Introduction

Since the identification of the gas nitric oxide (NO) as the endothelial-derived relaxing factor, NO has increasingly been recognized as being involved in numerous physiological and pathophysiological processes, in particular those related to inflammation and blood flow. NO can be synthetized by three isoforms of nitric oxide synthase (NOS): nNOS (neuronal NOS) and eNOS (endothelial NOS), which are both constitutive, Ca ²⁺ -dependent enzymes, and iNOS (inducible NOS), which produces large amounts of NO in inflammatory reactions ( ).

Since the first report of showed the protective effects of NOS inhibitors in mouse stroke models, the potential of NOS inhibitors in the treatment of neurological injuries has been emphasized not only in stroke but also in traumatic brain injury (TBI) ( ). While the role of NO in TBI pathophysiology—as in stroke—is widely accepted, inconsistent results have been reported regarding the effect of NOS inhibitors in experimental TBI, thus confirming the double-edged role of NO in brain injury ( ). As a result, NOS inhibitors are not currently in clinical development for the treatment of stroke or—with one exception—TBI.

Changes of NO Metabolism in the Brain After TBI

Numerous animal experiments in different models have shown that TBI causes the accumulation of markers of NOS activity such as nitrate/nitrite (NO _x ) and 3-nitrotyrosine (3-NT) in CSF or in brain tissue ( ). A time-dependent pattern of NO production has been proposed: immediately after injury (within minutes) a transient, significant increase of NO _x occurs as measured by microdialysis and NO-sensitive electrodes ( ). This is followed by a period of decreased NO production, which parallels reduction of the cerebral blood flow (CBF) ( ). The decrease might result from reduced activity of constitutive NOS (endothelial and neuronal). Given that TBI decreases CBF ( ) and NO is a cerebral vasodilator, activity of eNOS may be beneficial early after injury (in animal models up to 30 min postinsult). Indeed, treatment with l -arginine, the substrate of NOS, reduces brain lesion volume and this is associated with eNOS activation and improved CBF ( ). In addition, eNOS−/− mice have decreased CBF when compared with wild-type animals after TBI ( ). Expression of eNOS is necessary to mediate the vascular effect of l -arginine administration after TBI ( ). NO inhalation following experimental TBI in mice induces vasodilatation in hypoxic brain regions, thereby ameliorating regional ischemia, reducing brain damage, and improving neurological outcome ( ).

There is also evidence for the involvement of the other constitutive NOS, nNOS, in TBI pathophysiology. However, the activity of nNOS is difficult to distinguish from that of endothelial and mitochondrial NOS. In addition, TBI experiments with nNOS−/− mice are not available. Treatment with 7-nitroindazole, a relatively selective nNOS inhibitor, following experimental TBI reduces posttraumatic neurological deficits ( ). Pretreatment with 7-nitroindazole decreases the brain lesion volume following TBI ( ) and intracerebral injection of nNOS antiserum reduced blood–brain barrier permeability and cerebral edema ( ). Since NO plays an important role in glutamate-mediated neurodegeneration, nNOS might be the isoform primarily involved in excitotoxic neurodegeneration ( ).

Following TBI in both animals and humans, cerebral NO _x concentration starts to increase considerably after 3–6 h and peaks at 20–42 h postinjury. This delayed increase is most likely due to the activity of iNOS in the course of inflammatory reactions ( ). In iNOS−/− mice reduced motor activity, fewer cognitive deficits, and decreased brain lesion volume were found when compared with wild-type animals after cryogenic TBI ( ). In arteries isolated from rats with TBI, an increased expression of endothelial iNOS disrupts cerebrovascular tone ( ). In general, as in stroke or other conditions, the role of iNOS is considered detrimental in TBI; however, there are some open questions. Some reports suggest potentially beneficial effects of iNOS, particularly in repair processes after brain injury. have shown that iNOS−/− mice have increased cognitive deficits when compared with wild-type mice at 17–21 days after TBI. In addition, iNOS−/− mice have been demonstrated to have lower levels of ascorbate, an endogenous antioxidant, in the brain 72 h after TBI ( ). Animals receiving antisense iNOS oligodeoxynucleotides exhibited an exacerbation of TBI-induced CBF reduction ( ) and iNOS−/− mice showed a reduced recovery of CBF 72 h after TBI ( ). In summary, in animal models iNOS appears to cause detrimental effects during a discrete time window following TBI, while iNOS-mediated beneficial processes (repair) occur later in the time course ( ).

The formation of peroxynitrite from NO and superoxide is considered to be one important mediator of NO-induced toxicity. Since peroxynitrite is very reactive, it modifies proteins (in particular tyrosine residues to form 3-NT) causing their inactivation. Moreover, it triggers lipid peroxidation and induces DNA damage ( ). In addition to NO production also superoxide production is clearly increased in experimental TBI ( ). Therefore, enhanced peroxynitrite formation can be expected in TBI. Indeed, there is ample evidence for an increase of peroxynitrite production in brain tissue and CSF ( ). Both eNOS protein expression and upregulation of iNOS mRNA appear to coincide with the peroxynitrite-mediated protein damage in the closed cortical impact model, suggesting that both constitutive and iNOS isoforms contribute to peroxynitrite-mediated protein damage ( ). Increased formation of peroxynitrite has also been observed in the CSF of TBI patients ( ).

