Evaluation of mucosal immune profile associated with Zileuton nanocrystal-formulated BCS-II drug upon oral administration in Sprague Dawley rats

Abstract

Nanocrystal drug formulation involves several critical manufacturing procedures that result in complex structures to improve drug solubility, dissolution, bioavailability, and consequently the efficacy of poorly soluble Biopharmaceutics Classification System (BCS) II and IV drugs. Nanocrystal formulation of an already approved oral drug may need additional immunotoxic assessment due to changes in the physical properties of the active pharmaceutical ingredient (API). In this study, we selected Zileuton, an FDA-approved drug that belongs to BCS-II for nanocrystal formulation. To evaluate the efficacy and mucosal immune profile of the nanocrystal drug, 10-week-old rats were dosed using capsules containing either API alone or nanocrystal formulated Zileuton (NDZ), or with a physical mixture (PM) using flexible oral gavage syringes. Control groups consisted of untreated, or placebo treated animals. Test formulations were administrated to rats at a dose of 30 mg/kg body weight (bw) once a day for 15 days. The rats treated with NDZ or PM had approximately 4.0 times lower (7.5 mg/kg bw) API when compared to the micron sized API treated rats. At the end of treatment, mucosal (intestinal tissue) and circulating cytokines were measured. The immunological response revealed that NDZ decreased several proinflammatory cytokines in the ileal mucosa (Interleukin-18, Tumor necrosis Factor-α and RANTES [regulated upon activation, normal T cell expressed and secreted]). A similar pattern in the cytokine profile was also observed for the micron sized API and PM treated rats. The cytokine production revealed that there was a significant increase in the production of IL-1β and IL-10 in the females in all experimental groups. Additionally, NDZ showed an immunosuppressive effect on proinflammatory cytokines both locally and systemically, which was similar to the response in micron sized API treated rats. These findings indicate that NDZ significantly decreased several proinflammatory cytokines and it displays less immunotoxicity, probably due to the nanocrystal formulation. Thus, the nanocrystal formulation is more suitable for oral drug delivery, as it exhibited better efficacy, safety, and reduced toxicity.

Keywords:

• Nanocrystal drug formulation
• zileuton
• biopharmaceutics classification system (BCS-II)
• active pharmaceutical ingredients (API)
• physical mixture (PM)
• inflammatory cytokines
• immunotoxicity
• excipients
• mucosal immunity

Acknowledgements

The authors would like to thank Dr. Joshua Kanungo (NCTR) and Dr. Nathan Koonce (NCTR) for reviewing the manuscript and providing valuable comments and suggestions. We would also like to thank Toxicologic Pathology Associates (TPA) for proving immunohistochemistry work and PMN migration analysis. Additionally, we would like to thank animal care facilities for their help in completing the in vivo studies successfully. Dr. Banu Zolnik’s guidance during the selection of candidate molecule and constructive comments throughout the tenure of the study is highly appreciated.

Author contributions

KG, SK, DJB designed the study and KG, SK, RJ, AB performed the experiments, analyzed the data, and KG, SK, RJ interpreted the results of the experiments. The original manuscript was written by KG, SK and RJ and revised by KG, SK, DJB, SC. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s). This manuscript reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration.

Data availability statement

The datasets of the study will be available as per the guidelines of US-Food and Drug Administration data sharing policy.

Additional information

Funding

This research was funded in part by a Collaborative Opportunities for Research Excellence in Science (CORES) grant.