Although interspecies allometric scaling needs further refinements and has limitations, it is still a potential tool and rational option for the estimate of pharmacokinetic parameters in species for which there are no data available or to better interpret preclinical efficacy and safety trials. Allometric scaling can offer insight into possible mechanisms of species-dependent drug disposition.
ABSTRACT:Human CYP1A2 is an important drug-metabolizing enzyme, similar in sequence to CYP1A1 but with distinct substrate specificity. We have previously shown that residue 382 affected CYP1A1 and CYP1A2 specificities with alkoxyresorufins. To determine whether this residue is also important for the metabolism of other substrates, we have investigated phenacetin oxidation by single (T124S, T223N, V227G, N312L, and L382V) and multiple (L382V/ T223N, L382V/N312L, L382V/T223N/N312L, and L382V/T124S/ N312L) mutants of CYP1A2. The enzymes were expressed in Escherichia coli and purified. All the CYP1A2 mutants that contained the L382V substitution displayed much higher activities than the wildtype enzyme, with k cat values 3-fold higher, in contrast to other mutants, for which k cat decreased. Likewise, a significant increase in specificity, expressed as the k cat /K m ratio, was observed for the mutants containing the L382V substitution. The efficiency of coupling of reducing equivalents to acetaminophen formation was decreased for all the single mutants except L382V, for which the coupling increased. This effect was also observed with multiple CYP1A2 mutants containing the L382V substitution. Low activities of the four other single mutants were likely caused by dramatically increased uncoupling to water. In contrast, the increase in activity of the L382V-containing mutants resulted from decreased water formation. This finding is consistent with molecular dynamics results, which showed decreased phenacetin mobility leading to increased product formation. The results of these studies confirm the importance of residue 382 in CYP1A2-catalyzed oxidations and show that a single residue substitution can dramatically affect enzymatic activity.Cytochromes P450 (P450s) are heme-containing monooxygenase enzymes, which are involved in the metabolism of numerous exogenous and endogenous compounds. P450s are ubiquitous in living organisms, with at least 50 families and 82 subfamilies found in different species. Human CYP1A subfamily has two major isoforms: CYP1A1 and CYP1A2. CYP1A2, one of the major P450s in the human liver, was first characterized as a phenacetin O-deethylase (Distlerath et al., 1985). Currently, it is estimated that this enzyme metabolizes approximately 11% of all drugs in humans (Shimada et al., 1994). Despite the fact that CYP1A2 participates in the deactivation and detoxification of xenobiotics, the main interest in this enzyme is because of the metabolic activation of a large number of chemical carcinogens (Guengerich and Shimada, 1991;Lewis et al., 1994).In humans, CYP1A2 shares 72% amino acid sequence identity with CYP1A1, but the substrate specificities and inhibitor susceptibilities of these enzymes are different. For example, substrates such as phenacetin and 7-methoxyresorufin are primarily metabolized by CYP1A2 with high catalytic efficiency, whereas CYP1A1 displays weak capability to oxidize those substrates. On the other hand, 7-ethoxyresorufin is preferentially oxidized by CYP1A1 (Nerurkar et al.,...
The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this manuscript was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated 18O from 18O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product, but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.
c Extralabel drug use of penicillin G in food-producing animals may cause an excess of residues in tissue which will have the potential to damage human health. Of all the antibiotics, penicillin G may have the greatest potential for producing allergic responses to the consumer of food animal products. There are, however, no population pharmacokinetic studies of penicillin G for food animals. The objective of this study was to develop a population pharmacokinetic model to describe the time-concentration data profile of penicillin G across two species. Data were collected from previously published pharmacokinetic studies in which several formulations of penicillin G were administered to diverse populations of cattle and swine. Liver, kidney, and muscle residue data were also used in this study. Compartmental models with first-order absorption and elimination were fit to plasma and tissue concentrations using a nonlinear mixed-effect modeling approach. A 3-compartment model with extra tissue compartments was selected to describe the pharmacokinetics of penicillin G. Typical population parameter estimates (interindividual variability) were central volumes of distribution of 3.45 liters (12%) and 3.05 liters (8.8%) and central clearance of 105 liters/h (32%) and 16.9 liters/h (14%) for cattle and swine, respectively, with peripheral clearance of 24.8 liters/h (13%) and 9.65 liters/h (23%) for cattle and 13.7 liters/h (85%) and 0.52 liters/h (40%) for swine. Body weight and age were the covariates in the final pharmacokinetic models. This study established a robust model of penicillin for a large and diverse population of food-producing animals which could be applied to other antibiotics and species in future analyses.
ABSTRACT:Human cytochromes P450 1A1 and 1A2 play important roles in drug metabolism and chemical carcinogenesis. Although these two enzymes share high sequence identity, they display different substrate specificities and inhibitor susceptibilities. In the present studies, we investigated the structural basis for these differences with phenacetin as a probe using a number of complementary approaches, such as enzyme kinetics, stoichiometric assays, NMR, and molecular modeling. Kinetic and stoichiometric analyses revealed that substrate specificity (k cat /K m ) of CYP1A2 was approximately 18-fold greater than that of CYP1A1, as expected. Moreover, despite higher H 2 O 2 production, the coupling efficiency of reducing equivalents to acetaminophen formation in CYP1A2 was tighter than that in CYP1A1. CYP1A1, in contrast to CYP1A2, displayed much higher uncoupling, producing more water. The subsequent NMR longitudinal (T 1 ) relaxation studies with the substrate phenacetin and its product acetaminophen showed that both compounds displayed similar binding orientations within the active site of CYP1A1 and CYP1A2. However, the distance between the OCH 2 protons of the ethoxy group (site of phenacetin O-deethylation) and the heme iron was 1.5 Å shorter in CYP1A2 than in CYP1A1. The NMR findings are thus consistent with our kinetic and stoichiometric results, providing a likely molecular basis for more efficient metabolism of phenacetin by CYP1A2.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.