Abstract
An increasing number of therapies for spinal cord injury (SCI) are emerging from the laboratory and seeking translation into human clinical trials. Many of these are administered as soon as possible after injury with the hope of attenuating secondary damage and maximizing the extent of spared neurologic tissue. In this article, we systematically reviewed the available preclinical research on such neuroprotective therapies that are administered in a non-invasive manner for acute SCI. Specifically, we reviewed treatments that have a relatively high potential for translation due to the fact that they are already used in human clinical applications or are available in a form that could be administered to humans. These included: erythropoietin, NSAIDs, anti-CD11d antibodies, minocycline, progesterone, estrogen, magnesium, riluzole, polyethylene glycol, atorvastatin, inosine, and pioglitazone. The literature was systematically reviewed to examine studies in which an in vivo animal model was utilized to assess the efficacy of the therapy in a traumatic spinal cord injury paradigm. Using these criteria, 122 studies were identified and reviewed in detail. Wide variations exist in the animal species, injury models, and experimental designs reported in the preclinical literature on the therapies reviewed. The review highlights the extent of investigation that has occurred in these specific therapies, and points out gaps in our knowledge that would be potentially valuable prior to human translation.
Introduction
A 1. Applying the agent to the overlying dura (and depending on penetration through the dura, diffusion through the cerebrospinal fluid [CSF] within the intrathecal space and into the cord) 2. Injecting the agent into the intrathecal space (and depending on its diffusion through the CSF into the cord) 3. Injecting the agent directly into the spinal cord
Within the enormous scope of pre-clinical investigation for SCI, there are numerous compounds that have been evaluated after some form of direct application to the spinal cord. Here, we limit the scope of this systematic review to three therapeutic strategies that have received considerable attention in the past two decades and are in various stages of clinical translation to promote endogenous neuroregenerative repair following SCI: (1) the degradation of inhibitory chondroitin sulfate proteoglycans with chondroitinase ABC; (2) the neutralization of myelin-mediated inhibition of neurite outgrowth with anti-Nogo (IN-1) antibodies or other Nogo-related approaches; and (3) the inhibition of Rho activation. The present systematic review provides an overview of the body of pre-clinical evidence that supports, or fails to support, the translation of the aforementioned neuroregenerative biological therapeutic strategies into human trials for SCI.
Methods
A PubMed search was conducted on “the therapy” and “spinal cord injury” (e.g., “chondroitinase ABC and spinal cord injury”). From the list of studies generated through this fairly indiscriminate search, we then applied the following inclusion criteria to systematically review the pre-clinical literature on biological therapies applicable to SCI: 1. Studies that include testing of the therapy in an in vivo animal model of spinal cord injury (i.e., exclusively in vitro studies were excluded) 2. Studies in which the spinal cord is traumatically injured with a contusion or compression device or is partially or completely transected (i.e., non-traumatic local or global ischemia, photochemical reaction, traumatic root avulsion, or dorsal root entry zone models were excluded) 3. At least two peer-reviewed publications available on the therapy (i.e., biological therapies supported by less than two peer-reviewed publications were excluded)
The data from the studies that fit the criteria were then tabulated to depict the animal model, injury model, treatment's dose and timing, experimental groups tested in the study, number of animals used (or “n” per group), and reported behavioral and non-behavioral outcomes (e.g., histologic, biochemical, or physiologic outcomes). A summary statement about the body of literature was then generated.
Results
Using this selection process, we identified the following therapies: chondroitinase ABC, anti-Nogo approaches, and Rho antagonists (Table 1). The PubMed searches on these therapies were initially conducted in the spring/summer of 2008 by SCI researchers across Canada and an updated search was conducted in June 2009. By applying the previously described criteria (essentially, in vivo animal studies utilizing a traumatic model of spinal cord injury), the following studies were generated, and the tables for each of these respective therapies are listed below.
