Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a major dose-limiting side effect of several anticancer drugs. The prevalence of CIPN ranges from one-third to two-thirds of cancer patients. CIPN can persist for months to years after completion of chemotherapy. Despite the efficacious use of paclitaxel in the treatment of tumors, it can induce many sensory symptoms, such as paresthesia, numbness, tingling and burning pain, and mechanical and cold allodynia, which typically are present in the hands and feet. Similar to other types of chronic pain, paclitaxel-induced CIPN is comorbid with depression and anxiety in cancer survivors, and paclitaxel induces changes in affect-like behavior in cancer-free animal models, suggesting that paclitaxel can cause long-lasting changes in mood, reducing the quality of life. While adjuvant therapies, such as duloxetine, tricyclic antidepressants, and gabapentin are prescribed to treat CIPN symptoms, none of these compounds can consistently reverse or prevent the development of CIPN. With no FDA-approved medication to treat CIPN, the purpose of the dissertation was to: i) characterize and develop a mouse model of paclitaxel-induced CIPN, ii) identify putative targets for CIPN treatment, and iii) test novel compounds for their ability to prevent and reverse CIPN in C57BL/6J mice. In the first Aim, we demonstrate that paclitaxel induces time- and dose-dependent hypersensitivity (mechanical and cold), which is potentiated by combination therapy with the chemotherapeutic carboplatin. In addition, paclitaxel-treated mice show changes in affect-like behaviors (anxiety-like, depression-like). In the second Aim, we used the prototypic nicotinic receptor (nAChR) agonist nicotine to reverse or prevent paclitaxel-induced mechanical hypersensitivity and degeneration of Intra-Epidermal Nerve Fibers (IENFs). Further, we discovered that nicotine’s antinociceptive effects in this mouse model of CIPN are mediated by the nicotinic receptor subtype α7. The third Aim used genetic and pharmacological approaches to dissect the role of α7 on the development and maintenance of paclitaxel-induced CIPN. Null mutant α7 mice (KO) hastens the onset, increases the magnitude, and delays the recovery of paclitaxel-induced mechanical hypersensitivity, as compared to littermate wildtype controls, whereas the selective α7 silent agonist R-47 to reverses and prevents paclitaxel-induced CIPN in C57BL/6J mice. We also examined the impact of R-47 on the paclitaxel-induced reduction of intraepidermal nerve fiber (IENF), as well as microglial morphology in the dorsal horn of the spinal cord. The data show that R-47 prevents paclitaxel-induced changes in microglial morphology and mechanical hypersensitivity behavior, without producing tolerance upon repeated administration. Finally, R-47 induces preference using the conditioned place test in paclitaxel-treated mice but vehicle-treated animals, suggesting that R-47 is a viable candidate for ongoing, spontaneous pain, with limited risk of abuse potential. Overall, these results support that the α7 nAChR subtype is an important target for the treatment and prevention of CIPN.

