carcinoma cells, or primary human lung cancer cells. Most importantly, the paclitaxel-induced decreases in H460 proliferation and LLC viability are not significantly attenuated by 1 µM nicotine. Moreover, 1 µM nicotine does not interfere with paclitaxel-induced apoptosis of A549 cells. Finally, R-47 also suppresses CIPN without increasing lung tumor cell viability and colony formation, or interfering with the cytotoxicity of paclitaxel. Our in vitro findings are supported by studies demonstrating that nicotine does not enhance tumor volume or cause premature death in tumor-bearing mice. These data suggest that nAChRs may be promising drug targets for the prevention and treatment of CIPN. Citation Format: S Lauren Kyte, Wisam Toma, Ganeshsingh Thakur, M Imad Damaj, David A. Gewirtz. Targeting nicotinic acetylcholine receptors for the prevention and reversal of chemotherapy-induced peripheral neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 986. doi:10.1158/1538-7445.AM2017-986

Involvement of Charcot-Marie-Tooth disease gene mitofusin 2 expression in paclitaxel-induced mechanical allodynia in rats.

Paclitaxel induces peripheral neuropathy, which is dose-limiting and results in loss of quality of life. Therefore, the prevention and treatment of paclitaxel-induced peripheral neuropathy are major concerns in clinical cancer therapy. However, the detailed mechanisms have not been fully elucidated. It has recently been reported that allelic variability in the Charcot-Marie-Tooth disease (CMT) genes, mitofusin 2 (MFN2), Rho guanine nucleotide exchange factor 10 (ARHGEF10), and periaxin (PRX), affected paclitaxel-induced peripheral neuropathy in clinical cases. Therefore, we hypothesized that paclitaxel may induce peripheral neuropathy due to changes in Mfn2, Arhgef10, and Prx mRNA expression. Paclitaxel (6mg/kg) was administered intraperitoneally, on two consecutive days per week for 4 weeks in rats. Paclitaxel-induced peripheral neuropathy was measured by the von Frey test and acetone test, mechanical allodynia, and cold hyperalgesia, respectively, on days 0, 3, 10, 17, and 24. Mfn2, Arhgef10, and Prx mRNA expression in the spinal cord were analyzed by qRT-PCR on days 3 and 24. Paclitaxel induced mechanical allodynia from days 17-24, but did not induce cold hyperalgesia. In addition, paclitaxel reduced Mfn2 mRNA expression, but not Arhgef10 or Prx mRNA expression, on days 3 and 24. In addition, Mfn2 mRNA level was decreased before the appearance of mechanical allodynia. The results of the present study suggest that a reduction in Mfn2 mRNA expression contributes to paclitaxel-induced mechanical allodynia.

Small molecule inhibitors of PSD95–nNOS protein–protein interactions suppress formalin-evoked Fos protein expression and nociceptive behavior in rats

Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide. NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein–protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95–nNOS protein–protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within the lumbar spinal cord of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95–nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95–nNOS protein–protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.

CXCR1/2 pathways in paclitaxel-induced neuropathic pain.

Chemotherapy-induced peripheral neuropathy (CIPN) is a type of neuropathic pain that represents a frequent and serious consequence of chemotherapy agents. Over the last years, significant progress has been achieved in elucidating the underlying pathogenesis of CIPN. The interference of taxanes with microtubule has been proposed as a mechanism that leads to altered axonal transport and to permanent neurological damages. The inflammatory process activated by chemotherapeutic agents has been considered as a potential trigger of nociceptive process in CIPN.In this study we investigated the effect of reparixin, an inhibitor of CXCR1/CXCR2, in suppressing the development of paclitaxel-induced nociception in rats. Moreover, reparixin activity in reversing the neurotoxic effects induced by paclitaxel or GRO/KC in F11 cells was also analyzed.Reparixin administered by continuous infusion ameliorated paclitaxel-induced mechanical and cold allodynia in rats. In F11 cells, reparixin was able to inhibit the increase of acetyladed α-tubulin induced both by paclitaxel and GRO/KC. The subsequent experiments were performed in order to dissect the signal transduction pathways under GRO/KC control, eventually modulated by paclitaxel and/or reparixin. To this aim we found that reparixin significantly counteracted p-FAK, p-JAK2/p-STAT3, and PI3K-p-cortactin activation induced either by paclitaxel or GRO/KC.Overall the present results have identified IL-8/CXCR1/2 pathway as a mechanism involved in paclitaxel-induced peripheral neuropathy. In particular, the obtained data suggest that the inhibition of CXCR1/2 combined with standard taxane therapy, in addition to potentiating the taxane anti-tumor activity can reduce chemotherapy-induced neurotoxicity, thus giving some insight for the development of novel treatments.

Tempol Ameliorates and Prevents Mechanical Hyperalgesia in a Rat Model of Chemotherapy-Induced Neuropathic Pain.

