Many drug oxygenations are mainly mediated by polymorphic cytochromes P450 (P450s) and also by flavin-containing monooxygenases (FMOs). More than 50 years of research on P450/FMO-mediated drug ...oxygenations have clarified their catalytic roles. The natural product coumarin causes hepatotoxicity in rats via the reactive coumarin 3,4-epoxide, a reaction catalyzed by P450 1A2; however, coumarin undergoes rapid 7-hydroxylation by polymorphic P450 2A6 in humans. The primary oxidation product of the teratogen thalidomide in rats is deactivated 5'-hydroxythalidomide plus sulfate and glucuronide conjugates; however, similar 5'-hydroxythalidomide and 5-hydroxythalidomide are formed in rabbits in vivo. Thalidomide causes human P450 3A enzyme induction in liver (and placenta) and is also activated in vitro and in vivo by P450 3A through the primary human metabolite 5-hydroxythalidomide (leading to conjugation with glutathione/nonspecific proteins). Species differences exist in terms of drug metabolism in rodents and humans, and such differences can be very important when determining the contributions of individual enzymes. The approaches used for investigating the roles of human P450 and FMO enzymes in understanding drug oxidations and clinical therapy have not yet reached maturity and still require further development. SIGNIFICANCE STATEMENT: Drug oxidations in animals and humans mediated by P450s and FMOs are important for understanding the pharmacological properties of drugs, such as the species-dependent teratogenicity of the reactive metabolites of thalidomide and the metabolism of food-derived odorous trimethylamine to non-odorous (but proatherogenic) trimethylamine
-oxide. Recognized differences exist in terms of drug metabolism between rodents, non-human primates, and humans, and such differences are important when determining individual liver enzyme contributions with substrates in
and
systems.
Dietary-derived coumarin is of clinical interest for its potential hepatotoxicity in humans because such toxicity is especially evident in rats. In this study, the oxidative metabolism of coumarin to ...active coumarin 3,4-epoxide (as judged by the formation rates of o-hydroxyphenylacetic acid) and excretable 7-hydroxycoumarin was investigated in liver fractions from rats and humans. In rat liver microsomes, the formation rate of o-hydroxyphenylacetic acid (~6 pmol/min/mg microsomal protein) from coumarin at 10 μM was dependent on the presence of liver cytosolic fractions. Rat hepatocytes mediated similar formation rates of o-hydroxyphenylacetic acid and 7-hydroxycoumarin (~0.1 nmol/hr/106 cells) at 0.20–20 μM coumarin. Human hepatocytes mediated the biotransformation of coumarin to o-hydroxyphenylacetic acid at roughly similar rates to those of rat hepatocytes. In contrast, the formation rates of 7-hydroxycoumarin by human hepatocytes were around 10-fold higher at ~1 nmol/hr/106 cells. In the presence of human liver cytosolic fractions, the oxidative formation rate of o-hydroxyphenylacetic acid was relatively high in cytochrome P450 (P450) 1A2-rich human liver microsomes. The inhibitory effects of furafylline/α-naphthoflavone and 8-methoxypsoralen, P450 1A2 and 2A6 inhibitors, respectively, were seen on the rates of o-hydroxyphenylacetic and 7-hydroxylation formations, respectively, in pooled human liver microsomes. Human liver microsomes selectively inactivated for P450 1A2 and 2A6 showed low rates of o-hydroxyphenylacetic acid and 7-hydroxylation formation (~20–30% of control), respectively. Among the P450 isoforms tested, recombinant human P450 1A2 predominantly mediated o-hydroxyphenylacetic formation. These results suggested that the metabolic activation and deactivation of coumarin were mediated mainly by P450 1A2 and 2A6 enzymes, respectively. The metabolic oxidation of coumarin via 3,4-epoxidation forming o-hydroxyphenylacetic acid could inform individual human risk assessments of dietary-derived coumarin, for which hepatotoxicity is especially evident in rats.
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Human metabolic profiles for substances such as toxic food-derived compounds are usually allometrically extrapolated from traditionally determined in vivo rat concentration profiles. To evaluate ...internal exposures in humans without any reference to experimental data, physiologically based pharmacokinetic (PBPK) modeling could be used if the model input parameters could be estimated in silico. This approach would simplify the use of PBPK models for forward dosimetry after oral doses. In this study, the in silico estimation of input parameters for PBPK models (i.e., fraction absorbed × intestinal availability, absorption rate constants, and volumes of the systemic circulation) was updated for an panel of 355 chemicals (212 previously analyzed and 143 additional substances) using a light gradient boosting machine learning algorithms (LightGBM) based on between 11 and 29 in silico-calculated chemical descriptors. Simplified human PBPK models were then used to calculate virtual maximum plasma concentrations (Cmax) and areas under the concentration–time curve (AUC) based on two sets of input parameters, i.e., traditionally derived values from in vivo data and those calculated in silico using the current updated systems. Both sets of Cmax and AUC data were well correlated (r = 0.87 and r = 0.73, respectively; p < 0.01, n = 355). Therefore, input parameters for human PBPK models for a diverse range of compounds could be successfully estimated using chemical descriptors and in silico tools. This approach to pharmacokinetic modeling has potential for application in computational toxicology and in the clinical setting for assessing the potential risk of general chemicals.
