*5.3.4 ASA "receptors"*

The eminent facility for ASA to acetylate proteins has been known for decades and proteomic studies—*see above*—have shown its very marked extent. While the functional relationship between such activity and its effects are unclear the blockade of glucose6phosphate dehydrogenase (G6PD), affecting the pentose phosphate pathway, and disruption of mitochondrial respiration may explain platelet autophagy [41]. Clearly, as for SA in plants, caution is required in the strict definition of ASA receptors [39, 40].

#### *5.3.5 SA "receptors"*

While the above caveat applies the blunderbuss masking effect of acetylation is not a consideration. At least 15 SA binding proteins are described to date [39] but some intriguing examples point to effects on proteins with plant and bacterial homologues.

More recent work on peroxisome proliferator-activated receptors (PPARs) has also shown the importance of central nervous system actions. Peroxisomes are oxidative (H2O2 producing) organelles subserving redox regulation and metabolism of very long chain fatty acids. They are abundant in the CNS, where such fatty acids abound and their increase, when required, is receptor mediated. Studies have compared the anti-inflammatory effect of agonists of PPARα and PPARγ (themselves inactive at the site of inflammation) with the effect of dexamethasone and ASA. Only other agonists and ASA (which itself has generally no direct\* PPAR agonist effect) were found to diminish inflammation when given after the inflammatory insult in contrast to the effect of dexamethasone. The conclusion was that PPARα and PPARγ regulate inflammation through a mechanism similar to salicylates and distinct from that ascribed to steroids [53]. The authors postulated that activating PPARs in the CNS could elicit **the release of a salicylate-like compound, an endosalicylate**, which may subsequently cause the release of a physiological anti-

These results on CNS activity point to steroid/NSAID differentiation which is at

Given the ever increasing complexity of SA and ASA effects revealed by basic research it seems blinkered to increasingly restrict focus to platelet/COX effects in

We reiterate our conclusion that ASA is no mere anti-platelet prototype [54]. That is the case, we aver, for most of the protean pathophysiological effects of ASA and not solely in cancer chemoprevention. The evidence summarised here, particu-

The place of SA in the biosphere overall is, we think, as pivotal as it appears to be in plants. While a unifying hypothesis to explain its many roles is elusive we suspect they all ultimately relate to the need for evolving life to balance its requirements for oxygen and iron [55]. These authors concluded that the sequestration of iron to restrict its reaction with reactive oxygen species (ROS) is one of our major antioxidant defence mechanisms. They particularly emphasise, in that summary, how such sequestration remains critical to our ongoing resistance to bacterial

The huge increase in energy production arising from enzymatic reduction of oxygen enabled evolution of multicellular animal life. While that was an evolutionary milestone ability to use the resulting reactive oxygen species (ROS) for cell signalling and regulation may have been the first true breakthrough in development of complex life [56]. By then SA was already well established and poised to interact (with ROS) as required. In animals its many effects are unlikely to be less complex than the interactions steadily becoming clarified in plants [39]. *Many may depend upon the type of intricate relationships which initiate plant systemic acquired resistance (SAR) with an initial SA induced redox change. Subsequent SA concentration sensitive*

\* However ASA's apparently direct binding to PPARα may explain its stimulation of hippocampal

plasticity [52] and potential for prevention of Alzheimers.

larly our demonstration of the in-vivo synthesis of SA, points to it as a truly endogenous molecule in animals and man. Potential "preserved" receptors which have been described are there, we suggest, not simply to deal with ingested SA or

least partially dependent on how agents influence the anti-inflammatory and

immunomodulatory effect of melanocortins (ACTH and MSH).

inflammatory substance such as αMSH.

*Salicylic Acid Sans Aspirin in Animals and Man DOI: http://dx.doi.org/10.5772/intechopen.91706*

**6. Conclusions**

the biomedical field.

other exogenous precursors.

infection.

**189**

Human glyceraldehyde3-phosphate dehydrogenase (HsGADPH)—has been identified as a SA binding protein—as it is in plants. In addition to its central role in glycolysis GADPH participates in pathological processes, with effects on viral replication and neuronal cell death [47]. Its suppression, by low μM levels of salicylate, in a model of cell death comparable to that induced by reactive oxygen species (ROS) was found. The authors postulate that likely due to suppression of HsGADPH nuclear translocation, mirroring the effect of the anti-Parkinsonian drug Deprenyl.

The same group have also shown [39, 48] that SA targets human high mobility group box 1 (HMGB1), an abundant chromatin associated protein, present in all animal cells; fungi and plants have related proteins. Its diverse effects modulate inflammatory processes. *HMGB1's many activities and receptors likely account for its multiple roles in human disease which include sepsis, arthritis, atherosclerotic plaque formation and cancer.* The effect of SA on HMGB1 occurs at concentrations far lower than those required to inhibit COX enzyme activity; an effect on COX2 is on synthesis rather than activity.

