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Age-related diseases deprive individuals of a higher quality of life and therefore therapeutics for their treatment provide significant potential. An overview of the observations of nitrones as potential therapeutics in several age-related diseases is presented. Treatment of acute ischemic stroke is one condition where a nitrone (NXY-059) is in late phase 3 clinical trials now. Also presented is a summary of the most recent work we have accomplished on the anticancer activity of the nitrones in a hepatocellular carcinoma. The mechanistic basis of action of these compounds in several animal models is not yet understood at the molecular levels; however, it does appear clear that their anti-inflammatory properties are central to their action, which is based on their ability to down-regulate exacerbated signal transduction processes.
Successful aging can be defined as maximizing lifespan while maintaining the highest quality of life. Lifespan is a measurable parameter but many other subjective parameters are also important in successful aging including freedom from or control of age-related diseases. Consider the two aging paradigms shown in Fig. 1. The area under the curve of both are roughly equivalent. However in one, even though it covers a much larger lifespan, a greater fraction of time is spent in a significantly less-than-optimum quality of life condition when compared to the shorter life paradigm that occupies a longer period of time in a much higher quality of life state. Many age-related diseases become manifest in the second portion of life causing drastic lowering of the quality of life. Therefore focus on abating age-related diseases will yield significant benefit perhaps as much or more than when lifespan issues only are considered.
Our goal for some time has been to understand redox parameters in the etiology of some age-related diseases. From this effort we have discovered that specific nitrones not only have helped us in understanding age-related disease processes but serendipitously have proven to be candidates for therapeutics in some cases. This report provides a perspective on these issues. Specifically this report will present results of studies on the antiaging activity of nitrones followed by a summary of our discovery of their neuroprotective activity in stroke and the current state of the clinical studies. The potential of nitrones in other age-related diseases will then be presented including our recent findings on the anticancer activity of phenyl-tert-butyl-nitrone (PBN) in a hepatocellular carcinoma model. Finally, mechanism of action will be discussed in the context of understanding of the etiology and pathobiology of the age-related disease processes.
Nitrones in aging studies
The popular notion that free radicals are important in aging has led to many studies and the development of methodologies designed to help probe into the complex processes involved. This is only one of several reasons why nitrone-based free radical traps have seen increased use. The nitrone functionality, i.e. X − CH = NO − Y, in general will react with a free radical, R•, to produce a free radical adduct as shown in the equation:
X − CH = NO − Y + R• → X − CHR − NO• − Y.
One of the more commonly used nitrones is α-phenyl-tert-butyl-nitrone (PBN) where X = phenyl group and Y = tert butyl group. When a reactive free radical reacts with a nitrone, the trapped free radical adduct is a nitroxyl free radical and therefore is a much more stable free radical than the highly reactive and very unstable R•. By use of electron paramagnetic resonance (EPR) methods it is possible in some cases to identify R• based on the signature EPR spectrum the nitrone trapped radical presents. This property has made the nitrones useful in characterizing free radical intermediates. Because nitrones react with highly reactive free radicals to render them less reactive, this represents a potential means of controlling free radical processes. Perhaps this is one of the reasons why several studies have been carried out to test the notion that nitrones influence lifespan.
Several studies indicate that PBN has lifespan-enhancing properties (Edamatsu et al., 1995; Sack et al., 1996; Saito et al., 1998; Floyd et al., 2001, 2002a). Each of these studies has technical issues that lead one to question the results obtained. Nevertheless, the most rigorous study was conducted by Cutler's group where 50 C57BL/6 J mice were administered PBN (or not) in the drinking water starting when the animals were 24.5 months old (Saito et al., 1998). Starting at this old age PBN significantly increased the mean lifespan (29.0 vs. 30.1 months P < 0.005) and maximum lifespan (31.7 vs. 33.3 months). Presently a long-term antiaging study in mice starting when they were 2 months old is being done by Dr Richard Miller at the University of Michigan where PBN is compared head to head with several other antiaging candidate chemicals. The experimental results are not available yet.
