Preclinical and clinical evaluation of the kappa opioid receptor as a therapeutic target in multiple sclerosis

Abstract

Multiple sclerosis (MS) is a chronic, inflammatory, and neurodegenerative disease for which there is no cure. Aotearoa, New Zealand is a high-risk country for MS with an overall prevalence of 73.1 per 100,000 population, compared to the global prevalence of 35.9. The progression of MS treatments is impeded by the dual-faceted presentation of the disease as it is characterised by maladaptive periods of both neuroinflammation and demyelination. Designing pharmacotherapies that target, and positively modulate, both aspects of MS pathology is critical for future drug development. Within the last decade, research has identified the kappa opioid receptor (KOR), encoded by the OPRK1 gene, as a promising target for modulating both remyelination and neuroinflammatory processes. Preclinical studies have demonstrated that KOR agonists, particularly U50,488, promote the differentiation of oligodendrocyte precursor cells (OPCs) into mature, myelinating oligodendrocytes (OLs). This is a key process in restoring myelin in the central nervous system. U50,588, however, is associated with adverse clinical side effects, including dysphoria and sedation, which limits its therapeutic utility. As such, the translational relevance of KOR-targeted therapeutic strategies remains unclear, particularly in the context of MS pathology. The aim of this thesis was to investigate preclinical efficacy and clinical relevance of KOR signalling in MS by using in vitro, in vivo, and postmortem human tissue-based approaches. First, an OPC-enriched culture system was established to assess the effects of KOR agonists on OPC differentiation. These studies demonstrated that KOR activation, namely via U50,488, tifluadom, and the novel compound LDK-276, significantly improved differentiation rates of OPCs compared to vehicle in a ligand-dependent manner. Further pharmacological investigations revealed that intracellular signalling pathways implicated in KOR function, including PI3K, MEK/ERK, and p38 MAPK, are also required for basal OPC survival and differentiation. This highlights the complexity of targeting KOR signalling, as pathway modulation may impact fundamental cellular processes. To assess the functional relevance of these findings in vivo, the cuprizone and rapamycin model of demyelination was utilised, alongside serum neurofilament-light chain (NfL) as a clinically relevant biomarker of neuroaxonal injury. Significantly elevated levels of circulating NfL levels were observed following 12 weeks of cuprizone and rapamycin administration (termed maximum disease; MD), confirming axonal damage in this model. NfL concentrations remained elevated following six weeks of treatment with nalfurafine, LDK-276, or vehicle during the recovery phase, with no significant reduction relative to MD levels. Treatment with KOR agonists did, however, significantly increase levels of remyelination compared to vehicle. This suggests that KOR signalling effects may occur on a different temporal scale than what was measured in this experimental paradigm, or that KOR activation alone may be insufficient to promote neuroaxonal recovery. To provide anatomical and clinical context, the distribution and cell-type localisation of OPRK1 transcript were characterised in postmortem human brain tissue from both healthy and MS-diagnosed donors using RNAScope® fluorescent in situ hybridisation (fISH). This work represents the first detailed spatial characterisation of OPRK1 expression in human MS brain tissue. OPRK1 expression was found to be predominantly neuronal, with limited localisation to other glial cell types, a pattern which was largely preserved between healthy and MS-diagnosed donors. Within different lesion types, variation in OPRK1 expression displayed region-dependent variability, with patterns largely reflecting differences between grey and white matter rather than consistent changes in transcript up- or downregulation. These findings suggest that KOR signalling remains biologically relevant in the MS brain and may inform future approaches in the development of targeted therapeutics. The findings of the current research demonstrate that, while KOR activation promotes OPC differentiation in vitro, its functional effects in vivo and relevance to human disease are more complex and context dependent. This work advances our understanding of KOR signalling in MS and underscores the importance of incorporating human relevant systems when evaluating therapeutic targets. KOR-targeted therapies, therefore, may hold potential as part of combination strategies aimed at promoting remyelination and repair within the complex pathological environment of the MS brain.