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- Age-Dependent Changes in Cytochrome P450 Abundance and Composition in
Human Liver [Articles]-
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Authors:
Subash, S; Prasad, B.
Pages: 1363 - 1372
Abstract: Cytochrome P450 (CYP) superfamily represents the major drug-metabolizing enzymes responsible for metabolizing over 65% of therapeutic drugs, including those for pediatric use. CYP-ontogeny based physiologically based pharmacokinetic (PBPK) modeling has emerged as useful approach to mechanistically extrapolate adult pharmacokinetic data to children. However, these models integrate physiological differences in the pediatric population including age-dependent differences in the abundances of CYP enzymes. Conventionally, developmental changes in CYP enzymes have been reported using protein abundance and activity data from subcellular fractions such as microsomes, which are prone to high technical variability. Similarly, the available pediatric pharmacokinetic data suffer from the lack of specific CYP substrates, especially in younger children. In the present study, we used viable hepatocytes from 50 pediatric (age, 1 day–18 years) and 8 adult human donors and carried out global proteomics-based quantification of all major hepatic CYP enzymes, including orphan enzymes that have not been studied previously. While CYPs 2B6, 3A5, 4A11, 4F3, and 4V2 did not show a significant association with age, all other quantified isoforms either increased or decreased with age. CYPs 1A2, 2C8, 2C18, and 2C19 were absent or barely detected in the neonatal group, while CYP3A7 was the highest in this group. The>1 to 2 years age group showed the highest total abundance of all CYP enzymes. The age-dependent differences in CYP enzymes reported in this study can be used to develop ontogeny-based PBPK models, which in turn can help improve pediatric dose prediction based on adult dosing, leading to safer drug pharmacology in children.SIGNIFICANCE STATEMENTWe quantified the age-dependent differences in the abundances of hepatic CYP enzymes using a large set of viable pediatric and adult hepatocytes using quantitative global proteomics. We report for the first time the ontogeny in the abundance of CYP enzymes in human hepatocytes, especially, orphan CYPs 20A1, 27A1, 51A1, 7B1, and 8B1 and CYP4 subfamily of enzymes. Our study provides important data about CYP ontogeny that can be used for the better prediction of pediatric pharmacokinetics using physiologically based pharmacokinetic modeling.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001608
Issue No: Vol. 52, No. 12 (2024)
- Utility of Common In Vitro Systems for Predicting Circulating Metabolites
[Articles]-
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Authors:
Freiberger, E. C; Thompson, M. P, Zhang, X, Underwood, E. B, Lynch, T. L, Jenkins, G. J, Wagner, D. S.
Pages: 1373 - 1378
Abstract: In vitro systems such as cultured hepatocytes are used early in drug development as a proxy for in vivo data to predict metabolites in human and the potential preclinical species. These data support preclinical species selection for toxicity studies as well as provide early evidence for potential active and reactive metabolites that can be generated in human. Although in vivo data would be best to select preclinical species for a given compound, only in vitro systems are available when selecting toxicity study species. However, as with any in vitro system, the correlation to actual in vivo results can be variable. Understanding the reliability of predicting in vivo metabolites from the various available in vitro assays and determining which system may be most predictive would help de-risk drug development teams’ selection process. In this manuscript, we address these questions: can in vitro systems predict circulating metabolites' If so, is predictivity quantitative or indicative of what levels may be seen circulating' Of the currently available in vitro systems, is one better than the others at generating predictive metabolites' To address the first two issues (general in vitro/in vivo predictivity, and whether any in vitro/in vivo correlations are quantitative), we used historical data from Abbott/AbbVie to compare in vitro metabolite profiles with metabolite profiles from in vivo absorption, distribution, metabolism, excretion, and clinical studies. In this retrospective analysis of historic metabolite profiling data, in vitro systems predicted ~50% of circulating metabolites present in vivo, across preclinical species and human, with no correlation between apparent concentrations in vitro versus in vivo. To address the final question, we selected 10 commercially available compounds with published metabolism data and incubated them in five common in vitro systems (microsomes, liver S9, suspension hepatocytes, HμREL cocultured hepatocytes, and hepatocyte spheroids); the new in vitro metabolite profiling data were compared against published in vivo data to determine whether any individual system was more accurate in generating known major human circulating metabolites. Suspension hepatocytes and cocultured hepatocytes marginally outperformed the other systems. Current in vitro systems have value early in development when in vivo studies are not feasible and are required for regulatory filings to support preclinical toxicology species selection but should not be treated as wholly representative of a given drug’s in vivo metabolism.SIGNIFICANCE STATEMENTThis is a comprehensive assessment of historic metabolism data quantitating the success rate of in vitro to in vivo predictivity. Reliability of in vitro systems for metabolite profiling is important for early drug development, and understanding predictivity will help give appropriate context to the data. New data were also generated to compare common in vitro liver models to determine whether any could be definitively identified as more predictive of human circulating metabolites than others.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001732
Issue No: Vol. 52, No. 12 (2024)
- Absorption, Distribution, Metabolism, and Excretion of Icenticaftor
(QBW251) in Healthy Male Volunteers at Steady State and In Vitro
Phenotyping of Major Metabolites [Articles]-
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Authors:
Glaenzel, U; Huth, F, Eggimann, F, Hackling, M, Leuthold, L. A, Meissner, A, Bebrevska, L.
