---
title: "Ketogenic diet alters hepatic CYP activity and may increase ozanimod exposure in mice"
id: "plos-one-8-ketogenic-diet-induced-changes-in-hepatic-drug-metabolism-with-potential"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-8-ketogenic-diet-induced-changes-in-hepatic-drug-metabolism-with-potential"
content_type: "clinical_feed_article"
specialty: "Pharmacology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357797"
published_at: "2026-09-08T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Ketogenic diet alters hepatic CYP activity and may increase ozanimod exposure in mice
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-8-ketogenic-diet-induced-changes-in-hepatic-drug-metabolism-with-potential
- **Specialty:** [Pharmacology](https://medichelpline.com/clinical-feed/pharmacology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357797)
- **Published At:** 2026-09-08T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The study evaluated whether a 4-week **ketogenic diet (KD)** modifies hepatic drug-metabolizing enzymes and affects the pharmacokinetics of **ozanimod** in female C57BL/6 mice. - Mice received either a KD (90% calories from fat) or a control diet (10% fat) for four weeks; ozanimod was administered intragastrically (5 mg/kg) for pharmacokinetic assessment. - KD induced metabolic ketosis and a significant rise in plasma cholesterol; other plasma metabolic and inflammatory markers changed only modestly. - KD shifted **gut microbiota composition** as assessed by whole-metagenome shotgun sequencing. - Hepatic cytochrome P450 (CYP) enzymes were altered: **CYP1A** mRNA expression and activity were significantly increased in KD-fed mice; pooled samples showed decreased **CYP2C** activity; **CYP3A** activity trended lower but did not reach significance. - Ozanimod systemic exposure tended to be higher in KD-fed mice, with an approximately 17% increase in area under the concentration–time curve; this increase did not achieve statistical significance. - The authors conclude KD modulates hepatic CYP expression and activity and may influence ozanimod pharmacokinetics, highlighting diet as a potential contributor to interindividual variability in drug response. - Data supporting the study are deposited in public repositories: Sequence Read Archive (PRJNA1336998) and gene expression data on Zenodo. - Funding and competing interest statements: multiple Czech grants supported the work; the authors declared no competing interests.
## Clinical Analysis & Structured Key Points
Ketogenic diet–induced changes in hepatic drug metabolism with potential implications for ozanimod pharmacokinetics in mice | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Peer Review Reader Comments Figures Figures Abstract Ketogenic diet (KD) is increasingly considered as an adjunctive therapeutic approach across a range of diseases, but its effects on the pharmacokinetics of concomitantly administered drugs remain unclear. Such interactions may be particularly relevant in multiple sclerosis, where KD is being explored as a complementary strategy alongside disease-modifying therapies, such as ozanimod. We therefore investigated whether KD affects ozanimod metabolism and pharmacokinetics and explored potential factors that may contribute to such effects. Specific pathogen-free female C57BL/6 mice were fed either a control diet containing 10% of calories from fat or a ketogenic diet containing 90% of calories from fat for 4 weeks. Metabolic, inflammatory, and hormonal parameters were determined in plasma. Gut microbiota composition was analyzed by whole-metagenome shotgun sequencing. In parallel, hepatic cytochrome P450 (CYP) enzymes were evaluated by mRNA expression and activity together with ozanimod pharmacokinetics. KD induced the expected metabolic adaptation to ketosis and led to a significant increase in plasma cholesterol accompanied by changes in gut microbiota composition. Other metabolic and inflammatory parameters showed only modest changes. In addition, KD altered the expression and activity of hepatic CYP enzymes, including enzymes involved in ozanimod metabolism: CYP1A activity and mRNA expression were significantly increased in KD-fed mice, whereas lower CYP2C activity was observed in pooled samples and CYP3A activity showed a non-significant trend toward lower values. Ozanimod exposure tended to be higher in KD-fed