Background: Chemotherapy Induced Peripheral Neuropathy (CIPN) is a debilitating neuropathy caused by commonly used chemotherapeutics. Clinically, the problem of CIPN is compounded by difficulties with diagnosis and limited treatment options. The pathophysiology of CIPN remains elusive, with current mechanistic postulates focused mainly on the peripheral nervous system. However, animal and human models of non-CIPN neuropathic conditions have shown the brain to be central to the development and maintenance of painful neuropathy. Moreover, evidence suggests that aberrant activity in key regions of the brain and brainstem could denote individual vulnerability for chronic pain states. The impact of the brain on CIPN development is unknown. Assessment of drug efficacy using brain imaging can provide sensitive readouts and is increasingly used in clinical trials. Aims: Firstly, to prospectively explore the structure and function of the brain in cancer patients prior to chemotherapy administration, using functional magnetic resonance imaging (fMRI), in order to determine whether baseline differences exist between patients who progress to CIPN as compared to those who do not. Secondly, to develop a pilot study using fMRI to investigate a topical treatment for CIPN, in order to assess the feasibility of setting up a study with this kind of design. Methods: To address the first aim of this thesis a prospective cohort study (the CIPN fMRI Study) was developed. Cancer patients scheduled to receive neurotoxic chemotherapy treatment including oxaliplatin, carboplatin, carbotaxol, or cisplatin, were recruited from three NHS trusts in Scotland, to undergo a high resolution (3 tesla) functional MRI scan, at a single time point prior to commencement of chemotherapy. During the scan structural, resting state and functional data were collected. Functional data involved the presentation of punctate stimuli (using a 256mN von Frey filament), above the patients’ right medial malleolus. While receiving the punctate stimuli, patients viewed images that had neutral or positive emotional content or a baseline coloured image with no content. Sample size was based on previously successful pain fMRI studies and pragmatic estimates. Acute CIPN was defined clinically by common toxicity criteria as necessitating a chemotherapy dose reduction or cessation. Data were analysed using FMRIB’s Software Library (FSL) version 5, 2015. Standard data pre-processing (brain extraction, registration, B0 unwarping, motion correction, and denosiing with FIX) was carried out. Structural analysis was conducted using FIRST. Resting state analysis utilised FSL’s MELODIC tool, and a non-parametric group comparison was made following a dual regression approach. FEAT was used for both first and second level functional analyses. Group comparisons were made using a mixed effects analysis (z threshold 2·3 and 2, regions considered significant at p < 0·05, cluster corrected). The group was split by sex to explore known sex differences in pain processing. To address the second aim of this thesis, a pilot fMRI randomised controlled trial (MINT3 Study) was designed. Approvals from ethics and research and development were sought and obtained. Data collection forms were developed. An fMRI experiment was proposed and a single pilot scan was conducted and analysed. Results: 30 patients were recruited for the CIPN fMRI study (mean age 60·4 years, 95% Confidence Interval: 57.4-63.4, 17 women). Two patients had lung cancer, nine had gynecological malignancies and 18 had colorectal cancer. 17 patients developed acute CIPN. Structural analysis showed that patients who developed CIPN had a smaller volume of the Nucleus Accumbens (NAc). Resting state analysis did not show clear differences between those who developed CIPN and those who did not. Finally, functional analysis showed that patients who did not develop CIPN had greater activation in the superior frontal gyrus when viewing positive emotional images as compared to those who did progress to CIPN. Region of interest analysis showed that female patients who developed CIPN had greater activity in their mesencephalic pontine reticular formation (MPRF). Male patients who progressed to CIPN had decreased activity in their thalamus. Feasability of the MINT3 study set up and fMRI paradigm was assessed. Interpretation Differences in brain structure and function are evident between patients who developed CIPN and those who did not. Crucially, the regions identified, in particular the NAc, have been postulated to denote a vulnerability for progression to pain states. Although the findings need further confirmation they suggest a paradigm shift in terms of CIPN as a clinical problem. Specifically, it appears that certain individuals can be considered as having increased risk of CIPN development prior to chemotherapy administration. This risk relates to the baseline structure, and function of their brains. Finally, the set up of the MINT3 fMRI study showed that this kind of study design is acceptable in terms of ethical and R&D approvals and a single healthy volunteer pilot.

