Дисертації з теми "Bone cancer pain"

Pain from bone metastases is multifaceted with clinical descriptors including ongoing pain, hypersensitivity to external stimuli and intermittent episodes of breakthrough pain characterized as a sudden and abrupt onset of severe pain on a background of well-controlled pain. Moreover, cancer-induced bone pain remains inadequately managed due to a myriad of side effects associated with the current pain relieving regimens, which primarily rely on administration of opiates. Despite advances made in cancer therapeutics, these patients experience an inferior quality of life with incapacitating pain with limited daily activities. Development of long-term novel, non-opiate mechanism-based therapeutics with limited side effects is considered beneficial in elevating the patients' quality of life. First part of this dissertation encompasses the role of p38 MAPK in a mouse model of cancer-induced bone pain in which breast cancer cells were injected and sealed into the femur. Our data demonstrated that both acute and prolonged inhibition of p38 MAPK blocked cancer-induced spontaneous pain but had no effect on the evoked pain indicating important differences in mechanisms mediating ongoing pain as opposed to evoked pain. Undermanaged control of breakthrough pain is attributed to poor understanding of underlying mechanisms and how they may differ from ongoing pain due, in part, to lack of a pre-clinical model in which these mechanisms can be studied. We have established a rat model of cancer-induced bone pain to examine ongoing pain and pain relief using conditioned place preference paradigm as well as breakthrough pain using palpation-induced conditioned place aversion. We have shown that while peripheral afferent input from the tumor-bearing tibia mediates cancer-induced ongoing pain and initiation of breakthrough pain, it does not contribute to the maintenance of breakthrough pain. These data suggest that molecular targets mediating these two mechanisms may be different. This hypothesis was confirmed by our findings in this model that acute blockade of interleukin-6 blocked movement-evoked breakthrough pain in tumor-bearing rats, but failed to block tumor-induced ongoing pain. Hence, we provide a platform to manipulate treatments that can be given alone or in combination with opiates in such a way that patients receive adequate control of breakthrough pain.

Cancer-induced bone pain (CIBP), due to bony metastases, is a major clinical problem, significantly reducing quality of life in cancer patients. Current therapies often provide inadequate analgesia or unacceptable side effects. The aim of this thesis was to characterise behaviours of a preclinical model of CIBP and test novel analgesic interventions in this model. A secondary aim was to investigate the involvement of the N-methyl-D-Aspartate (NMDA) receptors and TRP channels (TRPM8, TRPV1 and TRPV4) in CIBP. Investigation of CIBP in a preclinical model may lead to better pain management in CIBP patients. The results presented here demonstrate that this model of CIBP develops behaviours that may be indicative of mechanical allodynia, thermal sensitivity, movement-evoked pain, ongoing pain and spontaneous pain. This suggests that this model reflects the clinical condition of CIBP, where patients suffer from constant background pain with spontaneous and movement-related breakthrough pain. In this study it was found that radiotherapy significantly attenuated movement-evoked pain and thermal sensitivity to 20°C and 40°C. XRT also significantly reduced anxiety and risk assessment behaviours (grooming behaviour and number of protected stretch attends) compared to untreated CIBP. Duloxetine attenuated CIBP-induced mechanical allodynia, thermal sensitivity to 40°C and movement-evoked pain, whereas S,S-reboxetine attenuated thermal sensitivity to 40°C but did not effect CIBP-induced mechanical allodynia or movement-evoked pain. In addition, CB 65 attenuated movement-evoked pain and thermal sensitivity to 40°C. A single dose of gabapentin did not attenuate CIBP-induced mechanical allodynia, thermal sensitivity to 40°C or movement-evoked pain. These studies confirm that the CIBP model shows characteristics and pharmacological sensitivities consistent with known and predicted mechanisms and validate it as a useful model for assessing potential new treatments proposed for use in patients. Behavioural results suggest that NMDA receptors containing the NR2A subunit are involved in CIBP-induced movement-evoked pain. This suggests that NR2A antagonists may be useful for treating CIBP-induced movement-evoked pain. Additionally, results show that there is increased expression of NR2A in the laminae I, II and III in the dorsal horn of the spinal cord. XRT treated animals also showed increased expression of NR2A in laminae I and II. The selective involvement of NR2A in CIBP is different to other chronic pain states, for example, neuropathic pain states that appear to involve the NR2B subunit. The TRPV1 antagonist AMG 9810 did not attenuate mechanical allodynia, thermal sensitivity to 40°C or movement-evoked pain. Interestingly, the TRPM8 agonist icilin attenuated movement-evoked pain, which suggests that icilin might be useful in the treatment of movement-evoked pain. The TRPV4 antagonist RN 1734 attenuated mechanical allodynia, thermal sensitivity to 40°C and movement-evoked pain in CIBP. This suggests RN 1734 may be useful in the treatment of mechanical allodynia, thermal sensitivity to 40°C and movement-evoked pain in CIBP. Results show that the expression of TRPV4 is increased in DRG ipsilateral to the cancerbearing tibia. In conclusion, these results show that the preclinical model of CIBP investigated in this thesis is suitable for testing novel analgesic interventions. This thesis identified some useful targets for the analgesic treatment of CIBP and results suggest that many different mechanisms contribute to CIBP. A point to consider is that any robust effective treatment may need to target all (or at least several) of these mechanisms.

The most common cancer types have a high likelihood of metastasizing to the bone and can cause cancer-induced bone pain (CIBP). Current therapeutic options do not offer proper management and thus CIBP can severely affect a patient's quality of life. Dysregulation of the excitatory neurotransmitter, glutamate, may be involved in the complex and multifaceted mechanisms of CIBP. Because glutamatergic signaling promotes pain, a local rise in glutamate in the bone-tumor microenvironment may contribute to CIBP. Glutamate levels are regulated in part by the cystine/glutamate antiporter, system xc⁻. System xc⁻ is known to be expressed by many different cancer cell types. It functions by transporting cystine into cells and in return releasing glutamate into the extracellular space. Elevated glutamate levels driven by the upregulated expression of this antiporter may contribute to CIBP. Here we demonstrate that system xc⁻ is expressed on a spontaneously occurring murine mammary tumor cell line (66.1) and that treatment of these cells with the established inhibitor and anti-inflammatory agent, sulfasalazine, decreases glutamate secretion in a time and dose-dependent manner. Furthermore, in a novel model of breast CIBP, systemic sulfasalazine treatment not only reduces glutamate levels within the femur, but also significantly attenuates CIBP behaviors. Studies utilized 66.1 cells implanted into the femur intramedullary space of immunocompetent mice. Measurements of spontaneous and evoked pain were made 7 and 10 days post cancer cell inoculation. Systemic administration of sulfasalazine for 4 days (on days 7-10) significantly reduced spontaneous pain-related behaviors and glutamate in femur extrudate as compared to vehicle treated controls. In summary, we demonstrate that pharmacological inhibition of the system xc⁻ transporter attenuates CIBP related behaviors in mice. These data support a role for system xc⁻ in CIBP and validate it as an analgesic target. Further research is warranted to evaluate the potential repurposing of sulfasalazine as an antinociceptive agent for patients with CIBP.

