Plasma concentrations of melengestrol acetate in humans extrapolated from the pharmacokinetics established in in vivo experiments with rats and chimeric mice with humanized liver and physiologically based pharmacokinetic modeling

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Abstract

Some synthetic chemicals are suspected to be responsible for adverse effects on endocrine function. Sex hormones administered to farm animals are of particular interest because of their regulatory role in developmental processes. To predict concentrations in humans of the synthetic growth promoter melengestrol acetate (17α-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione), a forward dosimetry approach was carried out using data from no-observed-adverse-effect-level doses orally administered to mice or rats and from in vitro human and rodent experiments. Human liver microsomes preferentially mediated 2-hydroxylation of melengestrol acetate, but rodent livers produced additional unidentified hydroxymetabolites. Adjusted animal biomonitoring equivalents for melengestrol acetate from mouse and rat studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and human metabolic data with a simple physiologically based pharmacokinetic (PBPK) model. Melengestrol acetate elimination in humans was estimated to be slow compared with elimination in rodents. The disposition of melengestrol acetate in humans was evaluated using chimeric TK-NOG mice with humanized liver. The results suggest the usefulness of simplified PBPK modeling combined with in vitro and in vivo experiments and literature resources as well as a future interest in estimating by a full PBPK modeling using another bottom up system. This model may also be useful for risk evaluation and for simulating plasma concentrations resulting from exposure to low doses of melengestrol acetate and related compounds.

Highlights

► We developed a simple pharmacokinetic (PBPK) model of melengestrol acetate (MGA). ► Rat or mouse PBPK model of MGA was scaled to human model to predict in humans. ► MGA elimination in humans was estimated to be slow compared with those in rodents. ► Concentrations of MGA were evaluated by chimeric mice with humanized liver. ► The simplified human PBPK modeling is useful for risk evaluation of MGA.

Introduction

It is of global interest to develop simple, advanced, and accurate risk assessment systems to support the interpretation of data from human biomonitoring studies (Wild, 2005). Using dose estimates instead of external or applied doses can improve the characterization of dose–response relationships and the subsequent characterization of potential health risks. This improvement results from the direct relationship between internal dosimetry (shown in Fig. 1) and the biological response. Pharmacokinetic and/or toxicokinetic parameters for a variety of chemicals have been determined in animal toxicology studies, despite the species differences of drug-metabolizing enzymes (Clewell et al., 2004). Such data for humans is often very limited. Human health risk assessments use the dose–response relationship to characterize and quantify potential health risks. When relevant and reliable estimates of the internal dose of a compound or a key metabolite are available, the results of toxicology studies can often be better understood and evaluated in terms of the internal dose. It has been attempted to collect extensive information regarding specific physiologically based pharmacokinetic (PBPK) models found in the literature for predicting concentrations in various biological fluids following multiple dose exposures (Edwards and Preston, 2008, McLanahan et al., 2012). However, although simple, inexpensive, and reliable methods are needed for accurately evaluating the toxic risk (McLanahan et al., 2012), very few such methods have been established. For example, a nine-compartment PBPK model for acrylonitrile and its primary metabolite in humans has been reported (Sweeney et al., 2003); however, we proposed a simple and reliable PBPK model capable of both forward and reverse dosimetry approaches (Takano et al., 2010).

As one of candidate compounds with the concern of long-term intaking risk, the synthetic progesterone melengestrol acetate (17α-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione is of high potency as a progestin (Perry et al., 2005) and is used as a growth promoter for farm animals in several meat-exporting countries (Lauderdale et al., 1977, Perry et al., 2005). This has led to international disputes about the safety of meat originating from treated animals (Patterson et al., 1989). Although many studies have explored its safety with respect to both animals and meat consumers (Stephany, 2010), little is known about the fate of melengestrol acetate in humans (Cooper et al., 1967, Elce et al., 1967) or its potential endocrine-disrupting activity (Schiffer et al., 2001). Melengestrol acetate has been shown to be biotransformed to mono- and dihydroxymelengestrol acetate (including 2-hydroxylated and 6-hydroxymethylated metabolites) in rodents, but the hormonal or biological potential of these chemical species in humans has not been fully elucidated (Cooper et al., 1967, Elce et al., 1967, Metzler and Pfeiffer, 2001) under the concern of long-term risk of melengestrol acetate intake in no-observed-adverse-effect level. Recently developed TK-NOG mice transplanted with human liver cells can survive without ongoing drug treatment (Hasegawa et al., 2011, Yamazaki et al., 2012). Because such survival was not achieved in previous liver reconstruction models, it is now possible to evaluate the estimates obtained from simplified human PBPK modeling by comparing them with in vivo experimental results from mice with humanized liver.

The purpose of the present study was to predict melengestrol acetate plasma concentrations in humans by a forward dosimetry analysis (Fig. 1) using data from chemical doses administered to animals and from in vitro experiments with liver microsomes from humans and animals. The adjusted animal biomonitoring equivalents after orally administered doses in rat and mouse studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and human metabolic data with a simple PBPK model. According to the present PBPK analysis, the synthetic progesterone melengestrol acetate is cleared slowly in humans compared with clearance in rodents.

Section snippets

Chemicals, animals, and enzyme preparations

Male 6-week-old Sprague–Dawley rats (Charles River Laboratory Japan, Tokyo, Japan) and control TK-NOG and humanized TK-NOG mice (∼20–30 g body weight) (Hasegawa et al., 2011) were used in this study. In the chimeric mice, more than 80% of liver cells were estimated to have been replaced with human hepatocytes, as judged by measurements of human albumin concentrations in plasma (Hasegawa et al., 2011, Yamazaki et al., 2012). Plasma samples were collected 0.5, 1, 2, 4, 7, and 24 h after single oral

Metabolism of melengestrol acetate

The in vitro metabolism of melengestrol acetate was investigated using liver microsomes from pooled human livers, rats, mice, and chimeric mice with humanized liver. Typical chromatograms are shown in Fig. 3 after the incubation of melengestrol acetate (10 μM) with liver microsomes; four metabolite peaks (designated as peaks 1–4 in Fig. 3) and peak 5 (assigned to melengestrol acetate) were observed. The four metabolites were present in different proportions in all reaction mixtures. The rates of

Discussion

Melengestrol acetate has a higher affinity for the progestin receptor than progesterone does (Bauer et al., 2000, Pfaffl et al., 2002). Although several metabolites of melengestrol acetate, possibly containing mono- and dihydroxylated moieties, were noted in an LC profile of reactions mediated by rat liver microsomes (Metzler and Pfeiffer, 2001), none of them was adequately characterized with regard to chemical structure. Three major but unidentified metabolites of melengestrol acetate have

Conclusions

A simplified PBPK model for the synthetic progesterone melengestrol acetate and its primary metabolite was developed using a combination of algorithms, in vitro and in vivo experimentation, and literature resources. According to the present PBPK analysis, melengestrol acetate is cleared more slowly from plasma in humans than in rodents. The present study indicates that simplified PBPK modeling is useful for application of the forward dosimetry approach to melengestrol acetate and to other

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported in part by The Food Safety Commission of Japan; the Ministry of Education, Culture, Sports, Science and Technology of Japan; and JCIA’s LRI program.

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