Characterising Heparan Sulfate Mimetics as Potential Therapeutics in Alphaviral Diseases

Abstract

Arthritogenic alphaviruses such as chikungunya virus (CHIKV) and Ross River virus (RRV) are transmitted by mosquito vectors, and can cause musculoskeletal disease manifestations such as, excruciating pain and inflammation in joints and the surrounding muscle tissues, in infected individuals. Despite the recent findings of cartilage damage, erosion and focal necrosis in murine models of both chikungunya viral disease (CHIKVD) and Ross River viral disease (RRVD), the role of human chondrocyte/cartilage in alphaviral infection has never been previously explored. In this thesis, it was shown that, both the lower and the higher density cultures (high-density cell cultures and micromass pellet cultures) of primary chondrocytes, supported the RRV-T48 infection and produced the upregulation of soluble inflammatory host factors during the infection. Interestingly, the heterogeneity of chondrocyte infectibility was also noted between two human chondrocyte donors. Chondrocytes that were derived from one patient could not sustain an efficient RRV-T48 replication when compared to chondrocytes that were isolated from another patient. Although this infection profile was surprising, the sample pool for the donor chondrocytes was extremely small thus, other confounding factors may have also contributed to the difference in infection. Additionally, the presence and co-localisation of RRV antigens within the chondrocytes of murine joints, proved that RRV could get to the cartilage and either cause damage directly or trigger the body systems to potentiate the damage. The second half of the thesis focused on exploring a new class of heparan sulfate (HS)-modulating compounds, as targeted treatments for arthritogenic alphaviral infection/diseases. Current drugs for alphaviral diseases are non-specific and include the use of non-steroidal anti-inflammatories which only provide temporary or partial relief in patients. Recently pentosan polysulfate (PPS), a sulfated polysaccharide and a HSmimetic, was found to have reduced the recruitment of inflammatory infiltrates and to have protected the cartilage matrix from degradation in RRV-infected mice. Following which, in this thesis, the roles of other HS-modulating compounds such as polynuclear platinum complexes (PPCs) and HS-mimetic PG545, were investigated for the treatment and/or inhibition of arthritogenic infection. The HS-modulating compounds were characterised for 50% inhibitory doses, in addition to strength of association studies that were performed using the surface plasmon resonance (SPR) and the isothermal titration calorimetry (ITC) binding methods. Both the high signal responses generated in the SPR and the ITC, demonstrated that PG545 had a direct potent inhibitory effect on the RRV virus particles. Therefore, PG545 was further evaluated in the well-established murine model of acute RRVD, and in in-vitro systems. Increasing concentrations of the compounds were able to inhibit plaque formation in RRV, Barmah Forest virus (BFV), and in both the East/Central/South African (ECSA) and the Asian CHIKV strains, as determined by the 50% plaque inhibitory assays. In mice, disease state and compound efficacy were primarily characterised by assessing hind limb weakness, expression levels of soluble host factors, the components of the cartilage matrix, viral titre and histopathology. Prophylactic, standard treatments of PG545 to RRV-infected mice had significantly reduced viral loads in muscles and joints corresponding to a reduction in their clinical scores of limb weakness and immune infiltrate recruitment, at 10 day-post infection (dpi). Also, at peak disease (10dpi), RRV-infected PG545-treated mice had lower expression levels of host factors, IL-6 and CCL-2. Furthermore, PG545 treatment also demonstrated a reduction in the matrix degrading enzyme heparanase (HPSE) in both muscle and joint samples. Serum biochemistries also showed that PG545 treatment in RRV-infected mice, reduced RRV-induced elevations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). It was also noted that, the standard dosing of PG545 only caused minimal lymphoid tissue anomaly in mice, while the prolonged PG545 dosage (to shorten clinical disease) resulted in both transient hepatosplenomegaly and anaemia. Moreover, both the histopathology and gene expression data, demonstrated that the splenomegaly was due to the expansion of the red pulp and the atrophy of the white pulp. It should also be noted that the long half-life of PG545 (as previously published), could have also contributed to the drug-induced transient pathogenesis, seen in mice that were treated with PG545 for a prolonged duration. Taken together, these findings suggest that although PG545 has a direct inhibitory effect on both RRV-infection as well as the RRV-induced inflammatory disease in host organisms, careful dosing needs to be observed due to the stability and long half- life of compound.