Pain is the most feared symptom of cancer and can impact patients' lives more than the cancer itself. Despite improvements in cancer prevention and detection, pain is often the first sign of cancer, with an estimated 70-75% of advanced stage cancer patients presenting with skeletal metastases. Cancer metastasis to the bone is associated with persistent pain that increases in intensity over time. Current treatments follow the World Health Organization (WHO) analgesic ladder for cancer pain management suggesting non-steroidal anti-inflammatory drugs (NSAIDs) for mild to moderate pain and opioids for moderate to severe pain. However, estimates indicate as many as 50-80% of cancer patients worldwide receive inadequate pain management. Moreover, opioid doses required for these patients are associated with adverse side effects further diminishing quality of life. Development of improved non-opioid therapies is dependent on increased understanding of mechanisms driving cancer pain and its relief. The objective of this dissertation was to characterize a rat model of cancer-induced bone pain, to develop approaches to measure both ongoing and breakthrough pain and to investigate the contribution of underlying inflammatory mechanisms to pain, bone destruction and bone remodeling. Using female Fischer F344/NhSD rats, histocompatible MAT B III mammary adenocarcinoma cells were sealed into the intramedullary space of the right rear tibia for a time course of 13 days. Ongoing pain was characterized based on the WHO 3-step ladder for pain management utilizing novel behavioral and neurochemical assays. Morphine and peripheral nerve block were sufficient to control ongoing pain, whereas NSAID treatment failed to provide pain relief. Cancer-bearing rats selectively displayed movement-induced breakthrough pain to a background of morphine-controlled ongoing pain. Furthermore, we determined that breakthrough pain is initiated, but not maintained, by peripheral afferent input from the tumor-bearing tibia using lidocaine administration prior to or following movement. For the final part of this study, we investigated the role of transient receptor potential vanilloid 1 (TRPV1) and interleukin-6 (IL-6) blockade, as these have been shown to be important mediators in animal models CIBP. Acute blockade of TRPV1 channels by AMG9810 selectively reversed inflammatory-induced pain, but failed to control evoked or ongoing CIBP. Acute blockade of interleukin-6 signaling by TB-2-081, an IL-6 receptor antagonist, successfully reversed evoke pain responses, but like AMG9810, failed to control ongoing pain. Sustained administration of TB-2-081 reversed cancer-induced tactile hypersensitivity and tumor-induced bone remodeling of the tibia. Further in vitro analysis revealed TB-2-081 functions by inhibiting the Jak/STAT cascade on both tumor cells and osteoblasts, suggesting that blockade of IL-6 signaling can effectively modulate the bone microenvironment to reduce tumor burden and pain. Combined, our data introduce a rat model of breast cancer bone metastasis, in which the underlying mechanisms of ongoing and breakthrough CIBP can be effectively studied. From this, novel therapeutic agents can be developed and investigated to help improve quality of life in patients suffering from this disease.

BACKGROUND:Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs.RESULTS:In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden.CONCLUSIONS:Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.

Background: Cancer-induced bone pain (CIBP) is a major clinical problem and a considerable therapeutic challenge. Radiotherapy (XRT) is the gold standard treatment for CIBP, but only half of patients achieve adequate analgesia. Patients have increased morbidity, anxiety and depression and reduced performance and quality of life. Despite these issues, CIBP is a neglected area of clinical research. Animal models have increased current knowledge of the pathophysiology, but clinical research is needed to translate these findings from bench to bedside. Also lacking is a standardised, comprehensive tool to assess CIBP and clinical biomarkers to predict analgesic response to treatment. Aims: 1) To summarise current understanding of the pathophysiology, epidemiology, clinical features, assessment and management of malignant bone disease and CIBP. 2) To characterise CIBP using quantitative sensory testing as a measure of altered sensory processing. 3) To establish systematically the sensory, cognitive, affective and functional components of CIBP to develop a comprehensive assessment tool. 4) To explore whether clinical biomarkers can be developed to aid prediction of response to treatment for CIBP, in particular XRT. Results: Assessment of CIBP, characterising the multi-dimensional components, was clinically practical and acceptable to patients. Using objective measures of function, patients with CIBP were a frailer, less active population compared with healthy adults. Prior to treatment, pain was severe with relationships seen between CIBP and sensation, mood, fear avoidance, catastrophizing and function. Patients who dropped out prior to follow up were significantly less active, with higher levels of depression and fear avoidance behaviour. Sixty-nine percent of evaluable patients who completed two assessments (48% of all patients on an intention-to-treat basis), achieved an analgesic response to XRT for CIBP, as defined as an improvement of ≥ 30% in the Brief Pain Inventory worst pain score two months after treatment. All dimensions of pain, fear avoidance and catastrophizing improved significantly in responders, but not non-responders. Anxiety, depression and emotional distress fell by a greater degree in responders. No objective functional differences were seen after XRT. Clear evidence of altered sensory processing was seen at the site of CIBP with abnormalities in both mechanical and thermal parameters. XRT resulted in alterations in response to evoked stimuli in responders with a greater number of patients in whom sensation normalised after XRT compared with non-responders. Patients with a combination of altered sensation to thermal, pin prick and wind up stimuli showed the largest reduction in worst pain after XRT. Abnormal cool sensation at the site of CIBP was an independent predictor of analgesic response to treatment. Conclusion: Strong associations exist between CIBP, sensation, cognition, mood and function. Multi-dimensional assessment should be performed to improve quality of life. Translational research to provide targeted individualised treatment should be high on the research agenda. Future work should focus on thermal sensory processing as a potential clinical biomarker of response to palliative XRT for CIBP.

Metastatic bone cancer causes severe pain that is primarily treated with opioids. A recently developed model of bone cancer pain was used to evaluate the effects of sustained morphine treatment. In cancer-treated mice, morphine enhanced spontaneous and evoked pain; these effects were dose-dependent and naloxone-sensitive. SP and CGRP positive DRG cells did not differ between sarcoma or control mice, but were increased following morphine in both groups. Morphine increased ATF-3 expression only in DRG cells of sarcoma mice. Morphine did not alter tumor growth in vitro or in vivo but increased sarcoma-induced bone destruction and incidence of spontaneous fracture in a dose- and naloxone-sensitive manner. Morphine increased osteoclast activity suggesting enhancement of sarcoma-induced osteolysis. Thus, morphine treatment may "add-on" additional mechanisms of pain beyond those induced by sarcoma. Despite the potential clinical significance, the exact mechanisms of opioid-induced hypersensitivity remain unknown. The vanilloid 1 receptor (TRPV1) is a molecular integrator of noxious stimuli. Sustained morphine elicited thermal and tactile hypersensitivity in WT, but not TRPV1 KO mice. Sustained morphine enhanced capsaicin-induced flinching and plasma extravasation in rats, indicating increased activity of these receptors in the periphery. Immunohistochemical studies indicate increase in TRPV1 expression in DRG and sciatic nerve, but not spinal cord, suggesting increased trafficking of TRPV1 channel to the periphery. Possible mechanisms of this enhanced expression and function of TRPV1 channels is activation of p38 MAPK. Sustained intrathecal infusion of p38 MAPK inhibitor prevents morphine-induced hypersensitivity and enhanced capsaicin-induced flinching, indicating the role of p38MAPK in the development of morphine-induced pain, possibly through sensitization of TRPV1 receptors. Acute administration of p38 MAPK inhibitor reversed morphine-induced pain suggesting the importance of p38 MAPK in the maintenance of morphine-induced hypersensitivity, likely through activation of TRPV1 channel. Sustained morphine also up-regulates NGF content in skin, which is then transported to DRG neurons where phosporilation of p38MAPK takes place. Single injection of anti-NGF peptibody blocked the development of morphine-induced hypersensitivity, enhanced capsaicin-induced flinching and plasma extravasation. Co-treatment with these compounds blocks the development of morphine-induced hyperalgesia and may optimize treatment of chronic pain states, like bone cancer pain.

