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- Materials and methods
The efficacy of the amphipathic ketoamide calpain inhibitor SNJ-1945 in attenuating calpain-mediated degradation of the neuronal cytoskeletal protein α-spectrin was examined in the controlled cortical impact (CCI) traumatic brain injury (TBI) model in male CF-1 mice. Using a single early (15 min after CCI-TBI) i.p. bolus administration of SNJ-1945 (6.25, 12.5, 25, or 50-mg/kg), we identified the most effective dose on α-spectrin degradation in the cortical tissue of mice at its 24 h peak after severe CCI-TBI. We then investigated the effects of a pharmacokinetically optimized regimen by examining multiple treatment paradigms that varied in dose and duration of treatment. Finally, using the most effective treatment regimen, the therapeutic window of α-spectrin degradation attenuation was assessed by delaying treatment from 15 min to 1 or 3 h post-injury. The effect of SNJ-1945 on α-spectrin degradation exhibited a U-shaped dose–response curve when treatment was initiated 15 min post-TBI. The most effective 12.5 mg/kg dose of SNJ-1945 significantly reduced α-spectrin degradation by ~60% in cortical tissue. Repeated dosing of SNJ-1945 beginning with a 12.5 mg/kg dose did not achieve a more robust effect compared with a single bolus treatment, and the required treatment initiation was less than 1 h. Although calpain has been firmly established to play a major role in post-traumatic secondary neurodegeneration, these data suggest that even brain and cell-permeable calpain inhibitors, when administered alone, do not show sufficient cytoskeletal protective efficacy or a practical therapeutic window in a mouse model of severe TBI. Such conclusions need to be verified in the human clinical situation.
The complex biochemical signaling mechanisms that make up the secondary injury response following traumatic brain injury (TBI) constitutes a major factor in determining the extent of post-traumatic neurodegeneration and neurological outcome. The compromise of calcium homeostasis is a well-established biochemical sequelae of TBI (McIntosh et al. 1997). Inducers of calcium perturbations leading to intracellular calcium overload include trauma-induced glutamate excitotoxicity and subsequent activation of glutamate receptor-operated and voltage-dependent calcium channels. The resulting accumulation in calcium induces downstream activation of enzymes and cytotoxic signaling cascades including those mediated by the cysteine protease, calpain (McCracken et al. 1999).
Activated calpains cleave key cellular substrates including cytoskeletal, membrane-associated, and neurofilament proteins and are associated with increased cell death (Kampfl et al. 1997). The neuronal cytoskeletal protein α-spectrin is a well-characterized substrate of calpain that is degraded into a calpain-specific 145-kDa α-spectrin breakdown product (SBDP), a caspase 3-specific 120 kDa SBDP or a 150 kDa mixed calpain/caspase 3 SBDP that can be differentiated and quantified by western blot (Wang 2000). Analysis of α-spectrin degradation is widely used as a measurement of post-TBI calpain activity and an assessment of biochemical neuroprotection in TBI models (Saatman et al. 1996a; Buki et al. 1999; Kupina et al. 2001, 2003; Thompson et al. 2006) and more recently as a CSF biomarker for human TBI (Farkas et al. 2005; Brophy et al. 2009).
Calpain inhibition has represented a viable neuroprotective therapeutic target for TBI because of its downstream location in the cytotoxic signaling induced by early biochemical disruptions in glutamate and Ca2+ homeostasis (Bartus 1997; Saatman et al. 2010). Thus, delayed Ca2+-induced neuropathological events such as axonal damage and neuronal death can be interjected through calpain inhibition resulting in preservation of neuronal integrity and function. Although calpain activation is an early event following TBI, it has been shown to remain active for at least 24 h days following injury (Thompson et al. 2006; Deng et al. 2007), thus theoretically allowing for a wider therapeutic post-injury time window. Furthermore, inhibition of calpain appears relatively safe as the physiological levels of active calpain are very low and the proactive form of the enzyme is only hyper-activated by calcium during pathological conditions.
There are a multitude of calpain inhibitors that have been developed and evaluated in various models of TBI including AK295 (Saatman et al. 1996b), calpain inhibitor II (Posmantur et al. 1997), SJA6017 (Kupina et al. 2001), and MDL28170 (Thompson et al. 2010). Our recent pharmacological analysis of MDL28170 in the mouse CCI-TBI model revealed the compound to be able to partially decrease calpain-mediated cytoskeletal degradation in the mouse brain after CCI-TBI injury if initially administered 15 min post-TBI followed by a pharmacokinetically appropriate maintenance dosing regimen out to 4 h. However, the anti-calpain efficacy was insufficient to produce a statistically significant effect if treatment was delayed for more than 1 h. (Thompson et al. 2010).