Nitrones are protective in stroke models
Our seminal observations demonstrating that PBN was neuroprotective in a Mongolian gerbil model were made in 1988 (Floyd, 1990, 1997; Carney et al., 1991; Carney & Floyd, 1991; Floyd & Carney, 1992). These were serendipitous observations because we were attempting to use PBN as an in vivo trap to characterize the possible secondary free radicals formed in an ischemia-reperfusion model of stroke. The expected results of trapping and then characterizing secondary free radicals during ischemic brain reperfusion were a failure; surprisingly however, we discovered that PBN was neuroprotective even if administered after reperfusion had begun in the gerbil stroke model (Floyd, 1997). These observations set in motion an effort to commercialize nitrones for the treatment of stroke that with time led to the selection of a lead compound, a PBN derivative known as NXY-059, which is now nearing the end of the phase 3 clinical trials for acute ischemic stroke. NXY-059 is the disodium salt of the 2,4-disulfonate PBN derivative with the formal chemical name of disodium 4-[(tert-butylimino)methyl] benzene-1,3-disulfonate N-oxide. NXY-059 has undergone significant developmental research effort involving demonstration of its effectiveness in experimental rodent models of stroke (Kuroda et al., 1999; Sydserff et al., 2002; Yoshimoto et al., 2002; Green et al., 2003; Maples et al., 2004; Green & Ashwood, 2005) as well as a primate model where marmosets were subjected to permanent occlusion of the right middle cerebral artery (Marshall et al., 2001, 2003). The window of opportunity when NXY-059 was effective was 3–6 h after starting reperfusion in a transient focal cerebral ischemia in rats (Kuroda et al., 1999). In another study where transient as well as permanent middle cerebral artery occlusion (MCAO) of rats were tested, significant protection was observed when NXY-059 was given starting 2 h after transient MCAO or after 4 h of permanent MCAO (Sydserff et al., 2002). It was demonstrated in the permanent MCAO model of rats that the amount of neuroprotection was linearly dose-dependent on NXY-059 and that protection from permanent MCAO required much higher levels of drug than that required in the transient MCAO model (Sydserff et al., 2002). NXY-059 was effective in marmosets if given immediately after permanent MCAO (Marshall et al., 2001) or when administered at 4 h after MCAO (Marshall et al., 2003).
Data presented in Fig. 2 demonstrate only one example of the results obtained when the marmosets were tested on a Valley Staircase Task where the animals had to reach their ipsi-lesioned (right) arm or their contra-lesioned (left) arm through a center slot in a plexiglass barrier and retrieve food rewards that were placed at ascending height levels of shelves (stairs) radiating upwards and outwards, presenting increasing difficulty of retrieving, from both the left or right side of the center slot (Marshall et al., 2001, 2003). The data presented are those from NXY-059 or vehicle (saline) treated marmosets 4 h after permanent occlusion of the right middle cerebral artery. Clearly the left arm was not used as extensively as the right arm in the tests but NXY-059 significantly enhanced the use of the left arm at 10 weeks and it also enhanced use of even the right arm at 3 weeks after the stroke. In addition to the demonstrated effectiveness in the movement performance test shown in Fig. 2, NXY-059 was also effective in preventing brain damage (evaluated histologically) to the lesioned brain. NXY-059 reduced the overall infarct size by 28% (saline, 324 ± 46 mm3; NXY-059, 234 ± 30 mm3) and demonstrated protection in the cortex, white matter and subcortical structures (Marshall et al., 2003). These results were vital in helping decide to continue the studies in humans. Several safety phase 1 and phase 2 clinical studies have been performed on NXY-059 in humans (Lees et al., 2001; Strid et al., 2002; Lees et al., 2003). Low doses of NXY-059 (250 mg over 1 h followed by 85 mg h−1 for 71 h or 500 mg over 1 h followed by 170 mg h−1 for 71 h) were given to patients with acute stroke. These doses yielded plasma levels of 25 and 45 µmol L−1 with no toxicity or safety issues noted (Lees et al., 2001). Another safety study with acute stroke patients was done where the NXY-059 dosage was significantly increased to 915 mg over 1 h followed by 420 mg h−1 for 71 h as well as at a much higher dose of 1820 mg over 1 h followed by 844 mg h−1 for 71 h (Lees et al., 2003). The plasma levels increased to 260 ± 79 µmol L−1 in the highest dose tested and the study showed that NXY-059 was well tolerated and showed no significant safety or toxicity issues. The phase 3 clinical studies started in May 2003. They are still ongoing but are expected to be finished in 2007 at which time the results will be evaluated to determine if NXY-059 (clinical name Cerovive) will be marketed as a therapeutic compound for patients suffering from acute ischemic stroke (http://www.renovis.com).