Pages: 1379 - 1387
Abstract: Icenticaftor (QBW251) is a potentiator of the cystic fibrosis transmembrane conductance regulator protein and is currently in clinical development for the treatment of chronic obstructive pulmonary disease and chronic bronchitis. An absorption, distribution, metabolism, and excretion study was performed at steady state to determine the pharmacokinetics, mass balance, and metabolite profiles of icenticaftor in humans. In this open-label study, six healthy men were treated with unlabeled oral icenticaftor (400 mg b.i.d.) for 4 days. A single oral dose of [14C]icenticaftor was administered on Day 5, and unlabeled icenticaftor was administered twice daily from the evening of Day 5 to Day 12. Unchanged icenticaftor accounted for 18.5% of plasma radioactivity. Moderate to rapid absorption of icenticaftor was observed (median time to reach peak or maximum concentration: 4 hours), with 93.4% of the dose absorbed. It exhibited moderate distribution (Vz/F: 335 L) and was extensively metabolized, principally through N-glucuronidation, O-glucuronidation, and/or O-demethylation. The metabolites M8 and M9, formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively, represented the main entities detected in plasma (35.3% and 14.5%, respectively) in addition to unchanged icenticaftor (18.5%). The apparent mean terminal half-life of icenticaftor was 15.4 hours in blood and 20.6 hours in plasma. Icenticaftor was eliminated from the body mainly through metabolism followed by renal excretion, and excretion of radioactivity was complete after 9 days. In vitro phenotyping of icenticaftor showed that cytochrome P450 and uridine diphosphate glucuronosyltransferase were responsible for 31% and 69% of the total icenticaftor metabolism in human liver microsomes, respectively. This study provided invaluable insights into the disposition of icenticaftor.SIGNIFICANCE STATEMENTThe absorption, distribution, metabolism, and excretion of a single radioactive oral dose of icenticaftor was evaluated at steady state to investigate the nonlinear pharmacokinetics observed previously with icenticaftor. [14C]Icenticaftor demonstrated good systemic availability after oral administration and was extensively metabolized and moderately distributed to peripheral tissues. The most abundant metabolites, M8 and M9, were formed by N-glucuronidation and O-glucuronidation of icenticaftor, respectively. Phenotyping demonstrated that [14C]icenticaftor was metabolized predominantly by UGT1A9 with a remarkably low Km value.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001751
Issue No: Vol. 52, No. 12 (2024)
- Candesartan Has No Clinically Meaningful Effect on the Plasma
Concentrations of Cytochrome P450 2C8 Substrate Repaglinide in Humans
[Articles]-
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Authors:
Piha, M. O. W; Cajanus, K, Engström, M. T, Neuvonen, M, Bergmann, T. K, Niemi, M, Backman, J. T, Filppula, A. M, Tornio, A.
