**5.1 Bacteria**

with aspirin use in man and only the crustacean body fluid samples examined did

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

As some bacteria, notably *Mycobacterial*, *Yersinia* and *Pseudomonas* species, synthesise SA to enhance iron chelation (see Section 5.1) the possibility of a gastrointestinal particularly colonic, bacterial source for SA was assessed in two animal models. Pooled serum from six mice treated with neomycin 100 mg/kg/day for 4 days had a serum SA concentration which was, at 0.309 μmol/L, slightly higher than the level of 0.268 μmol/L measured in six untreated animals. Other measurements were done on serum samples from Sprague-Dawley rats delivered by caesarean section, raised in a sterile environment and fed sterilised food. A group of 8 such germ free animals had a pooled serum SA level which was, at 0.166 μmol/L, 2.5 greater than the level of 0.069 μmol/L in serum from a group of control

A preliminary study followed serum SA and urinary SA + SU over 3 days on a water/milk diet (confirmed salicylate free on analysis) in a subject free of aspirin for

In six patients who had total colectomy or rectal excision following standard preoperative bowel preparation low level serum SA (range 0.012–0.085 μmol/L) was detected and urinary SA + SU excretion persisted (median lowest level 0.613 μmol/ 24 h; range 0.184–7.607) in all subjects for up to 5 days postoperatively, rising only on refeeding [28]. These results also, of course, have some relevance to Section 4.2.

Benzoic acid (BA) is a natural constituent of plants, with high levels found in fruits and vegetables. In plants synthesis of SA derives—at least partially—from phenylalanine via cinnamic and benzoic acids. Prior work, using formula diet feeding, also demonstrated that hippuric acid, the main metabolite of BA, may be formed endogenously in man, while a Sprague-Dawley rat radiolabeled experiment showed phenylalanine as the likely precursor [32]. Sodium benzoate as a food preservative also contributes to human intake and very high doses have been used in hepatic encephalopathy. These considerations led us to determine whether addition of BA to a very carefully standardised diet produced any change in serum or

A preliminary study, over 4 days in two subjects, suggested that BA, 1 or 2 g/day

on days 3 and 4 might be associated with a modest increase in urinary SA + SU excretion. Subsequently a labelled study was undertaken over 3 days in six individuals (4 M, 2F) who received 1 g of uniformly ring-labelled 13C BA with each of their main meals on day 2. They replicated their carefully recorded day 1 diet throughout and had regular blood sampling with complete urine collections. The **total** SA + SU urinary excretion increased, but not significantly (p = 0.052) and only in the 8–16 h sample after the first dose of BA. While no 13C was detected in samples prior to ingestion of the BA, the 13C isotope was confirmed in the 8–16 h urine sample from all six subjects. Its presence was determined by preliminary GC fractionation before subjecting the relevant fractions to derivatization and GC–MS. The 13C isotope accounted, by selective ion monitoring, for 0.4–10.9% (median 3.4%) in the SA derivative and 6.8–43.1% (median 33.9%) in the SU derivative. In addition considerable amounts of the expected 13C6-labelled hippuric acid were found [28].

at least 2 weeks. Excretion of SA + SU continued at a rate of 2.1 μmol/24 h throughout and serum SA did not fall—over the 72 h of the study—below 0.1 μmol/L

**4.3 SA in diet-restricted and fasting human subjects**

(20 the limit of detection of the assay) [28].

**4.4 SA formation from benzoic acid**

urinary salicylates—see **Figure 1**.

**184**

not contain SA.

animals [28].

This complex area is here only briefly overviewed in relation to its potential for pointing to possible effects of SA preserved into animals.

Para-aminosalicylic acid (PAS), the earliest truly effective anti-tuberculous agent, was long thought an analogue for para-aminobenzoic acid and so an inhibitor of folic acid biosynthesis. That was before the discovery of the mycobacterial siderophore (iron binding molecule) mycobactin, and that SA (also formed, as an extracellular metabolite, by mycobacteria in iron deficient conditions) is its direct precursor. It appears PAS primarily inhibits the conversion of SA into myobactin. Possible secondary roles for SA are the transfer of Fe2+ across the cell membrane, either for direct incorporation into various porphyrins and apoproteins, or for storage of iron within the cytoplasm in bacterioferritin (both roles also potential targets for PAS) [33].

There are many kinds of bacterial siderophores but SA or one of its hydroxylated metabolites (2,3DHBA) are at the core of the aryl- capped molecules found in *E. coli* (Enterobactin); *Yersinia* sp. and *Klebsiella pneumoniae* (Yersinibactin); *Pseudomonas* sp.(Pyochelin); *Vibrio* sp. (Vibriobactin/Vulnibactin) and *Acinetobacter baumanii* (Acinetobactin) [34]. These authors described a probe for the initial aryl acid activation enzymatic step in the synthetic pathways of these "bactins" (*via a nonribosomal peptide synthetase pathway initiated by adenylation*) and suggested lack of human homologues makes this a potential drug target—but see Section 5.3.4.

Intriguingly investigation into the bioinorganic chemistry of bacterial siderophores has revealed that many have functional capacities other than mere iron homeostasis. Examples include interactions with other metals such as zinc, copper and boron; signalling agents (referred to as "ferrimones") in the regulation of genes related to iron metabolism; protection—by those with catecholate structures—from oxidative stress and an antibiotic function in sideromycins [35].

Finally bacterial growth in the presence of salicylate can be both beneficial and detrimental. On the one hand an intrinsic multiple antibiotic resistance phenotype can be induced and on the other reduced resistance to some antibiotics might result and bacterial virulence factors may be affected [36]. While the in vivo consequences of these observations is speculative the findings highlight, the authors suggest, the ability of salicylate to alter gene expression; they claim that the only life form not yet (then) shown to be affected by salicylate is the Archaea!
