A more versatile cellular delivery system for adenosine

A more versatile cellular delivery system for adenosine was generated by engineering mouse embryonic stem cells to lack both doripenem of Adk (Fedele et al., 2004). Using a step-wise differentiation protocol (Okabe et al., 1996) Adk−/− ES cells were differentiated into transplantable adenosine-releasing neural precursor cells. These cells were highly effective in reducing stroke-induced brain injury in a mouse model of middle cerebral artery occlusion (Pignataro et al., 2007c). In a more recent approach Adk−/− ES cell derived neural precursor cells were transplanted into the infrahippocampal cleft of rats 1 week prior to the onset of hippocampal kindling (Li et al., 2007). In contrast to wild-type graft recipients and sham-treated control animals, Adk−/− graft recipients were characterized by a profound reduction of kindling induced epileptogenesis (Li et al., 2007). Even after 48 kindling stimulations, recipients of adenosine-releasing cells did not develop generalized stages 4 and 5 seizures, while generalized seizures in control animals first occurred around the 30th stimulation. These results, obtained in the rat hippocampal-kindling model, suggest a powerful antiepileptogenic effect achieved by focal augmentation of the adenosine system. The rat-kindling model is widely considered to be a model of epileptogenesis, and therapies, which suppress kindling development, have predictive value for antiepileptogenic effects (Lothman and Williamson, 1994, McNamara et al., 1985, Racine, 1978). On the other hand, the kindling model is a simplified model of epileptogenesis and does not reflect all histopathological changes observed in human TLE, which develop over months to years, while kindling epileptogenesis develops over weeks. The potential of Adk−/− ES cell derived neural precursors (NPs) to prevent epileptogenesis has recently been further substantiated in the mouse model of CA3 selective epileptogenesis (Li et al., 2008). Adk−/− ES derived NPs were transplanted into the infrahippocampal cleft of mice 24h after intraamygdaloid KA-injection. At this time point, the animals already had the epileptogenesis triggering CA3 selective injury. However, in contrast to wild-type graft recipients or sham-treated control animals Adk−/− NP recipients did not develop spontaneous seizures. Remarkably, when analyzed 3 weeks after transplantation, astrogliosis was significantly reduced, but most importantly ADK expression levels were close to normal (Li et al., 2008). These data further support the conclusion that ADK-deficient brain implants have the potential to prevent epileptogenesis. In an effort to engineer human stem cells for therapeutic adenosine release, human mesenchymal stem cells were engineered to release adenosine based on lentiviral expression of a miRNA directed against ADK. Human mesenchymal stem cell grafts with a knockdown of ADK significantly reduced acute brain injury and duration of SE in a mouse model of KA-induced epileptogenesis (Ren et al., 2007). Since epileptogenesis in patients is not a completed pathophysiological state, but rather a dynamic process leading to progressively increasing frequency and severity of seizures, to pharmacoresistance and eventually to cognitive deficits (Blumcke et al., 2002, Elger et al., 2004, Engel, 2002), the time course of disease progression might provide a sufficient time window for therapeutic augmentation of the adenosine system to prevent the progression of the disease.
Adenosine in human epilepsy The ADK hypothesis of epileptogenesis presented here is largely based on rodent models of ictogenesis and epileptogenesis. Clinical data in support of this hypothesis are still limited though several studies are worth mentioning. Microdialysis studies performed in patients with intractable complex partial epilepsy have demonstrated that following a seizure, extracellular adenosine levels increased 6–31-fold with the increase significantly greater in the epileptogenic hippocampus (During and Spencer, 1992). Likewise, adenosine metabolites were increased in cerebrospinal fluid following SE (Chin et al., 1995). These findings led to the concept that adenosine is an endogenous mediator of seizure arrest and postictal refractoriness (During and Spencer, 1992). Remarkably, and in agreement with upregulated ADK in epileptogenic hippocampus, in these studies basal adenosine levels sampled before the onset of seizures were lower in the epileptogenic hippocampus compared to the non-epileptic hippocampus (During and Spencer, 1992). Findings on A1R expression in human temporal lobe epilepsy are controversial; both down- and upregulation of A1R have been described (Angelatou et al., 1993, Glass et al., 1996). More recent findings from human studies suggest energetic dysfunction and mitochondrial dysfunction to be implicated in epileptogenesis (Kunz, 2002, Pan et al., 2005, Williamson et al., 2005). Although not further addressed in these studies, energetic and mitochondrial dysfunction may be directly linked to adenosine, which has been described as a retaliatory metabolite adjusting energy consumption to energy supplies (Newby et al., 1985). A recent study used the purinergic drug allopurinol as adjunctive therapy in a double-blind and placebo-controlled trial in intractable epilepsy (Togha et al., 2007). This study demonstrated increased seizure reduction in the allopurinol group compared to control; however data were statistically significant only after 4 months of treatment, and allopurinol-induced side effects were common. Despite these caveats, this study demonstrates that modulation of the purinergic system might be beneficial in intractable epilepsy.