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 analgesic effect of 600 mg and 1500 mg of a pain killing drug to metastatic bone pain, and associated side effects, were assessed. The experimental design was a double-blind cross over clinical trial involving 44 patients known to suffer from metastatic bone pain. Each patient received the active drug in one of two dosages and the placebo in a random order, each lasting about 14 consecutive days. The data consisted of daily measurements of several pain and side effect variables. A few covariates were available. It was found that the patient and the investigator achieved a high degree of agreement on the blinded preference of the active drug to the placebo. A multivariate analysis of variance (MANOVA) on three different summary scores (mean, median, trmean) calculated on the daily measurements for which the patient received the active drug and on those for which the patient received the placebo was conducted. It was found that for the group of pain variables the order of application and the treatment do not have a significant effect marginally, but that they interact significantly. Variation between subjects was also significant. For the group of side effect variables, however, only significant variation between subjects was found. This suggests that the drug does not have noticeable overall side effects. To account for correlations among the response measurements within each patient, the methodology of generalized estimating equations was used to assess the significance of the effects of the predictors. Although the results are less reliable as they depend on the asymptotic behaviour of statistics, it was found that regardless of the level of correlation within patient response measurements, only the interaction of order of application with treatment has a significant effect on each of the pain variables. All the statistical analyses were carried out using Minitab, SAS, Matlab and Splus. / Thesis / Master of Science (MS)

A grande aplicação dos radiofármacos está em medicina nuclear diagnóstica representando 95% dos procedimentos realizados, porém, nos últimos anos, tem crescido consideravelmente a sua aplicação em procedimentos terapêuticos. Os radionuclídeos que emitem particulas ionizantes (α, β e elétrons Auger) são indicados para tratamento de tumores. Tumores malignos são responsáveis por aproximadamente 12% dos óbitos e representam a terceira causa de mortalidade no Brasil. O 188Re é um dos mais atrativos radioisótopos para uma variedade de aplicações terapêuticas em medicina nuclear, oncologia e intervenções cardiológicas, é totalmente favorável e conveniente pelo fato de que ele é livre de carregador e pode ser obtido de forma econômica na forma de um gerador de 188W/188Re, além de possuir uma meia-vida fisica de 16,9 horas e 100% de emissão de radiação β-. A partir da década de 2000 vêm sendo realizadas diversas investigações envolvendo marcações de moléculas com 188Re. Os tumores metastáticos são a forma mais comum de malignidade esquelética. Em casos metastáticos os principais objetivos do tratamento são a prevenção de fraturas patológicas e promover a sobrevida com o máximo de preservação de função permitindo que o paciente mantenha o máximo possível de mobilidade e controle da dor. O objetivo deste trabalho foi realizar a comparação das marcações de diversos fosfonatos (Metileno disfofonato de Sódio MDP, Ácido Etilenodiaminotetrametilenofosfônico EDTMP, e do diclorometilenobifosfonato de sódio - Clodronato) com 188Re para terapia de metastáses ósseas. Fosfonatos são inibidores da reabsorção óssea osteoclástica e são efetivos neste tratamento. As marcações do MDP, EDTMP e Clodronato com 188Re foram otimizadas utilizando como agente redutor o cloreto estanoso (SnCl2 2H2O) e como agente estabilizante o ácido ascórbico. As variáveis estudadas foram massa do ligante, massa do SnCl2.2H2O, massa do ácido ascórbico, tempo, pH e temperatura da reação. Os resultados mostraram que se obteve um excelente rendimento de marcação de 98% para o 188Re-MDP, de 83% para o 188Re-EDTMP e 85% para o 188Re-Clodronato. / The wide application of radiopharmaceuticals in nuclear medicine, representing 95% of procedures performed is in diagnosis, but in recent years, its application in therapeutic procedures has grown considerably. The radionuclides that emit ionizing particles (α, β, and Auger electrons) are indicated for the treatment of tumors. Malignant tumors account for approximately 12% of deaths and represent the third cause of mortality in Brazil. 188Re is one of the most attractive radioisotopes for a variety of therapeutic applications in nuclear medicine, oncology and cardiology interventions, is fully favorable and convenient because it is carrier free and can be obtained inexpensively in the form of a generator of 188W/188Re, and has a physical half-life of 16.9 hours and 100% of β-radiation emission. From the 2000s several investigations have been conducted involving molecules labeled with 188Re. Metastatic tumors are the most common form of malignancy in the skeleton. In metastatic cases the main goals of treatment are the prevention of pathological fractures and the promotion of survival with maximum preservation of function allowing the patient to maintain the greatest possible mobility and pain control. The objective of this study was to compare the labeling of various phosphonates (Sodium methylene diphosphonate - MDP, Ethylenediaminetetramethylene phosphonic acid - EDTMP, and sodium dichloromethylenediphosphonate - clodronate) with 188Re for bone metastases therapy. Phosphonates are inhibitors of osteoclastic bone resorption and are effective in treatment. The labeling of MDP, EDTMP and Clodronate with 188Re was optimized using stannous chloride (SnCl2·2H2O) as reducing agent, and ascorbic acid as stabilizing agent. The variables studied were the ligand mass, SnCl2.2H2O mass, mass of ascorbic acid, time, pH and temperature of the reaction. The results showed an excellent labeling yield of 98% for 188Re-MDP, 83% for 188Re-EDTMP and 85% for 188Re-Clodronate.