mice, resulting in an approximately 17% increase in area under the concentration–time curve, although this effect did not reach statistical significance. In conclusion, our findings demonstrate that KD altered the expression and activity of hepatic CYP enzymes and revealed a non-significant trend toward increased ozanimod exposure. These observations highlight the potential importance of considering dietary interventions as a factor contributing to variability in drug response. Citation: Frybortova V, Satka S, Jourova L, Anzenbacher P, Zapletalova I, Kraus M, et al. (2026) Ketogenic diet–induced changes in hepatic drug metabolism with potential implications for ozanimod pharmacokinetics in mice. PLoS One 21(9): e0357797. https://doi.org/10.1371/journal.pone.0357797 Editor: Marcia B. Aguila, Universidade do Estado do Rio de Janeiro, BRAZIL Received: April 23, 2026; Accepted: August 21, 2026; Published: September 8, 2026 Copyright: © 2026 Frybortova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The datasets generated and analyzed during the current study are available in the Sequence Read Archive repository and can be accessed online at https://www.ncbi.nlm.nih.gov/sra/PRJNA1336998 . The data from gene expression analysis are available online at https://zenodo.org/records/17250497 . Funding: This study was supported by grants from the Czech Science Foundation (23-05645S), Palacky University students’ projects: IGA_LF_2026_009 and IGA_LF_2026_006, the Czech Academy of Sciences under the Lumina quaeruntur fellowship (LQ200202105), and the Ministry of Education, Youth and Sports of the Czech Republic grant Talking Microbes-understanding microbial interactions within One Health framework (CZ.02.01.01/00/22_008/0004597) and by grants CZ-OPENSCREEN-LM-2023052 and EATRISCZ-LM2023053. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have no competing interests to declare. Introduction The global burden of chronic autoimmune diseases continues to rise, despite major advances in pharmacology, which still primarily provides symptomatic relief rather than addressing the underlying disease mechanisms. Consequently, there is growing interest in exploring novel preventive or adjuvant strategies. One promising approach involves the use of therapeutic diets designed to modulate metabolic pathways, reshape the gut microbiome, influence immune responses, and reduce inflammation [ 1 ]. The ketogenic diet (KD) is a nutritional protocol characterized by a very low intake of carbohydrates and a high intake of fats. Originally established in the 1920s as a therapeutic intervention for epilepsy [ 2 ], KD has since evolved into a widely used weight-loss strategy and a contemporary lifestyle trend [ 3 ]. In recent years, KD has also been explored as a potential adjuvant therapy in several chronic conditions, including metabolic disorders, neurodegenerative diseases, and cancer [ 4 – 6 ]. Experimental studies have begun to uncover underlying mechanisms, suggesting that KD may exert neuroprotective and immunomodulatory effects. In the context of multiple sclerosis (MS), KD may therefore influence two key aspects of disease pathology: chronic inflammation and neurodegeneration. In addition, emerging clinical studies in patients with MS suggest that ketogenic dietary interventions are feasible and well tolerated and may be associated with improvements in quality of life, fatigue, and selected clinical outcomes [ 7 , 8 ]. The range of treatment options for MS is expanding, with more than 15 standard pharmacological therapies currently available [ 9 ]. Ozanimod (Zeposia®) is a recently approved sphingosine-1-phosphate receptor modulator indicated for relapsing-remitting MS and ulcerative colitis [ 10 , 11 ]. By selectively targeting S1PR1 and S1PR5, ozanimod modulates immune cell trafficking and thereby reduces autoimmune inflammation [ 12 ]. Ozanimod is generally well tolerated and exhibits a distinct pharmacokinetic profile characterized by extensive hepatic metabolism [ 13 ]. Its biotransformation involves several cytochrome P450 (CYP) enzymes, particularly CYP1A1, CYP3A4, and CYP2C8, as well as monoamine oxidase B (MAO-B) [ 14 ]. Although ozanimod itself exhibits pharmacological activity, its therapeutic efficacy largely depends on the formation of active metabolites generated through hepatic metabolism [ 15 ]. These metabolic pathways are essential for both the