(M1130-05-33) Physicochemical and Biopharmaceutical Characterization of Locally Bioavailable COX-2 Inhibitor for Familial Adenomatous Polyposis

Purpose: Familial adenomatous polyposis (FAP) is a rare (1 in 8,300 at birth) genetic condition characterized by the development of many tens to thousands of polyps in the rectum and colon, which eventually develop into colorectal cancer (CRC) [1,2,3]. Colectomy, typically performed between 15 and 25 years of age, is currently the only available treatment for FAP patients aimed at preventing CRC [4]. Several new and existing candidates and combinations have been tested in humans or are currently in phase I/II/III clinical trials for reducing polyp growth in FAP patients. However, most agents (including conventional cyclooxygenase-2 inhibitors such as celecoxib), even when effective, have severe or sometimes fatal adverse effects, making them unsafe for long-term chemoprevention in pediatric patients with FAP. To date, there is no FDA-approved treatment for FAP. To develop much-needed safe and effective therapy for this condition, it is essential that the local tissue (colonic) bioavailability of the drug is maximized while minimizing its systemic bioavailability to prevent on-target off-organ toxicities. Here, we employed PK-directed drug design to generate multiple analogs of the cyclooxygenase-2 (COX-2) inhibitor 6a1 (Figure 1), utilizing the concept of Locally Bioavailable Drugs (Patent # US 11,202,773 B2).

Methods: 6a1 analogs were designed by making structure-based modifications in two key regions (Figure 1). Region 1 aimed to enhance COX-2 inhibitory activity, and Region 2 was designed to improve solubility and/or glucuronidation, thereby reducing systemic bioavailability and enhancing local bioavailability through enterohepatic recirculation and hydrolysis in the distal gastrointestinal tract. Structure verification was performed using NMR and mass spectroscopy. Purity and stability were determined by UPLC equipped with a UV detector. In vitro COX-2 inhibitory activities of the analogs were measured at three different concentrations (ranging from 0.02 to 200 µM) as the percentage inhibition of human recombinant COX-2 enzyme-mediated prostaglandin E₂ (PGE₂) production, using a validated LC-MS/MS method [5]. IC₅₀ values for the inhibition of COX-2 by 6 selected analogs were also determined. Solubility of analogs was estimated using the Turbidimetric Solubility method. Extent of metabolism and glucuronidation: sulfation ratio was measured at 10 µM substrate concentration by overnight incubation with Caco-2 cells.

Results: The results showed that most analogs were >83% pure (except 6a1-07 and 6a1-09) and stable (Table 1). Substituting R2 with sulfate (6a1-02/6c1-02) and ester of valine (6a1-03/6c1-03) made unstable compounds, which converted back to 6a1 and 6c1 upon storage both as powder and in 50% MeOH solution at 4°C after 4 weeks. Fluoro (6a1-05), chloro (6a1-06), and bromo (6a1-08) substitutions enhanced COX-2 inhibition activity (Figure 2), but decreased solubility (Table 1), with 6a1-08 exhibiting the greatest improvement (2-fold higher than 6a1). The overnight incubation with Caco-2 cells resulted in 70-99% metabolism, accompanied by significant changes in the glucuronide-to-sulfate ratio of the analogs, based on R1 and R2 substitutions. Chloro-, and bromo- substitutions improved the glucuronide-to-sulfate ratio from 15:85 (for 6a1) to 45:55 (for 6a1-06) to 60:40 (for 6a1-08).

Conclusion: We established the structure-activity and structure-metabolism relationships of 6a1 analogs, providing valuable insights into LBD lead optimization that balances COX-2 inhibition activity and a Phase II metabolism profile (higher glucuronide-to-sulfate ratio).

References: 1. Vasen HF, Moeslein G, Alonso A, Aretz S, Bernstein I, Bertario L, et al. Guidelines for the clinical management of familial adenomatous polyposis (FAP). Gut 2008; 57:704-713.
2. Galiatsatos P, Foulkes WD. Familial adenomatous polyposis. Official journal of the American College of Gastroenterology| ACG 2006; 101:385-398.
3. Dinarvand P, Davaro EP, Doan JV, Ising ME, Evans NR, Phillips NJ, et al. Familial adenomatous polyposis syndrome: an update and review of extraintestinal manifestations. Archives of pathology & laboratory medicine 2019; 143:1382-1398.
4. Kyriakidis F, Kogias D, Venou TM, Karlafti E, Paramythiotis D. Updated Perspectives on the Diagnosis and Management of Familial Adenomatous Polyposis. Appl Clin Genet. 2023 Aug 14;16:139–153.
5. Yun C, Dashwood WM, Kwong LN, Gao S, Yin T, Ling Q, Singh R, Dashwood RH, Hu M. Accurate quantification of PGE2 in the polyposis in rat colon (Pirc) model by surrogate analyte-based UPLC-MS/MS. J Pharm Biomed Anal. 2017 Jul 25;148:42–50.

Acknowledgements: Acknowledgement: Rashim Singh is supported by the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health under award number K12TR004522.


Figure 1 Region 1 and 2 in the structure of 6a1 for structural modifications to make LBD Analogs


Figure 2 COX-2 Inhibitory Activity (IC50) of LBD Analogs