Many common cancers, including breast, prostate and lung cancers, have a propensity to metastasize to bone. Although these cancers go undetected in their native tissues, bone metastases often produce excruciating pain, the etiology of which is poorly understood. Cancer-induced bone pain (CIBP) is not well-controlled with existing medications, severely compromising patient quality of life. While CIBP is multifaceted, increased level of the excitatory neurotransmitter glutamate in the bone-tumor microenvironment may contribute to the pain state. Here, we demonstrate for the first time a relationship between reactive oxygen/nitrogen species, glutamate in the bone-tumor microenvironment and pain behaviors. The murine mammary adenocarcinoma cell line 66.1 is found to release glutamate via the cystine/glutamate antiporter system xc⁻. In a syngeneic model of breast CIBP in which 66.1 cells are inoculated into the femur intramedullary space, administration of sulfasalazine, an established system xc⁻ inhibitor and anti-inflammatory agent, reduces femur glutamate level and attenuates CIBP-related behaviors. Peroxynitrite, a reactive nitrogen species known to be generated in breast tumors, is shown to drive 66.1 system xc⁻ functional expression and tumor cell glutamate release. The elimination of peroxynitrite with the redox modulators FeTMPyP or SRI10 not only modulates tumor cell system xc⁻ functional expression in vitro and in vivo, significantly altering glutamate levels, but also assuages CIBP. In sum, we demonstrate that pharmacological inhibition of system xc⁻ transport attenuates CIBP-related behaviors. These data support a role for tumor-derived glutamate in CIBP and validate system xc⁻ an analgesic target in this pain state.

Whilst pain serves a physiological function, chronic diseases such as osteoarthritis and cancer of the bone involve central neurones and peripheral nociceptors becoming sensitised to heighten or extend the pain experience temporally, which is detrimental to the quality of life. Mechanisms underlying central sensitization of neurones are examined in this thesis in two rodent models of pain: osteoarthritis (OA) and cancer-induced bone pain (CIBP) using pharmacology, mapping of receptive field size for lamina I neurones in OA animals and TENS. Pharmacological studies focused on NMDA and P2X3 and P2X2/3 receptors using ketamine, ifenprodil and a novel agent: AF-353. Ketamine reduced the neuronal responses to noxious stimuli in OA animals to levels observed in naïve animals. However effects were significantly stronger in CIBP animals where doses 10-fold lower produced the same effect suggesting post-synaptic NMDA receptors have a significant role in the maintenance of CIBP. Ifenprodil, which acts at NR2B subunit containing receptors, which are presynaptically located and may thus control release of neurotransmitters from the afferent terminal, more strongly inhibited electrical stimuli evoked responses in OA animals. Inhibition of natural stimuli evoked neuronal responses were similar in both models suggesting the importance of primary afferent drive in maintenance of central sensitization in OA and CIBP. AF-353 which inhibits the actions of ATP on pre-synaptically located P2X3 and P2X2/3 receptors was found to profoundly inhibit neuronal responses to noxious thermal stimuli in CIBP suggesting that thermal-stimuli induced hyperalgesia has a central component driven by the purinergic system. Finally, TENS a nonpharmacological intervention was found to have no significant effect on evoked responses of lamina V neurones in CIBP animals questioning its suitability in managing chronic pain. Differing pharmacology appears to contribute to the maintenance of central sensitization in OA and CIBP suggesting that more targeted therapy may be more clinically appropriate.

Many cancerous solid tumors, such as breast, metastasize to the bone and induce bone pain (CIBP). CIBP is often severe due to an enhanced inflammation in the bone microenvironment, rapid bone degradation, and disease progression. Opioids are prescribed to manage this pain but may enhance bone loss and induce analgesic tolerance, further compromising patient quality of life. Angiotensin-(1-7) (Ang-(1-7)), a product of Angiotensin II cleavage by Angiotensin-Converting Enzyme 2, binds and activates the Mas receptor, a G(q/11)-protein coupled receptor (MasR). Angiotensin-(1-7)/MasR activation modulates the inflammatory signaling after acute tissue insult resulting in altered production of reactive oxygen species and further inflammatory response, yet no studies have investigated whether Ang-(1-7)/MasR play a role in CIBP. Therefore, we hypothesized that Ang-(1-7), acting at the MasR, inhibits CIBP by reducing proinflammatory cytokines/chemokines in an immunocompetent murine model of breast cancer-induced bone pain. Murine breast cancer cells, 66.1, were implanted into the femur of mice and allowed to establish and proliferate for 7 days. Cancer inoculation increased spontaneous and evoked pain behaviors by day 7 that were significantly reduced after a single injection of Ang-(1-7) and after chronic administration (p<0.01); co-administration of a MasR antagonist reversed this reduction. Cytokine/chemokine profiling of bone marrow extrudates from Ang-(1-7) mice of the cancer studies revealed significant increases in the relative expression of C5/C5a, IL-1ra, IL-16, M-CSF, MIG, with concomitant decreases in the expression of MIP-1α compared to vehicle controls (p<0.05). Ang-(1-7) administration normalized levels of IL-1ra and MIP-1α in the bone-tumor micro-environment. Data here suggest that modifying the cancer-induced inflammatory response in the bone-tumor micro-environment with Ang-(1-7) through the Mas receptor significantly attenuates CIBP, which may be an alternative therapeutic strategy for the nearly 70% of advanced stage cancer patients who experience excruciating pain.