Pages: 1388 - 1395
Abstract: In vitro evidence shows that the acyl-β-D-glucuronide metabolite of candesartan inhibits cytochrome P450 (CYP) 2C8 with an inhibition constant of 7.12 μM. We investigated the effect of candesartan on the plasma concentrations and glucose-lowering effect of repaglinide, a sensitive clinical CYP2C8 index substrate. In a randomized crossover study, ten healthy volunteers ingested 8 mg of candesartan or placebo daily for three days, and on day 3, they also ingested 0.25 mg of repaglinide one hour after candesartan or placebo. We measured the plasma concentrations of repaglinide, candesartan, and candesartan acyl-β-D-glucuronide, and blood glucose concentrations for up to nine hours after repaglinide intake. Candesartan had no effect on the area under the plasma concentration-time curve and peak plasma concentration of repaglinide compared with placebo, with ratios of geometric means of 1.02 [P = 0.809; 90% confidence interval (CI) 0.90–1.15] and 1.13 (P = 0.346; 90% CI 0.90–1.43), respectively. Other pharmacokinetic variables and blood glucose concentrations were neither affected. Candesartan acyl-β-D-glucuronide was detectable in seven subjects, in whom the peak concentration of repaglinide was 1.32-fold higher in the candesartan phase than in the placebo phase (P = 0.041; 90% CI 1.07–1.62). Systemic concentrations of candesartan acyl-β-D-glucuronide were very low compared with its CYP2C8 inhibition constant (ratio
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001798
Issue No: Vol. 52, No. 12 (2024)
- Characterization of Nonclinical Drug Metabolism and Pharmacokinetic
Properties of Phosphorodiamidate Morpholino Oligonucleotides, a Novel Drug
Class for Duchenne Muscular Dystrophy [Articles]-
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Authors:
Goey, A. K. L; Mukashyaka, M. C, Patel, Y, Rodino-Klapac, L. R, East, L.
Pages: 1396 - 1406
Abstract: Eteplirsen, golodirsen, and casimersen are phosphorodiamidate morpholino oligomers (PMOs) that are approved in the United States for the treatment of patients with Duchenne muscular dystrophy (DMD) with mutations in the DMD gene that are amenable to exon 51, 53, and 45 skipping, respectively. Here we report a series of in vivo and in vitro studies characterizing the drug metabolism and pharmacokinetic (DMPK) properties of these three PMOs. Following a single intravenous dose, plasma exposure was consistent for all three PMOs in mouse, rat, and nonhuman primate (NHP), and plasma half-lives were similar for eteplirsen (2.0–4.1 h) and golodirsen (2.1–8.7 h) across species and more variable for casimersen (3.2–18.1 h). Plasma protein binding was low (
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001819
Issue No: Vol. 52, No. 12 (2024)
- 3-epi-18{beta}-Glycyrrhetinic Acid or Its Glucuronide, the Metabolites of
Glycyrrhizinic Acid with Individual Differences, Correlated with
Diagnostic Marker for Licorice-Induced Pseudoaldosteronism in Humans
[Articles]-
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Authors:
Sakoda, R; Ishiuchi, K, Yoshino, T, Tsunoo, Y, Namiki, T, Ogawa-Ochiai, K, Minamizawa, K, Fukunaga, K, Watanabe, K, Makino, T.
Pages: 1407 - 1416
Abstract: Licorice is a crude drug that is used in traditional Japanese Kampo medicine and is also used as a sweetener. Occasionally, it causes pseudoaldosteronism (PsA) as a side effect. The major symptoms include hypokalemia, hypertension, edema, and low plasma aldosterone levels. PsA might be caused by the metabolites of glycyrrhizinic acid (GL), a component of licorice. The development of PsA markedly varies among individuals; however, the factors that cause these individual differences remain unknown. In this study, 78 patients who consumed Kampo medicines containing licorice were enrolled, and their laboratory data, including serum potassium levels, plasma aldosterone concentrations (PAC), and the concentrations of GL metabolites in the residual blood and/or urine samples were evaluated. Of the 78 participants, 18β-glycyrrhetinic acid (GA), 3-epi-GA, 3-oxo-GA, 18β-glycyrrhetinyl-30-O-glucuronide (GA30G), and 3-epi-GA30G were detected in the serum samples of 65, 47, 63, 62, and three participants, respectively. Of the 29 urine samples collected, GA30G and 3-epi-GA30G were detected in 27 and 19 samples. 3-epi-GA30G is a newly found GL metabolite. Moreover, 3-epi-GA, 3-oxo-GA, and 3-epi-GA30G were identified in human samples for the first time. High individual differences were found in the appearances of 3-epi-GA in serum and 3-epi-GA30G in urine, and the concentrations of these metabolites were correlated with serum PsA markers. The inhibitory titers of 3-epi-GA, 3-oxo-GA, GA30G, and 3-epi-GA30G on human 11β-hydroxysteroid dehydrogenase type 2 were almost similar. These findings suggest that 3-epi-GA and/or 3-epi-GA30G are associated with individual differences in the development of PsA.SIGNIFICANCE STATEMENTIn this study, we detected 3-epi-18β-glycyrrhetinic acid (3-epi-GA) in human serum for the first time. We also identified 3-epi-18β-glycyrrhetinyl-30-O-glucuronide (3-epi-GA30G) as a novel glycyrrhizinic acid (GL) metabolite in human urine. These GL metabolite levels showed correlations with markers of PsA. Additionally, there are individual differences in whether they appear in the serum/urine. In conclusion, 3-epi-GA/3-epi-GA30G correlates with individual differences in the development of PsA.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001840
Issue No: Vol. 52, No. 12 (2024)
- Differential Selectivity of Human and Mouse ABCC4/Abcc4 for Arsenic
Metabolites [Articles]-
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Authors:
Whitlock, B. D; Ma, Y, Conseil, G, OBrien, A. R, Banerjee, M, Swanlund, D. P, Lin, Z. P, Wang, Y, Le, X. C, Schuetz, J. D, Cole, S. P. C, Leslie, E. M.
