The ketogenic diet (KD) is a high-fat, low-carbohydrate nutritional regimen increasingly investigated as an adjunctive therapy across multiple conditions. KD produces metabolic changes including ketosis, altered lipid profiles, hepatic effects, and shifts in the gut microbiome. Because many drugs undergo extensive hepatic biotransformation, dietary interventions such as KD may modify drug-metabolizing enzyme expression or activity and thereby alter systemic drug exposure. Ozanimod, a sphingosine-1-phosphate receptor modulator used in relapsing-remitting multiple sclerosis and ulcerative colitis, is extensively metabolized in the liver by multiple pathways that include cytochrome P450 (CYP) enzymes. The present study tested the hypothesis that KD affects hepatic drug-metabolizing capacity and consequently the pharmacokinetics of ozanimod in mice.
Specific-pathogen-free female C57BL/6 mice (8–10 weeks old, average weight 19.6 ± 1.4 g) were randomized by weight to one of two dietary groups for four weeks. The ketogenic diet provided 90% of calories from fat and 10% from protein (Research Diets D10070801). The composition-matched control diet provided 10% of calories from fat, 80% from carbohydrates, and 10% from protein (Research Diets D19082304). Animals were housed under standard environmental conditions with a 12-hour light–dark cycle. No formal blinding was reported.
For pharmacokinetic experiments, ozanimod was formulated in vehicle (5% DMSO, 5% Tween 20, 90% 0.1N HCl) and given as a single intragastric dose of 5 mg/kg after four weeks on the respective diets. Plasma and tissue sampling for pharmacokinetic analysis were performed according to the study protocol described by the authors (details in the original methods). Hepatic tissue was collected for gene expression and enzyme activity assays.
KD-fed mice developed the expected metabolic adaptation to ketosis. The diet produced a significant increase in plasma cholesterol. Other measured metabolic, inflammatory, and hormonal parameters exhibited only modest changes compared to control-diet mice. The manuscript reports these findings as part of the characterization of systemic effects of KD in this experimental setting.
The study assessed gut microbiota composition using whole-metagenome shotgun sequencing. KD induced changes in microbial community composition relative to the control diet. Sequence data generated in the study are available in the Sequence Read Archive under accession PRJNA1336998.
Hepatic CYP enzymes were evaluated at the mRNA level and by measuring enzyme activity. KD altered the expression and activity of several hepatic CYP enzymes. Notably, CYP1A mRNA expression and enzymatic activity were significantly increased in KD-fed mice. Measurements indicated decreased CYP2C activity in pooled samples from KD animals. CYP3A activity showed a non-significant trend toward lower values in KD-fed mice. These changes are relevant because CYP1A, CYP2C, and CYP3A enzymes participate in the biotransformation of many drugs, including pathways implicated in ozanimod metabolism.
Ozanimod systemic exposure tended to be higher in KD-fed mice compared with controls. The reported effect corresponded to an approximate 17% increase in area under the concentration–time curve (AUC) in the KD group; however, this difference did not reach statistical significance. The manuscript frames this observation as a non-significant trend toward higher ozanimod exposure associated with KD-induced alterations in hepatic enzyme expression and activity.
The findings demonstrate that a 4-week KD in female C57BL/6 mice modifies hepatic drug-metabolizing enzyme expression and activity, with a clear increase in CYP1A and reductions in some other CYP activities. These enzyme-level changes coincide with shifts in gut microbiota and systemic lipid changes (notably higher plasma cholesterol). Although ozanimod exposure showed only a non-significant increase (~17% higher AUC) in KD-fed mice, the combined data indicate that dietary interventions such as KD can be a potential source of variability in drug pharmacokinetics and, by extension, in therapeutic response.
The study was performed in female C57BL/6 mice, and translation to humans requires caution. Some pharmacokinetic effects did not achieve statistical significance. The authors did not report formal blinding procedures. Detailed datasets are publicly available: microbiome sequence data at the Sequence Read Archive (PRJNA1336998) and gene expression data on Zenodo (links provided in the original article).
A ketogenic diet induced metabolic adaptation to ketosis, increased plasma cholesterol, altered gut microbiota composition, and modified hepatic CYP expression and activity in mice. These KD-associated changes corresponded with a non-significant trend toward increased systemic exposure to ozanimod. The results support consideration of dietary status as a potential factor contributing to interindividual variability in drug metabolism and pharmacokinetics.