Mini review
Safety testing of metabolites: Expectations and outcomes

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Abstract

Metabolites arising from chemical entities, old or new, are often mediators of toxicity. Frequently, metabolites are investigated in test animals, with the expectation that the resultant toxicity or activity will mimic the exposure of their formed counterparts. This communication described observations that showed discrepant kinetics between formed and preformed metabolites in the liver, intestine, and kidney, major drug removal organs. Differences in the observed areas under the curve (AUCs) or the extraction ratios (Es) of formed and preformed metabolites in the liver had been attributed to zonal, enzyme heterogeneity, membrane barriers, or transporters. Preformed and formed metabolite also differed in their handling by the kidney; only the preformed and not the formed metabolite would be filtered. In the intestine, differences in the absorption of the precursor and the metabolite and the flow pattern in the intestine would bring about discrepancy in the time-courses of the formed vs. preformed metabolites. Analytical solutions of the AUCs of the metabolites and extraction ratios, based on physiological modeling of the liver, kidney, and intestine, showed that the AUC of the preformed, administered metabolite was dependent only on metabolite parameters, whereas the AUC of the formed metabolite was modulated additionally by the metabolic, secretory and intestinal absorptive intrinsic clearances of the precursor drug. Hence, administration of the synthetic metabolite would not reflect the toxicity associated with the metabolite formed via bioactivation. However, data on preformed metabolite may be used for simultaneous fitting by a combined model of drug and metabolite. Such a strategy is shown to be successful in risk assessment of environmental chemicals. Upon refinement of the resultant model with data on metabolite transport and handling by modeling and simulations, the resultant model would be more robust to provide improved predictions on metabolite toxicity pursuant to drug administration.

Introduction

Metabolites are recognized as mediators of toxicity and/or contributors of biologic activity. The determination of risk necessitates a thorough investigation of what the toxic species is and its behavior. Metabolite administration may be required when the metabolite is toxic or active, and especially when the metabolite is unique, namely the metabolite exists only in man and not in rodents and non-rodents that are used for testing. The synthesis and administration of the synthetic, preformed metabolite to animals and humans have been suggested in the assessment of toxicity and pharmacologic activity.

In February 2008, the US Food and Drug Administration issued a Guidance that classified metabolites as major ones when the metabolite in the circulation exceeds 10% of drug exposure [1], whereas in a previous publication, the Metabolite In Safety Testing (MIST) document had suggested 25% exposure [2]. The meaning of %exposure is, however, vague. If the comparison was based on the ratio of areas under the curve (AUC) of the metabolite/drug, the inference is likely based on a similarity in the volumes of distribution of the formed and preformed metabolites, and of parent compound. When the metabolite and parent drug are structurally dissimilar and of different lipophilicities and pKa's, the assumption would not hold. Others proposed to base the estimates on unbound concentration of the metabolite in circulation, or amounts of metabolites in the excreta [3], [4]. Regardless of the basis, the rationale for administration of the synthetic metabolite is the expectation that the toxicity and activity of the preformed and formed metabolites are identical and that the kinetics of the preformed metabolite would mimic that of the formed metabolite.

However, it is often impossible to ascertain the identity of the toxic species. The lack of toxicity noted after the administration of the synthetic or preformed metabolite does not preclude the metabolite as the culprit of toxicity [1], [3], [4]. The reasons are many (for review, see [5], [6]). Metabolites must be stable enough to be quantified by conventional, analytical methods. Metabolites formed within eliminating organs, especially those that are reactive, will undergo further metabolism or be excreted immediately. The primary metabolite (Mi) that is formed from the precursor compound (P), qualified as {mi,P} to distinguish this from the preformed metabolite {pmi}, may undergo excretion that is mediated by excretory proteins (or transporters) or encounter enzymes for metabolism before the metabolite has a chance to leave the organ or tissue (Fig. 1). The sequential removal of the metabolite formed in situ the formation organ constitutes what “sequential first-pass removal” of the metabolite, and reduces the appearance of the metabolite systemically by the fraction that is removed [7]. From physiologically based pharmacokinetic (PBPK) modeling of the liver, kidney, and intestine, the areas under the curve of the formed metabolite (AUC{mi,P}) obtained after administration of the precursor (P) and that of the administered preformed metabolite, pmi (AUC{pmi}) revealed that differences existed between the AUCs and extraction ratios of the formed and preformed metabolites. This review provided the background information needed in the comparison of the kinetics or time-course between formed and preformed metabolites. Solutions on area under the curves and extraction ratios of formed and preformed metabolites based on physiological models on the liver, kidney and intestine, were described. Observations that compared the fates of the formed vs. preformed metabolites in organ/tissue perfusion studies were reported.