Nitrones are protective in other experimental age-related disease models
Experimental hearing loss
Hearing loss occurs during aging but it is also enhanced due to exposure to some environmental toxins such as carbon monoxide, cyanide, trimethyltin and acrylonitrile and it is enhanced by the combination of high levels of noise and these environmental toxins (Fechter et al., 1997, 2004; Rao & Fechter, 2000). Certain therapeutics such as aminoglycosides (Bates, 2003), cisplatin (Fechter et al., 1997) and high levels of dexamethasone or high levels of glucocorticoids to the intrauterine fetus (Canlon et al., 2003) also cause hearing loss. It is not known if the mechanisms involved in age-related hearing loss are the same as those caused by certain therapeutics as well as by noise plus environmental toxins, but there is ample evidence that the latter involves free radical processes (Bates, 2003; Fecher et al., 1997, 2004; Rao & Fecher, 2000). For this reason PBN was used to ascertain if it would alter hearing loss. It has been shown to be effective in mitigating hearing loss that occurs in guinea pigs or rats exposed to carbon monoxide and high level noise (Fechter et al., 1997; Rao & Fechter, 2000) as well as that caused by exposure of rats to acrylonitrile and high level noise (Fechter et al., 2004). PBN was also protective in the hearing loss caused by acoustic trauma in dexamethasone-treated rats (Canlon et al., 2003).
Retinal light damage
Damage to the retina by light occurs throughout life and may contribute to age-related loss in vision. Experimental exposure of albino rats to high intensity light (2700 lux) for 24 h causes significant loss in photoreceptor cells and loss of vision. PBN administration has been shown to prevent this damage in rats (Ranchon et al., 2003; Tomita et al., 2005). It was demonstrated that PBN given as an intraperitoneal injection of 50 mg kg−1 was taken up rapidly into the eye and then decreased exponentially with a half-life of about 2 h (Ranchon et al., 2001). Protection by PBN was demonstrated histologically by measurement of the thickness of the outer nuclear layer of photoreceptor cells as well as by electroretinograms (Ranchon et al., 2001). Retinitis pigmentosia (RP) is a disease causing loss of vision due to mutations in rodopsin resulting in death of photoreceptor cells. Several transgenic models of RP are now available. The effect of PBN was tested in two models where rodopsin was mutated in two positions, at the proline 23 position to histidine (P23H) or where rodopsin had a termination codon at serine 334 (S334ter). PBN was unable to prevent the loss of vision in these two animal models (Ranchon et al., 2003). However, when the animals were exposed to high light-mediated damage, PBN showed protection in the P23H mutant but not the S334ter mutant (Ranchon et al., 2003). The mechanistic basis of how PBN mediates protection from retinal light damage has been studied (Tomita et al., 2005). PBN markedly prevented caspase 3 activation in the photoreceptor cells leading to their apoptosis. PBN was shown to prevent light-mediated activation of activator protein-1 (AP-1) as well as c-fos activation (Tomita et al., 2005).
Many age-related neurodegenerative diseases involve neuroinflammation. In this condition damage to bystander neurons is caused by growth factors, proinflammatory cytokines and neurotoxins such as nitric oxide produced by activated microglia and astrocytes (Floyd, 1999a,b). An example of a disease that has a strong neuroinflammatory aspect is Alzheimer disease (Floyd, 1999b). Additionally, it is likely that brain aging per se exhibits a much less active but nonetheless persistent ‘smouldering’ neuroinflammatory state (Floyd, 1999b). We have conducted two experimental studies that clearly demonstrate that PBN acts to suppress neuroinflammatory processes (Floyd et al., 2000; Biegon et al., 2002).
In one experimental model we induced brain damage to the CA1 region of the hippocampus by administering kainic acid (KA) (Floyd et al., 2000). Three hours after KA administration brain neuroinflammatory processes increased significantly in the target tissue. PBN administration 30–90 min after giving KA caused almost complete suppression of these processes. It was demonstrated that KA induced p38 MAP kinase activation at 3 h in the hippocampus. Additionally the p65 component of the NFκB transcription factor was activated by KA in the hippocampus region. Both p38 activation as well as NFκB activation were suppressed entirely by administering PBN 30 min after KA injection. Additionally PBN almost completely suppressed the KA-induced seizures and the deaths caused by this neurotoxin (Floyd et al., 2000).
