A nano-liposome formulation of the PARP inhibitor Talazoparib enhances treatment efficacy and modulates immune cell populations in mammary tumors of BRCA-deficient mice

Abstract

Two recently approved PARP inhibitors provide an important new therapeutic option for patients with BRCA-mutated metastatic breast cancer. PARP inhibitors significantly prolong progression-free survival in patients, but conventional oral delivery of PARP inhibitors is hindered by limited bioavailability and off-target toxicities, thus compromising the therapeutic benefits and quality of life for patients. Here, we developed a new delivery system, in which the PARP inhibitor Talazoparib is encapsulated in the bilayer of a nano-liposome, to overcome these limitations. Methods: Nano-Talazoparib (NanoTLZ) was characterized both in vitro and in vivo. The therapeutic efficacy and toxicity of Nano-Talazoparib (NanoTLZ) were evaluated in BRCA-deficient mice. The regulation of NanoTLZ on gene transcription and immunomodulation were further investigated in spontaneous BRCA-deficient tumors. Results: NanoTLZ significantly (p<0.05) prolonged the overall survival of BRCA-deficient mice compared to all of the other experimental groups, including saline control, empty nanoparticles, and free Talazoparib groups (oral and i.v.). Moreover, NanoTLZ was better tolerated than treatment with free Talazoparib, with no significant weight lost or alopecia as was observed with the free drug. After 5 doses, NanoTLZ altered the expression of over 140 genes and induced DNA damage, cell cycle arrest and inhibition of cell proliferation in the tumor. In addition, NanoTLZ favorably modulated immune cell populations in vivo and significantly (p<0.05) decreased the percentage of myeloid derived suppressor cells in both the tumor and spleen compared to control groups. Conclusions: Our results demonstrate that delivering nanoformulated Talazoparib not only enhances treatment efficacy but also reduces off-target toxicities in BRCA-deficient mice; the same potential is predicted for patients with BRCA-deficient breast cancer.

Keywords: BRCA-deficient breast cancer; Nanoparticle; PARP inhibitor; Talazoparib; immunomodulation.

Figure 1

Characterization of Nano-Talazoparib (NanoTLZ). A. Physicochemical characterization of NanoTLZ via dynamic light scattering and (inset) transmission electron microscopy after staining with 1.0% uranyl acetate illustrates a monodisperse formulation with an average diameter of 75 nm. B and C. NanoTLZ is stable in size, zeta potential and polydispersity, for up to 2 months in storage at 4°C. D. W780 and W0069 cells were treated with NanoTLZ or free Talazoparib (TLZ) for 6 days. Cell viability was detected by the MTS assay. E. W780 and W0069 cells were treated with NanoTLZ or free Talazoparib (TLZ) for 48 hrs. NanoTLZ and TLZ increased the expression of γH2AX, cleaved-caspase 3 (c-caspase 3) and cleaved-PARP in these BRCA-deficient breast cancer cells.

Figure 2

Pharmacokinetics and pharmacodynamics of NanoTLZ. A. Mice bearing orthotopic BRCA-mutated human HCC1937 xenografts were injected with 1 mg/kg NanoTalazoparib (i.v.). Plasma drug concentrations were detected via HPLC. A two-compartment model was fit to the plasma data using PKSolver. B. Tumor PAR levels were detected via ELISA. ****, p<0.0001 for all time points compared to control C. Brca1^Co/Co;MMTV-Cre;p53^+/- mice (N=4) were injected with a single dose of Cy5-encapsulated nanoparticles, and the fluorescent signal was detected using a IVIS spectrum imaging system 24 hrs after injection. Major organs were dissected after in vivo imaging (left) and biodistribution of nanoparticles in these organs was detected (right). Blue arrows point to tumors. Representative images are shown. D. W780 cells were treated with 5% empty nanoparticles (vehicle) or Cy5-encapsulated nanoparticles for 1-2 hrs. Fluorescent Cy5 signal was detected with a fluorescence microscope. 200x magnification.