Pages: 1417 - 1428
Abstract: Millions of people globally are exposed to the proven human carcinogen arsenic at unacceptable levels in drinking water. In contrast, arsenic is a poor rodent carcinogen, requiring>100-fold higher doses for tumor induction, which may be explained by toxicokinetic differences between humans and mice. The human ATP-binding cassette subfamily C (ABCC) transporter hABCC4 mediates the cellular efflux of a diverse array of metabolites, including the glutathione (GSH) conjugate of the highly toxic monomethylarsonous acid (MMAIII), monomethylarsenic diglutathione [MMA(GS)2], and the major human urinary arsenic metabolite dimethylarsinic acid (DMAV). Our objective was to determine if mouse Abcc4 (mAbcc4) protected against and/or transported the same arsenic species as hABCC4. The anti-ABCC4 antibody M4I-10 epitope was first mapped to an octapeptide (411HVQDFTA418F) present in both hABCC4 and mAbcc4, enabling quantification of relative amounts of hABCC4/mAbcc4. mAbcc4 expressed in human embryonic kidney (HEK)293 cells did not protect against any of the six arsenic species tested [arsenite, arsenate, MMAIII, monomethylarsonic acid, dimethylarsinous acid, or DMAV], despite displaying remarkable resistance against the antimetabolite 6-mercaptopurine (>9-fold higher than hABCC4). Furthermore, mAbcc4-enriched membrane vesicles prepared from transfected HEK293 cells did not transport MMA(GS)2 or DMAV despite a>3-fold higher transport activity than hABCC4-enriched vesicles for the prototypic substrate 17β-estradiol-17-(β-D-glucuronide). Abcc4(+/+) mouse embryonic fibroblasts (MEFs) were ~3-fold more resistant to arsenate than Abcc4(–/–) MEFs; however, further characterization indicated that this was not mAbcc4 mediated. Thus, under the conditions tested, arsenicals are not transported by mAbcc4, and differences between the substrate selectivity of hABCC4 and mAbcc4 seem likely to contribute to arsenic toxicokinetic differences between human and mouse.SIGNIFICANCE STATEMENTToxicokinetics of the carcinogen arsenic differ among animal species. Arsenic methylation is known to contribute to this, whereas arsenic transporters have not been considered. Human ATP-binding cassette subfamily C member 4 (hABCC4) is a high-affinity transporter of toxicologically important arsenic metabolites. Here we used multiple approaches to demonstrate that mouse Abcc4 does not protect cells against or transport any arsenic species tested. Thus, differences between hABCC4 and mAbcc4 substrate selectivity likely contribute to differences in human and mouse arsenic toxicokinetics.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001852
Issue No: Vol. 52, No. 12 (2024)
- CYP P450 and non-CYP P450 Drug Metabolizing Enzyme Families Exhibit
Differential Sensitivities towards Proinflammatory Cytokine Modulation
[Articles]-
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Authors:
de Jong, L. M; Harpal, C, Berg, D.-J. v. d, Hoekstra, M, Peter, N. J, Rissmann, R, Swen, J. J, Manson, M. L.
