(T1530-04-19) Lymphatic Targeting of Cyclosporine Improves Survival and Ameliorates Lupus-Like Autoimmune Disease in NZB/W Mice

Purpose: Systemic lupus erythematosus (SLE) is a chronic polygenic autoimmune disease affecting over 1.5 million Americans, significantly impacting their quality of life (LFA, 2021). Cyclosporine (CsA) is a potent immunosuppressant that was first used in the management of SLE decades ago, but it is limited by its low bioavailability, narrow therapeutic index, and dose-dependent nephrotoxicity (Busauschina et al., 2004; Italia et al., 2006). We have developed a biodegradable, ligand-conjugated nanoparticle (P2Ns-GA) that targets transferrin receptors non-competitively, facilitating the direct delivery of CsA to the lymphatic system while overcoming the limitations of CsA (Ganugula et al., 2017). Using this novel delivery strategy, we significantly improved the lymph node bioavailability of CsA and enhanced its efficacy in Mrl-lpr mouse model of SLE (Ganugula et al., 2020). Owing to the heterogenic nature of lupus in humans, no single mouse model of SLE captures all the clinical presentations and underlying pathogenic events seen in humans. Hence, we aim to assess the efficacy of our CsA delivery system and its ability to improve survival in another mouse model of SLE, the NZB/W mouse, a model characterized by hypergammaglobulinemia, autoantibody production, and inflammation of the kidneys which are features seen in human SLE.

Methods: Female C57BL/6 or NZB/W mice aged 25 weeks were randomly assigned into five groups: Healthy (C57BL/6), Untreated (NZB/W), CsA (10mg/kg of commercial CsA), P2Ns-CsA (10mg/kg CsA equivalence) and P2Ns-GA-CsA (10mg/kg CsA equivalence). The CsA group was dosed orally, every day, while P2Ns-CsA and P2Ns-GA-CsA were dosed orally, 3 times per week. Urine and blood samples were collected at week 0 (week 25 of age) and week 14 (week 39 of age) of treatment. Fifty percent survival was recorded in the untreated NZB/W mice at week 39 of age. The study was terminated at this point, with all major organs and tissues collected, to enable the assessment of the effect and mechanism of the treatment on the surviving animals. The levels of total IgG, anti-dsDNA, VCAM 1, and Blood Urea Nitrogen were assessed in plasma while urinary creatine, albumin, and urea were assessed in urine. Lupus nephritis was assessed by measuring the levels of various kidney injury biomarkers in the serum and urine.

Results: Fifty percent of the NZB/W mice survived at 38 weeks of age. The various treatments improved survival in NZB/W mice (Figure 1). Compared to C57BL/6 mice, the levels of both total IgG and anti-ds DNA IgG were increased over 2-fold and 20-fold respectively. Treatment with P2Ns-GA-CsA significantly reduced the levels of total IgG and anti-ds DNA IgG in NZB/W mice by about 50 percent and 70 percent respectively (Figure 2 a and b). VCAM-1, a protein implicated in SLE was elevated in NZB/W mice when compared with the C57BL/6 mice. Mice treated with P2Ns-GA-CsA showed a significant reduction in the level of VCAM-1 in NZB/W mice (Figure 2c). Similarly, P2Ns-GA-CsA significantly reduced the level of Blood Urea Nitrogen in NZB/W mice (Figure 2d). The urinary levels of urea and albumin/creatinine ratio which were elevated in NZB/W mice were diminished by P2Ns-GA-CsA. Additionally, treatment of P2Ns-GA-CsA significantly reduced the urinary levels of kidney injury biomarkers such as Clusterin, Cystatin C, NGAL, and Osteopontin. A similar pattern was observed in the serum levels of the kidney injury biomarkers.

Conclusion: This study offers critical insight into the pharmacological potentials of P2Ns-GA-CsA in NZB/W mice. Despite that P2Ns-GA-CsA was administered less than half the time CsA (conventional CsA) was administered, P2Ns-GA-CsA improved survival in NZB/W mice and alleviated the severity of SLE by ameliorating hypergammaglobulinemia, suppressing autoantibody production, and reducing the levels of the kidney injury biomarkers. This shows that the novel delivery strategy improves the bioavailability of CsA and facilitates its efficacy at a much lower dose, thus preventing the dose-dependent nephrotoxicity of CsA. However, further analysis is in progress to determine the mechanism by which P2Ns-GA-CsA alleviates the severity of SLE in the NZB/W mouse model of SLE.

References: Lupus Foundation of America (2021). Lupus facts and statistics. https://www.lupus.org/resources/lupus-facts-and-statistics
Busauschina, A., Schnuelle, P., & van der Woude, F. J. (2004). Cyclosporine nephrotoxicity. Transplantation Proceedings, 36(2, Supplement), S229-S233. https://doi.org/https://doi.org/10.1016/j.transproceed.2004.01.021
Ganugula, R., Arora, M., Saini, P., Guada, M., & Kumar, M. N. V. R. (2017). Next Generation Precision-Polyesters Enabling Optimization of Ligand–Receptor Stoichiometry for Modular Drug Delivery. Journal of the American Chemical Society, 139(21), 7203-7216. https://doi.org/10.1021/jacs.6b13231
Ganugula, R., Arora, M., Zou, D., Agarwal, S. K., Mohan, C., & Kumar, M. (2020). A highly potent lymphatic system-targeting nanoparticle cyclosporine prevents glomerulonephritis in mouse model of lupus. Sci Adv, 6(24), eabb3900. https://doi.org/10.1126/sciadv.abb3900
Italia, J. L., Bhardwaj, V., & Ravi Kumar, M. N. V. (2006). Disease, destination, dose and delivery aspects of ciclosporin: the state of the art. Drug Discovery Today, 11(17), 846-854. https://doi.org/https://doi.org/10.1016/j.drudis.2006.07.015

Acknowledgements: Acknowledgements: This study is supported by NIH-R01AI155908 and LRA-1040394


Figure 1. Treatment with P2Ns-GA-CsA improves the survival of NZB/W mice. (A) Survival table (B) Survival Plot.


Figure 2. Treatment with P2Ns-GA-CsA alleviated the severity of SLE in NZB/W mice. (A) Total IgG estimated in Plasma by ELISA at 39-week of age (B) Anti-dsDNA estimated in Plasma by ELISA at 39-week of age (C) VCAM-1 estimated in Plasma by ELISA at 39-week of age (D) Blood Urea Nitrogen estimated in Plasma by ELISA at 39-week of age E) Urinary Urea estimated in Urine by ELISA at 39-week of age and F) Albumin/Creatinine Ratio estimated in Urine by ELISA at 39-week of age. Data was expressed as mean ± SEM (n = 6); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.


Figure 3. Treatment with P2Ns-GA-CsA prevented Kidney injury in NZB/W mice. (A) Cystatin C (B) NGAL (C) Clusterin and (D) Osteopontin estimated in serum by Multiplex Assay at 39 weeks of age. (E) Cystatin C (F) NGAL (G) Clusterin and (H) Osteopontin estimated in urine by Multiplex Assay at 39-week of age. Data was expressed as mean ± SEM (n = 6); *P < 0.05, **P < 0.01, ***P < 0.001.