In the second experimental model we induced brain inflammation by direct injection of lipolysaccharide (LPS) into the cisterna magna of the rat brain (Biegon et al., 2002). This is a well-known method of inducing acute brain inflammation. The brain inflammation-suppressing effect of PBN was assessed by administering PBN (65 mg kg−1 day−1) in the drinking water starting 24 h after LPS injection and continuing for 6 days after which the rats were killed. Parameters of brain inflammation and NMDA receptor activation were assessed by radiometric methods. Activated microglia was assessed using [3H]PK11195 and additionally [125I]iodoMK801 as a marker of open channel, activated state of NMDA receptors. LPS caused a two- to threefold increase in microglial activation in the temporal and entorhinal cortex, hippocampus and sustantia innominata. PBN caused an approximately 25% decrease in this microglial activation. The largest effect of LPS was a > 50% loss of activation of the NMDA receptors in the temporal and entorhinal cortex, hippocampus and substantia innominata. Furthermore, we demonstrated that PBN completely reversed this loss. Loss of NMDA receptor function occurs in the brain inflammation that occurs in meningitis. It is also noted in Alzheimer disease (AD) and may be responsible in part for the cognitive deficits of AD (Biegon et al., 2002).
Anticancer activity of nitrones
We have discovered that PBN inhibits the development of hepatocellular carcinoma (HCC) in the choline-deficient HCC model (Floyd et al., 2002b; Nakae et al., 2004). Table 1 shows a detailed summary of the data illustrating the results we obtained in a 70-week carcinogenesis trial. Several important points are worth noting. First, PBN significantly suppressed development of HCC as well as hepatocellular adenoma (HCA) in this model. Second, PBN, whether administered in the first 26 weeks, last 44 weeks or for the entire 70 weeks, significantly suppressed HCA and HCC development. Third, PBN itself was not carcinogenic when administered for the total 70 weeks (group 6 of Table 1). We have also shown that 4-hydroxy-PBN, the P450 metabolite of PBN, also has anticancer activity in this model (unpublished observations). Our first observations on the action of PBN in the choline-deficiency model were made over a 12-week treatment period (Nakae et al., 1998). The data (Table 2) showed that this nitrone caused a potent suppression of the size of the preneoplastic lesions. PBN had much less effect on the number of lesions. The amount of 8-hydroxy-2′-deoxyguanosine (8-OHdG) present in the liver DNA decreased markedly with increasing doses of PBN, but was not as sensitive to PBN administration as the size of the lesions.
We have conducted another series of experiments essentially as the one shown in Table 2 except the rats were on the dietary treatments for 16 weeks and, in addition to PBN, we also examined four different congeners of PBN, i.e. the 4-OHPBN, 3-OHPBN, 2-OHPBN and 2-SPBN, i.e. 2-sulfo-PBN (Nakae et al., 2003). We observed that PBN, 4-OHPBN and 3-OHPBN were very active in suppressing the size of the preneoplastic lesions but were less effective in decreasing the number of lesions. However, 2-OHPBN and 2-SPBN were not active in suppressing the size of the preneoplastic lesions. The most striking observation we made was that PBN as well as 4-OHPBN and 3-OHPBN increased apoptosis of the cells in the preneoplastic lesions per se but had the opposite effect on the cells of the surrounding ‘normal’ tissue. These data are shown in Table 3.
The remarkable enhancement of apoptosis in cells within the preneoplastic lesion caused by PBN and active derivatives (Table 3) correlated strongly with the decrease in preneoplastic lesion size (Table 2) and therefore we postulate that the anticancer activity of PBN (and other active derivatives) is due to its ability to enhance death of the ‘committed’ preneoplastic cells. This working hypothesis, however, does not explain the mechanistic basis of the anticancer action of PBN. Our first clue as to the mechanistic basis of how PBN may be acting to enhance apoptosis of preneoplastic cells was a chance observation that nitric oxide (NO) is produced by hepatocytes of livers from rats that are on a choline-deficient (CD) diet (Kotake et al., 2005). NO production by a population of hepatocytes from rats on CD diet was inhibited by administrating PBN. Administration of the nitric oxide synthase catalytic inhibitors aminoguanidine as well as N-nitroarginine completely suppressed NO production. The suppression of NO production by PBN was shown to be due to inhibition of iNOS expression. PBN has been shown to inhibit the enhanced expression of iNOS in several biological systems where this gene is up-regulated due to specific challenges. Other observations which reinforce the view that induction of iNOS (with enhanced NO) in CD livers is suppressed by PBN include (i) enhanced iNOS expression in CD liver demonstrated by Western blotting is suppressed by PBN; (ii) enhanced NO levels in the bile of CD livers as assessed by the MDG-Fe trapping method (Kotake et al., 1999) is suppressed by PBN; and (iii) enhanced iNOS expression in CD livers as assessed by immunofluorescence is suppressed by PBN (unpublished observations).