activation and elimination of the drug and may be modulated by individual-specific factors such as diet, inflammation, or the gut microbiome. KD has been shown to alter the pharmacokinetics of several drugs [ 16 ], raising concerns about its potential to interfere with the metabolism and efficacy of pharmacological treatments. This is particularly relevant in the context of MS, where KD has been suggested as a possible adjunct to standard therapies such as ozanimod. In addition to its metabolic effects, KD has been associated with increased circulating β-hydroxybutyrate, altered lipid metabolism, hepatic inflammation, and shifts in gut microbiota composition [ 1 , 17 – 19 ]. Previous studies have shown that these changes may alter hepatic CYP1A, CYP2C, CYP2E1, and CYP3A expression or activity [ 20 – 22 ], suggesting that KD may influence ozanimod biotransformation. Despite the increasing clinical interest in KD, its effects on hepatic enzyme function and xenobiotic metabolism remain insufficiently characterized [ 23 ]. Because ozanimod depends on hepatic biotransformation for both elimination and the formation of active metabolites, KD-induced changes in CYP activity, inflammation, or gut microbiota may influence both its systemic exposure and pharmacological activity. We hypothesized that KD may alter hepatic drug-metabolizing capacity and thereby influence the pharmacokinetics and efficacy of ozanimod. To test this hypothesis, we examined whether KD affects ozanimod pharmacokinetics in mice and explored potential mechanisms underlying this effect, including changes in hepatic CYP expression and activity, inflammatory status, and gut microbiota composition. Materials and methods Animals Fifty-two 8- to 10-week-old specific-pathogen-free (SPF) C57BL/6 female mice were used, with an average body weight of 19.6 ± 1.4 g (mean ± SD). The mice were obtained from the breeding colonies of the Institute of Microbiology of the Czech Academy of Sciences. Mice were fed for 4 weeks either a ketogenic diet (KD, D10070801, Research Diets, New Brunswick, NJ, USA) with 90% of calories derived from fat and 10% from protein, or a composition-matched control diet (CD, D19082304, Research Diets, New Brunswick, NJ, USA), provided by the same manufacturer, containing 10% of calories from fat, 80% from carbohydrates, and 10% from protein. All animals were kept in a room with a 12 h light-dark cycle at 22 ± 2 °C. Animals were assigned to experimental groups before the start of the study using weight-based randomization to achieve comparable baseline body weight distribution between groups. No formal blinding procedures were incorporated into the study design. For pharmacokinetic experiments, ozanimod was diluted in 5% DMSO, 5% Tween 20, and 90% 0.1N HCl, and applied as a single intragastric dose of 5 mg/kg per mouse after 4 weeks on the respective diets. A dose of 5 mg/kg was selected to enable observation of the drug’s pharmacokinetic profile, as this dose had been employed in prior studies where its non-toxic effect was demonstrated [ 24 , 25 ]. At every given time point (0, 2, 4, 6, 8, and 24 hours after ozanimod application), three mice were euthanized and plasma samples were collected. In subsequent experiments, mice were assigned to each diet group (ketogenic or control) to assess the effects of the ketogenic diet on plasma metabolic parameters, inflammatory and hormonal markers, and the mRNA expression and activity of drug-metabolizing enzymes (8 mice per group). After 4 weeks on the diets, liver, plasma, and fecal samples were collected. Animals remained on their respective experimental diets ad libitum throughout the experiment and no fasting period was implemented prior to euthanasia, blood glucose, ketone, or lipid measurements. Mice were euthanized by isoflurane overdose, followed by cervical dislocation. Blood samples were collected by cardiac puncture through both atria into syringes coated with 0.5 mol/L EDTA (pH 8). Livers, without gallbladders, were removed and stored at −80 °C. Blood plasma was separated by centrifugation (2,500 × g, 15 min, 4 °C), and plasma samples were stored at −80 °C. The experiments were approved by the Committee for the Protection and Use of Experimental Animals of the Institute of Microbiology of the Czech Academy of Sciences (approval ID: 18–2023-P). Glucose and β-hydroxybutyrate measurements Plasma glucose and β-hydroxybutyrate