Chondroitinase ABC
This systematic review revealed 23 studies, which utilized chondroitinase ABC (ChABC) in an in vivo model of SCI (Table 2). As would be expected, the majority of studies involved the use of the rat species, although one mouse model (Carter et al., 2008) and one cat model (Tester and Howland, 2008) were tested as well. The injury models typically were that of “sharp” or “crush” injuries, such as a hemisection, over-hemisection, transection, dorsal crush, or forceps compression, reflecting the mechanism of action of ChABC and the desire to measure axonal sprouting/growth in response to it. Two studies employed an NYU impactor for a thoracic contusion injury (Ikegami et al., 2005; Iseda et al., 2008). Notably, Iseda and colleagues (2008) actually compared ChABC in both a contusion and a hemisection model and reported that ChABC promoted sprouting only in the hemisection, but not the contusion, SCI model.
aCSF, artificial cerebrospinal fluid; BBB, Basso, Beattie and Bresnahan locomotor test; BMS, Basso Mouse Scale; C5, cervical vertebra 5; ChABC, chondroitinase ABC; CSPG, chondroitin sulfate proteoglycan; CST, corticospinal tract; CTB labeled, cholera toxin subunit B; DREZ, dorsal root entry zone; GAG, glycosaminoglycan; GAP, growth-associated protein; GSK-3ββ, glycogen synthase kinase-3ββ; GFAP, glial fibrillary acidic protein; ICV, intracerebroventricularly; IT, intrathecally; MAb, monoclonal antibody; Mg, Matrigel; NT-3, Neurotrophin-3; NPSC, neural precursor cells; OEG, olfactory-ensheathing glia; PI, post-injury; PNG, peripheral nerve graft; q48h, interval 48 hours; SC, Schwann cell; SCI: spinal cord injury; SD rats, Sprague-Dawley rats; T8, thoracic vertebra 8; YFP-H, yellow fluorescent protein.
The majority of injuries occurred in the thoracic spine, although eight of the 24 articles employed cervical injury models. The mode and timing of administration in this series of articles varied substantially. The ChABC was typically either injected directly into the cord at various depths and distances from the injury site or delivered via intrathecal injection/infusion. The dosing regimen ranged from immediate, single application (e.g., Tan et al., 2006; Yick et al., 2003, 2004) to continuous infusion over many weeks (e.g., Massey et al., 2008).
A number of researchers have described the use of ChABC as a supplement to a cell transplant therapy such as Schwann cells, olfactory-ensheathing glia, peripheral nerve transplants, fetal cells transplants, or neural precursor cells (Chau et al., 2004; Fouad et al., 2005, 2009; Houle et al., 2006; Ikegami et al., 2005; Vavrek et al., 2007). The inclusion of these studies in this table could be debated as the ChABC was utilized as an adjunct (and by itself these are less informative about the therapeutic applicability of ChABC as a stand-alone therapy). Nonetheless, we include them here as they fulfilled the basic criteria of being in vivo assessments within a traumatic SCI model.
In keeping with the interest in ChABC's putative mechanism of action, 13 of the 24 studies were solely focused on anatomical/histologic outcomes, with no measurement of behavioral outcome (Carter et al., 2008; Chau et al., 2004; Fouad et al., 2009; Iaci et al., 2007; Ikegami et al., 2005; Iseda et al., 2008; Massey et al., 2006, 2008; Shields et al., 2008; Tan et al., 2006; Tom and Houlé, 2008; Vavrek et al., 2007; Xia et al., 2008; Yick et al., 2003). In general, independent laboratories report ChABC promoting increased axonal sprouting (particularly serotonergic fibers) using either anterograde or retrograde tracing techniques. As an adjunct treatment to a transplantation therapy (e.g., Schwann cells, peripheral nerve graft), ChABC reportedly improved axonal growth and/or serotonergic sprouting (Chau et al., 2004; Fouad et al., 2005; Houle et al., 2006; Shields et al., 2008; Tom and Houlé, 2008). Xia and colleagues (2008) reported a reduced cystic cavity, suggestive of some neuroprotective effect. Carter and colleagues (2009) reported the prevention of neuron atrophy in the cortical regions that contained CSNs projecting to the thoracic spinal cord. In the same article, the effect on the intracellular signaling was demonstrated.
As for behavioral outcomes, the use of cervical injury models by a number of authors allowed for the assessment of forelimb functional recovery. In the cervical injury models, evidence for improvements in forelimb function with ChABC was modest. While Yick and colleagues (2004), Houle and colleagues (2006), and Garcia-Alias and colleagues (2008) reported some improved forelimb performance with ChABC treatment (the latter in a number of metrics), significant improvements with ChABC alone were not observed by others (Kim et al., 2006). In thoracic injury models, behavioral recovery with ChABC treatment alone was found to be modest in some studies (Caggiano et al., 2005; Huang et al., 2006; Tester and Howland, 2008), while others described no improvements (Barritt et al.). Fouad and colleagues (2009) reported the positive effects of ChABC on bladder function and morphology. In combination with a cell transplant therapy, behavioral recovery was greater when the transplant was combined with ChABC than without (Fouad et al., 2006; Kim et al., 2006), suggesting that the ChABC may play an important role as an adjunct therapy for such transplants.