Many common cancers, including breast, prostate and lung, have a predilection to metastasize to the bone, bringing not only bone destruction but severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain and drug-induced side effects; furthermore, recent findings suggest that patients are severely under-treated for their cancer pain. Strong analgesics, namely opiates, are the first-line therapy in alleviating cancer-related pain despite severe side effects including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which can bring highly undesirable side-effects including nephrotoxicity and osteonecrosis of the jaw. Thus novel therapeutics are needed to treat the pain of metastatic cancer patients. Animal models of cancer-induced bone pain (CIBP) have revealed that the neurochemistry of cancer has distinctive features from other chronic pain states. These include factors released from the cancer cells, tumor activated macrophages and increased osteoclast degredation of bone within the bone microenvironment, all acting to sensitize free nerve endings.One possibility of inhibiting cancer-mediated pain inducing factors includes agonism of the Cannabinoid 2 receptor agonists. Cannabinoid CB2 receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB2 agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB1/CB2 agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Osteolytic sarcoma within the femur produced spontaneous and touch evoked behavioral signs of pain within the tumor-bearing limb. The systemic administration of AM1241 both acutely and for 7 days significantly attenuated spontaneous and evoked pain in the inoculated limb. Sustained AM1241 significantly reduced bone loss and decreased the incidence of cancer-induced bone fractures. In addition, CB2 agonists significantly reduce breast cancer-induced bone pain, bone loss and breast cancer proliferation in part via cytokine/chemokine suppression. Studies utilized the spontaneously-occurring syngenic murine mammary cell line (66.1) implanted and sealed into the femur intramedullary space. Measurements were made of spontaneous pain, bone loss and cancer proliferation. The central and systemic administration of the CB2 agonist JWH015 for seven days significantly attenuates pain. Pharmacological characterization with cannabinoid 1 and 2 antagonists demonstrates that the effects JWH015 on pain were mediated by the CB2 receptor. We and others have found that bone induced cancer pain increases the expression of GFAP and Iba1 in the lumbar spinal cord which are markers of astrocytes and microglia respectively, compared to control animals. After administration of JWH015 (i.t), the release of spinal pro-inflammatory cytokines, IL-6 and TNFá, are reduced suggesting that modulation of glial cytokines may be one mechanism by which CB2 agonists can attenuate pain centrally. On the other hand, systemic administration of JWH015 reduces cancer-induced elevation of cytokines in the tumor microenvironment, suggesting a mechanism by which CB2 agonist is attenuating pain peripherally. Additionally, systemic administration improves bone modification, as demonstrated via micro-computed tomography and bone serum markers while decreasing femoral tumor burden. In vitro, JWH015 reduced cancer cell proliferation and other inflammatory mediators shown to promote pain, bone loss and proliferation. These results suggest CB2 agonists as a novel treatment for breast cancer-induced bone pain, where disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone-pain and increased survival without the major side effects of current therapeutic options. Another future therapeutic option for metastatic bone cancer pain may include cathepsin inhibitors. Cysteine cathepsins (B, C, F, H, K, L, O, L2/V, W, X/Z) are highly expressed in many human cancers and have been associated with poor patient prognosis. In the RIP1-Tag2 transgenic model of pancreatic cancer, mice treated with VBY-825, a reversible inhibitor of cathepsins S, B, V, L, K showed a significant reduction in tumor incidence and growth. Here we demonstrate the cathepsin inhibitor VBY-825 reduces cancer-induced pain behaviors. Additionally, tumor bearing animals treated with VBY-825 demonstrate a reduction in bone resorption, possibly mediated through a reduction in osteoclast activity. These results indicate that a cathepsin inhibitor targeting multiple cathepsins, such as VBY-825, could be a novel therapeutic for bone metastases.Part of the failure to palliate cancer pain is due to a poor understanding of the etiology of cancer pain. Preclinical studies have just begun to scratch the surface on how such cancers may interact with the bone microenvironment to result in pain and bone loss. Further studies are desperately needed at both the preclinical and clinical level to determine the unique molecular profile of cancer pain that may lead to the development of superior therapeutics for CIBP. The studies presented herein provide preclinical evidence that warrant the investigation of these compounds in the clinic as treatment for cancer-induced bone pain.

Cancer-induced bone pain is described as persistent, dull pain that increases in intensity with disease progression. Cancer patients experience ongoing pain and breakthrough pain, defined as transient periods of moderate to severe pain that break through ongoing pain medication. A better understanding of differences in mechanisms driving these aspects of cancer-induced pain may lead to development of more effective pain therapeutics with fewer side effects than currently used treatments. To address this issue, we have developed a preclinical rat model where breast cancer cells are injected in the right tibia of female rats, allowing for cancer growth, metastases, and subsequent bone degradation as revealed by radiographic analyses. To measure ongoing pain, we adapted the conditioned placed preference (CPP) paradigm, which uses negative reinforcement to determine the rewarding aspects of pain alleviating manipulations. A conditioned placed avoidance (CPA) paradigm was used to measure movement-induced incident pain. Our data indicate that peripheral nerve block alleviates cancer-induced ongoing pain, revealing that afferent input maintains ongoing pain. Palpation of the tumor-bearing limb produces CPA, likely reflecting movement-induced breakthrough pain as observed in cancer patients. Such results contribute to the overall goal of developing better therapeutics, thereby improving the quality of life for cancer patients.

Metastatic bone cancer originates from breast malignancies causing severe pain and bone destruction in patients. Amongst the novel therapies under clinical development for the treatment of bone metastases are cathepsin inhibitors. Cysteine cathepsins (B, C, F, H, K, L, O, L2/V, W, X/Z) are highly expressed in many human cancers and have been associated with poor patient prognosis. In the RIP1-Tag2 transgenic model of pancreatic cancer, mice treated with VBY-825, reversible inhibitor of cathepsins S, B, V, L, K showed a significant reduction in tumor incidence and growth. In this study, we evaluate the efficacy of the cathepsin inhibitor, VBY-825 as treatment for cancer-induced bone pain. Breast cancer cells, 66.1, were injected within the intramedullary space of the femurs of female mice. After seven days of inoculation, the animals were treated with VBY-825 or vehicle (5% dextrose) subcutaneously for seven days. Spontaneous pain behaviors were significantly attenuated in cancer-induced mice treated with VBY-825, compared to vehicle treated animals. Additionally, cancer-induced animals treated with VBY-825 demonstrated both an improvement in bone integrity and reduction of tumor burden. These results indicate that a cathepsin inhibitor targeting multiple cathepsins, such as VBY-825, could be a novel therapeutic for bone metastases.

Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.

Cancer-induced bone pain (CIBP), resulting from primary sarcoma in the bone or metastatic spread to bone, is a common complaint among cancer patients and a major clinical problem. Recent innovations in rodent models of CIBP have facilitated investigation of novel drug treatments and of the mechanisms underlying this condition. The aim of this project was to characterize behavioural and dorsal horn neuronal alterations in a rat model of CIBP, and to study the effects of these alterations on different pharmacological systems. Following intratibial injection of MRMT1 mammary carcinoma cells to induce CIBP, behavioural testing revealed that cancer animals developed significant mechanical and cold allodynia, as well as ambulatory-evoked pain, commencing on post-operative day 9. Sham-operated animals (injection of cell medium alone) showed no behavioural alterations. In vivo electrophysiological characterization of superficial dorsal horn (SDH) neuronal responses to natural (mechanical, heat, brush and cold) and electrical stimuli in the halothane anaesthetized rat revealed neuropathological alterations correlating temporally with the behavioural alterations. There was a marked shift in SDH neuronal populations such that in shams the ratio of wide dynamic range (WDR) to nociceptive specific (NS) cells was 26%:74% whereas in animals with CIBP it was 47%:53%. Furthermore, WDR cells in animals with CIBP had significantly increased responses to mechanical, thermal, and electrical-evoked stimuli compared to shams. These first studies show that the spinal cord is hyperexcitable in CIBP, probably driven by changes in populations of SDH neurons, and that there is a clear temporal link between behavioural and neuronal alterations proving the latter to be a viable substrate for pharmacological testing in this pain model. Acute electrophysiological study using the selective 5-HT3 receptor antagonist ondansetron showed that descending facilitatory serotonergic pathways are enhanced in CIBP. Gabapentin and morphine worked both acutely and chronically to normalise the evoked dorsal horn neuronal responses in rats with CIBP. Chronic behavioural studies showed that these drugs also significantly reduced pain behaviour to the pre-operative baseline response and, furthermore, gabapentin shifted the abnormal WDR:NS SDH cell ratio back towards the sham ratio. These investigations therefore confirmed that pain behaviour in CIBP is strongly linked to changes in populations and excitability of SDH neurones, and highlighted gabapentin as a possible novel treatment for CIBP in the clinic.