levels were determined at days 0, 1, 2, 3, 5, 7, 10, 12, 14, 21, and 28 from the tail vein using Abbott FreeStyle Optium Neo Blood Glucose and Ketone monitoring system (Abbott Diabetes Care, USA). To minimize stress from frequent blood sampling, a rotational design was applied, in which 2 animals per group were sampled at each individual sampling time, with a minimum of 2 days between consecutive bleeds for each animal. To account for diurnal oscillations in ketone levels driven by natural feeding and fasting cycles [ 26 ], measurements were taken 2–4 times daily across both light and dark phases (except on days 0 and 5). For graphical depiction, these intra-day measurements were averaged into daily means. Ferric Reducing Antioxidant Power (FRAP) assay The antioxidant capacity was assessed using the Ferric Reducing Antioxidant Power (FRAP) assay, which is based on the reduction of Fe 3+ to Fe 2+ ions under acidic conditions. This reduction leads to the formation of a blue-colored ferrous complex with 2,4,6-tris(2-pyridyl)-1,3,5-triazine, and the increase in absorbance is measured at 593 nm. The assay was performed following the method described previously [ 27 ], with ascorbic acid used as the reference standard. The final reaction mixture contained 200 μL of the working FRAP reagent – prepared in a 10:1:1 ratio of 300 mmol/L acetate buffer (pH 3.6), 10 mmol/L TPTZ in 40 mmol/L HCl, and 20 mmol/L FeCl 3 – and 10 μL of the test sample, standard, or blank. Results were expressed as the molar concentration of ascorbic acid equivalent to the antioxidant activity exhibited by the sample. Plasma lipid profile Total plasma cholesterol was measured using the enzymatic Kit from BioSystems (REF11539). The red-colored product was detected at 510 nm. High-density lipoproteins (HDL) were measured using the same protocol as total cholesterol, with the addition of preliminary steps involving the precipitation of (very)low-density lipoproteins using phosphotungstic acid and Mg 2+ , followed by centrifugation. Due to limited sample volume, pooled samples from each dietary group were used. Triglycerides (TAG) were measured using the enzymatic Kit from BioSystems (REF11529). The colored product was measured photometrically at 510 nm. Enzyme-Linked Immunosorbent Assay (ELISA) Levels of leptin, IL-6, IL-1β, and TNF-α were analyzed in plasma and whole liver homogenates using ELISA murine leptin Kit (900-K76K), ELISA murine IL-6 Kit (900-K50K), ELISA murine IL-1β Kit (900-K47K), and ELISA murine TNF-α Kit (900-K54K) from PeproTech according to the manufacturer’s instructions. Due to limited sample volume, pooled samples from each dietary group were used. Determination of ozanimod in murine plasma The pharmacokinetic profile of ozanimod was determined using a previously validated HPLC method with fluorescent detection [ 28 ]. Briefly, mouse plasma was mixed with internal standard, nabumetone, precipitated with acetonitrile containing 0.1% HCl, centrifuged, evaporated under nitrogen flow at 40 °C, and finally reconstituted in 100 µL of mobile phase. HPLC studies were performed using the Shimadzu LC-20 HPLC system (Shimadzu, Kyoto, Japan) equipped with UV/fluorescence detection. Separation was achieved on a Chromolith HighResolution RP-18e monolithic column (100 × 4.6 mm; Merck, Darmstadt, Germany), fitted with a HighResolution RP-18 endcapped guard column (5 × 4.6 mm; Merck, Darmstadt, Germany). The mobile phase consisted of 16 mmol/L sodium acetate (pH 4.7) and acetonitrile (1.7/1; v/v). Data were analyzed using LabSolutions software (Shimadzu, Kyoto, Japan). Gene expression analysis Total RNA was isolated from murine liver tissues stored in RNA later using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The concentration and purity of total RNA were quantified spectrophotometrically using the NanoPhotometer® N60 (Implen, Munich, Germany). 