A grande aplicação dos radiofármacos está em medicina nuclear diagnóstica representando 95% dos procedimentos realizados, porém, nos últimos anos, tem crescido consideravelmente a sua aplicação em procedimentos terapêuticos. Os radionuclídeos que emitem particulas ionizantes (α, β e elétrons Auger) são indicados para tratamento de tumores. Tumores malignos são responsáveis por aproximadamente 12% dos óbitos e representam a terceira causa de mortalidade no Brasil. O 188Re é um dos mais atrativos radioisótopos para uma variedade de aplicações terapêuticas em medicina nuclear, oncologia e intervenções cardiológicas, é totalmente favorável e conveniente pelo fato de que ele é livre de carregador e pode ser obtido de forma econômica na forma de um gerador de 188W/188Re, além de possuir uma meia-vida fisica de 16,9 horas e 100% de emissão de radiação β-. A partir da década de 2000 vêm sendo realizadas diversas investigações envolvendo marcações de moléculas com 188Re. Os tumores metastáticos são a forma mais comum de malignidade esquelética. Em casos metastáticos os principais objetivos do tratamento são a prevenção de fraturas patológicas e promover a sobrevida com o máximo de preservação de função permitindo que o paciente mantenha o máximo possível de mobilidade e controle da dor. O objetivo deste trabalho foi realizar a comparação das marcações de diversos fosfonatos (Metileno disfofonato de Sódio MDP, Ácido Etilenodiaminotetrametilenofosfônico EDTMP, e do diclorometilenobifosfonato de sódio - Clodronato) com 188Re para terapia de metastáses ósseas. Fosfonatos são inibidores da reabsorção óssea osteoclástica e são efetivos neste tratamento. As marcações do MDP, EDTMP e Clodronato com 188Re foram otimizadas utilizando como agente redutor o cloreto estanoso (SnCl2 2H2O) e como agente estabilizante o ácido ascórbico. As variáveis estudadas foram massa do ligante, massa do SnCl2.2H2O, massa do ácido ascórbico, tempo, pH e temperatura da reação. Os resultados mostraram que se obteve um excelente rendimento de marcação de 98% para o 188Re-MDP, de 83% para o 188Re-EDTMP e 85% para o 188Re-Clodronato. / The wide application of radiopharmaceuticals in nuclear medicine, representing 95% of procedures performed is in diagnosis, but in recent years, its application in therapeutic procedures has grown considerably. The radionuclides that emit ionizing particles (α, β, and Auger electrons) are indicated for the treatment of tumors. Malignant tumors account for approximately 12% of deaths and represent the third cause of mortality in Brazil. 188Re is one of the most attractive radioisotopes for a variety of therapeutic applications in nuclear medicine, oncology and cardiology interventions, is fully favorable and convenient because it is carrier free and can be obtained inexpensively in the form of a generator of 188W/188Re, and has a physical half-life of 16.9 hours and 100% of β-radiation emission. From the 2000s several investigations have been conducted involving molecules labeled with 188Re. Metastatic tumors are the most common form of malignancy in the skeleton. In metastatic cases the main goals of treatment are the prevention of pathological fractures and the promotion of survival with maximum preservation of function allowing the patient to maintain the greatest possible mobility and pain control. The objective of this study was to compare the labeling of various phosphonates (Sodium methylene diphosphonate - MDP, Ethylenediaminetetramethylene phosphonic acid - EDTMP, and sodium dichloromethylenediphosphonate - clodronate) with 188Re for bone metastases therapy. Phosphonates are inhibitors of osteoclastic bone resorption and are effective in treatment. The labeling of MDP, EDTMP and Clodronate with 188Re was optimized using stannous chloride (SnCl2·2H2O) as reducing agent, and ascorbic acid as stabilizing agent. The variables studied were the ligand mass, SnCl2.2H2O mass, mass of ascorbic acid, time, pH and temperature of the reaction. The results showed an excellent labeling yield of 98% for 188Re-MDP, 83% for 188Re-EDTMP and 85% for 188Re-Clodronate.

