The research discussed above led our group to investigate a role for CDKi drugs in inflammation. Our research paradigm has been that inflammation should be driven down resolution pathways by the induction of apoptosis in granulocytes followed by their effective clearance by professional phagocytes such as macrophages (Leitch et al., 2008). We initially examined the in vitro effects of R-roscovitine, NG-75 and hymenialdisine on human neutrophils at different time points and drug concentrations. These CDKi drugs are structurally diverse but their uniform effect was to promote neutrophil apoptosis in a time- and concentration-dependent manner as evidenced by annexin-V binding and morphological assessment. Neutrophils treated with R-roscovitine and the caspase inhibitor zVAD-fmk failed to enter apoptosis, suggesting that R-roscovitine was acting in a caspase-dependent manner. This was evident at 4 h post-incubation when caspase 3 cleavage was already detectable. The most intriguing in vitro result was the ability of the CDKi drugs to overcome diverse survival factors including db-cAMP, GM-CSF and LPS. These survival factors utilize the major inflammatory signalling pathways: PI3K, NF-κB, JAK/STAT and MAPK to augment neutrophil survival. Given that the major hurdle to development of anti-inflammatory agents is the redundancy conferred by these same signalling pathways, this result underlined the potential of CDKi therapy for treatment of inflammatory disease. Inflammation research demands in vivo experimentation as it is impossible to recreate the inflammatory milieu in vitro and hence to predict the efficacious translation of an agent that has been successful in vitro. R-roscovitine was investigated in three mouse models of neutrophil-dominant inflammation including: carrageenan-induced pleurisy, bleomycin lung injury and passively induced arthritis. In the carrageenan pleurisy model, 100 mg·kg−1 of R-roscovitine administered intra-peritoneally reduced an established inflammatory infiltrate to near-basal levels consistent with those found in an untreated mouse pleural cavity. There was a reduction in populations of inflammatory cells (including neutrophils, monocytes and macrophages), oedema formation and pro-inflammatory cytokines. This effect was reversed in vivo by administration of the caspase inhibitor zVAD-fmk. In mice with established bleomycin lung injury, there was a reduction in BAL neutrophil numbers assessed after 3 days, a reduction in histopathological lung inflammation after 7 days and an effect on bleomycin-induced lethality. Finally, in mice with established passively induced arthritis, there was an improvement in clinical scores of arthritis following R-roscovitine administration (Rossi et al., 2006). These in vivo findings suggest encouraging pleiotropic effects of CDKi drug on granulocyte recruitment, survival and removal. Pleiotropic effects of CDKi drug are supported by work from Liu et al. who have shown that CDK4 is important in leukocyte recruitment and adhesion. They studied CDK4−/− knockout mice with bleomycin lung injury and utilized siRNA to CDK4 and CDKi to show that CDK4 inhibition inhibited leukocyte recruitment in the mouse model and leukocyte adhesion in EC matrix models (Liu et al., 2008). In addition, Sekine et al. have now demonstrated positive effects of flavopiridol and a specific CDK4,6 inhibitor on animal models of rheumatoid arthritis (Sekine et al., 2008). Their findings suggest lymphocyte-independent effects of CDKi drugs (including down-regulation of fibroblast proliferation and growth) responsible for improvement in joint histology and clinical arthritis scores in various mouse models. Findings in inflammatory joint and lung disease models are mirrored in kidney disease models where the CDKi drug R-roscovitine has entered phase 1b clinical trials for inflammatory kidney disease. Glomerulonephritides are characterized by inflammation and progressive, scarring destruction of key functional kidney units leading to renal dysfunction and failure. Preclinically, CDKi drugs have been shown to protect renal tubular epithelium from enhanced apoptosis while inhibiting the abnormal proliferation of tubular epithelial and mesangial cells. In vitro and in vivo work has demonstrated that R-roscovitine can restore normal kidney function in animal models of IgA-mediated glomerulonephritis, crescentic glomerulonephritis, lupus nephritis and collapsing glomerulonephropathy (Gherardi et al., 2004; Milovanceva-Popovska et al., 2005; Zoja et al., 2007; Obligado et al., 2008). The work with NZBxNZW mice affected by early or established proliferative lupus nephritis is particularly interesting, as while leucocyte-driven inflammation was shown to be reduced, there was also evidence of a direct effect of CDKi against autoimmune T- and B-lymphocyte responses. It is perhaps less surprising that CDKi should work in this setting, given the proliferative potential/state of differentiation of lymphocytes, but nonetheless, the possibility of pleiotropic action against autoimmune inflammation is an exciting one.
Further evidence for the utility of CDK inhibition has been provided by research into the properties of endogenous CDKis. The physiological CDKi p21 (WAF1, SD1, Cip1), a specific inhibitor of CDK2, 4 and 6, has been shown to negatively regulate macrophage activation by reducing TNF-α and IL-1β production in response to LPS. Additionally, p21−/− mice have an increased susceptibility to LPS-induced shock which is associated with elevated levels of IL-1β (Lloberas and Celada, 2009; Scatizzi et al., 2009). In an inflammatory lung disease model, p21 was over-expressed in the lungs of mice subjected to bleomycin injury by an intra-tracheal adenoviral transfer method (Inoshima et al., 2004). p21 expression in lung epithelial cells led to a reduction in lung inflammation, preservation of epithelial cells and reduced fibrosis. In rheumatoid arthritis patients, p21 gene transfer was shown to down-regulate expression of inflammatory mediators and tissue-degrading proteinases such as: IL-6, -8, type I IL-1 receptor, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-3alpha, cathepsins B and K, and matrix metalloproteinases-1 and -3 (Nonomura et al., 2003).
In summary, CDK inhibition promotes neutrophil apoptosis in vitro even in the presence of powerful survival factors and promotes resolution of inflammation, in vivo, in various animal models of neutrophil-dominant inflammation. Neutrophil apoptosis has been shown to be central to the resolution of inflammation by caspase inhibition which reversed the beneficial impact of CDKi drugs. In addition, the anti-proliferative and anti-apoptotic effects of CDKi drugs protect epithelia against inflammatory insult. CDKi drugs also prevent lymphocyte proliferation and pro-inflammatory signalling indicating potential effects against chronic and autoimmune, inflammatory disease. An understanding of the mechanism of action by which these powerful anti-inflammatory effects are achieved may allow optimization of CDK inhibition and suggest further targets for pharmacological intervention.