Background Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting toxicity with significant sequelae impacting prognosis and quality of life. The natural history and pathophysiological mechanisms of CIPN are unclear. Equally, the lack of systematic approach to diagnosis and assessments contribute to difficulty identifying at risk patients with implications on symptom burden. Effective management of CIPN is also difficult due to limited treatment options. To try and address this challenging clinical problem, this thesis aimed to adopt a translational approach to: 1) characterisation and early identification of the development of CIPN in cancer patients receiving neurotoxic chemotherapy and 2) explore topical treatment options in patients with chronic peripheral neuropathic pain. Methodology In the CIPN study, a mixed cohort of colorectal, gynaecological and lung cancer patients receiving neurotoxic chemotherapy (platinum agents and taxanes) were assessed prospectively, at baseline (prior to initiating chemotherapy), during cycles (every 3 weeks) and post-treatment (every 3 months) for up to 12 months (cumulative 261 assessments). Comprehensive longitudinal clinical characterisation consisted of the integration of quantitative sensory testing (QST), objective measure of function (grooved pegboard test), patient-reported outcomes and in vivo confocal microscopy to provide insight into the clinical course and potential psychophysical biomarkers of CIPN during and after chemotherapy. In the pilot intervention study, patients with chronic, complex cancer treatment related peripheral neuropathic pain received a single application of high concentration 8% capsaicin patch. Assessments conducted at baseline, 4 weeks and 12 weeks included patient-reported outcomes and QST with an exploratory application of in vivo confocal microscopy in a case. Results In the CIPN study, 33 patients when compared to 33 age and gender matched healthy controls displayed thermal hyperalgesia, sensorimotor impairment and increased resting heart rate prior to initiating neurotoxic chemotherapy. Characterisation of somato-sensory profile demonstrated dysfunction of the various types of primary afferent nerves (Aβ, Aδ and C). Assessing the change over time from baseline to during cycles and post-treatment follow up, revealed signs and symptoms as early as cycle 2 with an increase in the later cycles and 3 months post-treatment follow up. A greater burden was observed at 12 months in comparison to baseline. Significant changes were observed in QST parameters indicating both small and large fibre deficits. Interesting associations were observed for example with tactile deficits in the upper and lower limb and patient-reported outcomes. The repeated measures model provided an opportunity to distil the relationship between subjective and objective measures of CIPN. The subclinical findings at baseline however did not translate to obvious predictors of CIPN development. The exploratory use of in vivo confocal microscopy (45 healthy controls, 9 patients) demonstrated correlation with current assessment tools (QST). Analysis from the pilot intervention study of 20 patients revealed clinically significant improvement in pain in a subset at 4 and 12 weeks post-treatment. Conclusion Overall the combination of subjective and objective measures utilised in the prospective characterisation of this mixed cohort of cancer patients provided a useful paradigm for qualifying and quantifying the trajectory of CIPN related peripheral nerve damage and symptom burden with additional contribution from the novel in vivo confocal microscopy work. In capturing the varied spectrum of phenotypes, this approach may provide insight into the complexities of the underlying neurobiological mechanisms. The baseline subclinical sensory, motor and autonomic nerve dysfunction implicate a cancer-mediated process possibly contributing to CIPN. Although the preliminary investigation of baseline predictors of CIPN was inconclusive, thermal pain threshold warrant further investigation. These findings highlight the need to further address prediction and risk stratification in larger studies. The exploratory intervention study suggests that patients with chronic neuropathic pain may receive some benefit in pain severity, function and mood with effect continuing at 12 weeks post-treatment. This research warrants further investigation in larger cohorts.

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My thesis introduces the topic of nociceptive signalisation and processes involved in the formation and spreading of neuropathic pain. This study focuses on the mechanisms of nociceptive synaptic transmission mechanisms in the level of spinal dorsal horn and its modulation by paclitaxel, a chemotherapeutic drug inducing neuropathic changes. The attention is put especially on the possibility of glial activity participation in paclitaxel side effects. This idea stems from the existing hypothesis of the functional connection between TLR4 and TRPV1 receptor activity. TRPV1 is well known for its participation in chemical, thermal and nociceptive sensory transmission. Minocycline antibiotic is considered as an inhibitor of microglial activation therefore it was used for blocking neuroinflammation. The experimental part is comparing an impact of substances applied to the model of tachyphylaxis used for monitoring of nociceptive transmission changes according to decreasing activity of TRPV1 receptors. Electrophysiological recording of miniature excitatory postsynaptic currents from neurons in the Rexed laminae I. and II. of spinal dorsal horn was used. The results of my measurements show that minocycline is able to suppress acute effects of paclitaxel application in vitro if the spinal slice is incubated...

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第21306号 / 人健博第62号 / 新制||人健||5(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 黒木 裕士, 教授 恒藤 暁, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM