Ticagrelor is a selective adenosine diphosphate receptor antagonist used as an antiplatelet agent. Clinical use shows substantial inter-individual variability in efficacy. Because gut microbiota can influence drug absorption, metabolism, and overall pharmacokinetics, this study aimed to determine whether intestinal flora mediate ticagrelor pharmacokinetics. The investigators combined human pharmacokinetic profiling, fecal microbiome analysis, in vitro co-incubation experiments, and pseudo-germ-free (PGF) rat pharmacokinetic studies to address this question.
Twenty-two healthy volunteers (aged 19–36 years) who had not taken medications for at least 2 weeks were enrolled. Each subject received a single oral 90 mg dose of ticagrelor swallowed whole with 240 mL water. Blood samples were collected before dosing and at 20, 40 minutes, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, 24, 36, and 48 hours post-dose. Plasma was processed and stored at −80 °C until assay. Hierarchical clustering was used to identify subject groups with different pharmacokinetic parameters.
Fecal samples collected prior to ticagrelor dosing were used for microbial DNA extraction and 16S rRNA amplicon sequencing. Libraries were sequenced on an Illumina NovaSeq platform and processed via the EasyAmplicon pipeline. Denoising and ASV generation used unoise3, with taxonomic annotation against the Silva database. Alpha-diversity metrics (ACE, Chao, Simpson, Shannon) and beta-diversity (PCoA at the ASV level) were computed, and LEfSe was applied for biomarker discovery.
Human and rat fresh fecal samples were processed to produce gut microbiota suspensions, stored anaerobically with glycerol stocks at −80 °C. Activated stocks were incubated in mGAM broth and then co-incubated anaerobically with ticagrelor solution (final incubation at 37 °C for 24 h). Negative controls contained mGAM broth with ticagrelor but without microbiota. Incubations were stopped with cold methanol containing internal standard, centrifuged, and analyzed by UPLC-MS/MS to detect ticagrelor and potential metabolites.
Specific-pathogen-free Sprague–Dawley rats were randomized to control or PGF groups (n = 8 per group). PGF rats received an antibiotic cocktail by gavage and in drinking water for 2 weeks (ampicillin, neomycin sulfate, metronidazole, vancomycin) to deplete intestinal flora. Fecal DNA was extracted and quantified to validate the pseudo-sterility model.
Rats were fasted for 12 hours before experiments. Intragastric administration of ticagrelor at 18 mg/kg (0.5% CMC-Na vehicle) was performed with blood sampling at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 7.5 and 24 h. Intravenous administration of 3 mg/kg ticagrelor (2% Tween 80 vehicle) included sampling at 0, 0.25, 0.5, 1, 2, 3, 4, 5, 6 and 24 h. Plasma was separated and stored at −80 °C for analysis.
Plasma concentrations of ticagrelor and its major metabolite AR-C124910XX were measured by UPLC-MS/MS on an AB Sciex Qtrap 6500 system using multiple-reaction monitoring. Non-compartmental pharmacokinetic analysis was performed using DAS 2.0 software. Data are reported as mean ± SD. Group comparisons used unpaired two-tailed t-tests or Mann–Whitney tests with p < 0.05 indicating statistical significance.
Measured plasma concentration–time profiles over 48 hours demonstrated considerable variability among the 22 subjects. For ticagrelor, AUC (0–48) ranged approximately three-fold among individuals (reported range 1,429 to 5,582 ng·h/mL), and AUC (0-∞) showed a similar three-fold range (1,568 to 5,746 ng·h/mL). Cmax varied up to seven-fold (137 to 953 ng/mL). The metabolite AR-C124910XX also displayed notable individual differences in AUC and Cmax. Hierarchical clustering divided subjects into Group H (higher AUC; n = 12) and Group L (lower AUC; n = 10).
Comparison of pre-dose fecal microbiota between Group H and Group L showed no statistically significant differences in alpha-diversity indices (ACE, Chao, Simpson, Shannon) or in beta-diversity by PCoA at the ASV level. Taxonomic abundance comparisons and LEfSe analysis did not reveal consistent compositional differences that correlated with the higher versus lower ticagrelor exposure groups.
Co-incubation experiments using activated human and rat fecal microbiota suspensions and ticagrelor under anaerobic conditions, followed by UPLC-MS/MS quantification, indicated that gut microbiota did not directly metabolize ticagrelor in the experimental system. Negative controls and microbiota-containing incubations did not demonstrate microbiota-mediated transformation of ticagrelor to detectable metabolites.
Pharmacokinetic comparison between PGF and control rats after oral and intravenous ticagrelor administration showed no significant differences in AUC (0–48), AUC (0-∞), or Cmax for ticagrelor or AR-C124910XX. These in vivo findings support the in vitro observation that depletion of intestinal flora does not alter ticagrelor systemic exposure in this rat model.
Across human pharmacokinetic profiling, fecal 16S rRNA sequencing, in vitro co-incubation, and PGF rat pharmacokinetics, this study found no evidence that gut microbiota affect the pharmacokinetics of ticagrelor or its metabolite AR-C124910XX. While humans exhibited marked inter-individual PK variability (three-fold differences in AUC and greater variation in Cmax), these differences were not explained by fecal microbial composition or by direct microbial metabolism in the experiments performed. The study methods included standardized UPLC-MS/MS quantification and validated microbiome and PGF models. Details on potential alternative explanations for human PK variability (for example, absorption differences, host metabolism, or transporter activity) were not established within the presented data and were not reported as resolved by this study.
Overall, the combined human, in vitro, and PGF rat data support the conclusion that intestinal microbiota do not have a measurable impact on ticagrelor pharmacokinetics under the conditions examined.