1000 ng of RNA was converted to single-stranded cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland). The real-time qPCR was performed on the LightCycler 1536 Instrument (Roche) using commercial TaqMan Gene primers, shown in Table 1 (Thermo Fisher Scientific, Waltham, MA, USA). The 1536-well plates were pipetted using the Echo Liquid Handler (Labcyte, Dublin, Ireland). The mRNA expressions were calculated using the 2 (-ΔΔC(T)) method [ 29 ], and the values of target genes were normalized to the values for the housekeeping gene hypoxanthine guanine phosphoribosyl transferase ( Hprt ). The data from gene expression analysis are available online at Zenodo: https://doi.org/10.5281/zenodo.17250497 . Download: PNG larger image TIFF original image Table 1. TaqMan Gene Expression Assays (Thermo Fisher Scientific). https://doi.org/10.1371/journal.pone.0357797.t001 Liver microsomal fractions and cytochrome P450 enzyme activity assays Every microsomal fraction was prepared from the whole mouse liver by differential centrifugation according to the established protocol [ 30 ] and then stored at −80 °C. Concentrations of CYP enzymes were determined spectrophotometrically using carbon monoxide [ 31 ], and total protein content was measured using a bicinchoninic acid assay from Thermo Fisher Scientific (cat: 23228). The activities of enzymes, orthologues to human CYPs, were measured in liver microsomal fractions according to the established methods [ 31 , 32 ]. Generally, individual incubation mixtures contain potassium phosphate buffer (pH 7.4), NADPH-generating system (NADP + , isocitrate, isocitrate dehydrogenase and MgCl 2 ), liver microsomes and the specific substrates: ethoxyresorufin (CYP1A); coumarin (CYP2A), 7-ethoxy-4-trifluoromethylcoumarin (CYP2B), diclofenac (CYP2C), bufuralol (CYP2D), chlorzoxazone (CYP2E) and diazepam (CYP3A). For the determination of metabolites, a Shimadzu LC-20 HPLC system with UV or fluorescence detection was used. The analyses were performed with a LiChrospher RP-18 column (5 μm), 250 × 4 mm (Merck). Gut microbiota analysis Fecal samples were collected after 4 weeks of dietary intervention and placed in company-provided tubes containing preservative solution, then sent to TransnetYX (Cordova, TN, USA) for DNA extraction, library preparation, and shotgun whole-metagenome sequencing using Illumina NextSeq 2000 instrument at a sequencing depth of 2 million 2x150 bp read pairs per sample. The data of composition of the gut microbiome, species relative abundances, alpha and beta diversity were analyzed using the One Codex platform (One Codex, San Francisco, CA, USA). Detailed information regarding the proprietary read quality filtering, taxonomic assignment criteria, and species-level abundance estimations can be found at: https://docs.onecodex.com/en/articles/6891294-transnetyx-sequencing-and-analysis-methods . Raw sequencing data are available at https://www.ncbi.nlm.nih.gov/sra/PRJNA1336998 . Plotting and statistical analysis The data were analyzed using GraphPad Prism 8.4.3 (GraphPad Software, Inc.) and RStudio 2025.05.1 Build 513 (RStudio, Inc.). Due to the small sample size (n = 8), non-parametric tests were used for statistical evaluation, specifically the Mann-Whitney U test. For analyses involving simultaneous evaluation of multiple parameters, a correction for multiple comparisons using the Benjamini, Krieger, and Yekutieli was applied, and differences were considered statistically significant at q < 0.05. Longitudinal blood glucose and β-hydroxybutyrate data were analyzed using a two-way mixed-effects model with REML estimation and Geisser-Greenhouse correction; significance is reported for the diet factor. Statistical evaluation of microbiome diversity metrics and differential taxonomic abundances was performed using Mann-Whitney U test, PERMANOVA or multiple t-test with Holm-Sidak correction for multiple comparisons. Pharmacokinetic AUC values were calculated by the trapezoidal rule and compared between groups using Bailer’s method, appropriate for destructive sampling designs, with a two-sided t-test and Welch-Satterthwaite degrees of freedom. Details of the statistical tests used are provided in the figure legends. Results Ketogenic diet induces metabolic adaptation As an initial step, we evaluated selected metabolic parameters to confirm metabolic adaptation to KD. While the glucose levels dropped, particularly within the first week ( Fig 1A ), the concentration of circulating BHB increased significantly throughout the entire study period, as expected under KD conditions ( Fig 1B ). In the livers of KD-fed mice, the mRNA levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 ( Hmgcs2 ) and 3-hydroxy-3-methylglutaryl-CoA lyase ( Hmgcl ) were significantly elevated, reflecting the diet’s impact on ketone body synthesis ( Fig 1C ). Altogether, these changes in
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