Section snippets

Sequential elimination of the formed metabolite within metabolite formation organs

The immediate, sequential removal of the primary metabolite, Mi, during its time of genesis within the organ reduces its rate of appearance systemically by the fraction that is removed, the extraction ratio of formed metabolite, or E{mi,P} [7]. The rate of appearance of the metabolite in the circulation is the apparent availability of the formed metabolite (F{mi,P}, or [1  E{mi,P}]), multiplied to the rate of formation of the metabolite [7] (Fig. 1). The concept of immediate, sequential

The liver

The liver is the most important organ for drug bioactivation. It is endowed with the richest abundance of Phase I enzymes, represented by the cytochrome P450s: CYP3A4 CYP2D6, CYP2C19, and CYP1E1 and other oxidases, hydrolases, as well as phase II enzymes, the uridine diphosphate glucuronosyltransferases (UGTs), sulfotranssferases (SULTs), and glutathione S-transferases (GSTs) for conjugation. Normally, drug metabolism brings inactivation, but there are some products that are pharmacologically

The kidney

The kidney is comprised of nephrons, functional units for filtration, secretion, and reabsorption. It consists of basolateral and luminal (apical or brush border) transporters capable of drug excretion and excretion as well as sufficient enzymes for metabolism (Fig. 5). The important basolateral influx transporters consist of the organic anion transporters (OATs) and organic cation transporters (OCTs), whereas the oligopeptide transporters, PEPT1 and PEPT2, and ASBT are reabsorptive

The intestine

The intestine is noted for its absorptive function because of the presence of villi and microvilli and transporters for organic anions and cations (for reviews, see [55], [56]). There exist ample drug metabolizing enzymes such as the CYPs for oxidation and UGTs, SULTs, and GSTs for conjugation (Fig. 7). Absorption occurs with ASBT, PEPT1, and OATP2B1. Exsorption transporters exist at the apical membrane via the 170 kDa P-glycoprotein and MRP2 that mediate efflux of drug back to the lumen,

Common features on modeling of formed and preformed metabolite kinetics

This review has provided some solutions of formed and preformed metabolite kinetics and a comparison of the AUC{mi,P} and AUC{pmi} within the single eliminating organs. Needless to say, processes of binding, basolateral influx and efflux, cellular metabolism and apical excretion interact simultaneously to result in net removal and clearances. These solutions (Table 3, Table 4, Table 5) are useful for the deduction of mechanisms in drug–drug interactions, notably to distinguish what happened

Whole body: multiple organs for metabolite formation and sequential metabolism

There exists a general approach based on whole body physiologically based pharmacokinetic modeling to examine metabolite kinetics. The transition of modeling from a single organ to the whole body is achieved via inclusion of all of the eliminating organs in addition to the non-eliminating organs, which may be conveniently lumped as highly or poorly perfused tissues according to the flow characteristics to the tissue [89], [90], [91], [92], [93]. A physiologically based pharmacokinetic model is

Concluding remarks

As shown in this review, the kinetics of the preformed metabolite may not be able to directly predict the kinetics and toxicity of the formed metabolite following drug use. Metabolites formed from precursor drugs may behave differently from the administered preformed metabolite species, unless the drug and metabolite are independent of transporter-mediation and exhibit flow-limited distribution, such that enzymes involved in metabolite formation and removal are readily accessible within a

Conflict of interest

None

Glossary for transporters and enzymes

ASBT
apical sodium-dependent bile acid transporter, SLC10A2
BCRP
breast cancer resistance protein, ABCG2
BSEP
bile salt export pump, ABCB11
CYP
cytochrome P450
GLUT1
glucose transporter 1, SLC2A1
GST
glutathione S-transferase
MCT1
monocarboxylic acid transporter 1, SLC16A1
MDR1
multidrug resistance protein 1, ABCB1, also known as P-gp or P-glycoprotein
MRP2–6
multidrug resistance-associated protein isoforms 2–6, ABCC2–6
NT1,2
nucleoside transporter 1 and 2, SLC28A1–2
NTCP
sodium-dependent taurocholate

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