An example of a bacterial homologue enzyme, found in mice, is responsible for synthesis of 2,5DHBA—the iron binding moiety of a mammalian siderophore [49]; that enzyme is a homologue of bacterial EntA which catalyses 2,3DHBA production during enterobactin biosynthesis (Section 5.1). 2,5DHBA can, of course, also derive from the metabolism of SA or benzoic acid.

Other orthologs of a plant SA receptor—NAD(P)-reductase like proteins—have been characterised in the human neuroblastoma SK-N-SH cell line and mouse brain tissue [50]. Their results may point, the authors claim, to the existence of a thermoregulation system that is evolutionary conserved.

#### *5.3.6 ASA and SA as NSAIDs*

The few direct studies to validate the assertion 5.3.2a above compared salsalate (which yields only SA on absorption) with SA, generally at the higher doses used in rheumatic diseases. Given what we know about the distribution and relative inhibition of COX1 and COX2 it's not surprising that at comparable doses effects were similar with a predictable lower gastrointestinal toxicity of SA [51]. The authors suggested that, when ASA was originally marketed, commercial forces equated taste and tolerability/toxicity! These prominent rheumatologists concluded that "nonacetylated salicylates should be preferred to ASA in rheumatology". They clearly supported the German proverb:- "Bitter im Mund, gesund im Korper."

While the NSAID categorisation originally served to differentiate the side effects of SA and steroids, very early work had shown a CNS effects specific to salicylates. Later studies—stimulated by discovery of the antipyretic/anti-inflammatory actions of the neuropeptide α-melanocyte stimulating hormone (αMSH)—clearly demonstrated peripheral effects of salicylates introduced into the CNS by injection into the lateral ventricle. These experiments showed that CNS doses which had no effect systemically had a marked effect on the mouse model of inflammation used. The effect was restricted to the salicylates; central injection of an anti-inflammatory dose (when given intra-peritoneally) of indomethacin had no effect: neither did intraventricular dexamethasone or prostaglandin E2 [43].

*Salicylic Acid Sans Aspirin in Animals and Man DOI: http://dx.doi.org/10.5772/intechopen.91706*

More recent work on peroxisome proliferator-activated receptors (PPARs) has also shown the importance of central nervous system actions. Peroxisomes are oxidative (H2O2 producing) organelles subserving redox regulation and metabolism of very long chain fatty acids. They are abundant in the CNS, where such fatty acids abound and their increase, when required, is receptor mediated. Studies have compared the anti-inflammatory effect of agonists of PPARα and PPARγ (themselves inactive at the site of inflammation) with the effect of dexamethasone and ASA. Only other agonists and ASA (which itself has generally no direct\* PPAR agonist effect) were found to diminish inflammation when given after the inflammatory insult in contrast to the effect of dexamethasone. The conclusion was that PPARα and PPARγ regulate inflammation through a mechanism similar to salicylates and distinct from that ascribed to steroids [53]. The authors postulated that activating PPARs in the CNS could elicit **the release of a salicylate-like compound, an endosalicylate**, which may subsequently cause the release of a physiological antiinflammatory substance such as αMSH.

These results on CNS activity point to steroid/NSAID differentiation which is at least partially dependent on how agents influence the anti-inflammatory and immunomodulatory effect of melanocortins (ACTH and MSH).

### **6. Conclusions**

*5.3.5 SA "receptors"*

homologues.

Deprenyl.

synthesis rather than activity.

*5.3.6 ASA and SA as NSAIDs*

**188**

from the metabolism of SA or benzoic acid.

moregulation system that is evolutionary conserved.

While the above caveat applies the blunderbuss masking effect of acetylation is not a consideration. At least 15 SA binding proteins are described to date [39] but some intriguing examples point to effects on proteins with plant and bacterial

*Drug Repurposing - Hypothesis, Molecular Aspects and Therapeutic Applications*

Human glyceraldehyde3-phosphate dehydrogenase (HsGADPH)—has been identified as a SA binding protein—as it is in plants. In addition to its central role in glycolysis GADPH participates in pathological processes, with effects on viral replication and neuronal cell death [47]. Its suppression, by low μM levels of salicylate, in a model of cell death comparable to that induced by reactive oxygen species (ROS) was found. The authors postulate that likely due to suppression of

HsGADPH nuclear translocation, mirroring the effect of the anti-Parkinsonian drug

