Academic foreign compound metabolism – ‘quo vadis’?

Keywords:

• Foreign compound metabolism
• drug metabolism
• xenobiochemistry

In the early days of academic foreign compound metabolism, the biologically-assisted chemical alteration of a compound, it was usually agreed acceptable to identify the structure of metabolites down to 5% or 2% of the given dose if possible. Anything less was regarded as beyond the limits of detection during that time, and any extraction procedures with subsequent concentration may have led to the creation of metabolic artefacts (metabonates; Beckett 1971), a problem still encountered today (Verpoorte et al. 2022). The compounds that were identified were usually then ranked arbitrarily into ‘major’ and ‘minor’ metabolites. Regulatory authorities have since decreed to industry, in alignment with current opinion, that drug metabolites should be identified and, in some cases, their potential toxicity investigated if they represent 10% or more of the dosed product (Gross and Wilson 2009; Schadt et al. 2018; Food and Drug Administration 2020). In actuality drug metabolite identities are usually determined to below this level. Modern technology is quite able to deliver these results, with some techniques measuring down to the femtogram range or further. This is extremely useful when measuring microscopic or exceptionally dilute samples. Indeed, for known active metabolites there is no threshold value even from regulatory perspectives and in these instances as much detail as possible must be sought.

Ostensibly, it appears that the absolute number of drug metabolite molecules is not critical. The dosage of different pharmaceutical agents varies enormously. Some (e.g. warfarin, colchicine) are given in milligram quantities whereas others (e.g. ampicillin, sulfasalazine) may be prescribed in gram amounts. The molecular weights of these compounds mentioned are roughly similar (308.3, 399.4, 365.4, 398.4 gmol⁻¹, respectively) so there may be a thousand-fold difference in the number of molecules (of each particular drug) administered. In terms of these number of molecules, 10% of a drug given in gram doses correlates with 100 times the total dose of a drug given at the milligram level. With such varying drug dosage regimens, citing a fixed percentage of an administered dose appears somewhat arbitrary where the absolute number of molecules are concerned. However, these differing number of drug molecules may have an equivalent potency with respect to their particular biological activity, be it therapy or toxicity. Additionally, in this context, such a difference blends into insignificance when the total number of molecules to which the organism is exposed is taken into consideration. Just 1% of a 1 mg dose of warfarin still contains 10¹⁵ molecules. These factors affect the absolute amounts of metabolites produced; finding a minor metabolite for a low dose drug may test the limits of detection.

Many of the xenobiotic molecules entering the body may be chemically altered, become biotransformation products, but to what limits should such metabolites be sought? Shortly after the emergence of chlorpromazine onto the clinical scene (1951/2), three metabolites were suspected, the major one being the sulfoxide (Williams 1959). A few years later 17 metabolites were reported (Huang 1967), then 26 whilst 168 theoretical metabolites were proposed (Green and Forrest 1966). It is quite probable that these predicted metabolites (and more) exist in miniscule quantities and have so far avoided detection (or not been looked for). A more recent example is that of evobrutinib, a tyrosine kinase inhibitor (Caldwell et al. 2019), which has been reported to provide 40 Phase I and 18 Phase II metabolites (Scheible et al. 2021). Only one of these 58 metabolites accounts for more than 10% of the administered dose and this has been further investigated (Scheible et al. 2023). Soon a xenobiotic may be reported to have several hundred metabolites, but to what avail? With the exception of those amounting to over 10% or so of the dose one could view the pursuit of the remainder as obsession with minutiae, merely pedantic ‘stamp collecting’. However, if a minor metabolite is suspected to be, or has been shown to be, reactive especially in a toxicological manner, this ‘stamp collecting’ is essential. Complications arise if the troublesome metabolite is not isolable. The substance may be retained covalently bound to macromolecules within the organism, may be inseparable from endogenous material during analysis or may have ‘disappeared’ by shedding its intrinsic label (radioactive, heavy isotope) during metabolism or be in a disguised form such as a dimer, polymer or unexpected conjugate. All of these factors, and more, may cause a metabolite to go ‘missing’ and precipitate unforeseen consequences (Wang et al. 2023).