Many cancerous solid tumors metastasize to the bone and induce pain (cancer-induced bone pain [CIBP]). Cancer-induced bone pain is often severe because of enhanced inflammation, rapid bone degradation, and disease progression. Opioids are prescribed to manage this pain, but they may enhance bone loss and increase tumor proliferation, further compromising patient quality of life. Angiotensin-(1-7) (Ang-(1-7)) binds and activates the Mas receptor (MasR). Angiotensin-(1-7)/MasR activation modulates inflammatory signaling after acute tissue insult, yet no studies have investigated whether Ang-(1-7)/MasR play a role in CIBP. We hypothesized that Ang-(1-7) inhibits CIBP by targeting MasR in a murine model of breast CIBP. 66.1 breast cancer cells were implanted into the femur of BALB/cAnNHsd mice as a model of CIBP. Spontaneous and evoked pain behaviors were assessed before and after acute and chronic administration of Ang-(1-7). Tissues were collected from animals for ex vivo analyses of MasR expression, tumor burden, and bone integrity. Cancer inoculation increased spontaneous pain behaviors by day 7 that were significantly reduced after a single injection of Ang-(1-7) and after sustained administration. Preadministration of A-779 a selective MasR antagonist prevented this reduction, whereas pretreatment with the AT(2) antagonist had no effect; an AT(1) antagonist enhanced the antinociceptive activity of Ang-(1-7) in CIBP. Repeated Ang-(1-7) administration did not significantly change tumor burden or bone remodeling. Data here suggest that Ang-(1-7)/MasR activation significantly attenuates CIBP, while lacking many side effects seen with opioids. Thus, Ang-(1-7) may be an alternative therapeutic strategy for the nearly 90% of patients with advanced-stage cancer who experience excruciating pain.

Development of animal models has furthered our understanding of mechanisms underpinning cancer-induced bone pain (CIBP). Cancer-induced bone pain is a unique pain state with mechanisms akin to those of chronic inflammatory and neuropathic pain. I aim to highlight the role of both neuronal and non-neuronal mechanisms in a rat model of cancer-induced bone pain (MRMT-1). Anti-convulsant drugs are useful in the treatment of neuropathic pain and have been shown to be effective in animal models of inflammatory pain. Carbamazepine reduced neuronal excitability seen at time of peak behaviour after MRMT-1 but not sham injection, highlighting the possible role of sodium channel activity in maintaining the neuronal excitability in cancer-induced bone pain. Furthermore carbamazepine reduced pain-like ipsilateral hypersensitivity after MRMT-1 injection in awake animals further indicating the usefulness of sodium channel antagonism in cancer-induced bone pain. Peptidergic C fibre population, which co-express calcitonin-gene related peptide (CGRP) and the receptor for nerve growth factor trkA, carry sensory information from bone. It is for these reasons that modulation of CGRP signalling in the MRMT-1 model of cancer-induced bone pain has been investigated using "drug X" (donated from Pfizer UK) which interrupts CGRP signalling in nociceptive pathways. Subcutaneous administration of drug X reduced ipsilateral pain-like behaviours seen following MRMT-1 injection, suggesting that CGRP antagonism may be a useful target in the treatment of cancer-induced bone pain. Microglia have been implicated in the pathophysiology underlying neuropathic pain and have become activated in models of cancer-induced bone pain. OX42 labelled microglial in the lumbar dorsal horn ipsilateral to MRMT-1 increase between days 5 and 11 post surgery. Microglial inhibition using minocycline between days 0 and 9 but not between days 5 and 9 post-MRMT-1 injection delays pain-like behaviour in the post-operative period, indicating that early microgliosis is key in the initiation of cancer-induced bone pain.

Many common cancers have a predisposition for bone metastasis. Tumor occupation of bone is both destructive and a source of debilitating pain in cancer patients. As a result, cancer-induced bone pain (CIBP) is the single most common form of clinical cancer pain. Opioids remain the golden standard for the management of CIBP; however, >30% of cancer patients do not experience adequate pain relief with opioids. Furthermore, clinical reports have suggested that opioids can exacerbate bone loss and increase the likelihood of skeletal-related events. To date, there is no known direct mechanism for opioid-induced bone loss (OIBL). We hypothesized that opioid off-target activation of toll-like receptor 4 (TLR4), an innate immune receptor that is expressed in bone, mediates an increase bone loss and associated CIBP. In the 66.1-BALB/cfC3H murine model of breast cancer bone metastasis, TLR4 expression is upregulated in tumor-burdened bone. Chronic morphine treatment exacerbated spontaneous and evoked pain behaviors in a manner paralleled by bone loss: we identified an increase in spontaneous fracture and osteolysis markers including serum collagen-type I (CTX) and intramedullary receptor activator of nuclear κ-B ligand (RANKL). Administration of (+)naloxone, a non-opioid TLR4 antagonist, attenuated both exacerbation of CIBP and morphine-induced osteolytic changes in vivo. Morphine did not alter tumor burden in vivo or tumor cell growth in vitro. Importantly, morphine produced the in vitro differentiation and activation of osteoclasts in a dose-dependent manner that was reversible with (+)naloxone, suggesting that morphine may contribute directly to osteolytic activation. To improve opioid management of CIBP, we then posited and evaluated three novel adjunct therapeutic targets: cannabinoid receptor-2, adenosine 3 receptor and sphingosine-1-phosphate receptor 1. These pharmacological targets were identified as having a multiplicity of anti-cancer, osteoprotective and/or neuroprotective effects in addition to analgesic efficacy in chronic pain. Targets were tested in the 66.1-BALB/cfC3H model of CIBP and demonstrated to have stand-alone efficacy as antinociceptive agents. Taken together, this work provides a cautionary evaluation of opioid therapy in cancer-induced bone pain and seeks to mitigate opioid side effects through the identification of innovative adjunct therapies that can ultimately improve quality of life in patients suffering from cancer pain.