The same group have also shown [39, 48] that SA targets human high mobility group box 1 (HMGB1), an abundant chromatin associated protein, present in all animal cells; fungi and plants have related proteins. Its diverse effects modulate inflammatory processes. *HMGB1's many activities and receptors likely account for its multiple roles in human disease which include sepsis, arthritis, atherosclerotic plaque formation and cancer.* The effect of SA on HMGB1 occurs at concentrations far lower than those required to inhibit COX enzyme activity; an effect on COX2 is on

An example of a bacterial homologue enzyme, found in mice, is responsible for synthesis of 2,5DHBA—the iron binding moiety of a mammalian siderophore [49]; that enzyme is a homologue of bacterial EntA which catalyses 2,3DHBA production during enterobactin biosynthesis (Section 5.1). 2,5DHBA can, of course, also derive

Other orthologs of a plant SA receptor—NAD(P)-reductase like proteins—have been characterised in the human neuroblastoma SK-N-SH cell line and mouse brain tissue [50]. Their results may point, the authors claim, to the existence of a ther-

The few direct studies to validate the assertion 5.3.2a above compared salsalate (which yields only SA on absorption) with SA, generally at the higher doses used in rheumatic diseases. Given what we know about the distribution and relative inhibition of COX1 and COX2 it's not surprising that at comparable doses effects were similar with a predictable lower gastrointestinal toxicity of SA [51]. The authors suggested that, when ASA was originally marketed, commercial forces equated taste and tolerability/toxicity! These prominent rheumatologists concluded that "nonacetylated salicylates should be preferred to ASA in rheumatology". They clearly

While the NSAID categorisation originally served to differentiate the side effects of SA and steroids, very early work had shown a CNS effects specific to salicylates. Later studies—stimulated by discovery of the antipyretic/anti-inflammatory actions of the neuropeptide α-melanocyte stimulating hormone (αMSH)—clearly demonstrated peripheral effects of salicylates introduced into the CNS by injection into the lateral ventricle. These experiments showed that CNS doses which had no effect systemically had a marked effect on the mouse model of inflammation used. The effect was restricted to the salicylates; central injection of an anti-inflammatory dose (when given intra-peritoneally) of indomethacin had no effect: neither did

supported the German proverb:- "Bitter im Mund, gesund im Korper."

intraventricular dexamethasone or prostaglandin E2 [43].

Given the ever increasing complexity of SA and ASA effects revealed by basic research it seems blinkered to increasingly restrict focus to platelet/COX effects in the biomedical field.

We reiterate our conclusion that ASA is no mere anti-platelet prototype [54]. That is the case, we aver, for most of the protean pathophysiological effects of ASA and not solely in cancer chemoprevention. The evidence summarised here, particularly our demonstration of the in-vivo synthesis of SA, points to it as a truly endogenous molecule in animals and man. Potential "preserved" receptors which have been described are there, we suggest, not simply to deal with ingested SA or other exogenous precursors.

The place of SA in the biosphere overall is, we think, as pivotal as it appears to be in plants. While a unifying hypothesis to explain its many roles is elusive we suspect they all ultimately relate to the need for evolving life to balance its requirements for oxygen and iron [55]. These authors concluded that the sequestration of iron to restrict its reaction with reactive oxygen species (ROS) is one of our major antioxidant defence mechanisms. They particularly emphasise, in that summary, how such sequestration remains critical to our ongoing resistance to bacterial infection.

The huge increase in energy production arising from enzymatic reduction of oxygen enabled evolution of multicellular animal life. While that was an evolutionary milestone ability to use the resulting reactive oxygen species (ROS) for cell signalling and regulation may have been the first true breakthrough in development of complex life [56]. By then SA was already well established and poised to interact (with ROS) as required. In animals its many effects are unlikely to be less complex than the interactions steadily becoming clarified in plants [39]. *Many may depend upon the type of intricate relationships which initiate plant systemic acquired resistance (SAR) with an initial SA induced redox change. Subsequent SA concentration sensitive*

<sup>\*</sup> However ASA's apparently direct binding to PPARα may explain its stimulation of hippocampal plasticity [52] and potential for prevention of Alzheimers.

*oligomer/monomer transformation permits nuclear translocation of a cytosolic messenger to activate immune-associated genes* [40]*.*

Re-focus on the importance of the SA moiety of ASA should also lead to further evaluation of SA derivatives which are more active than SA itself in interaction with particular "binding protein/receptors" [47, 48]. At a more basic level we have previously pointed out that, particularly to extend its use in prophylaxis, the risk/ benefit profile of ASA may be improved with an SA/ASA combined formulation [54].

Given what we have learned on this investigative journey it is somewhat paradoxical that we embarked upon it driven by desire to capitalise on the hydroxylation of ASA as a biomarker of oxidative "stress" in man—see Section 2.
