Jill C. Fehrenbacher, PhD - US grants

DESCRIPTION (provided by applicant): Epidemiological studies indicate a higher prevalence of painful disorders in females than in males. Although gender-related differences are numerous, considerable evidence implicates estrogens as critical factors in sex-dependent differences in pain, especially in conditions where inflammation is present (see review by Fillingim and Ness, 2000). Numerous studies have investigated the genomic effects of estradiol on nociceptive responses;however, the discovery that estradiol can activate intracellular signaling pathways through non-genomic mechanisms opens up the possibility that the steroid hormone might also induce posttranslational changes of ion channels or receptors in trigeminal neurons to alter the sensitivity of neurons to thermal, mechanical, or chemical stimuli. Our preliminary data indicate that both estradiol and G-1, a specific agonist for the GPR30, elicit increases in inflammation-induced orofacial thermal hyperalgesia in female rats, but have no effect on thermal sensitivity in saline-injected animals. Likewise, acute treatment with estradiol augments the stimulated release of the nociceptive neuropeptide iCGRP from inflamed peripheral tissues, but has no effect on release from noninflamed biopsies. Inflammation induced by injection of the vibrissal pad with CFA augments the local concentration of estradiol, suggesting that this putative inflammatory mediator is present in vivo at relatively high concentrations around the nerve terminals. Finally, we demonstrate that not only are estradiol levels increased with inflammation, but the GPR30 receptor expression within the trigeminal ganglia is also increased subsequent to vibrissal pad inflammation in trigeminal ganglia isolated from female (OVX + E2 replacement) rats. These data suggest that inflammation and estrogen may interact to facilitate nociceptive signaling and contribute to increased pain perception in females. We will examine the expression and localization of the receptor in trigeminal ganglia, infraorbital nerve, and vibrissal pad skin from control and inflamed rats using both mRNA and protein as endpoints. Furthermore, we will ascertain whether the GPR30 has a functional role in modulating nociceptor sensitivity by means of in vivo behavioral studies and in vitro studies examining the sensitivity of trigeminal neurons. Because the pharmacology of the GPR30 distinguishes it from other estrogen receptors, the findings here may provide an opportunity for the development of therapeutics which can selectively reverse GPR30 activity to attenuate inflammatory pain without affecting the actions of estrogens at the classical receptors. PUBLIC HEALTH RELEVANCE This study will examine the contribution of a newly discovered receptor for estrogens to differences in pain between males and females. During inflammation, there is an increase in the amount of estrogens and of these novel receptors in tissues from female rats. We will examine whether these increases contribute to enhanced pain during inflammation in females.

? DESCRIPTION (provided by applicant): Although inflammation-induced peripheral sensitization (i.e. increased sensitivity of sensory neurons) can resolve as an injury heals, under pathological conditions this sensitization is maintained and contributes to chronic inflammatory pain. Studies of the cellular mechanisms mediating this maintenance of peripheral sensitization have focused on transcriptional changes that alter protein expression or post-translational modulation of various proteins, especially ion channels. To date, however, these studies have not resulted in new therapeutic approaches for treating chronic inflammatory pain. For this R21 application, we propose a novel mechanism for maintaining sensitization of sensory neurons, i.e. inflammation-induced DNA damage. This damage could result in an alteration in the phenotype of neurons from normal to the sensitized state. Recent studies performed in our laboratory provide support for examining this mechanism, since we have shown that augmenting DNA repair mechanism reverses toxicity in sensory neurons induced by cancer therapies. Furthermore, our preliminary data suggest that inflammation and the inflammatory mediators LPS, MCP-1, and, PGE2, can produce DNA damage in sensory neurons. Thus, we hypothesize that inflammation and inflammatory mediators produce oxidative DNA damage in sensory neurons that contributes to hypersensitivity and that augmenting the base excision repair pathway protects neurons from this damage and thus attenuates the enhanced excitability. To test this hypothesis we propose two specific aims. In studies for the first aim, w will determine whether CFA-induced inflammation or long-term exposure to inflammatory mediators (LPS, MCP-1 or PGE2) in isolated sensory neurons produces reactive oxygen species (ROS) and DNA damage in sensory neurons. We also will determine whether antioxidants or increasing APE1 repair activity (by overexpressing it in sensory neurons) prevents or reverses the DNA damage. In aim 2, we will determine whether augmenting APE1 activity with overexpression in sensory neurons prevents or reverses peripheral sensitization induced by CFA injection into the rat hindpaw or by long-term exposure to inflammatory mediators (LPS, MCP-1 or PGE2) in isolated sensory neurons. If we demonstrate that DNA repair reverses peripheral sensitization that occurs during inflammation, our findings have important implications for elucidating a novel therapeutic target for treating chronic pain.

