(M1330-01-04) The Non-GLP Dose Range-Finding Study on Lymphatic-Targeting of Cyclosporine in Mrl-lpr Mouse Model of SLE

Purpose: Cyclosporine (CsA), a calcineurin inhibitor and potent immunosuppressant currently approved for use in preventing the rejection of transplanted organs, has proven to be effective in the treatment of lupus nephritis (Alamilla-Sanchez et al., 2021; Moroni et al., 2006). However, the use of CsA has been limited in the management of lupus owing to its nephrotoxicity, poor bioavailability, and narrow therapeutic index (Busauschina et al., 2004; Italia et al., 2006). To improve the bioavailability and therapeutic efficacy of cyclosporine while preventing its nephrotoxicity, we developed a biodegradable, ligand-conjugated nanoparticle (P2Ns–Gambogic Acid (GA) that targets transferrin receptors non-competitively, facilitating the direct delivery of CsA to the lymphatic system (Ganugula et al., 2017; Ganugula et al., 2020). We aim to assess how the efficacy and safety of P2Ns–GA-CsA changes across the various dose levels and determine the most appropriate dose of P2Ns–GA-CsA which will alleviate the severity of SLE without causing further damage or adverse effects.

Methods: Female Mrl or Mrl-lpr mice aged 8 weeks were randomly assigned into seven groups: G1- Healthy (Mrl), G2- Untreated (Mrl-lpr), G3- CsA (15mg/kg of commercial CsA), G4- P2Ns-CsA (15mg/kg CsA equivalence) and P2Ns-GA-CsA (G5- 5mgkg, G6-10mg/kg, and G7-15mg/kg CsA equivalence). The CsA group was dosed orally, every day, for 14 weeks while P2Ns-CsA and P2Ns-GA-CsA groups were dosed orally, 3 times per week for 14 weeks. Urine and blood samples were collected at week 0 (week 8 of age), week 7 (week 15 of age), and week 14 (week 22 of age) of treatment. After the last dose, the animals were sacrificed with all major organs and tissues collected, weighed, and stored appropriately. Splenomegaly and lymphadenopathy were assessed by weighing the spleen and cervical lymph node. The levels of anti-dsDNA, total IgG, VCAM 1, and Blood Urea Nitrogen were assessed in plasma while urinary creatine, albumin, and urea were assessed in urine via ELISA. Renal histology was assessed by H & E staining.

Results: The appearance of lymphadenopathy was evident in the Mrl-lpr mice as the disease progressed. Treatment with P2Ns-GA-CsA at 5mg and 10mg/kg CsA equivalence significantly reduced the enlargement of the cervical lymph nodes (Figure 1 a & b) and spleen in Mrl lpr mice (Figure 1c). Compared to Mrl mice, total IgG and anti-ds DNA IgG levels were increased over 30-fold and 40-fold, respectively. Treatment with P2Ns-GA-CsA at 5mg and 10mg/kg significantly reduced the levels of total IgG and anti-ds DNA IgG in Mrl-lpr mice by about 30 percent and 50 percent respectively (Figure 2 a and b). VCAM-1, a protein implicated in SLE was elevated in Mrl-lpr mice compared to the Mrl mice. Only the mice treated with P2Ns-GA-CsA at 10mg/kg showed a significant reduction in the level of VCAM-1 in Mrl-lpr mice (Figure 2c). Similarly, P2Ns-GA-CsA at 10mg/kg and 15mg/kg significantly reduced the level of Blood Urea Nitrogen in Mrl-lpr mice (Figure 2d). The urinary levels of urea, albumin, and creatinine, (Figure 2 e, f, & g) which were elevated in Mrl-lpr mice were significantly diminished by all the different treatments. However, only the Mrl-lpr mice treated with P2Ns-GA-CsA (G5- 5mgkg, G6-10mg/kg, and G7-15mg/kg CsA equivalence) showed a significantly reduced urinary Albumin/Creatinine ratio (Figure 2h). The hematoxylin and eosin (H&E) stained kidney sections showed high inflammatory cell infiltration which was reduced by P2Ns-GA-CsA at 5mg and 10mg/k (Figure 3). Taken together, P2Ns-GA-CsA at 10mg/kg was the most promising treatment in the study.

Conclusion: These studies offer critical insight into the pharmacological potentials and limitations of P2Ns-GA-CsA. P2Ns-GA-CsA alleviated the severity of SLE, ameliorating lymphadenopathy and splenomegaly in Mrl-lpr mice and reducing the levels of the disease biomarkers. At 5mg/kg, treatment with P2Ns-GA-CsA showed therapeutic efficacy which was more significant at 10 mg/kg. At 15mg/kg, P2Ns-GA-CsA seems to be showing some toxicity. However, further analysis is ongoing to determine the exact effect of the treatment at this dose. Additionally, further analysis is ongoing to determine the various mechanisms by which P2Ns-GA-CsA at 10mg/kg alleviates the severity of SLE in the Mrl-lpr mouse model of lupus.

References: Alamilla-Sanchez, M. E., Alcala-Salgado, M. A., Alonso-Bello, C. D., & Fonseca-Gonzalez, G. T. (2021). Mechanism of Action and Efficacy of Immunosupressors in Lupus Nephritis. Int J Nephrol Renovasc Dis, 14, 441-458. https://doi.org/10.2147/ijnrd.S335371
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
Moroni, G., Doria, A., Mosca, M., Alberighi, O. D., Ferraccioli, G., Todesco, S., Manno, C., Altieri, P., Ferrara, R., Greco, S., & Ponticelli, C. (2006). A randomized pilot trial comparing cyclosporine and azathioprine for maintenance therapy in diffuse lupus nephritis over four years. Clin J Am Soc Nephrol, 1(5), 925-932. https://doi.org/10.2215/cjn.02271205

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


Figure 1. Treatment with P2Ns-GA-CsA ameliorated lymphadenopathy and splenomegaly in Mrl-lpr mice. (A) Cervical Lymph Node Weight (B) Representative images of the cervical lymph node from group 1 (G1) till group 7 (G7). (C) Spleen weight. Data was expressed as mean ± SEM (n = 6); *P < 0.05, **P < 0.01.


Figure 2. Treatment with P2Ns-GA-CsA alleviated the severity of SLE in Mrl-lpr mice. (A) Total IgG estimated in Plasma by ELISA at 22-week of age, (B) Anti-dsDNA estimated in Plasma by ELISA at 22-week of age, (C) VCAM-1 estimated in Plasma by ELISA at 22-week of age, (D) Blood Urea Nitrogen estimated in plasma by ELISA at 22-week of age, E) Urinary Urea estimated in urine by ELISA at 22-week of age, F) Urinary Albumin estimated in urine by ELISA at 22-week of age, G) Urinary Creatinine estimated in urine by ELISA at 22-week of age, and H) Urinary Albumin/Creatinine Ratio estimated at 22-week of age. Data was expressed as mean ± SEM (n = 6); *P < 0.05, **P < 0.01, ****P < 0.0001.


Figure 3. Treatment with P2Ns-GA-CsA alleviated the renal inflammation. Representative images of H & E stained kidney section from group 1 (G1) till group 7 (G7).