Throughout this study, I studied the novel cancer agent, VR23 for its potential as an anti-inflammatory agent. Further, I investigated the mechanism by which VR23 acts to down-regulate inflammatory markers.
In Chapter 1, I investigated the ability of VR23 to down-regulate pro-inflammatory markers in an acute inflammatory cell model. I observed previously that the level of
macrophages in a mouse spleen treated with VR23 was much lower than those treated with other compounds such as paclitaxel. From this data and the fact that VR23 was created as a hybrid molecule of HCQ, we thought that VR23 would have anti-inflammatory capabilities. I found that in an acute inflammatory cell model VR23 can effectively down-regulate pro-inflammatory cytokines such as IL-6, TNF-α, IL-1β, and IL-8. In addition, VR23 presented potent anti-inflammatory capabilities in reducing IL-6 and MCP-1 in chronic anti-inflammatory cell models, including HFLS cells from RA patients. Lastly, VR23 showed effectiveness in an acute lung injury mouse model by reducing cytokine secretion, cell recruitment, and myeloperoxidase activity. In Chapter 1, I found that VR23 indeed possesses quite potent anti-inflammatory property. It also furthered our understanding of VR23 and its effectiveness in specifically reducing IL-6 and MCP-1 in both acute and chronic inflammatory settings. This study showed the potential for VR23 to be used as a novel anti-inflammatory drug, in addition to increasing its effectiveness as an anti-cancer drug.
In Chapter 2, I investigated the mechanism by which VR23 is able to reduce
inflammation. In Chapter 1, I showed that VR23 effectively down-regulates the levels of IL-6 and its down-stream gene expression. This Chapter focuses on unraveling the potential for VR23 to disrupt the IL-6-STAT3 pathway as a mechanism for how it may prevent inflammation. With a focus on IL-6 related pathways, I examined the effects of VR23 on LPS induced p-STAT3, CREB, and SOCS3 and I found that VR23 effectively down-regulates p-STAT3. In the IL-6 induced inflammatory model, I further corroborated this by demonstrating VR23’s potency at reducing p-STAT (Y705), while not affecting upstream markers of the IL6-STAT3 pathway.
Hydroxychloroquine, a well-known DMARD being frequently used at clinics, showed no
6R specific inhibitor, LMT-28. Further, the gene expression of down-stream IL-6-STAT3 pathway proteins, MCP-1 and c-myc were also down-regulated by VR23. To determine if VR23 could affect the IL-6-STAT3 pathway in a chronic RA model, I examined its capabilities in primary HFLS from RA patients and healthy donors. VR23 demonstrated effectiveness in preventing the phosphorylation of STAT3 in HFLS cells from RA patients, while the clinically used DMARD, HCQ, was not effective. Systemic inflammation often involves the production and circulation of CRP [254], which is induced by IL-6 stimulation in hepatocytes [255]. In this model I showed that VR23 has the potential to prevent the phosphorylation of STAT3, while reducing the CRP cytokine levels. Hydroxychloroquine is known for its ability to increase the pH of acidic organelles, which in part is the mechanism that helps to mitigate inflammation.
VR23 was tested in parallel with HCQ to determine their abilities to inhibit phagocytosis and increase the pH of acidic organelles. Interestingly, we found that VR23 was very effective at inhibiting phagocytosis in comparison to HCQ. In addition, VR23 was able to easily increase the pH of acidic organelles, while HCQ actually decreased the lysosomal pH. I found that VR23 has the ability to mitigate acidic organelle’s pH in a relatively shorter time frame. This is a contrast with HCQ which requires quite a long time to show its effects in elevating lysosomal pH.
This study unraveled the effects of VR23 on IL-6, by demonstrating its potency in preventing the phosphorylation of STAT3 (Y705) in monocytes, HFLS cells, and hepatocytes. It also further showed the ability of VR23 to increase the pH of acidic organelles which may be attributed to its effects on the phosphorylation of STAT3. In the future, VR23 could be analyzed in a collagen-induced arthritis mouse model to further understand its effects on mitigating p-STAT3 and IL-6 in sera and synovial tissue.
In Chapter 3, I investigated VR23 treated cells to determine if cells metabolize it and whether the metabolites are active. An LCMS-based study revealed that VR23 is metabolized into two major products, CPQ and DK23, in human cells. I confirmed the identities of the two metabolites by synthesizing CPQ and DK23 and analyzing them by LCMS. I then examined them for their anti-inflammatory activities. DK23 showed anti-inflammatory activity, of which potency was comparable to that of VR23 at their respective IC50 concentrations. In contrast, CPQ was inactive in terms of mitigating inflammation. Specifically, DK23, as an analog of VR23,
showed similar anti-inflammatory effects as VR23 in reducing MCP-1, IL-6, p-STAT3 (Y705) in HFLS-RA cells. These findings should be corroborated in the future with more RA patient samples, as limited samples were available. Perhaps importantly, the VR23 metabolism study led us to the discovery of the DK23 novel anti-inflammatory agent. Thus, it is warranted to further characterize DK23 including in vivo toxicology, solubility and the route of administration, etc. in order to develop it as a potential anti-inflammatory and anti-rheumatic drug.
Overall, my study has demonstrated great potential for VR23 as a novel
anti-inflammatory agent. VR23 has the potential to be used in both acute and chronic settings of inflammation, specifically for RA. VR23 is superior as an anti-inflammatory to the currently available DMARDs, as its mechanism is more specific and it has a low toxicity profile. Further, in comparison to hydroxychloroquine, VR23 appears more effective as it can reduce
inflammatory markers more effectively and in a timely manner. VR23 works to prevent the phosphorylation of STAT3 and its down-stream genes, leading to the reduction of pro-inflammatory cytokines such as IL-6 and MCP-1, which play a critical role in RA disease progression. This allows VR23 to have similar specificity to biologics, with less systemic toxic side effects. DK23, the major active metabolite of VR23, demonstrates similar anti-inflammatory effects as VR23, which functions through the regulation of the IL6-STAT3 pathway. The
validation of DK23’s activity furthers our understanding of VR23 as a novel anti-inflammatory agent. These findings also expand the potential for DK23 in its ability to be used on its own as a novel anti-inflammatory compound for chronic inflammatory conditions. VR23 and its
metabolite, DK23, have great potential as novel compounds for the treatment of chronic inflammatory conditions such as RA by being effective, selective, and low in toxicity.