PROJECT SUMMARY/ABSTRACT Chemotherapy-induced peripheral neuropathy (CIPN) is a major side effect of many efficacious anticancer drugs, including platinum drugs, taxanes, proteasome inhibitors, vinca alkaloids, epothilones, and immunomodulators. Their neurotoxic side effects can be so debilitating that treatment may need to be reduced or stopped. However, unlike other major side effects of chemotherapy (e.g. nausea, hair loss, bone marrow failure), no standard, effective treatments exist to prevent or reverse CIPN. This is largely because the cellular mechanisms for CIPN have not been identified and the symptoms of CIPN including numbness, decreased blood flow to extremities, loss of proprioception, loss of tendon reflexes, pain, allodynia, and/or increased sensitivity to cold vary greatly in patients. Because CIPN is debilitating and may be irreversible, identification of key targets to prevent neurotoxicity without compromising the tumor-killing effects of anticancer drugs is critical in developing a first-in- class therapeutic that can directly affect a patient's ability to receive optimal treatment. Our previous studies examining the hypothesis that DNA damage of sensory neurons contributes to CIPN laid the foundation for the proposed work, which is poised to develop a drug candidate. We demonstrated that reducing DNA base excision repair (BER) activity by reducing expression of the apurinic/apyrimidinic endonuclease/redox factor (APE1) augmented the neurotoxicity produced by anticancer treatment, whereas supplementing APE1's repair activity attenuated the neurotoxicity. It is likely that, in non-dividing cells like neurons, DNA damage could alter the function of sensory neurons in ways that manifest as the symptoms observed in CIPN. Consequently, DNA repair would be critical for proper genetic expression of the right types and amounts of proteins, a crucial element of genomic maintenance. For the proposed studies, we will examine whether augmenting APE1 repair activity in vivo will prevent chemotherapy-induced alterations in sensory neuronal function (manifested as CIPN) without jeopardizing the cancer treatment. Using tumor bearing mice, we will examine whether a small molecule (E3330) which was identified to enhance APE1's DNA repair function in neurons can prevent (aim 1) or reverse (aim 2) DNA damage and alterations in the function of sensory neurons caused by cisplatin, oxaliplatin or carboplatin. Furthermore, we will examine whether the small molecule (E3330) will compromise the anticancer efficacy of the platinum drugs by examining DNA damage and tumor survival following treatment (aim 3). Because E3330 has been found to act as a single agent and in combination with other cancer therapeutic drugs to decrease tumor cell growth, this molecule has the potential to offer a ?win-win? scenario; block tumor cell growth while protecting against neuronal dysfunction. Additionally, E3330 will enter a phase 1 clinical trial for solid tumors followed by phase 1b/phase 2 trials for various indications that include platinums in their SOC (e.g. colon, pancreatic). Therefore, it requires further preclinical study using an in vivo paradigm to demonstrate effectiveness in the context of neuronal protection and CIPN models.

PROJECT SUMMARY / ABSTRACT As cancer treatments continue to become more effective with increases in patient survival, we are recognizing clinical consequences of therapy that negatively impact the course of therapy and the quality of life of patients and survivors. Of major clinical significance is chemotherapy-induced peripheral neuropathy (CIPN), which can be severe enough to necessitate reducing or stopping treatment and thus can compromise therapy. Furthermore, CIPN can continue long after therapy is stopped and is irreversible in a significant number of patients. Compounding this problem is a lack of effective treatments available to prevent or reverse CIPN. The lack of effective prevention or treatment for CIPN is a direct consequence of not understanding the mechanisms that cause the neurotoxicity. As such, examining the provocative question of ?What are the molecular and/or cellular mechanisms that underlie the development of cancer therapy-induced severe adverse sequelae?? will be addressed in our studies using animal models and an array of endpoints measuring changes in sensory neuronal function which parallel clinical symptoms of CIPN. Most CIPN develops over time with few if any acute symptoms after initial therapy, but increases in severity with continued therapy. The delay in onset of neuropathy suggests that there is an aggregate effect of drugs over time that results in a long-term alteration in neuronal function. Consequently, it is important to examine the mechanisms by which cumulative exposure to chemotherapeutics might result in neurotoxicity. Previously, we demonstrated that reducing the activity of the DNA base excision repair (BER) pathway by reducing expression of the apurinic/apyrimidinic endonuclease/redox factor (APE1/Ref-1 or APE1) exacerbated neurotoxicity produced by anticancer treatment, whereas augmenting the repair activity of APE1 attenuated the neurotoxicity. These data support the notion that DNA damage is a critical mechanism by which the function of sensory neurons is altered by chemotherapeutics. Indeed, it is likely that in post-mitotic cells (e.g. neurons) DNA damage could result in abnormal protein production that is maintained unless the DNA damage is repaired, reversing the aberrant transcriptional effects of the neurotoxins. Therefore, we hypothesize that APE1 is a critical protein for protecting neurons from cancer therapies and that augmenting APE1 DNA repair activity will prevent and reverse chemotherapy-induced alterations in sensory neuronal function. Furthermore, fully understanding the DNA damage and the mechanisms by which the BER pathway reverses this damage will lead to the identification of novel targets for CIPN prevention or therapy. To address these hypotheses, we propose three aims which will determine whether augmenting APE1 repair activity in vivo prevents or reverses DNA damage in sensory neurons and the subsequent alterations in sensory neuronal function caused by anticancer drug administration as well as determining the mechanisms mediating APE1-induced neuroprotection of isolated sensory neurons.