Figure 3

NanoTLZ prolonged the overall survival and was more effective in inducing tumor regression compared to free Talazoparib in BRCA-deficient mice. Brca1^Co/Co;MMTV-Cre;p53^+/- mice were started on treatment when tumors were 4 mm in diameter. The treatments were either control (saline, i.v.), empty nanoparticles (vehicle, i.v.), NanoTLZ (i.v., 0.33 mg/kg), free Talazoparib (i.v.TLZ, i.v., 0.33 mg/kg), or free Talazoparib (oral TLZ, gavage, 0.33 mg/kg). Treatment was given three times a week. Mice were sacrificed when the tumor reached 10 mm in diameter. A. Growth curves of individual tumors treated with saline or NanoTLZ. (N= 6 in saline and 9 in NanoTLZ groups) B. NanoTLZ significantly prolonged the overall survival of BRCA-deficient mice compared to controls, oral TLZ and i.v. TLZ. N=5 in saline and vehicle groups, N=8 in NanoTLZ and TLZ treatment groups. Symbols in red: *, p<0.05, **, p<0.0.1, ***, p<0.001 vs. NanoTLZ. Symbols in black: ***, p<0.001 vs. saline. C. NanoTLZ significantly improved the progression free survival of BRCA-deficient mice compared to i.v. TLZ and oral TLZ. N=8. **, p<0.0.1, ***, p<0.001 vs. NanoTLZ. D. All tumors were classified into three groups: regressing (tumor volume decreased more than 50%), no change (tumor volume did not increase or decrease by more than 50%), active growth (tumor continued to grow and tumor volume increased more than 50%). N=6-19/group. *, p<0.05. vs. saline; ^#, p<0.05 vs. NanoTLZ.

Figure 4

NanoTLZ is better tolerated than free Talazoparib in BRCA-deficient mice. A. Brca1^Co/Co;MMTV-Cre;p53^+/- mice were treated three times a week and weighed prior to each treatment. Initial weight, the weight when the mice were started on treatment; final weight, weight after 10 injections of the corresponding treatment. Data presented as mean ± SEM. N=5 in saline group and N=8 in the NanoTLZ and Talazoparib treatment groups (i.v. TLZ and oral TLZ). *, p<0.05 vs. initial weight. B. Representative pictures of alopecia observed in free Talazoparib treatment groups.

Figure 5

RNAseq analysis of tumors from BRCA-deficient mice treated with NanoTLZ or i.v. Talazoparib. When Brca1^Co/Co;MMTV-Cre;p53^+/- mice developed tumors 4 mm in diameter, they were treated with either saline, NanoTLZ (0.33 mg/kg), or Talazoparib (i.v. TLZ, 0.33 mg/kg) by i.v. for 5 doses (3 times a week). Total RNA was isolated from the tumors and processed for RNAseq analysis. A. Cluster analysis of differentially expressed genes in each group. N=3 mice/group. B. Venn diagram of differentially expressed genes in NanoTLZ and i.v. Talazoparib group. C. Validation of gene expression by real-time PCR. Data presented as mean ± SEM. N=5 mice/group. *, p<0.05 vs. Saline. D. Protein expression of PCNA, CyclinD1, CyclinE1, cleaved-caspase (c-caspase) 3 in tumors treated with saline or NanoTLZ. Vinculin was used as the loading control. E. Immunohistochemistry of PCNA and γH2AX expression in tumor sections, 400x magnification.

Figure 6

NanoTLZ modulates immune cell populations in BRCA-deficient mice. Brca1^Co/Co;MMTV-Cre;p53^+/- mice bearing tumors (4 mm in diameter) were treated with 5 doses of saline, empty nanoparticle (vehicle), free TLZ (i.v. TLZ) or NanoTLZ, and tumor, spleen and mammary gland without visible tumors were collected for flow cytometry. The percentage of immune populations with significant changes in spleen, mammary gland, or tumor are shown from A to C, respectively. N=5 mice/group. *, p<0.05 vs. saline. D. The changes of total immune cells (CD45) and T cells (CD3) in mammary gland were confirmed by immunohistochemistry. The changes in myeloid-derived suppressor cells (MDSCs, Gr-1) in the spleen were also validated using immunohistochemistry, 200x magnification. The change of Foxp3 expression around the tumor was shown by IHC at 400x magnification.