Presumably all possible biochemical metabolites exist fleetingly at some level, even down to a single molecule, their production being a function of probability. Inherent physio-chemical properties, fortuitous spatio-temporal orientation during molecular collisions together with momentary energetic favourability in alignment with overall thermodynamic and kinetic considerations dictate such events (Nicholson and Wilson 2003). Chemical aspects concerning the reactivities of specific sites within the molecule that may undergo metabolism are also a major and perhaps an overriding consideration. Over the years several compounds have furnished metabolites that, in the first instance, may not have been expected. However, as these metabolites develop a structural dissimilarity to the administered compound, they may become virtually unrecognisable. Simply degradation products, fragments recycled through the machinery of intermediary metabolism. When does a metabolite cease to become an accepted metabolite? It is true that a multitude of metabolites may each account individually for a small, and perhaps insignificant, percentage of the administered dose, but collectively they summate and the organism is exposed to this combined array of metabolites. The potential problems of synergy and a ‘cocktail effect’ must be borne in mind. Although it is generally accepted that threshold levels are required for virtually all physicochemical reactions to initiate a measurable response, some carcinogenic theories believe that direct genotoxicity may require only one molecule. However, presumably for one molecule to breach the myriad biological defences and recovery systems to produce a clinical effect, it may require a relentless attack by a vast number of like molecules, virtually a ‘forlorn hope’ of chemical assault.

Of course, drug metabolism is only part of xenobiochemistry. Many molecules investigated in academia for their metabolic fate are not useful as drugs and have little relevance, importance or interest to the pharmaceutical, agricultural or commercial industries; they are academic explorations. When xenobiochemistry was in its infancy, a remark often heard at meetings was that this ‘new discipline’ was of scant consequence and was ‘simply putting Beilstein through a rat’ (Beilstein – organic chemical database). Today, with the exception of a few atypical reactions, emulatory quirks and esoteric permutations, all of the routes of xenobiotic metabolism appear to have been elucidated. Some may disagree with this statement; indeed, the present authors hope that there are still more surprises to unfold. The study of foreign compound metabolism has therefore followed the time-honoured pathway of scientific exploration and endeavour. Initial research carried out by talented amateurs working in home-based laboratories followed by input from academics, who expand the field and delineate the main areas. Then, as the discipline becomes ‘mainstream’, the involvement of industry with the financial resources to make practical applications of the knowledge gained. Where does this leave xenobiochemistry? Given that the talented amateurs will find it very difficult to contribute to modern science, with its requirement for expensive machines, and the collective of multinational pharmaceutical companies (‘big pharma’) must necessarily focus on pursuing profit from new products, only academics have the occasional luxury of speculation about the natural world. Despite financial restraints, the function of academics remains what it has always been, to advance basic study and to use imagination to integrate aspects of pure science with everyday life. Academic accomplishments in this field have now diversified and the emphasis has changed. Evaluating quantitative differences in metabolite formation led to the study of polymorphic variation in metabolic capacity, the upsurgence within genetic research permitted the uncovering of genetic and epigenetic associations between drug metabolism enzymes and transporter molecules and the advances in the various omics technologies allowed the resurrection of the importance of the microbiome. These are among the more recent ventures. They all involve setting xenobiochemistry, and more broadly pharmaco-toxicology, in the context of modulation by biochemical and physiological factors. With an increased understanding that what we are able to measure is only the above-sea-level tip of an iceberg, this must lead to a more nuanced approach. Will the gamut of personalised medicine with the associated capacity to re-balance the gut microbiome and the immune system be the next frontier? Time will tell.

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The authors report no declarations of interest.

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Data sharing is not applicable to this article as no new data were created or analysed in this study.

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