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Publication: In Vitro

Abstract
The principal active constituent of the botanical drug candidate PBI-05204, a supercritical CO2 extract of Nerium oleander, is the cardiac glycoside oleandrin. PBI-05204 shows potent anticancer activity and is currently in phase I clinical trial as a treatment for patients with solid tumors. We have previously shown that neriifolin, which is structurally related to oleandrin, provides robust neuroprotection in brain slice and whole animal models of ischemic injury. However, neriifolin itself is not a suitable drug development candidate and the FDA-approved cardiac glycoside digoxin does not cross the blood–brain barrier. We report here that both oleandrin as well as the full PBI-05204 extract can also provide significant neuroprotection to neural tissues damaged by oxygen and glucose deprivation as occurs in ischemic stroke. Critically, we show that the neuroprotective activity of PBI-05204 is maintained for several hours of delay of administration after oxygen and glucose deprivation treatment. We provide evidence that the neuroprotective activity of PBI-05204 is mediated through oleandrin and/or other cardiac glycoside constituents, but that additional, non-cardiac glycoside components of PBI-05204 may also contribute to the observed neuroprotective activity.

Finally, we show directly that both oleandrin and the protective activity of PBI-05204 are blood brain barrier penetrant in a novel model for in vivo neuroprotection. Together, these findings suggest clinical potential for PBI-05204 in the treatment of ischemic stroke and prevention of associated neuronal death.

Discussion

We have shown that the cardiac glycoside oleandrin can provide robust neuroprotection in a brain tissue-based model for ischemic injury, reminiscent of that which we have previously established for the related cardiac glycoside, neriifolin, in this brain slice model as well as in two independent whole-animal models of ischemic stroke (Wang et al. 2006). Interestingly, the effectiveness of oleandrin in inhibiting human cancer cell growth is associated the expression of the a3 subunit of the Na+, K+-ATPase (Yang et al. 2009), and whereas three of the four known a-subunit Na+, K+-ATPase isoforms are expressed in brain (all but a4), the a3 isoform shows primarily neuronal expression in the adult CNS (McGrail et al. 1991; Watts et al. 1991). Together with similar neuroprotective properties we previously reported for digoxin, digitoxin, and ouabain in the brain slice model (Wang et al. 2006), we suggest that this emerging pharmacology of neuroprotection indicates the Na+, K+-ATPase (Kaplan 2002) as a likely target of action of oleandrin, as well as of the botanical drug PBI-05204, derived from an extract of the Nerium oleander plant, whose principal active component is oleandrin.
Intriguingly, PBI-05204 was originally developed as a clinical candidate for the treatment of cancer, and represents an increasing appreciation of the utility of target engagement with the Na+, K+-ATPase enzyme in cancer therapy (Newman et al. 2008; Prassas and Diamandis 2008). The benefits of cardiac glycosides in cancer and focal ischemia may be mediated through inhibition of the classically known function of Na+, K+-ATPase, namely, the active transport of Na+ and K+ ions across the plasma membrane (Kaplan 2002). As the Na+, K+-ATPase accounts for 40–70% of ATP consumption in the brain (Astrup 1982; Clausen et al. 1991; Friberg and Wieloch 2002), one possible neuroprotective mechanism in stroke is that inhibition of Na+, K+-ATPase preserves ATP levels at a time when catastrophic decreases in ATP levels are leading to necrotic and other forms of neuronal cell death (Golstein and Kroemer 2007; Besancon et al. 2008). Such a mechanism as part of a metabolic ‘arrest’ of transmembrane ionic gradients was proposed by Hochachka (1986) as a defense strategy against hypoxia across the animal kingdom.

Additionally, while excess Ca2+ entry is a principal component of excitotoxicity (Choi 1995; Pettmann and Henderson 1998), drastic decreases in cytoplasmic Ca2+ can also be damaging, especially in the penumbra of stroke (Franklin and Johnson 1992; Johnson et al. 1992; Lee et al. 2000). In fact, abnormally low levels of intracellular Ca2+ have been reported in models of transient ischemia in a 1–3 day period corresponding to the progression of cell death in the penumbra (Connor et al. 1999). Inhibition of the Na+, K+-ATPase would likely lead to increases in neuronal intracellular Ca2+ by the same mechanism as the inotropic effect of digitalis compounds in treating congestive heart failure, namely, the secondary inhibition of the Na+/Ca2+ exchanger via erosion of the Na+ transmembrane gradient (Hardman et al. 1996). In fact, the Na+/Ca2+ exchanger itself has been directly implicated in neuronal survival in stroke models, although the mechanism remains unclear (reviewed in Besancon et al. 2008).

However, it is also possible that modulation of the pleiotropic signaling functions of the Na+, K+-ATPase may be involved in the neuroprotective effects reported here.

There is a growing appreciation, for example, that Na+, K+-ATPase does not operate in isolation as a simple transmembrane pump, but rather forms the central core of a complex signalosome which has been described as a vesicular, multimolecular signaling complex that is assembled in caveolae and delivers signals to specific intracellular components such as mitochondrial membranes (Garlid et al. 2009). Other activities associated with the Na+, K+-ATPase signalosome include elevated intracellular Ca2+, activation of Src and the ERK1/2 pathways, as well as activation of phosphoinositide 3-kinase and protein kinase B (Akt) (Schoner and Scheiner-Bobis 2007). Additional involvement of one or more of these alternative, Na+, K+-ATPase signalosome-associated pathways in oleandrin-mediated neuroprotection cannot be excluded at present.

We have also provided indirect evidence for additional non-cardiac glycoside components contained in PBI-05204 that provide neuroprotection in the brain slice stroke assay.

In future studies, it will be of interest to determine whether molecular components mediating these neuroprotective effects, distinct from oleandrin and other cardiac glycoside constituents of PBI-05204, can be isolated and identified. Alternatively, it is possible that these additional components in PBI-05204 may act as co-factors in cardiac glycoside action at the Na+, K+-ATPase, perhaps binding to different sites in the extended Na+, K+-ATPase receptor complex or modulating enzyme-associated signalosome activities and function.

Finally, although a full, in vivo study of PBI-05204 in whole-animal models of stroke was beyond the scope of the present report, we have shown here that lasting neuroprotective benefit can be conferred by PBI-05204 via its systemic delivery in an in vivo setting in which the BBB remains intact. In contrast, digoxin, the only cardiac glycoside in current clinical usage in the U.S., does not cross the BBB. Although this approach could not be used to determine directly an in vivo time window for therapeutic benefit following focal ischemia, we could show in explanted brain slice assays that the neuroprotective benefit of PBI-05204 is maintained for up to a 4–6 h delay of compound administration after OGD. Collectively, these attributes of PBI-05204-mediated neuroprotection suggest its potential use in the treatment of clinical stroke, and extend previous reports from us and others implicating inhibition of the Na+, K+-ATPase in providing neuroprotection against ischemic injury (Wang et al. 2006; Tzen et al. 2007; Gottron and Lo 2010). PMID: 21950737

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