Anti-Nogo approaches
The approach of targeting myelin inhibition, and specifically what is now known as Nogo and its downstream pathways, is one of the longest-studied therapeutic strategies in SCI, with the first description of the effect of anti-Nogo (IN-1) monoclonal antibody on corticospinal tract sprouting emerging in 1990, nearly 20 years ago. Everything published over the subsequent decade on this strategy came from Dr. Martin Schwab and his colleagues in Zurich as they sought to characterize the effects of this treatment. These studies employed rat and primate species and a sharp partial transection injury (e.g., dorsal hemisection, over-hemisection, unilateral pyramidotomy) of the thoracic or upper cervical spine, with an IN-1 secreting hybridoma implanted into the dorsal frontro-parietal cortex typically at the time of spinal cord injury. Many of these studies examined histologic outcomes exclusively, with a focus on whether the IN-1 antibody was stimulating axonal regeneration/sprouting (which typically it was). Behavioral outcomes were also improved in some studies. Certainly, there was compelling evidence from this single laboratory to support the promise of the IN-1 antibody therapy, providing a rationale to pursue the translation of this approach.
With the cloning of Nogo in 2000, the strategy surrounding this aspect of myelin inhibition has evolved into different approaches. The IN-1 monoclonal antibody approach (which involved the transplant of a hybridoma secreting IgM antibody) has been supplanted by an intrathecally applied anti-Nogo IgG antibody approach, which has now entered clinical trials. This intrathecally applied anti-Nogo IgG treatment has been tried in both rat and primate SCI models, with immediate post-injury administration leading to both anatomic and functional improvements (Freund et al., 2006, 2007, 2009; Liebscher et al., 2005; Wannier-Morino et al., 2008). This anti-Nogo IgG intrathecal approach has been translated into human clinical trials, with a European and Canadian trial being initiated in 2007.
Another approach being developed by Dr. Stephen Strittmatter and his colleagues at Yale University is the competitive antagonism of the Nogo receptor with a synthetic Nogo-66(1-40) peptide (Nogo extracellular peptide, residues 1-40), otherwise known as “NEP1-40” or “Nogo-66 receptor antagonist peptide.” This has been applied both intrathecally and systemically (via subcutaneous injection) in mouse and rat studies utilizing both cervical and thoracic partial transection spinal cord injury models. In this acute treatment paradigm, NEP1-40 has been reported to promote both histologic improvements (i.e., enhanced sprouting/regeneration), and modest behavioral improvements (Atalay et al., 2007; Cao et al., 2008; Grandpre et al., 2002; Li and Strittmatter, 2003). It has also been reported by Li and Strittmatter (2003) to be effective in a subacute treatment paradigm with a 7-day delay post-injury prior to intervention. Steward and colleagues (2008) attempted to reproduce these findings with NEP1-40 in an NIH-funded replication study, and while there was a weak suggestion that NEP1-40 “created a situation that was slightly more conducive to axon regeneration,” the robust sprouting/regeneration and improved behavioral recovery reported by Li and Strittmatter were not observed.
An additional approach, also pioneered by Strittmatter and colleagues, is the use of a soluble Nogo receptor ectodomain, which has been administered intrathecally at the time of injury in rodent thoracic contusion and partial transection models (Li et al., 2004; Wang et al., 2006). In both cases, there was improved behavioral recovery. In the study by Wang and colleagues (2006), a 3-day delay in treatment did not nullify the behavioral improvements.
Measured within the context of the SCI research community, the pre-clinical body of work behind the anti-Nogo approaches is considerable (Table 3). There are good examples of treatments that have been extensively studied, but the majority of investigation has resided within single laboratories. The significant benefits of NEP1-40 (Li and Strittmatter, 2003) were not observed in a formal, NIH-funded replication study (Steward et al., 2008), suggesting the need for further investigation to characterize the robustness of this intervention. The anti-Nogo antibody treatment has not undergone a similar replication study, although commercial intellectual property issues will likely preclude such an investigation. A recent study that evaluated the corticospinal tract after a dorsal hemisection injury in two different Nogo-deficient mutant mouse lines found enhanced regeneration in neither (Lee et al., 2009). Interpreting the implications of such negative findings in this loss-of-function experiment with the positive findings from Dr. Schwab's laboratory with the administration of anti-Nogo antibodies is difficult given the obvious differences in experimental paradigm.