Pages: 1429 - 1437
Abstract: Compromised hepatic drug metabolism in response to proinflammatory cytokine release is primarily attributed to downregulation of cytochrome P450 (CYP) enzymes. However, whether inflammation also affects other phase I and phase II drug metabolizing enzymes (DMEs), such as the flavin monooxygenases (FMOs), carboxylesterases (CESs), and UDP glucuronosyltransferases (UGTs), remains unclear. This study aimed to decipher the impact of physiologically relevant concentrations of proinflammatory cytokines on expression and activity of phase I and phase II enzymes, to establish a hierarchy of their sensitivity as compared with the CYPs. Hereto, HepaRG cells were exposed to interleukin-6 and interleukin-1β to measure alterations in DME gene expression (24 h) and activity (72 h). Sensitivity of DMEs toward proinflammatory cytokines was evaluated by determining IC50 (potency) and Imax (maximal inhibition) values from the concentration-response curves. Proinflammatory cytokine treatment led to nearly complete downregulation of CYP3A4 (~98%) but was generally less efficacious at reducing gene expression of the non-CYP DME families. Importantly, FMO, CES, and UGT family members were less sensitive toward interleukin-6 induced inhibition in terms of potency, with IC50 values that were 4.3- to 7.4-fold higher than CYP3A4. Similarly, 18- to 31-fold more interleukin-1β was required to achieve 50% of the maximal downregulation of FMO3, FMO4, CES1, UGT2B4, and UGT2B7 expression. The differential sensitivity persisted at enzyme activity level, highlighting that alterations in DME gene expression during inflammation are predictive for subsequent alterations in enzyme activity. In conclusion, this study has shown that FMOs, CESs, and UGTs enzymes are less impacted by IL-6 and IL-1β treatment as compared with CYP enzymes.SIGNIFICANCE STATEMENTWhile the impact of proinflammatory cytokines on CYP expression is well established, their effects on non-CYP phase I and phase II drug metabolism remains underexplored, particularly regarding alterations in drug metabolizing enzyme (DME) activity. This study provides a quantitative understanding of the sensitivity differences to inflammation between DME family members, suggesting that non-CYP DMEs may become more important for the metabolism of drugs during inflammatory conditions due to their lower sensitivity as compared with the CYPs.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001867
Issue No: Vol. 52, No. 12 (2024)
- Quantitative Prediction of Drug-Drug Interactions Caused by CYP3A
Induction Using Endogenous Biomarker 4{beta}-Hydroxycholesterol [Articles]
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Authors:
Takubo, H; Taniguchi, T, Nomura, Y.
Pages: 1438 - 1444
Abstract: Evaluation of the CYP3A induction risk is important in early drug development stages. This study focused on 4β-hydroxycholesterol (4β-HC) as an endogenous biomarker of drug-drug interactions (DDIs) caused by CYP3A induction. We investigated a new approach using 4β-HC for quantitative prediction of DDIs caused by CYP3A induction based on the mechanistic static pharmacokinetic (MSPK) model. The induction ratio, i.e., the ratio of plasma 4β-HC or 4β-HC/cholesterol (4β-HC/C) with and without a coadministered CYP3A inducer, and the ratio of the area under the plasma concentration-time curve (AUCR), i.e., the ratio of the AUC of plasma CYP3A substrate drugs with and without a coadministered CYP3A inducer, were collected. The scaling factor (d) in the MSPK model was calculated from the induction ratio of 4β-HC or 4β-HC/C based on the systemic term in the MSPK model. The AUCR of 18 CYP3A substrates with and without coadministration of seven CYP3A inducers were then predicted by substituting the calculated d value into the MSPK model. This approach showed that approximately 84% of the predicted AUCR values were within a twofold range of the observed values, showing that this approach can be a good tool to quantitatively predict DDIs caused by CYP3A induction.SIGNIFICANCE STATEMENTA concise approach to predict drug interactions with adequate accuracy is preferable in the early drug development stage. In this study, a new approach using 4β-hydroxycholesterol for quantitative prediction of drug-drug interactions caused by CYP3A induction was investigated. The predictability was verified using seven CYP3A inducers and 18 substrates.
PubDate: 2024-11-15T07:28:24-08:00
DOI: 10.1124/dmd.124.001876
Issue No: Vol. 52, No. 12 (2024)
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