Physiologically based pharmacokinetic (PBPK) modeling has the potential to play significant roles in estimating internal chemical exposures. The three major PBPK model input parameters (i.e., ...absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearances) were generated in silico for 212 chemicals using machine learning algorithms. These input parameters were calculated based on sets of between 17 and 65 chemical properties that were generated by in silico prediction tools before being processed by machine learning algorithms. The resulting simplified PBPK models were used to estimate plasma concentrations after virtual oral administrations in humans. The estimated absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearance values for the 212 test compounds determined traditionally (i.e., based on fitting to measured concentration profiles) and newly estimated had correlation coefficients of 0.65, 0.68, and 0.77 (p < 0.01, n = 212), respectively. When human plasma concentrations were modeled using traditionally determined input parameters and again using in silico estimated input parameters, the two sets of maximum plasma concentrations (r = 0.85, p < 0.01, n = 212) and areas under the curve (r = 0.80, p < 0.01, n = 212) were correlated. Virtual chemical exposure levels in liver and kidney were also estimated using these simplified PBPK models along with human plasma levels. These results indicate that the PBPK model input parameters for humans of a diverse set of compounds can be reliability estimated using chemical descriptors calculated using in silico tools.
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Cynomolgus monkeys (Macaca fascicularis, an Old World Monkey) have been widely used as a non-human primate model in preclinical studies because of their genetic and physiological ...similarity to humans. This trend has been followed by common marmoset (Callithrix jacchus, a New World Monkey). However, drug-metabolism properties in these non-human primates have not been fully understood due to limited information on cytochrome P450 (P450) enzymes, major drug-metabolizing enzymes in humans. Multiple forms of cynomolgus monkey P450 enzymes have been identified and characterized in comparison to those of humans, including a cynomolgus monkey specific form, P450 2C76. Similarly, marmoset P450 1A/B, 2A, 2C, 2D, and 4F enzymes were recently identified and characterized to understand drug metabolism properties. In this research update, updates for marmoset, cynomolgus monkey, and human P450 cDNAs are provided. Marmoset and cynomolgus monkey P450 enzymes showed high sequence homology to their human counterparts and generally had similar substrate recognition functionality to human P450 enzymes; however, they also possibly contribute to limited specific differences in drug oxidative metabolism partly due to small differences in amino acid residues. These findings provide a foundation for successful use of non-human primates as preclinical models and will help to further understand molecular mechanisms of human P450 function. In addition to the P450 enzymes, flavin-containing monooxygenases, another monooxygenase family, in these non-human primates have been found to be involved in the oxidation of a variety of compounds associated with pharmacological and/or toxicological effects in humans and are also described.
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Aniline and its dimethyl derivatives reportedly become haematotoxic after metabolic N-hydroxylation of their amino groups. The plasma concentrations of aniline and its dimethyl derivatives after ...single oral doses of 25 mg/kg in rats were quantitatively measured and semi-quantitatively estimated using LC–tandem mass spectrometry. The quantitatively determined elimination rates of aniline; 2,4-dimethylaniline; and 3,5-dimethylaniline based on rat plasma versus time curves were generally rapid compared with those of 2,3-; 2,5-; 2,6-; and N,2-dimethylaniline. The primary acetylated metabolites of aniline; 2,4-dimethylaniline; and 3,5-dimethylaniline, as semi-quantitatively estimated based on their peak areas in LC analyses, were more extensively formed than those of 2,3-; 2,5-; 2,6-; and N,2-dimethylaniline. The areas under the curve of unmetabolized (remaining) aniline and its dimethyl derivatives estimated using simplified physiologically based pharmacokinetic models (that were set up using the experimental plasma concentrations) showed an apparently positive correlation with the reported lowest-observed-effect levels for haematotoxicity of these chemicals. In the case of 2,4-dimethylaniline, a methyl group at another C4-positon would be one of the determinant factors for rapid metabolic elimination to form aminotoluic acid. These results suggest that rapid and extensive metabolic activation of aniline and its dimethyl derivatives occurred in rats and that the presence of a methyl group at the C2-positon may generally suppress fast metabolic rates of dimethyl aniline derivatives that promote metabolic activation reactions at NH2 moieties.