Metastatic bone cancer is the most common cause of pain in patients with malignant tumors. Prolonged opioid treatment remains the primary method to treat pain in these patients. Sustained morphine exposure enhances both bone cancer-induced pain and bone loss in mice implanted with sarcoma cells. Sustained treatment of bone marrow cultures with morphine results in COX-2 dependent upregulation of RANKL and PGE2, and suppression of OPG. This results in increased osteoclastogenesis which was dependent on COX-2 and OPG/RANKL regulatory axis. Treatment with morphine does not induce any direct changes in osteoclasts or sarcoma cells. The in vitro data was validated in the animals where morphine induces an increase in the osteoclastogenesis and RANKL, and suppresses OPG. These data indicate that morphine enhances osteoclastogenesis by modulating the OPG/RANKL regulatory axis in osteoblasts through a COX-2 dependent mechanism.Prolonged opioid exposure induces an opioid-receptor dependent hyperalgesia in humans and in animals. Studying the direct effect of opioids on primary sensory neurons we demonstrate a modest increase in CGRP cellular content that was not opioid-receptor dependent. Although dynorphin A (2-13) and PGE2 enhanced the release of the neuropeptide, pretreatment with opioids does not influence the capsaicin or KCl evoked CGRP release. These date indicate that the neurochemical changes seen in vivo may be dependent on factors upregulated in the periphery and/or the CNS.It has been demonstrated that sensory neurons innervating the femur express markers of neuronal injury and the intramedullary region of the femur becomes devoid of nerve fibers as the tumor expands. In this study we demonstrate that the sarcoma cells generate high levels of ROS and release hydrogen peroxide into the surrounding space, which induces death and injury to both sensory neurons and glia. This death was prevented by the anti-oxidants BHA and catalase. The present study provides evidence that ROS released by cancer cells can directly lead to injury and death of sensory neurons. ROS induced injury may be one of the mechanism through which sensory neurons are injured in the murine bone cancer pain model.

Pain often accompanies cancer and most current therapies for treating cancer pain have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomyosin receptor kinase A (TrkA) has become an attractive target for attenuating chronic pain.In the present report, we use a mouse model of bone cancer pain and examine whether oral administration of a selective small molecule Trk inhibitor (ARRY-470, which blocks TrkA, TrkB and TrkC kinase activity at low nm concentrations) has a significant effect on cancer-induced pain behaviors, tumor-induced remodeling of sensory nerve fibers, tumor growth and tumor-induced bone remodeling. Early/sustained (initiated day 6 post cancer cell injection), but not late/acute (initiated day 18 post cancer cell injection) administration of ARRY-470 markedly attenuated bone cancer pain and significantly blocked the ectopic sprouting of sensory nerve fibers and the formation of neuroma-like structures in the tumor bearing bone, but did not have a significant effect on tumor growth or bone remodeling.These data suggest that, like therapies that target the cancer itself, the earlier that the blockade of TrkA occurs, the more effective the control of cancer pain and the tumor-induced remodeling of sensory nerve fibers. Developing targeted therapies that relieve cancer pain without the side effects of current analgesics has the potential to significantly improve the quality of life and functional status of cancer patients.

Les micro-ARNs (miRNAs) sont de petits ARNs (20-25 nt) qui ont un rôle important dans les mécanismes d'interférence ARN. Les miRNAs sont des inhibiteurs de l'expression génique qui interviennent au niveau post-traductionnel en s'hybridant à des sites spécifiques de leurs ARNm cibles. Ce mécanisme induit la dégradation de l'ARNm ou l'inhibition de sa traduction. Puisque l'hybridation partielle du miRNA est suffisante pour induire une inhibition, chaque miRNA peut avoir des centaines de cibles. Les miRNAs sont impliqués dans de nombreuses fonctions biologiques et en particulier dans processus neuronaux. Plus de la moitié des miRNAs connus sont exprimés dans le cerveau de mammifère avec une distribution spécifique du miRNA considéré. A l'échelle sub-cellulaire il y a également une distribution hétérogène des miRNAs. De plus, il a été montré récemment une implication des miRNAs dans la régulation de la traduction locale dans les neurones. En effet, des miRNAs et des protyeines impliquées dans la biogenèse et la fonction des miRNAs ont été retrouvés dans le soma, les dendrites et les axones. Il a été montré que la dérégulation des miRNAs été impliquée dans de nombreux mécanismes pathologiques. Cette thèse a pour objectif de révéler le rôle des miRNAs dans la plasticité synaptique. Nous avons étudié l'implication des miRNAs dans les mécanismes de la plasticité synaptique homéostatique et dans la plasticité dysfonctionnelle rencontrée en condition de douleur cancéreuse.Notre hypothèse était que la régulation de la traduction locale des récepteurs AMPA dans les dendrites en condition d'homéostasie synaptique implique les miRNAs. Par bio-informatique, qRT-PCR et test luciférase, nous avons identifié le miRNA miR-92a comme régulateur de la traduction de l'ARNm de GluA1. Des immunomarquages des récepteurs AMPA et des enregistrements des courants miniatures AMPA montrent que miR-92a régule spécifiquement l'incorporation synaptique de nouveau récepteurs AMPA contenant GluA1 en réponse à un blocage de l'activité synaptique. La douleur est un symptôme très fréquemment associé au cancer et constitue un challenge pour les médecins puisque aucun traitement spécifique et efficace n'existe. C'est sans doute le résultat d'un manque de connaissances des mécanismes moléculaires responsables de la douleur cancéreuse. En combinant les screening des miRNA et des ARNm, nous avons mis en évidence une voie de régulation impliquant miR-124, un miRNA enrichi dans le système nerveux. Ainsi, dans un modèle de douleur cancéreuse chez la souris, la diminution de miR-124 est associée à une augmentation de ces cibles : calpain 1, synaptopodine et tropomyosine 4. Toutes ces protéines ont précédemment été identifiées comme des molécules clef de la fonction et de la plasticité synaptique. Des experiences in vitro ont confirmé que miR-124 exercait une inhibition multiple de calpain 1, synaptopodine et tropomyosine 4. La pertinence clinique de cette découverte a été vérifiée par le screening du liquide cérébro-spinal de patients souffrant de douleur cancéreuse qui montre également une diminution de miR-124. Ce résultat suggère un fort potentiel thérapeutique du ciblage de miR-124 dans les douleurs cancéreuses. Enfin, l'injection intrathécale de miR-124 dans des souris cancéreuses a permis de normaliser l'expression de la synaptopodine et de stopper la douleur cancéreuse lors de la phase initiale de la maladie
MicroRNAs (miRNAs) are a type of small RNA molecules (21-25nt), with a central role in RNA silencing and interference. MiRNAs function as negative regulators of gene expression at the post-transcriptional level, by binding to specific sites on their targeted mRNAs. A process results in mRNA degradation or repression of productive translation. Because partial binding to target mRNA is enough to induce silencing, each miRNA has up to hundreds of targets. miRNAs have been shown to be involved in most, if not all, fundamental biological processes. Some of the most interesting examples of miRNA activity regulation are coming from neurons. Almost 50% of all identified miRNAs are expressed in the mammalian brain. Furthermore, miRNAs appear to be differentially distributed in distinct brain regions and neuron types. Importantly, miRNAs are reported to be differentially distributed at the sub-cellular level. Recently, miRNAs have been suggested to be involved in the local translation of neuronal compartments. This has been derived from the observations reporting the presence of miRNAs and the protein complexes involved in miRNA biogenesis and function in neuronal soma, dendrites, and axons. Deregulation of miRNAs has been shown to be implicated in pathological conditions. The present thesis aimed at deciphering the role of miRNA regulation in neuronal plasticity. Here we investigated the involvement of miRNA in synaptic plasticity, specifically in homeostatic synaptic plasticity mode. In addition, we investigated the involvement of miRNAs in the maladaptive nervous system state, specifically, in bone cancer pain condition.We hypothesized that local regulation of AMPA receptor translation in dendrites upon homeostatic synaptic scaling may involve miRNAs. Using bioinformatics, qRT-PCR and luciferase reporter assays, we identified several brain-specific miRNAs including miR-92a, targeting the 3’UTR of GluA1 mRNA. Immunostaining of AMPA receptors and recordings of miniature AMPA currents in primary neurons showed that miR-92a selectively regulates the synaptic incorporation of new GluA1-containing AMPA receptors during activity blockade.Pain is a very common symptom associated with cancer and is still a challenge for clinicians due to the lack of specific and effective treatments. This reflects the crucial lack of knowledge regarding the molecular mechanisms responsible for cancer-related pain. Combining miRNA and mRNA screenings we were able to identify a regulatory pathway involving the nervous system-enriched miRNA, miR-124. Thus, miR-124 downregulation was associated with an upregulation of its predicted targets, Calpain 1, Synaptopodin and Tropomyosin 4 in a cancer-pain model in mice. All these targets have been previously identified as key proteins for the synapse function and plasticity. Clinical pertinence of this finding was assessed by the screening of cerebrospinal fluid from cancer patient suffering from pain who presented also a downregulation of miR-124, strongly suggesting miR-124 as a therapeutic target. In vitro experiments confirmed that miR-124 exerts a multi-target inhibition on Calpain 1, Synaptopodin and Tropomyosin 4. In addition, intrathecal injection of miR-124 was able to normalize the Synaptopodin expression and to alleviate the initial phase of cancer pain in mice

