The DKPs are a group of modified cyclic di-peptides that can be derived from endogenous neuropeptides. The most tested DKPs are cyclic His-Pro and its modified analogues. Cyclic His-Pro is naturally derived from the tripeptide thyrotropin-releasing hormone (TRH) (Faden et al., 1989; 1999). Both TRH and cyclic His-Pro are neuroprotective after traumatic brain injury (TBI) and spinal cord injury. Several bioactive analogues of cyclic His-Pro also prevent neuronal injury and improve motor and cognitive function (Faden et al., 1989; 1999; Takami et al., 1991). Inspired by their findings, we hypothesized that cyclic-Gly-Pro, another endogenous diketopiperazine with nootropic action (Samonina et al., 2002), may be an additional metabolite of GPE. Such cyclic structures may render a molecule resistant to enzymatic breakdown and more lipophilic for better central uptake. We have therefore examined the protective effects of cyclic Gly-Pro and its analogue, NNZ 2591, which has been modified to overcome potential disadvantages of the endogenous compound and to improve its potency.
The data suggest more potent neuroprotection by NNZ 2591 than by cyclic Gly-Pro after central administration, as the effective doses of NNZ 2591 are lower than those of cyclic Gly-Pro. The introduction of an allyl substituent at C-8a on the diketopiperazine skeleton may thus confer lipophilicity, thereby facilitating the ability of NNZ 2591 to cross the CSF-brain barrier (Guan et al., 2007) after the compound is delivered to the CSF.
The physiological role of the BBB is to selectively prevent large and/or hydrophilic substances from accessing the CNS (Pardridge, 2005). The central uptake of some molecules, regardless of their molecular size, is largely dependent on injury-induced BBB breakdown (Pardridge, 1991; Guan et al., 2004). However, central uptake of NNZ 2591 appears not to be injury dependent, as in normal control rats, the amounts of free NNZ 2591 detected in the CSF were similar to those found in the plasma (Figure 4, Guan et al., 2007).
Figure 4. The left panel shows the half-life of NNZ 2591 in the blood and the cerebrospinal fluid (CSF) after single i.v. administration in hypoxia-ischemia injured adult rats. The right panel shows the level of NNZ 2591 in the blood and the CSF 2 h after single administration of NNZ 2591 in normal rats. ##P < 0.01.
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Although the level of NNZ 2591 in blood fell to half of the initial value 2 h after a single intravenous dose, the CSF level remained the same between 0.5 and 2 h after a bolus injection, suggesting a threshold effect for central uptake (Figure 4; Guan et al., 2007). The window of CSF turnover, which is approximately 1 h in rats, is largely responsible for drug elimination from the CNS due to CSF absorption (Pardridge, 1991; Guan et al., 2004). The maintained CSF level to 2 h suggests a sustained central transfer of NNZ 2591 from plasma and that central uptake of NNZ 2591 compensates for the drug's elimination through CSF absorption. The compound is detectable 6 h after the administration.
Given that the BBB appears to be highly permeable to NNZ 2591, the effective dose range (0.6–3 mg·kg−1) used for peripheral administration is rather high compared with the dose range (2–20 ng rat-1) tested by central administration (Guan et al., 2007). An in vitro albumin binding assay suggests a potential protein binding-releasing of NNZ 2591 in the plasma, which may alter the pharmacokinetics of NNZ 2591 by influencing the level of free compound in the plasma (Guan et al., 2007). Furthermore, the neuroprotective effects of NNZ 2591 show complex dose dependency after central administration, with higher doses being ineffective (Guan et al., 2007). However, this dose dependency of NNZ 2591 is not as prominent after peripheral administration, as the three different doses tested were all effective. The possible binding-releasing process discussed previously may serve as a sustained release depot after the free compound is biotransformed or eliminated from plasma (Endrenyi, 1998).
Although a single treatment resulted in a modest histological improvement, repeated treatment with NNZ 2591 resulted in almost complete long-term protection when histology was examined 9 weeks later (Guan et al., 2007). Repeated treatment with NNZ 2591 also prevents HI injury-induced somatosensory-motor deficit (Guan et al., 2007). Faden et al. have also suggested that another diketopiperazine derived from thyrotropin-releasing hormone could improve recovery of motor and cognitive function following TBI in rats and mice (Faden et al., 2003; 2005). Given that the single treatment used by those authors presumably only prevented cell death within the first few hours after injury, repeated treatment may have targeted the more progressive brain injury which occurred several days after the initial insult. Long-lasting neuroprotective effects are critical for any treatment regimen, as the improved neuronal outcomes detected soon after the treatment may not always be maintained long term (Fisher and Schaebitz, 2000; Gladsrone et al., 2002). Delayed neuronal/glial injury can be mediated through necrotic, apoptotic and necroptotic pathways (Degterev et al., 2005); although the downstream cascades of these cell death pathways largely overlap (Yuan et al., 2003). Cleaved caspase-3 has been used as a marker for the caspase activation-dependent apoptotic pathway, whereas AIF is commonly used as a marker for apoptotic pathways that do not rely on activating caspases (Yuan et al., 2003). Treatment with NNZ 2591 inhibits the protein expression of caspase-3 but not AIF (Guan et al., 2007), suggesting that the inhibition of the caspase-mediated apoptotic pathway may mediate the neuroprotective effect of NNZ 2591.
Even after a broad receptor-binding screening, which included acetylcholine, GABA, glutamate, opoid, adrenergic and serotonergic receptors, the mode of action of DKP has not been identified (Faden et al., 2003). The neuroprotective mechanism of NNZ 2591 also needs further investigation. However, inhibition of reactive microglia and elevation of astrocytes may have a role in the protective effect of NNZ 2591 (Guan et al., 2007). Inhibiting both necrotic and apoptotic neuronal death pathways, without altering cerebral blood flow, has been suggested to be involved in neuroprotection of B35, another modified diketopiperazine, after TBI (Faden et al., 2003). Moreover, inhibiting glutamate-induced excitotoxicity, beta-amyloid induced cells death and promotion of survival factors may also mediate the neuroprotective activity of DKP in vitro (Faden et al., 2005).
The up-regulation of cell cycle proteins occurs in both mitotic and post-mitotic brain cells after brain injury during adulthood. The activation of cell cycle proteins induce cell proliferation in astrocyte and microglia, which are mitotic cells, and initiate caspase-related apoptosis in neurons, which are post-mitotic cells (Byrnes and Faden, 2007). The research from Faden's group also suggests that the effects of B35 on inhibiting neuronal apoptosis and microglial proliferation are mediated by inhibiting the expression of cell cycle proteins (Faden et al., 2005). Thus, inhibiting cell cycle protein may also be associated with the effect of NNZ 2591 on inhibiting caspase-3 activation-dependent apoptosis (Guan et al., 2007).