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Many drug oxidations and conjugations are mediated by a variety of cytochromes P450 (P450) and non-P450 enzymes in humans and non-human primates. These non-P450 enzymes include ...aldehyde oxidases (AOX), carboxylesterases (CES), flavin-containing monooxygenases (FMO), glutathione S-transferases (GST), arylamine N-acetyltransferases (NAT),sulfotransferases (SULT), and uridine 5′-diphospho-glucuronosyltransferases (UGT) and their substrates include both endobiotics and xenobiotics. Cynomolgus macaques (Macaca fascicularis, an Old-World monkey) are widely used in preclinical studies because of their genetic and physiological similarities to humans. However, many reports have indicated the usefulness of common marmosets (Callithrix jacchus, a New World monkey) as an alternative non-human primate model. Although knowledge of the drug-metabolizing properties of non-P450 enzymes in non-human primates is relatively limited, new research has started to provide an insight into the molecular characteristics of these enzymes in cynomolgus macaques and common marmosets. This mini-review provides collective information on the isoforms of non-P450 enzymes AOX, CES, FMO, GST, NAT, SULT, and UGT and their enzymatic profiles in cynomolgus macaques and common marmosets. In general, these non-P450 cynomolgus macaque and marmoset enzymes have high sequence identities and similar substrate recognitions to their human counterparts. However, these enzymes also exhibit some limited differences in function between species, just as P450 enzymes do, possibly due to small structural differences in amino acid residues. The findings summarized here provide a foundation for understanding the molecular mechanisms of polymorphic non-P450 enzymes and should contribute to the successful application of non-human primates as model animals for humans.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
This article reviews in vitro metabolic activities including Michaelis constants (Km), maximal velocities (Vmax) and Vmax/Km and drug-steroid interactions such as induction and cooperativity ...(activation) of cytochromes P450 (P450 or CYP) in human tissues, including liver and adrenal gland, for 14 kinds of endogenous steroid compounds, including allopregnanolone, cholesterol, cortisol, cortisone, dehydroepiandrosterone, estradiol, estrone, pregnenolone, progesterone, testosterone and bile acids (cholic acid). First, we considered the drug-metabolizing P450s. 6 -Hydroxylation of many steroids, including cortisol, cortisone, progesterone and testosterone, was catalyzed primarily by CYP3A4. CYP1A2 and CYP3A4, respectively, are likely the major hepatic enzymes responsible for 2-/4-hydroxylation and 16α-hydroxylation of estradiol and estrone, steroids that can contribute to breast cancer risk. In contrast, CYP1A1 and CYP1B1 predominantly metabolized estrone and estradiol to 2- and 4-catechol estrogens, which are endogenous ultimate carcinogens if formed in the breast. Some metabolic activities of CYP3A4, including dehydroepiandrosterone 7 -/16α-hydroxylation, estrone 2-hydroxylation and testosterone 6 -hydroxylation, were higher than those for polymorphically expressed CYP3A5. Next, we considered typical steroidogenic P450s. CYP17A1, CYP19A1 and CYP27A1 catalyzed steroid synthesis, including hydroxylation at 17α, 19 and 27 positions, respectively. However, it was difficult to predict which hepatic drug-metabolizing P450 or steroidogenic P450 will be mainly responsible for metabolizing each steroid hormone in vivo based on these results. Further research is required on the metabolism of steroid hormones by various P450s and on prediction of their relative contributions to in vivo metabolism. The findings collected here provide fundamental and useful information on the metabolism of steroid compounds.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Physiologically based pharmacokinetic (PBPK) modeling has the potential to estimate internal chemical exposures. Algorithms for predicting the input parameters for PBPK modeling, such as absorption ...rate constants (ka), were previously reported for 323 chemicals in rats. In this study, a currently updated system for estimating the fraction absorbed × intestinal availability of compounds, along with a modified estimation system that generates ka values, is reported, based on the previously analyzed 323 primary compounds, 10 secondary compounds, and 39 additional substances. The in silico estimation of input parameters for PBPK models (i.e., fraction absorbed × intestinal availability and ka) was adapted for an updated panel of 372 chemicals using machine learning algorithms based on between 16 and 18 in silico-calculated chemical properties. Simplified human PBPK models were then used to calculate virtual areas under the plasma concentration–time curve (AUC) based on two sets of input parameters, i.e., traditionally derived values from in vivo data and those calculated in silico using the current updated machine learning systems. The AUC data sets were well correlated; the current correlation coefficient increased from 0.61 to 0.82 (p < 0.01, n = 372). Therefore, the above-described computational methods constitute a new alternative approach that could contribute to chemical safety evaluations.
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