Au vu de la relative inefficacité des traitements actuels de la douleur cancéreuse osseuse (DCO) il est devenu nécessaire aujourd'hui d'identifier de nouvelles cibles (cellulaires et/ou moléculaires) pour développer de nouveaux outils thérapeutiques. Dans ce contexte, ces dernières années, de nombreuses études ont suggéré que les cellules gliales, principalement les astrocytes et la microglie, pourraient contribuer au développement et au maintien de la douleur chronique. D'autre part, dans des modèles d'études précliniques de la DCO, plusieurs auteurs ont récemment constaté une réactivité astrocytaire importante dans les cornes dorsales de la moelle épinière et ont montré que, si on empêche cette réactivité, les symptômes douloureux sont diminués. Cependant, la relation exacte existant entre la réactivité des cellules gliales et les symptômes douloureux en condition de DCO est inconnue. Afin de décrypter cette relation, nous avons dans un premier temps étudié le décours temporel des comportements douloureux et caractérisé l’état de sensibilisation centrale dans un modèle de DCO chez le rat induit par l'injection de cellules de carcinome glandulaire mammaire (MRMT-1) dans le tibia. Nous montrons par des approches radiologiques, comportementales (tests de douleur évoquée et de distribution pondérale dynamique) et immunohistochimiques (immunodétection de la protéine Fos après palpation non douloureuse de la patte) que les animaux cancéreux MRMT développent graduellement une tumeur osseuse (premiers signes au 10ème jour post-inoculation), une allodynie et une hyperalgésie mécaniques (à partir du 10ème jour) et thermiques (à partir du 14ème jour), un inconfort de la patte injectée (à partir du 14ème jour ) et des phénomènes de sensibilisation centrale. Dans un deuxième temps, nous avons recherché des indices structuraux et fonctionnels de réactivité gliale spinale dans notre modèle de DCO. L'objectif était donc de dater l'apparition de la réactivité gliale, et de déterminer la nature des cellules gliales impliquées : microglie et/ou astrocytes. Nous montrons par des approches immunohistochimiques qu’aucun signe morphologique de réactivité astrocytaire ni microgliale n’est observable pendant l’établissement et le maintien de la DCO alors que ces signes existent dans un modèle de douleur neuropathique (ligature de nerfs spinaux). De plus, par des approches moléculaire (qRT-PCR) et biochimique (technique du Bio-Plex) nous montrons que, parmi les 20 marqueurs structuraux et fonctionnels de réactivité gliale testés, seule l’expression de l’aquaporine 4 (un canal à eau spécifique des astrocytes) est significativement augmentée en condition de DCO. Nos résultats suggèrent donc que les astrocytes et les cellules microgliales jouent des rôles différents dans la douleur cancéreuse et dans la douleur neuropathique. Enfin, dans un troisième temps, nous avons cherché à mettre en évidence une implication des astrocytes dans la pathologie DCO au travers d’une caractérisation des transmissions glutamatergique et glycinergique, qui sont toutes deux fortement modulées par l’environnement astrocytaire. Par la quantification de l’expression de l’ARNm (qRT-PCR) et par dosage des taux d’acides aminés (électrophorèse capillaire), nous montrons que les principaux acteurs (transporteurs, récepteurs, agonistes et co-agonistes) de la transmission glutamatergique et glycinergique spinale ne subissent pas d’altération significative en condition de DCO. En conclusion, nous montrons que des symptômes douloureux chroniques peuvent se développer et se maintenir (1) sans signe d’astrogliose et de réactivité microgliale spinale ; et (2) sans altération de l’expression des principaux acteurs de la transmission spinale glutamatergique et glycinergique. Nos résultats invitent donc à revoir le lien très fort qui est fait actuellement entre douleur chronique et astrogliose
The relative lack of efficiency of current treatments used to relieve bone cancer pain prompts to the identification of new molecular and/or cellular targets for the development of new therapeutic strategies. In that context, a large number of recent studies have suggested the involvement of glial cells, among which astrocytes and microglial cells, in the onset and maintenance of chronic pain symptoms. In few animal models of bone cancer pain, several authors have recently evidenced an increased glial reactivity in spinal cord dorsal horn, and demonstrated that preventing astrocytic reactivity was sufficient to reduce pain symptoms in these models. However, the exact relationship of glial reactivity with bone cancer pain symptoms remains poorly understood. In order to decipher this link, we have first studied the temporal development of pain symptoms, and characterized the degree of central sensitization in a rat model of bone cancer pain induced by the injection of mammary gland carcinoma cells (MRMT-1) in the tibial bone. Using radiologic assessment of tumor development, behavioral measurements to quantify evoked (von Frey hairs) and spontaneous (dynamic weight bearing) pain and immunodetection of Fos after non nociceptive palpation of cancer bearing limb, we demonstrate that animals injected with MRMT-1 cells gradually develop a bone tumor (first detectable 10 days after inoculation), a mechanical allodynia and hyperalgesia (first noticeable at day 10), and later on a thermal allodynia and hyperalgesia (first detectable at day 14) as well as discomfort of the injected limb (day 14) and finally central sensitization phenomenons. Second, we have investigated the presence of structural and functional markers of spinal glial reactivity in our model of bone cancer pain. Our objectives were to date the onset of spinal glial reactivity, for microglial and astrocytic cells. Using immunohistochemical approaches, we show that none of the classical markers of astrocytic and microglial reactivity can be observed during the onset and the persistent phase of bone cancer pain whereas the markerswere easily identified in a neuropathic pain model (spinal nerve ligation). Furthermore, using molecular (qRT-PCR) as well as biochemical (Bio-Plex) approaches, we show that among the 20 structural and functional markers of glial reactivity tested, only aquaporin-4 displays increased mRNA levels in bone cancer pain model. Hence, our results suggest that astrocytes and microglial cells play different roles in bone cancer and neuropathic pain. Finally, we tried to evidence the involvement of astrocytes in bone cancer pain by characterizing glutamatergic and glycinergic synaptic transmission, both of which are heavily modulated by astrocytic environment. By quantifying mRNA levels (qRT-PCR) and measuring the level of inhibitory and excitatory amino acids (capillary electrophoresis), we show that the main actors (transporters, receptors, agonists and co-agonists) of glutamatergic and glycinergic transmissions in the spinal cord do not undergo any significant alteration in bone cancer pain conditions. We conclude that chronic painful symptoms may develop and persist (1) without any sign of astrogliosis or enhanced microglial reactivity in the spinal cord, and (2) without any alteration in the expression/levels of the main actors involved in glutamatergic and glycinergic transmission. These results therefore question the strong link that is frequently made between astrogliosis and chronic pain