SNJ-1945 ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl) -3-methylbutyl) carbamic acid 5-methoxy-3-oxapentyl ester) is a more recently identified α-keto amide calpain inhibitor that is based on the backbone of SJA6017 (Kupina et al. 2001) and was designed to improve membrane permeability and stability and to increase solubility thereby increasing overall bioavailability. SNJ-1945 was originally developed as a potential orally administered therapeutic for calpain-induced retinal degeneration and dysfunction (Oka et al. 2006; Shirasaki et al. 2006, 2008), but it has recently been shown to be protective in models of focal brain ischemia and cardiac dysfunction (Koumura et al. 2008; Yoshikawa et al. 2010). In this study, we investigated the pharmacological profile of SNJ-1945 in the mouse controlled cortical impact (CCI-TBI) model. We evaluated the dose–response, optimal treatment duration, and therapeutic window of SNJ-1945 in terms of inhibition of calpain-mediated cortical neuronal α-spectrin degradation at its previously determined 24 h post-TBI peak in the mouse CCI-TBI model (Thompson et al. 2006; Deng et al. 2007).
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- Materials and methods
In this study, we assessed the efficacy of SNJ-1945-mediated calpain inhibition in the mouse CCI-TBI model by measuring the degradation of α-spectrin into the calpain-specific 145-kDa SBDP (Wang 2000) at its 24 h peak (Thompson et al. 2006; Deng et al. 2007). We first performed a dose–response analysis using a single bolus of SNJ-1945 administered 15 min post-injury to identify the most effective single dose of SNJ-1945 required to attenuate 24 h α-spectrin degradation. This experiment revealed that 12.5 mg/kg was the ideal dose. However, a U-shaped dose–response pattern was observed, wherein higher doses caused a loss of efficacy. This was followed by testing a short term 4 h and an extended 12 h multiple dosing regimen to investigate whether repeated SNJ-1945 dosing would improve on the inhibition of α-spectrin degradation. It did not, and the extended dosing regimen also resulted in a loss of an effect. Finally, we determined the therapeutic window of the single 12.5 mg/kg dose of SNJ-1945 by delaying the initial treatment time. This last experiment revealed that the cytoskeletal protective action of SNJ-1945 is less than 1 h post-injury.
A major uncertainty concerning calpain inhibition as a therapeutic strategy for TBI is derived from the reported differences among the various pharmacological inhibitors regarding inhibitory selectivity versus simultaneous inhibition of other proteases (e.g., caspase 3, cathepsins, and proteosomal), selectivity for particular calpain isoforms, brain and cellular permeability, dose–response, optimal duration of treatment and therapeutic window in established models of TBI. Conflicting data across TBI experimental studies with various calpain inhibitors have lead neuroprotection researchers to question the potential of pharmacological calpain inhibition to reliably improve experimental outcomes to a degree that makes this approach translatable to human TBI trials. Moreover, the current limitations with the available calpain inhibitors, which may contribute to the reported inconsistencies in TBI outcome measurements, are mostly pharmaceutical (solubility, stability) and/or pharmacokinetic (blood brain barrier and/or neuronal permeability). While SNJ-1945 improves on those limitations, aqueous solubility still is not ideal and more importantly, the U-shaped dose–response might be problematic in clinical studies in which the effective dose range could be narrow. This dose–response pattern has not been described for any of the calpain inhibitors previously tested in TBI models, but this is the first study to carry out a detailed dose–response analysis. Having excluded the possibility of solubility limitations as a pharmaceutical explanation for the U-shaped dose–response, we are left with the idea that there may be an unknown complexity in SNJ-1945s cytoskeletal protective pharmacology, where lower doses protect against post-traumatic calpain-mediated cytoskeletal damage, while at higher doses some opposing action causes the effect to be lost.
A series of repeated dosing experiments were performed to maintain calpain inhibition for the duration of its established peak activity in the mouse CCI-TBI model. The failure of repeated administration of SNJ-1945 every 2 h up to 12 h to reduce α-spectrin degradation is likely because of intracellular drug concentrations reaching the concentration range associated with the right side of the U-shaped dose–response curve. The observed U-shaped dose–response curve for neuronal cytoskeletal protection suggests a sensitive threshold of calpain inhibition where too much inhibition may actually contribute to counter-productive actions in injured neurons. It is plausible that calpain exhibits multimodal effects that vary depending on the needs of the surrounding at risk neuronal population in the injured brain. For instance, it has been observed that there is a requirement for calpain activity in plasma membrane wound repair following mechanical stress in embryonic fibroblasts cultured from calpain small-subunit knockout mice (Mellgren et al. 2007). Moreover, the same investigators utilized isozyme-selective siRNAs to demonstrate that calpain-mediated plasma membrane repair was indeed specific to m- or μ-calpain and not other cytosolic proteases (Mellgren et al. 2009). Therefore, in this study, we speculate that the lack of effect on cytoskeletal degradation with higher doses of SNJ-1945 could be an indirect result of the inhibition of calpain-mediated membrane reparative effects, the results of which antagonize the neuroprotective effects of inhibition of calpain-mediated cytoskeletal proteolysis.