Chemotherapy-induced neuropathic pain is difficult to treat and prevent. Tempol decreases cellular superoxide radical levels and oxidative stress. The aims of our study were to investigate the analgesic and preventive effects of tempol on paclitaxel-induced neuropathic pain in rats and to identify the associated mechanisms of action. Neuropathic pain was induced with intraperitoneally injected paclitaxel on four alternate days in male Sprague–Dawley rats. Tempol was administered systemically as a single injection and a continuous infusion before or after the injection of paclitaxel. The mechanical threshold for allodynia, protein levels, and free radical levels were measured using von Frey filaments, Western blotting, and live cell imaging, respectively. After the rats developed neuropathic pain behavior, a single intraperitoneal injection and continuous infusion of tempol ameliorated paclitaxel-induced mechanical allodynia. Systemic infusion of tempol in the early phase of the development of pain behavior prevented the development of paclitaxel-induced pain behavior. Paclitaxel increased the levels of phosphorylated protein kinase C, phosphorylated nuclear factor κB, phosphodiesterase 4D (PDE4D), IL-1β, and monocyte chemoattractant protein-1 in the lumbar dorsal root ganglia; however, tempol decreased these levels. Paclitaxel also increased superoxide levels in a culture of primary dorsal root ganglion cells and tempol decreased these levels. In conclusion, tempol alleviates and prevents chemotherapy-induced neuropathic pain in rats by reducing the levels of inflammatory cytokines and free radicals in dorsal root ganglia.

Prophylactic Administration of Aucubin Inhibits Paclitaxel-Induced Mechanical Allodynia via the Inhibition of Endoplasmic Reticulum Stress in Peripheral Schwann Cells.

Paclitaxel is a chemotherapeutic agent that causes peripheral neuropathy as its major dose-limiting side effect. However, the peripheral neuropathy is difficult to manage. A study we recently conducted showed that repetitive administration of aucubin as a prophylactic inhibits paclitaxel-induced mechanical allodynia. However, the mechanisms underlying the anti-allodynic activity of aucubin, which is a major component of Plantaginis Semen, was unclear. In addition to mechanical allodynia, aucubin inhibited spontaneous and mechanical stimuli-induced firing in spinal dorsal horn neurons; however, catalpol, a metabolite of aucubin, did not show these effects. Furthermore, paclitaxel induced the expression of CCAAT/enhancer-binding protein homologous protein, a marker of endoplasmic reticulum (ER) stress, in the sciatic nerve and a Schwann cell line (LY-PPB6 cells); however, this effect was inhibited by aucubin. These results suggest that aucubin inhibits paclitaxel-induced mechanical allodynia through the inhibition of ER stress in peripheral Schwann cells.

CD8+ T Cells and Endogenous IL-10 Are Required for Resolution of Chemotherapy-Induced Neuropathic Pain.

Chemotherapy-induced peripheral neuropathy persists after completion of cancer treatment in a significant subset of patients, whereas others recover. Persistent neuropathy after completion of cancer treatment severely affects quality of life. We propose that understanding how neuropathy resolves will identify novel avenues for treatment. We identified a novel and critical role for CD8+ T cells and for endogenous IL-10 in recovery from paclitaxel-induced neuropathy in mice. Enhancing the capacity of CD8+ T cells to promote resolution or increasing IL-10 signaling are promising targets for novel interventions. Clinically, peripheral blood CD8+ T-cell function and/or the capacity of individuals to produce IL-10 may represent biomarkers of risk for developing persistent peripheral neuropathy after completion of cancer treatment.

Abstract 3574: Nicotine prevents chemotherapy-induced peripheral neuropathy in vivo, and fails to stimulate the growth of lung cancer cells or interfere with the effectiveness of chemotherapy in vitro

Abstract Chemotherapy has played a significant and unparalleled role in the treatment and survival of cancer patients. However, this pharmacological approach can lead to long-term symptoms of drug toxicity, including chemotherapy-induced peripheral neuropathy (CIPN), a result of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, tingling, cold sensitivity, or allodynia, and an overall decrease in quality of life. Paclitaxel (Taxol), a taxane that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59 to 78% of patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can present either during or after chemotherapy administration. Without the development of an efficacious treatment, CIPN can be a dose-limiting factor for chemotherapy or delay treatment, thereby influencing survival and quality of life. Our studies have revealed that the prototypical nicotinic acetylcholine receptor agonist, nicotine, is capable of reversing and preventing the development of paclitaxel-induced CIPN in vivo, and does not interfere with the cytotoxic properties of paclitaxel in vitro. Male C57BL/6J mice were infused with 24 mg/kg of nicotine via subcutaneous 7-day osmotic minipumps starting 48 hours prior to paclitaxel treatment, which consisted of 8 mg/kg intraperitoneal injections every other day for a total of four injections. The use of von Frey filament testing revealed that nicotine dose-dependently reverses and prevents paclitaxel-induced mechanical allodynia. In regards to the in vitro studies, MTT and MTS colorimetric assays showed that concentrations of nicotine ranging from 0.1 to 10 μM fail to significantly increase or decrease the viability of A549, H460, Lewis lung carcinoma, or human explant lung cancer cells. Similar results were obtained in ovarian cancer cell lines, where no significant difference in cell growth was observed following nicotine treatment of SKOV-3/DDP and OVCAR-3 cells. Most importantly, the paclitaxel-induced decreases in both H460 cell viability and growth were not significantly attenuated by nicotine. Moreover, 1 μM nicotine did not interfere with paclitaxel-induced sub-G1 DNA content or apoptosis of A549 cells. These findings suggest that nicotinic acetylcholine receptors may be promising drug targets for the prevention and treatment of CIPN. Citation Format: Sarah L. Kyte, Wisam Toma, M I. Damaj, Xianjun Fang, David A. Gewirtz. Nicotine prevents chemotherapy-induced peripheral neuropathy in vivo, and fails to stimulate the growth of lung cancer cells or interfere with the effectiveness of chemotherapy in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3574.