Although there has been much evidence concerning the probable role of nNOS inhibitors in perinatal asphyxia [Bolaños and Almeida, 1999
], there has been insufficient evidence to move forward to a clinical trial, especially as the benefit in ameliorating motor deficits in animals has not been shown until recently [Derrick et al., 2004
]. Since the effects of nNOS inhibition are opposite to those of eNOS inhibition [Bolaños and Almeida, 1999
], the most desirable property of a potential neuroprotectant is to have a wide margin between inhibition of nNOS and eNOS in the effective dose range. 7-NI is one of the most popular nNOS inhibitors, purportedly for its specificity [Moore et al., 1993
]. However, the margin for inhibition between nNOS and eNOS is at best 4-fold. It has been speculated that 7-NI may act through an unknown mechanism other than nNOS inhibition [Matsumura et al., 2008
A new series of nNOS inhibitors was designed using fragment hopping [Ji et al., 2008
, 2009b; Silverman et al., 2009
], a new fragment-based approach, for de novo inhibitor design focusing on ligand diversity and isozyme selectivity. This led to a series of compounds that has a much higher in vitro specificity than all nNOS inhibitors currently available [Ji et al., 2008, 2009a]. We selected one of the most effective of these compounds, JI-8 (compound 5 in Ji et al. [2009b]
), and compared its neuronal protection with the effect of 7-NI in our rabbit cerebral palsy model.
The dose of JI-8 was chosen based on previous experience [Ji et al., 2009b
], and 7-NI was given to the animals at the same molar concentration to compare equivalent doses. In the neurobehavioral tests, we found that the JI-8 group had significant improvement over the control group and was significantly better than the 7-NI group. Interestingly, the percentage of severe outcomes was not different between the JI-8 group and the 7-NI group. One possible explanation would be that JI-8 rescues those kits that would otherwise have been stillbirths, thus shifting the distribution to survival and to the left on the abscissa of figure . The other possibility would be that the optimal dose of JI-8 was not employed. Although JI-8 only showed transient inhibition of nNOS activity [Ji et al., 2009b
], the long-term effect of JI-8 still needs further investigation. Our data suggest that JI-8 is more potent than 7-NI, but the optimal dosing strategy still needs to be worked out to test for increased survival and decreased incidence of severely affected kits.
JI-8 significantly decreased nNOS activity in fetal brains. This suggests that JI-8 could pass both the placental and blood-brain barriers. 7-NI only showed limited protection, although it has to be said that its dose was much lower (1/100 to 1/50) than those used by other researchers [Moore et al., 1993
; Alderton et al., 2001
; Willmot et al., 2005
]. We also did not observe any inhibition of fetal brain nNOS activity by 7-NI. This implies that 7-NI is a much less potent nNOS inhibitor than JI-8, but it might be beneficial due to a mechanism other than inhibition of nNOS. The difference between 7-NI and JI-8 confirms findings in the existing literature that decreasing NO during HI is an important mechanism of neuroprotection. The most likely mechanism affected is that of oxidative stress from NO and NO-derived radicals. The developing fetal brain is most vulnerable to oxidative stress because of immature antioxidant enzyme systems [Mishra and Delivoria-Papadopoulos, 1988
; Khan and Black, 2003
], early development of prooxidant systems such as xanthine oxidase [Vettenranta and Raivio, 1990
] and NOS [Keilhoff et al., 1996
], and having a relative deficiency in tetrahydrobiopterin that predisposes to uncoupling of nNOS and the production of superoxide from nNOS [Vásquez-Vivar et al., 1999
]. NO reacts with available superoxide to form peroxynitrite [Beckman, 1991
] and reactive nitrogen species [Bolaños et al., 2009
], which are powerful biological oxidants [Knapp et al., 2001
]. In rat neuronal cultures, peroxynitrite is believed to cause neuronal damage following hypoxia-reoxygenation [Cazevieille et al., 1993
] and it leads to necrosis and apoptosis in motor neurons [Estevez et al., 1998
]. Low levels of peroxynitrite may result in apoptosis in cortical neurons, while high levels induce necrotic cell death [Bonfoco et al., 1995
]. Further studies are needed to establish the link between reactive nitrogen species and motor deficits.
During the experiment, JI-8 did not show any measurable effects on the blood pressure and heart rate of dams as both remained unchanged for 40 min after receiving the drug. However, even the low dose of 7-NI had some undesirable effects. It significantly decreased the dam's heart rate during the HI procedure following drug delivery (fig. ). This decrease may not be clinically significant but does not help in recovery. A decreased heart rate, if not compensated by increased cardiac output, has the potential to further compromise uterine blood flow. Furthermore, when we administered the 100-fold higher dose, JI-8 again did not show any detectable effects on blood pressure and heart rate. In contrast, when a 100-fold higher dose of 7-NI was delivered (in the concentration range used in other studies), we observed a significant drop in systolic blood pressure in the dams (fig. ). The later onset of cardiovascular effects on the rabbit dams suggests that 7-NI may be either inhibiting eNOS or acting through other, additional mechanisms.
We used the TRAP assay to discover whether there was any antioxidant effect of JI-8 or 7-NI. The procedure of our TRAP assay is also called the Trolox-equivalent antioxidant capacity assay [Huang et al., 2005
]. Usually, the results are read at the end of the reaction [Huang et al., 2005
], but we calculated the capacity of the solution based on the areas under the curve. Because the reaction rates are different, our method was more suitable to represent the true antioxidant capacity over time. JI-8 did not behave like a classic antioxidant as the reaction curve for JI-8 is quite different from that for Trolox (fig. ). There was a slow reaction during the TRAP assay. This can be explained by the aminopyridine group in its structure [Ji et al., 2009b
], which can react with ABTS·+
. Since the reaction with the artificial oxidant ABTS·+
is slow, whether JI-8 could function as a true antioxidant or scavenger of reactive nitrogen species in vivo needs further study.
In summary, we found that JI-8 showed superior neuroprotection to 7-NI in our rabbit cerebral palsy model. JI-8 had not only higher potency and specificity, but also had fewer side effects on the cardiovascular system. It is possible that JI-8 has some antioxidant capacity, but it does not behave like known antioxidants. With the development of newer nNOS inhibitors, further studies may reveal even more potent and specific drugs that may be suitable for ultimate translation to clinical studies.