(M1330-01-03) Plasma and Gastrointestinal Tissue Analysis of Prednisolone in Mice Following LPS-Induced Inflammation

Purpose: Prednisolone, a synthetic corticosteroid, is widely used to treat inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis ¹. However, data on its pharmacokinetics (PK) and tissue distribution in the gastrointestinal (GI) tract, particularly in preclinical models, remain limited. Accurate quantification of prednisolone in blood and in GI tissues has been particularly challenging due to the identical molecular weight of prednisolone and endogenous cortisone, requiring precise chromatographic separation ^{2, 3}. In this study, we optimized a previously reported mass spectrometric method to enable separation and quantification of prednisolone in plasma and GI tissues (jejunum, ileum, colon)^{2, 3}. This method was applied to assess the PK and GI tissue distribution of prednisolone in an LPS-induced inflammatory mouse model.

Methods: Prednisolone and cortisone were extracted from mouse plasma (40 µL) or GI tissue (50 mg) homogenates (colon, ileum, jejunum; homogenized in methanol at 5 µL/mg) followed by liquid-liquid extraction with 1 mL methyl tert-butyl ether (MTBE). Deuterated analog, Prednisolone-(9,11,12,12)-d₄ served as the internal standard (IS). Following centrifugation, the organic layer was evaporated under nitrogen and reconstituted in 100 µL methanol:water (1:1, v/v). Chromatographic separation was performed on a Zorbax SB-Phenyl column (2.1 × 100 mm) using a mobile phase of 0.1% formic acid in water (A) and acetonitrile (B) at 0.15 mL/min, with an 8-minute total run time. Detection was achieved using a triple quadrupole mass spectrometer with positive electrospray ionization, monitoring transitions m/z 361.160→343.138 for prednisolone, m/z 361.350→163.100 for cortisone and 365.110→347.180 for IS. Prednisolone (10 mg/kg, oral) was administered to mice pre-treated with LPS (2.5 mg/kg, i.p.). Plasma and GI tissue samples were collected at predetermined times (0-6 h) post-dose and analyzed by the developed liquid chromatography with tandem mass spectrometric method (LC-MS/MS).

Results: The lower limit of quantification (LLOQ) for prednisolone was 0.001 µM in both plasma (nmol/ml) and gastrointestinal tissues (nmol/g), with a linear calibration range of 0.001-10 µM. Prednisolone was successfully separated from endogenous cortisone, with retention times of 5.93 minutes for prednisolone and 6.42 minutes for cortisone. Following oral administration, peak concentrations (C_max) were observed at 1 hour post-dose, with values of 0.93 ± 0.18 nmol/mL in plasma, and relatively higher concentrations in jejunum (6.07 ± 1.29 nmol/g) and ileum (3.28 ± 1.12 nmol/g). The area under the concentration-time curve (AUC₀-ₜ) values were also highest in the jejunum and ileum with 10.5 ± 4.20 and 8.06 ± 4.71 nmol*h/g, respectively. The apparent tissue-to-plasma partition coefficients (K_p), calculated as AUCₜᵢₛₛᵤₑ/AUCₚₗₐₛₘₐ, were 4.55 for the jejunum and 3.50 for the ileum.

Conclusion: We developed a robust LC-MS/MS method to quantify prednisolone in plasma and gastrointestinal tissues, achieving separation from endogenous cortisone. To our knowledge, this is the first systematic study to characterize prednisolone distribution across gastrointestinal tissues while addressing the analytical challenge of separating it from endogenous cortisone. Following oral administration in LPS-treated mice, prednisolone exhibited the highest distribution in the jejunum and ileum, with significantly lower distribution in the colon. These findings suggest that prednisolone delivery could be further optimized to enhance targeting of the ileum and colon for improved treatment of inflammatory bowel disease.

References: (1) Lichtenstein, G. R.; Abreu, M. T.; Cohen, R.; Tremaine, W.; American Gastroenterological, A. American Gastroenterological Association Institute technical review on corticosteroids, immunomodulators, and infliximab in inflammatory bowel disease. Gastroenterology 2006, 130 (3), 940-987. DOI: 10.1053/j.gastro.2006.01.048 From NLM Medline.
(2) Ionita, I. A.; Fast, D. M.; Akhlaghi, F. Development of a sensitive and selective method for the quantitative analysis of cortisol, cortisone, prednisolone and prednisone in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2009, 877 (8-9), 765-772. DOI: 10.1016/j.jchromb.2009.02.019 From NLM Medline.
(3) Ionita, I. A.; Akhlaghi, F. Quantification of unbound prednisolone, prednisone, cortisol and cortisone in human plasma by ultrafiltration and direct injection into liquid chromatrography tandem mass spectrometry. Ann Clin Biochem 2010, 47 (Pt 4), 350-357. DOI: 10.1258/acb.2010.010027 From NLM Medline.

Representative chromatograms of prednisolone, cortisone and prednisolone-(9,11,12,12)-d4