(T1430-05-27) Evaluate pH-Triggered and Sustainable Drug Release for Dendrimer-Based Formulations of STAT3 Inhibitor

Purpose: Myeloid-derived suppressor cells (MDSCs) contribute to the immune-suppressive tumor microenvironment and are linked to poor clinical outcomes in many cancers¹. Transcription factor signal transducer and activator of transcription 3 (STAT3) is highly activated in MDSCs. As a ‘master regulator’ of immune suppression, STAT3 promotes angiogenesis, host immunosuppression, and tumor invasion.² Thus, inhibition of STAT3 has emerged as a promising therapeutic strategy for modulating the tumor microenvironment. Although several small-molecule STAT3 inhibitors have been developed, the clinical translation of STAT3 inhibitors has been hampered by several challenges, including poor water solubility, limited bioavailability, severe dose-limiting toxicity, poor pharmacokinetics (PK), off-target toxicity, and limited efficacy in clinical trials.³ 

Methods: LLL12 is a novel small molecule inhibitor of STAT3 that specifically targets and inhibits STAT3 activity by directly binding with STAT3 and inhibiting the phosphorylation of STAT3.^4-6 Dendrimers are exceptional drug delivery vehicles, serving as nanocarriers capable of navigating even the most challenging sites within the body. Our initial findings indicate that systemically injected hydroxyl (OH) dendrimers selectively target MDSCs and other myeloid cells within the tumor and lymphoid organs in vivo.⁷ Based on this observation, we have developed a systemic nanotherapeutic employing dendrimer-based drug delivery. When LLL12 is covalently conjugated to dendrimers, the hydrolyzable linkages between dendrimer and LLL12 enable controlled and sustained drug release at different rates under the acidic pH of the intracellular environment. This highly controllable approach renders dendrimer-drug conjugates an ‘inactive form’ in systemic circulation, and an ‘activated form’ with sustained drug release through dendrimer-mediated internalization by myeloid cells in the tumor microenvironment.

Results: We formulated a systemic dendrimer-conjugated STAT3 inhibitor (D-LLL12) with fast- and slow-release linker profiles by reacting LLL12-linkers with bi-functional dendrimers. The STAT3 inhibitor LLL12, featuring a fast-release linker profile was synthesized utilizing an azide terminal carbonate compound, while a slow-release linker profile was synthesized using an acid terminal compound. Concurrently, bi-functional dendrimers were synthesized utilizing 6-heptynoic acid for fast-release dendrimers and Fmoc-GABA-OH for slow-release dendrimers. Final D-LLL12 formulations were characterized for their physicochemical properties using NMR, HPLC and DLS to ensure successful formulation (Fig. 1). To investigate the stability and release profile of D-LLL12, we further quantified the drug released from D-LLL12 conjugate over time in various buffers (pH of 4.5-7.4) using HPLC. Our preliminary in vitro data reveals that D-LLL12 significantly improved the safe dosing window of the free drug (LLL12) without triggering apoptosis (Fig. 2).

Conclusion: We developed novel nanoformulations of the STAT3 inhibitor LLL12 based on hydroxyl surface PAMAM dendrimers – D-LLL12, which enables the systemic delivery of LLL12 with improved PK and safety profiles. Understanding the pH-responsiveness and charge of D-LLL12 can provide insights into their stability and mechanism of drug release leading to the development of a more effective nanotherapeutic.

References: 1. Takacs GP, Kreiger CJ, Luo D, Tian G, Garcia JS, Deleyrolle LP, Mitchell DA, Harrison JK. Glioma-derived CCL2 and CCL7 mediate migration of immune suppressive CCR2(+)/CX3CR1(+) M-MDSCs into the tumor microenvironment in a redundant manner. Front Immunol. 2022, 13, 993444.
2. Kim, J. E.; Patel, M.; Ruzevick, J.; Jackson, C. M.; Lim, M. STAT3 Activation in Glioblastoma: Biochemical and Therapeutic Implications. Cancers (Basel), 2014, 6, 376.
3. Beebe JD, Liu JY, Zhang JT. Two decades of research in discovery of anticancer drugs targeting STAT3, how close are we? Pharmacol Therapeut. 2018, 191, 74.
4. Zuo M, Li C, Lin J, Javle M. LLL12, a novel small inhibitor targeting STAT3 for hepatocellular carcinoma therapy. Oncotarget. 2015, 6, 10940.
5. Nie YL, Li YY, Hu S. A novel small inhibitor, LLL12, targets STAT3 in non-small cell lung cancer in vitro and in vivo. Oncol Lett. 2018, 16, 5349.
6. Lin L, Hutzen B, Li PK, Ball S, Zuo M, DeAngelis S, Foust E, Sobo M, Friedman L, Bhasin D, Cen L, Li C, Lin J. A novel small molecule, LLL12, inhibits STAT3 phosphorylation and activities and exhibits potent growth-suppressive activity in human cancer cells. Neoplasia. 2010, 12, 39.
7. Littrell CA, Takacs GP, Sankara CS, Sherman A, Rubach, KA, Garcia JS, Bell C, Harrison JK, Zhang F. Systemically targeting monocytic MDSCs using dendrimers and their cell-level biodistribution kinetics. 2024, bioRxiv, doi: 10.1101/2024.02.14.580395.

Acknowledgements and

Funding: UF Research Opportunity Seed Fund, American Brain Tumor Association Research Discovery Grant (DG2200050), College of Pharmacy PROSPR Award, NIH/NIGMS Grant project RM1 GM139690


Figure 1. DLS and HPLC studies


Figure 2. IC50 efficacy and toxicity studies