(T1530-07-38) Synthesizing Cisplatin Pt (IV) Derivatives for Reducing Systemic Toxicity and Enhancing Anticancer Efficacy in Triple-Negative Breast Cancer

Purpose: Triple negative breast cancer (TNBC) is one of the subtypes of aggressive mammary cancer. BRCA dysfunction in TNBC cells make them highly sensitive to DNA damaging drugs such as cisplatin. However, chemoresistance, non-specific biodistribution and severe systemic toxicities majorly limits the clinical applicability of cisplatin [1]. Here we have designed cisplatin Pt (IV) agents to strategically resolve these challenges (figure 1). In Pt (IV) derivatives, central Pt atom is conjugated with axial ligands, to make them kinetically inert and to provide an opportunity to enhance its anticancer activity. The axial ligands used were valproic acid (VA), tocopherol succinate (TOS) and chlorambucil (CBL). VA is established histone deacetylase inhibitor which improves DNA accessibility for cisplatin interaction [2]. TOS present anticancer activity by inhibiting anti-apoptotic proteins, Bcl-2 and Bcl-xL, thereby triggering mitochondrial cell death pathways [3]. While CBL is DNA alkylator drug, which may help to induce dual DNA damage [4]. The developed Pt(IV) derivatives can undergo reduction in presence of intratumoral reducing agents (glutathione, ascorbate, etc.) to release cisplatin and axial ligands for exhibiting their anticancer activity.

Methods: For synthesizing the Pt (IV) derivatives, cis,cis,trans-[PtCl₂(OH)₂(NH3)₂] referred as ‘oxoplatin’ was prepared and later was used to form axial ester bond with VA, TOS and CBL respectively. The formed derivatives were characterized using ¹H, ¹³C NMR and mass spectroscopy. In vitro physicochemical characterization like, log P determination (octanol/ water partitioning method), stability of derivatives (in 50 mM PBS, pH 7.2 with 5% BSA), drug-albumin interaction study (through CD spectroscopy) and reduction half-life determination (in 10 equiv. ascorbate and glutathione containing PBS media respectively) was performed. Animal cell culture studies including IC₅₀ determination (via MTT and sulphorhodamine B assay), synergy determination, cellular uptake, GSH assay, ROS production (using DCFH-DA), apoptosis assay (flow cytometer (Cytoflex, Beckman Coulter, USA)), disruption of cellular ion homeostasis, mitochondrial membrane potential analysis, nuclear staining, and caspase-3 assay was performed on MDA-MB-231 cells. At in vivo level, anticancer efficacy study (using 4T1 cell line induced tumor model in BALB/c mice) [5] and toxicity study (estimating AST, ALT, BUN and creatinine levels and organ histology imaging) was performed.

Results: Characteristic peaks in ¹H and¹³C NMR were observed and CP-VA, CP-TOS and CP-CBL showed m/z 621 [M+Cl]^-, 1357 [M-H]^‑ and 906.81 [M-H]⁺ respectively which confirmed the formation of CP-derivatives. Log P values of oxoplatin, CP-VA, CPTOS, and CP-CBL were found to be −2.27, −2.75, 0.28, 1.39, and 1.75, respectively. All three conjugates-maintained stability in PBS till the test period of 12 h with minimal loss of drug content ( < 10%). CP-VA showed ~30% reduction in drug content when incubated with albumin, whereas, other two derivatives showed better stability in BSA with a reduction of < 15% drug content on 12 h incubation (figure 2). The reduction potential of all three derivatives in presence of GSH and ascorbate was observed to be rapid where, >55% of Pt(IV) was reduced in 2 h in both ascorbate and GSH. However, in PBS, derivatives were stable with < 90% reduction, highlighting the selective stability of the derivatives in extracellular environment. The cell culture studies for Pt (IV) derivatives revealed [CP-VA: >5-fold; CP-TOS: >7-fold; CP-CBL: >9.5-fold] improved cellular drug uptake with strong synergism (CI value < 0.35 for all derivatives) and showed lower dependency on copper transporter 1 (Ctr1) for uptake than cisplatin. The results of enhanced in vitro activity were well corroborated by in vivo efficacy testing in the 4T1 cell-based TNBC model, showcasing ~2–7 folds higher tumor volume reduction for Pt (IV) derivatives than cisplatin (figure 3). In addition, designed derivatives significantly reduced the nephrotoxicity risk involved in cisplatin therapy indicated by systemic toxicity biomarkers and organ histopathology.

Conclusion: The results highlighted the potential of cisplatin (IV) agents in resolving cisplatin drug delivery challenges and denoted the critical role of axial ligand selection in Pt(IV) prodrug designing. Also, it indicated that developed cisplatin (IV) derivatives could open new avenues for safer synergistic chemotherapy in TNBC.

References: [1]. Poggio, Francesca, et al. Annals of oncology 29.7 (2018): 1497-1508.
[2]. Göttlicher, Martin, et al. The EMBO journal 20.24 (2001): 6969-6978.
[3]. Shiau, Chung-Wai, et al. Journal of Biological Chemistry 281.17 (2006): 11819-11825.
[4]. Di, Antonio, et al. Journal of the American Chemical Society 136.16 (2014): 5860-5863.
[5]. Bathara, Minal, et al. Molecular Pharmaceutics 17.7 (2020): 2473-2486.

Acknowledgements: The authors acknowledge Neon Laboratories Ltd., India, for providing cisplatin as a gift sample for research purposes and thank the director NIPER, S.A.S. Nagar, India for providing necessary research facilities. The authors did not receive any funding for this investigation. The authors declare no conflict of interest.


Figure 1. Schematic illustration of purpose and outcome of the present investigation


Figure 2. Illustrating the data for mentioned in vitro and cell culture studies


Figure 3. Illustrating the data for mentioned anticancer and toxicity studies