(M1530-07-42) Discovery of a Targeted Oral Prodrug for Prostate Cancer Therapy

Purpose: Prostate cancer is the second most common cancer among men globally ^[1]. Oral chemotherapy, also known as “therapy at home,” is always a preferred route compared to standard intravenous injection because it allows prolonged chemotherapeutic treatment and prevents multiple injections or hospital supervision^[2]-[3]. However, poor solubility, physiological absorption barriers, and drug instability in the GI tract create a great challenge for oral chemotherapy ^[4]. Among various pharmaceutical strategies, conjugation of active drug to small peptides is a novel strategy for oral delivery of anticancer drug which is facilitated by the specific transporter that is overexpressed on small intestine ^[5]. Novel pyrimidine derivative, TGX-221 is a selective, potent, inhibitor of PI3Kβ and holds a great promise as a potential chemotherapy drug against prostate cancer ^[6]; however poor water solubility and tumor specificity of TGX-221 limit its therapeutic application in cancer therapy ^[7]. In this project, we aim to conjugate modified TGX-221 with series of selected dipeptides as oral chemotherapy prodrugs.

Methods: Based on our previous studies we found that it is important to keep amino acid serine constant and change the second amino acid to find the best dipeptide to conjugate to modified TGX-221. We performed cellular uptake of 20 dye-labeled-dipeptides (Fam-dipeptides) in five prostate cancer cell lines to screen and find the best dipeptide to conjugate to modified TGX-221. Then, the selected dipeptides were synthesized by Gyros Protein peptide synthesizer and conjugated to modified TGX-221. Next, we performed cellular uptake in C4-2 cell line to find the best prodrug among selected 9 dipeptide-drug conjugate for further study and analyzed them with LCMS/MS. Also, prodrug stability was determined in mouse and human serum. Finally, prodrug activity in C4-2 prostate cancer cells was determined by cell titer glow viability assay.

Results: We successfully designed and synthesized TGX-221 prodrugs. To increase tumor specificity, we screened and found the series of dipeptides and conjugated them to the modified drug. Through cellular uptake study in C4-2 prostate cancer cell line, the most specific prodrug was selected which showed almost 18 times higher uptake compared to parental drug. Caco-2 permeability study showed 8 times higher prodrug uptake compared to TGX-221. Stability of the prodrug in human serum is higher than that in mouse serum. The best prodrug, TGX-SF, showed IC₅₀ of 48.6 mM, which is 3.7 times higher than parental prodrug.

Conclusion: This study suggests that TGX-SF is a highly promising anti-cancer compound with high specificity and water solubility compared to parental drug. It can be potentially used as a targeted oral chemotherapy prodrug against prostate cancer.

References: 1. 1. Wang, L., et al., Prostate Cancer Incidence and Mortality: Global Status and Temporal Trends in 89 Countries From 2000 to 2019. Front Public Health, 2022. 10: p. 811044.
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3. Thanki, K., et al., Oral delivery of anticancer drugs: challenges and opportunities. J Control Release, 2013. 170(1): p. 15-40.
4. Sparreboom, A., et al., Limited oral bioavailability and active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine. Proc Natl Acad Sci U S A, 1997. 94(5): p. 2031-5.
5. Dahan, A., E.M. Zimmermann, and S. Ben-Shabat, Modern prodrug design for targeted oral drug delivery. Molecules, 2014. 19(10): p. 16489-505.
6. He, C., et al., Characterization of a novel p110beta-specific inhibitor BL140 that overcomes MDV3100-resistance in castration-resistant prostate cancer cells. Prostate, 2017. 77(11): p. 1187-1198.
7. Jackson, S.P., et al., PI 3-kinase p110beta: a new target for antithrombotic therapy. Nat Med, 2005. 11(5): p. 507-14.