Ab, antibody; BBB, Basso, Beattie and Bresnahan locomotor test; C5, cervical vertebra 5; CGRP, calcitonin gene related protein; CSF, cerebrospinal fluid; CST, cortico-spinal tract; HRP, horseradish peroxidase; KO, knockout; MEP, motor-evoked potentials; PI, post-injury; SCI, spinal cord injury; SD rats, Sprague-Dawley rats; T8, thoracic vertebra 8.
Rho antagonists
The Rho pathway is recognized as an important biochemical signaling pathway in growth cone dynamics and neuronal apoptosis. Inhibition of Rho activation may therefore influence axonal sprouting/regeneration and secondary damage at the injury site. The strategy of inhibiting Rho within in vivo models of SCI has been explored by a number of investigators (Table 4). Dr. Lisa McKerracher and colleagues were the first research group to demonstrate that the application of Rho-kinase inhibitors, Y27632 and C3 transferase, directly to the SCI epicenter immediately after injury could significantly improve behavioral recovery within mice that had received a dorsal over-hemisection experimental lesion (Dergham et al., 2002). This C3 transferase approach was found to reduce RhoA activation after contusive thoracic SCI (Dubreuil et al., 2003).
BBB, Basso, Beattie and Bresnahan locomotor test; C5, cervical vertebra 5; GAP, growth-associated protein; GST, glutathione S-transferase; PI, post-injury; SCI, spinal cord injury; SD rats, Sprague-Dawley rats; T8, thoracic vertebra 8.
Interestingly, the experience of others with C3 transferase early on was not similarly positive. Sung and colleagues (2003) reported that their C3-treated animals were severely emaciated, and many were terminated. Fournier and colleagues (2003) observed no axonal regeneration/sprouting and delayed hindlimb locomotor recovery, despite significantly reduced scar formation with C3-treated animals compared to fusion protein control-treated animals. Nonetheless, design improvements to create a C3-fusion protein consisting of the C3 transferase coupled to a transport sequence (i.e., BA-210) facilitate the ability for this biological compound to cross plasma membranes and gain increased distribution throughout the injured spinal cord. This more permeable form of C3 transferase has been translated into human clinical trials under the name Cethrin® (BioAxone Therapeutique, Montreal, Quebec, Canada). Rights to Cethrin have since been licensed to Alseres Pharmaceuticals (Hopkinton, MA). A recent report by McKerracher's group studied BA-210 in a rodent contusion injury model and examined different time windows of intervention within a mouse hemisection model (Lord-Fontaine et al., 2008). The authors reported improved behavioral outcomes with the BA-210 in both injury models, with an effective time window extending 24-h post-injury.
The other approach that has been studied is that of Y27632, a selective Rho-kinase inhibitor (downstream of Rho). This has been investigated both as an oral drug and as a directly applied agent. In mouse and rat contusion and partial transection models of SCI, this was found to promote behavioral recovery (Dergham et al., 2002; Fournier et al., 2003; Sung et al., 2003; Tanaka et al., 2004). Chan and colleagues (2005) demonstrated that low doses of Y27632 were detrimental to the rodent cord after dorsal hemisection, while higher doses tended to accelerate recovery.
Discussion
This article reviews three therapeutic approaches, which entail the direct application of a biological therapy to the injured spinal cord or overlying dura; two have already been commercialized and have moved forward into clinical trials (the anti-Nogo antibody trial [Novartis] and BA-210 [Cethrin]). The anti-Nogo antibody approach that evolved from the IN-1 antibody work has been fairly extensively studied, albeit almost exclusively by Dr. Schwab and Novartis. The C3 approach went into clinical trial as BA-210 after the studies of Dergham and colleagues (2002) and Dubreil and colleagues (2003) were reported. At this stage, replication studies of these patented technologies are unlikely to occur, although the NIH-funded replication of the initial NEP1-40 results has demonstrated how difficult it can be to demonstrate the robustness of the effect.