Our results clearly show enhanced apoptosis of liver preneoplastic cells if iNOS expression is inhibited by PBN and NO production is diminished. Therefore the lack of NO is associated with apoptosis of these cells or, stated another way, the presence of NO prevents the apoptosis of preneoplastic liver cells. Several studies of livers containing tumors (Salvucci et al., 2001; Torok et al., 2002; Radisavljevic, 2003), as well as of isolated hepatocytes (Kim et al., 1997a, 1997b, 2000) demonstrate that NO prevents apoptosis by forming critical S-nitrosylation bonds which inactivate specific caspases, probably caspase 8 (Kim et al., 2000) or 9, thereby inactivating them and preventing the initiation of apoptosis. In addition to this antiapoptotic role of NO, there are several other critical enzymes which NO can inactivate to promote cancer development. These include OGG1 (Jaiswal et al., 2001), the DNA repair enzyme that repairs 8-OHdG lesions in DNA, methionine adenosyltransferase, MAT I/III, that is inactivated by S-nitrosylation (Avila et al., 1997, 1998; Ruiz et al., 1998; Castro et al., 1999; Perez-Mato et al., 1999; Corrales et al., 2002) and PTEN, the tumor suppressor gene product (unpublished observations). Therefore if these four enzymes are inactivated by NO this puts an enhanced pressure on the cells to progress toward cancer: first, by preventing their apoptosis through inhibition of caspases; second, by enhancing the levels of 8-OHdG thereby enhancing the potential for mutations; third, by decreasing the level of S-adenosylmethionine which enhances liver cell proliferation (Avila et al., 1997, 1998; Ruiz et al., 1998; Castro et al., 1999; Perez-Mato et al., 1999); and fourth, by decreasing PTEN activity leading to enhanced Akt-mediated pro-oncogenic cell growth (Cantley & Neel, 1999). Figure 3 presents a summary of this important concept.
Mechanism of action of nitrones
There have been many reports and reviews discussing the mechanism of action of the nitrones but in the final analysis there are several points that are important to emphasize. First, a definitive chemical mechanism to explain the many observations on nitrones in different biological models has not emerged, despite numerous attempts. Second, the concept that nitrones simply act as a sponge to absorb free radicals is generally untenable (see review, Floyd, 1997), although there is the possibility that this may be the case in reference to the action of NXY-059 in stroke since this compound can reach over 200 µm in plasma (Maples et al., 2004). Third, it is very clear that PBN acts as a potent anti-inflammatory agent by suppressing up-regulated signal transduction pathways in several biological models including the brain (Floyd, 1999a,b) and eye (Tomita et al., 2005). It is not clear, however, whether suppression of intracellular oxidants is involved. Fourth, the mechanistic basis of the anticancer action of PBN appears to be its suppression of iNOS induction and the resulting enhancement of NO production. Suppression of iNOS expression does, however, relate to its known action of suppressing exacerbated signal transduction pathways as discussed above. It is clear that specific nitrones may have different actions even in a specific biological model. Therefore further work is required to establish the role of specific nitrones in reference to particular age-related diseases and models. This approach can be expected to yield new understanding of the etiology of the age-related diseases.
The carcinogenesis data summarized was that collected with funds from the NIH R01CA82506. The commercialization of the nitrones was first begun by Centaur Pharmaceuticals Inc. of which the author was cofounder and the further commercialization presently being done by Renovis Inc. with which the author acts as a consultant. The author is the inventor of several patents related to the application of nitrones to the treatment of several age-related diseases. The author thanks many scientific colleagues and those involved in industry over the years that helped make this work possible.