Recently, we described experiments with the calpain inhibitor MDL28170 in the presently employed mouse CCI-TBI model in which we demonstrated neuroprotection in terms of a decrease in cortical α-spectrin proteolysis, but without a reduction in cortical contusion lesion volume (Thompson et al. 2010). Although the reduction in α-spectrin degradation with MDL28170 in that study was modest, the dose–response did not appear to follow a U-shaped curve as an extended treatment regimen of four doses over 4 h and 45 min after TBI proved more effective than administration of two doses over 45 min post-TBI. In addition, a statistically significant reduction in α-spectrin degradation remained when treatment initiation was delayed from 15 min to 1 h post-injury, but not 3 h.
It should be cautioned that this study does not include a histological evaluation of the ability of SNJ-1945 to reduce cortical contusion lesion volume or any behavioral measures related to motor or cognitive outcome. However, as noted above, our previous work with the calpain inhibitor MDL28170 showed that compound was ineffective in reducing lesion volume despite its reduction of calpain-mediated α-spectrin degradation (Thompson et al. 2010). Concerning the lack of an effect on lesion volume, it is plausible that the cytoskeletal protective effect of calpain inhibition is region and perhaps neuronal population specific. Effective inhibition of calpain-mediated α-spectrin proteolysis is likely limited to the area adjacent to the cortical contusion lesion. This area might be referred to as the ‘traumatic penumbra’ analogous to the term ‘ischemic penumbra’, which refers to the area at risk surrounding the core of the infarcted zone in focal stroke studies. Intuitively, in TBI (as well as in ischemic stroke), activation of secondary injury cascades would be delayed in this area compared to the epicenter of contusion lesion site where cell death pathways are more rapidly initiated following injury. Indeed, post-traumatic calpain activation and α-spectrin degradation have been detected as early as 15 min after TBI, which limits the possibility of pharmacologically attenuating acute calpain-mediated proteolysis at the contusion site, where secondary injury mechanisms are more intensely activated and damage evolves relatively rapidly after CCI-TBI (Kampfl et al. 1996; Saatman et al. 1996a).
Whether SNJ-1945 would decrease cortical lesion volume, the decrease in post-traumatic α-spectrin degradation clearly represents neuronal and mainly axonal cytoskeletal neuroprotection since α-spectrin is present in neuronal processes as a neurofilament-stabilizing protein. Consistent with this assertion, MDL28170, which also reduced post-traumatic α-spectrin degradation in the CCI-TBI model (Thompson et al. 2010), has also been reported in the rat fluid percussion TBI model to attenuate corpus callosal axonal degeneration as evidenced by increase axonal sparing and function (Ai et al. 2007).
Despite the axonal cytoskeletal protective effects of SNJ-1945 shown in this report, enthusiasm for the further neuroprotective evaluation of this particular calpain inhibitor appears limited due in part to the narrow U-shaped dose–response curve that may be because of negative effects of the compound on calpain-mediated membrane repair that may predominate at the upper end of the dose–response curve. Furthermore, the compound's ability to demonstrably inhibit cytoskeletal degradation is associated with a therapeutic window that is too short to be relevant to future clinical testing in TBI patients. While slightly longer, the therapeutic window for MDL28170 is also less than 3 h post-injury (Thompson et al. 2010). Although these characteristics do not completely eliminate calpain inhibition as a therapeutic approach, it would seem that their potential for TBI neuroprotection when administered alone is associated with serious limitations across the class of compounds that are direct-acting calpain inhibitors.
We have recently demonstrated in other studies in the mouse CCI-TBI paradigm that treatment with either the mitochondrial neuroprotectant NIM811 (Mbye et al. 2009) or the lipid peroxidation inhibitor U-83836E (Mustafa et al. 2011), which both act to protect mitochondrial calcium buffering, can indirectly limit post-traumatic calpain activation and attenuate α-spectrin degradation with as much as a 12 h post-traumatic therapeutic window. Thus, the approach of protecting cellular homeostatic mechanisms that indirectly limit calpain activation by controlling intracellular calcium levels may be a promising approach for inhibiting the neurodegenerative effects of calcium overload-activated calpain activation in TBI than trying to inhibit calpain directly beyond the first few hours after injury. Nevertheless, it is possible that the addition of a direct calpain inhibitor such as SNJ-1945 might further improve the protective effects of mitochondria protectants or antioxidant compounds, as a combinatorial neuroprotective approach. This remains to be determined in future studies.