Les cancers de la prostate (CaP), du sein et des poumons sont souvent de diagnostic difficile notamment en raison de leur caractère initial asymptomatique. Rarement douloureux aux stades primaires, ces cancers ont une forte propension à métastaser vers le microenvironnement osseux. Cliniquement, à ce stade, cela se traduit par des douleurs qui sont à la fois très invalidantes et résistantes aux traitements antalgiques de référence, y compris aux morphiniques. La difficulté provient en partie du caractère multifactoriel de ces douleurs qui associent composantes nociceptive et neuropathique. Parmi les acteurs de cette douleur, un rôle essentiel est attribué au dérèglement du système nerveux (périphérique et central) et à la croissance tumorale qui affecte tous deux le microenvironnement osseux.Le glutamate, neurotransmetteur excitateur majeur du système nerveux, est récemment apparu comme une cible potentielle dans la prise en charge de certains cancers, dont le CaP. Le riluzole, une molécule anti glutamatergique, possédant déjà une autorisation de mise sur le marché dans le traitement de la SLA, a démontré un effet antalgique dans plusieurs modèles de douleur inflammatoire et neuropathique et un effet antiprolifératif in vitro. C’est pourquoi, nous avons recherché si cette molécule pouvait avoir des effets bénéfiques dans le traitement des douleurs osseuses induites par le CaP, et comment il pouvait influer sur le développement du CaP métastatique osseux. Nous avons utilisé un modèle de douleur osseuse par injection intratibiale de cellules humaines de CaP, les cellules PC3-luc, et administré de riluzole dans l’eau de boisson. Chez ce modèle, le riluzole a démontré un effet antalgique significatif impliquant le canal TREK-1, canal sélectif pour l’ion potassium. De plus, le riluzole diminue significativement la viabilité des cellules in vitro et ralentit la croissance tumorale in vivo sans affecter le remodelage osseux. L’effet antiprolifératif du riluzole impliquerait une augmentation de l’expression des canaux TREK-1 dans les cellules PC3 participant à leur hyperpolarisation.En conclusion, ce travail permet de mettre en évidence l’intérêt du riluzole en tant que molécule d’intérêt pour le traitement des douleurs osseuses du CaP dont le mécanisme d’action implique certainement le canal potassique fond TREK-1
Prostate (PCa), breast and lung cancer are often difficult to diagnose due to their initial asymptomatic nature. Rarely painful in the primary stages, these cancers have a high propensity to metastasize to the bone microenvironment. Clinically, at this stage, this translates into pain that is both very disabling and resistant to standard analgesic treatments, including morphine. The difficulty comes from the multifactorial nature of this pain, which combines nociceptive and neuropathic components. Among the factors attributed to cancer pain, an essential role is attributed to the disruption of the nervous system (peripheral and central) and to tumor growth, both of which affect the bone microenvironment.Glutamate, a major excitatory neurotransmitter of the nervous system, has recently emerged as a potential target in the management of solid cancers, including PCa. Riluzole, an anti glutamatergic molecule, which is authorized for the treatment of ALS, has demonstrated an analgesic effect in several inflammatory and neuropathic pain models and an antiproliferative effect in vitro. Therefore, we investigated whether this molecule could have beneficial effects in the treatment of PCa-induced bone pain, and how it could influence the development of bone metastatic PCa. We used a bone pain model by intratibial injection of human PCa cells, PC3-luc cells, and administered riluzole in the drinking water. In this model, riluzole demonstrated a significant analgesic effect involving the TREK-1 channel, a selective channel for potassium ions. In addition, riluzole significantly decreased cell viability in vitro and slowed tumor growth in vivo without affecting bone remodeling. The antiproliferative effect of riluzole would imply an increase in the expression of TREK-1 channels in PC3 cells participating in their hyperpolarization.In conclusion, this work highlights the importance of riluzole as a molecule of interest for the treatment of PCa bone pain whose mechanism of action certainly involves the TREK-1 potassium channel

碩士
國立臺灣大學
生命科學系
103
Cancer-induced bone pain (CIBP) is a common pain in patients with advanced cancer. When cancer metastasizes to the bone, it can cause persistent and unbearable pain which often cause patient’s physical and mental suffering. Although CIBP is one of the most serious clinical problems, the pathophysiological mechanism of CIBP has not been elucidated. Opioid, such as morphine, is commonly used in cancer pain management. The aim of this study is to combine the positron emission tomography-computed tomography (PET/CT) imaging and pain behavior tests to investigate the pain- and morphine analgesia-related brain regions in the CIBP mice. We injected 4T1 mouse breast cancer cells into left femur bone marrow cavity of the BALB/c mice, using 18F-NaF as tracer to evaluate the development of cancer cells in the bone environment. Mice in sham control group were injected with phosphate buffered saline. Then, we measured pain related behaviors with limb use observation, von Frey filaments test and acetone stimulus on the day before surgery, Day 7, Day 10 and Day 14 after the surgery to confirm pain development. Morphine doses (10, 15, 30 mg/kg, i.p.) were administered on Day 16 after the surgery. In addition, we investigated spontaneous pain and morphine-analgesic effect on CIBP mice brain by 18F-fluorodeoxyglucose (FDG) PET/CT. In the PET imaging study, each mouse was scanned 3 times: before bone surgery, Day 14, after the surgery, and Day 16, 30 min after the 15 mg/kg morphine treatment. Our results showed that the CIBP mice showed significant spontaneous pain, mechanical allodynia and cold allodynia on 14 days after the 4T1 cancer cells injection. Morphine dose 15 mg/kg was sufficient to relieve spontaneous pain, mechanical and cold allodynia of the CIBP mice between 30 to 60 minutes post-treatment. In PET study, brain glucose metabolic activity of sensory and motor cortex in mice increased during movement, and the results were reversed during sleeping. In order to prevent these results interfering the analysis of CIBP-related brain regions, we removed the activity parameters, and the results showed that in the CIBP condition, glucose metabolic activity were significant increased in bilateral insular cortex and bilateral S2. Morphine analgesia effect may be produced by the observed suppressing contralateral insular cortex and contralateral S2 brain regions, as well as activation of the habenula and PAG. Our data suggest that contralateral insular cortex and contralateral S2 may play an important role in the CIBP.