Mini reviewSafety testing of metabolites: Expectations and outcomes
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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PBPK Modeling to Estimate Metabolite Formation From First-Pass Organs: Intestine and Liver
2017, Comprehensive Medicinal Chemistry IIIFixing clearance as early as lead optimization using high throughput in vitro incubations in combination with exact mass detection and automatic structure elucidation of metabolites
2013, Drug Discovery Today: TechnologiesCitation Excerpt :Although this could be seen in some cases as a way to prolong the residence time of an active molecule it is more often associated with increased risk. With the publication of a guidance for industry in 2008 the FDA provided recommendations on when and how to assess the safety of metabolites [9,10]. In some cases reactive metabolites are generated.
Definition of metabolism-dependent xenobiotic toxicity with co-cultures of human hepatocytes and mouse 3T3 fibroblasts in the novel integrated discrete multiple organ co-culture (IdMOC) experimental system: Results with model toxicants aflatoxin B1, cyclophosphamide and tamoxifen
2012, Chemico-Biological InteractionsCitation Excerpt :An alternative approach is comparative cytotoxicity evaluation in metabolically competent (i. e. hepatocytes) and incompetent cells [11,38]. Approaches for the evaluation of the formation of stable metabolites by the liver which may cause toxicity in non-hepatic tissues include pre-incubation of the xenobiotic with metabolite generating systems such as liver microsomes and hepatocytes, followed by cytotoxic evaluation in a metabolically incompetent cell type [57,65,69] or a full scale toxicological evaluation of purified or synthesized metabolites such as the “Metabolites in Safety Testing (MIST)” approach currently endorsed by the U. S. FDA for pharmaceutical development [4,49,58]. The results presented here suggest that IdMOC with the metabolically competent human hepatocytes and the metabolically incompetent 3T3 cells represents an effective approach for the definition of the role of hepatic metabolism on xenobiotic toxicity.
Overview: Evaluation of metabolism-based drug toxicity in drug development
2009, Chemico-Biological InteractionsSafety Testing of Drug Metabolites
2009, Annual Reports in Medicinal ChemistryCitation Excerpt :If new target organ toxicity ensues from this situation, how do we put that into perspective [1,4]? Pang and others [15–17]have demonstrated that kinetic differences can be observed between metabolites formed in vivo and those that are synthesized and then orally administered. These are but some of the vexing issues that remain as we attempt to comply with the MIST Guidance.