In conclusion, in animal models of TBI, the time course of NO production as well as activity pattern and regulation of NOS isoforms reflects the complex pathophysiology of TBI. Within the time course of TBI pathophysiology, both lack and excess of NO can contribute to secondary damage.

In humans information on the role of NO in TBI is naturally limited. However, increased cerebral NO production as measured by NO _x levels has been demonstrated in cerebrospinal fluid (CSF), in cerebral microdialysate, and in brain tissue after TBI (see Table 8.1 ) and the end products of NO, nitrate/nitrite, levels are correlated with TBI severity ( ). In addition, also in humans, iNOS expression appears to be increased after TBI ( ). The majority of the studies showed a correlation of increased NO _x levels to outcome. However, some studies did not, which can be explained, on one hand, by methodological drawbacks such as low number of patients and intrinsic problems of the analytes microdialysate (dependent on location of the probe) and CSF (no fixed time points, contamination by blood). On the other hand, this might reflect the complex temporal pattern of NO function in the injured brain, as inconsistent results have been reported by the same group ( ).

NOS Inhibitors in Experimental TBI

In agreement with a deleterious role of NO in TBI—at least within a specific time window—various inhibitor studies suggest that inhibition of NOS reduces secondary damage and is, thus, beneficial. Isoenzyme-specific as well as nonspecific inhibitors have been shown to reduce neuronal cell death following TBI ( ). Various selective iNOS inhibitors, such as amino-guanidine, l -N(6)-(1-iminoethyl) lysine hydrochloride and 1400W reduce motor deficits and the extent of brain lesion 24 and 72 h after TBI ( ).

The importance of the temporal sequence of NO production is also supported by inhibitor studies; while studies with preinjury administration of nonspecific inhibitors frequently show no changes or even a worsening of brain injury ( ), studies with postinjury administration of iNOS-specific inhibitors show beneficial effects on post-TBI recovery ( ). One study, however, showed worse recovery from TBI after l -N(6)-(1-iminoethyl) lysine hydrochloride if administered immediately after the injury ( ).

Knowledge of the time window and of the stage of development of the area at risk is required for the successful administration of therapies based on NOS inhibition. This and issues concerning dosing and inhibitor specificity make the development of such therapies complex and demanding. To date, with the exception of the tetrahydrobiopterin antagonist VAS203, none of these animal studies referred to earlier have translated into human clinical trials.

Tetrahydrobiopterin Antagonists as NOS Inhibitors

Activity of all NOS isoforms depends on the presence of the cofactor 5,6,7,8-tetrahydrobiopterin (BH4) ( ). The biochemical function of BH4 is still not fully elucidated, but a role in the catalytic mechanism of NOS (redox activity, modification of the heme environment) is discussed ( ). Other suggested functional roles of BH4 in NOS include enhancement of dimer formation and subsequent suppression of uncoupling, the production of superoxide by NOS in the absence of cofactor BH4 and substrate. Two identical but highly anticooperative binding sites for BH4 have been identified in the protein complex of all NOS isomers ( ).

Since BH4 is essential for NOS activity, the development of pterin-binding site antagonist has been discussed as an alternative approach to arginine-derived antagonists to modulate NOS activity ( ). One of the most potent pterin-based inhibitors is 2-amino-5,6,7,8-tetrahydrobiopterin (VAS203) ( Fig. 8.1 ) ( ). VAS203 competes with exogenous BH4 and inhibits enzyme activity with an IC ₅₀ of approximately 1 μmol/L but cannot displace the cofactor BH4 once bound to the high-affinity site ( ). Thus, in the presence of BH4, VAS203 decreases NOS activity to a basal level (20–40% of the fully activated level) and NO production is not completely suppressed ( ). In the absence of exogenous or endogenous BH4, VAS203 can be incorporated into the high-affinity binding site with a considerably lower IC ₅₀ value of 13 nmol/L ( ).

VAS203 as representative of the antipterin class of NOS inhibitors: (A) cofactor 5,6,7,8-tetrahydro- l -biopterin (BH4); (B) 4-amino-5,6,7,8-tetrahydro- l -biopterin (VAS203).

Therefore, in cell culture experiments using endothelial cells inhibition of NO production by VAS203 was weak, because VAS203—other as in cell-free assays— has to compete with endogenous BH4 present in cells ( ). In contrast, the cofactor antagonist is incorporated in situ directly into the high-affinity binding site in the case of de novo synthesized iNOS resulting in a more effective inhibition of iNOS ( ).

While being an antagonist with respect to the catalytic mechanism, the antipterins including VAS203 can act as agonists of BH4 in other respects such as stabilization of the dimeric enzyme and inhibition of uncoupling ( ).