1.4.1 The development of CDDO and its derivatives
The synthetic triterpenoid, CDDO and its derivatives including CDDO-Im and CDDO-methyl ester (CDDO-Me), have been shown to be potent inducers of Nrf2. The structures of the three compounds are shown in figure 1.3. The key role that Nrf2 target genes play in the response to oxidative stress, together with the anti-inflammatory and anti-proliferative properties of the triterpenoids, mean that CDDO-derived compounds have been widely investigated as potential chemotherapeutic as well as chemopreventive agents.
CDDO is an analogue of the naturally occurring oleanolic acid. Oleanolic acid and its isomer, ursolic acid, are two triterpenoids that are known to have weak anti-inflammatory and anti-carcinogenic properties (Huang et al., 1994; Nishino et al., 1988), and CDDO and its derivatives were originally developed as anti-inflammatory agents. However,
21 subsequent studies have also highlighted their anti-oxidant and anti-proliferative properties (Dinkova-Kostova et al., 2005; Suh et al., 1999).
Initial work showed that the synthetic triterpenoids have anti-cancer properties in a variety of in vitro models including human breast cancer, lung cancer and leukaemia cell lines (Ito
et al., 2000; Konopleva et al., 2004; Lapillonne et al., 2003; Zou et al., 2004). CDDO-Im has been shown to have anti-proliferative properties and induce differentiation in cell models as well as to decrease tumour burden in a mouse model of melanoma (Place et al., 2003). Furthermore, CDDO and CDDO-Me have been used in Phase I clinical trials for treatment of leukaemia as well as for solid tumours (Vannini et al., 2007). The potential utility of the triterpenoids extends beyond the treatment of cancer, with studies investigating the anti- diabetic effects of CDDO-Me (Saha et al., 2010), as well as the potential benefits of CDDO analogues in the treatment of Huntington’s disease and emphysema (Stack et al., 2010; Sussan et al., 2009).
22
Figure 1.3: The structure of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its methyl ester (CDDO-Me) and imidazolide (CDDO-Im) derivatives. All three compounds have been shown to be potent inducers of Nrf2.
CDDO-Me
CDDO-Im
CDDO
23 1.4.2 The synthetic triterpenoids and Nrf2 induction
Initial evidence for triterpenoid activation of Nrf2 came from a study in which CDDO was shown to potently induce the phase II response in embryonic fibroblasts (Dinkova-Kostova
et al., 2005), a response that was abolished in cells from the Nrf2 null mouse. Results from the paper also strongly suggest that Keap1 is a molecular sensor for the triterpenoids. In subsequent work, Liby et al. demonstrated a time-dependent induction of mRNA levels of the Nrf2-regulated gene, haem oxygenase-1 (HO-1), following treatment of U937 human leukaemia cells with CDDO and CDDO-Im, and also an increase in Nrf2 protein levels (Liby
et al., 2005). HO-1 protein induction was also seen in vivo via western blot in a variety of organs including the liver, lung and stomach in an experiment using CD-1 mice treated with 2 μmol CDDO-Im.
The parent compound oleanolic acid has also been shown to induce expression of Nrf2 mRNA and the mRNA of Nrf2 target genes when dosed once daily for 4 days to mice and rats (Liu et al., 2008), while other triterpenoid compounds including the avicins, have been associated with induction of the Keap1-Nrf2 pathway (Haridas et al., 2004).
In a 2007 paper by Yates et al., an NQO1-ARE-luciferase reporter gene assay was used in association with in vivo bioluminescent imaging to determine NQO1 transcriptional activation in a mouse model following treatment with CDDO derivatives. CDDO-Me was seen to induce Nqo1 transcripts in the liver, small intestine and lung after a single dose, while CDDO-Me dosed on three consecutive days also maintained or further increased the levels of Nqo1 expression seen after a single dose, suggesting sustained induction of the Nrf2 pathway using CDDO-Me is possible.
CDDO-Im has been shown to be protective against paracetamol-induced hepatotoxicity in WT but not in Nrf2 KO mice (Reisman et al., 2009). CDDO-Im attenuated the rise in ALTs seen in WT mice 6h after administration of a 500mg/kg dose of paracetamol. A 380% increase was detected in Nrf2 protein levels in nuclear extracts from livers of WT mice treated with CDDO-Im dosed once daily for three days (1mg/kg; i.p.). The synthetic triterpenoids have also been shown to offer Nrf2-dependent protection against damage to other organs. For example, CDDO-Im has been shown to induce an Nrf2-mediated protective response in the kidneys of mice treated with ferric nitrilotriacetate (FeNTA), a compound associated with a high incidence of renal adenocarcinomas in rodents (Tanaka
24
et al., 2008a). Furthermore, CDDO-Im was protective in Nrf2 WT mice against cisplatin- induced renal toxicity (Aleksunes et al., 2010a). Nrf2 has a protective role against neuronal and capillary degeneration in retinal ischemia–reperfusion injury: CDDO-Me is protective in WT but not in Nrf2 KO mice (Wei et al., 2011).
1.4.3 The synthetic triterpenoids also modulate other signalling pathways
A number of other signalling pathways have also been implicated in the documented effects of CDDO and its derivatives. The inflammatory properties are, in part, mediated through modulation of NF-κB signalling and CDDO, CDDO-Im and CDDO-Me have all been shown to inhibit both the constitutive as well as the inducible activation of NF-κB (Shishodia et al., 2006). This may be through inhibition of IKK-β as both CDDO-Me and CDDO-Im have been shown to inhibit IKK-β, an effect that is mediated by oxidation of Cys- 179 (Ahmad et al., 2006; Yore et al., 2006). The inhibitory effects of the CDDO analogues on the transcription of TNF-α, INF-γ, inducible COX-2 and iNOS (Honda et al., 1998; Place et al., 2003; Suh et al., 1998; Suh et al., 1999), are also likely to contribute to the potent anti- inflammatory properties of the compounds.
Other intracellular targets for the synthetic triterpenoids have also been identified including the PPAR-γ receptor (Wang et al., 2000) and STAT signalling (Liby et al., 2006), with CDDO-Me shown to inhibit activation of the JAK1/STAT3 pathway (Ahmad et al., 2008). Consequently, while Nrf2 activation may have a role in the therapeutic properties of CDDO and its derivatives, the relative contribution of other pathways remains to be determined.