The fact that these therapies involve some form of direct application to the spinal cord influences their clinical translation in a number of ways. For one, the main target of these agents is to promote axonal growth/sprouting/plasticity by altering the inhibitory SCI environment or the response of axons to this environment. Yet most of the agents are tested in an acute injury paradigm with immediate application at the time of injury, and hence, sorting out the behavioral responses attributable to axonal growth versus those attributable to some form of neuroprotection is difficult. This was, for example, quite evident in the study by Dergham and colleagues (2002), where improved behavioral recovery was seen almost immediately after injury and would therefore be difficult to attribute to the promotion of axonal regeneration. The subsequent study by Dubreuil and colleagues (2003) pointed out that RhoA activation occurred locally at the site of injury after acute SCI and suggested a mechanism by which immediately applied C3 transferase (and also BA-210, or Cethrin) might have a neuroprotective effect. Conceivably, the anti-Nogo and ChABC approaches may be expected to have some additional neuroprotective effect in the rodent spinal cord as well, given that they are administered very early after injury in these pre-clinical studies.
From the patients' perspective, it matters little whether improved function is related to some form of neuroprotection or some effect on axonal growth. However, at a translational and operational level, the distinction between neuroprotection versus neuroregenerative mechanisms has substantial relevance, given that the need to directly apply these treatments to the exposed spinal cord or dura imposes significant practical challenges to the translation of these therapies. The time delay may be significant before such treatments can be then applied to an acutely injured patient, owing to transport time and availability of imaging and operating room facilities. So if it is indeed a neuroprotective mechanism of action that is being sought, the direct application to the spinal cord clearly has some limitations with respect to how quickly the therapy can be instituted. Incorporating that inherent delay in clinical treatment into experimental pre-clinical studies is simple enough (one just waits before applying the therapy); more difficult is taking the time window of intervention in human studies and extrapolating that to rodent studies (and vice versa). How similar or different the temporal pattern of relevant biological processes is between rodents and humans is unclear, although work by Dr. Lynne Weaver and colleagues would suggest that the time course of cellular invasion into the injured cord is reasonably similar between the two (Fleming et al., 2006). Irrespective of this, when reviewing the studies included in these tables, one cannot help but feel that more pre-clinical studies with delays of intervention are needed. In 21 of 23 ChABC studies, 22 of 24 anti-Nogo studies, and 8 of 9 anti-Rho studies (i.e., 91% of all studies), the intervention was applied immediately after the injury was induced.
The other important consideration in the translation of these directly applied biologic therapies is their biodistribution within the injured spinal cord. It is difficult to ascertain the degree and kinetics of the agents' distribution into the spinal cord, either via diffusion through the cerebrospinal fluid within the intrathecal space (e.g., anti-Nogo antibodies) or diffusion through the dura and then across the CSF (e.g., BA-210). For example, in most ChABC studies, a sharp partial transection model is employed (hence opening up the dura), and then the ChABC is directly injected into and/or infused onto the cord. How to translate the findings of such studies into a clinically relevant therapeutic paradigm is not clear. Similarly, partial transection injuries have been used for most anti-Nogo studies, so determining what the biodistribution of the antibody within a contused spinal cord is unclear. Finally, for an extradural application of a protein such as Cethrin, while the biodistribution in a rodent model of cord injury may be acceptable (where the CSF space is very small), how such a protein distributes to the injured human spinal cord when it must diffuse through a substantial amount of CSF is unknown. These are all important considerations for such directly applied therapies.
In summary, in this article we systematically reviewed the preclinical animal model data on ChABC, anti-Nogo approaches, and anti-Rho approaches. The question of whether any one of these approaches is ready for human translation has already been answered to some extent, as both the anti-Nogo antibody and the Rho antagonist Cethrin are well into clinical trials. Despite this, there remain questions about the time window of applicability and the biodistribution of these agents within the injured cord. Further pre-clinical work on these therapies is warranted in order to refine and optimize the treatment paradigms for human study.
Footnotes
Acknowledgments
This work was supported by the Rick Hansen Institute (formerly The Canadian SCI Solutions Network). BKK holds a CIHR New Investigator Award, and WT is the Rick Hansen Man in Motion Chair of Spinal Cord Injury Research.
Author Disclosure Statement
No conflicting financial interests exist.