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.

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.

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.

Metastatic bone cancers cause severe symptoms including pain that compromises patient functional status, quality of life, and survival. Current treatment strategies have limited efficacy and dose-limiting side effects. Cancer-induced bone pain (CIBP) is a unique pain state that shares features with but is distinct from the pathology of neuropathic and inflammatory pain. This dissertation investigates how CIBP is generated and maintained by the direct effects of cancer cells on their metastatic microenvironment and the peripheral nervous system, including unique signaling properties and gene expression changes. In particular, we found that genetic knockdown of the functional subunit xCT of the system xC- cystine/glutamate antiporter can reduce CIBP, further elucidating this as a therapeutic of interest. We found that the neuroprotective voltage-gated calcium channel inhibitors progesterone and pregabalin markedly reduce mechanical hypersensitivity and excitability in sensory neurons of the dorsal root ganglion (DRG) in male rat models of neuropathic pain, but that these effects and less pronounced in females. In cancer pain, these sex differences are reversed, with females but not males demonstrating a delay in time-to-onset of mechanical hypersensitivity. We also analyzed gene expression at the DRG by RNA-Sequencing of rat models of CIBP. Our findings uncovered differential gene expression between CIBP and sham controls and between ipsilateral and contralateral DRGs in CIBP model rats. These studies have identified several promising avenues for therapeutic research for CIBP. / Dissertation / Doctor of Philosophy (PhD) / The tools we have right now to manage severe and chronic pain are insufficient. Patients with advanced cancers including bone cancer can suffer from very severe pain. This pain is generated in a number of ways including by the tumour itself releasing chemicals that activate pain-sensing nerves, by the destruction of the bone in and around the tumour, and by the sensitization of the nervous system, which can make pain worse and longer lasting. We have taken three approaches to researching cancer pain and to investigating new treatments. We have found that by reducing the amount of glutamate that cancer cells can release into their environment, we can reduce cancer pain in mice. We also found that treating rats with pregabalin and progesterone can change nerve signaling and reduce neuropathic pain, but that this effect is most pronounced in male rats with neuopathic pain and smaller in female rats with neuropathic pain, and even smaller in rats with cancer pain. We also analyzed expression of all the protein-coding genes in dorsal root ganglia from rats with cancer pain and found that there are many differences from rats without pain. Some of these differences may be promising new research targets. Going forward this research has provided important evidence necessary for next steps to develop new therapies and research strategies for cancer pain.