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Background: Current treatment strategies for rheumatoid arthritis (RA) involve disease-modifying antirheumatic drugs (DMARDs) that treat symptoms rather than the underlying drivers of disease. This study investigates a novel drug candidate composed of a citrullinated cyclic peptide aptamer in a hyaluronic acid/chitosan nanoparticle (APT001). The citrullinated peptide was chosen as a potential T and B cell epitope based on theoretic and experimental evidence and showed association with disease activity in RA (1) and the nanoparticle was designed for uptake by neutrophils and monocytes for drug delivery to the site of inflammation (2). Previously, we showed that APT001 can reduce signs of arthritis in rodent models of RA.(3)
Objectives: This study aims to investigate the therapeutic efficacy, safety profile, and mode-of-action of APT001 in a rat model of collagen-induced arthritis (CIA).
Methods: Rats were dosed with APT001 subcutaneously (s.c.) at 5 mg/kg or 2.5 mg/kg for 3 weeks and followed for additional 4 weeks observation period. The effect of APT001 was evaluated by degree of paw swelling, histopathological examination, and RNA sequencing. Toxicology was assessed by hematoxylin and eosin (H&E) staining of liver, kidney, brain, spleen, and lung. Biodistribution was explored using NIR microscopy and APT001 labelled with Cy5.5 in the peptide component.
Results: APT001 at 5 mg/kg completely mitigated joint swelling (score 0) already after the 3 weeks treatment period. Disease activity score at week 7 was 0 in both APT001 treatment groups, 6.4 in untreated rats, 2.2 in rats treated with nanoparticle without peptide, and 1.8 in rats treated with peptide without nanoparticle. Cartilage damage score by H&E staining was 3 in untreated group versus 0 in group treated with APT001 5 mg/kg. The number of pathologic TRAP positive cells was 12 in untreated group versus 4 in group treated with APT001 5 mg/kg.
RNA sequencing revealed upregulation in 1459 and downregulation in 1424 DEGs in the joints. In ontology and pathway analyses cell metabolism and cell division were most affected with significant decrease in the IL-17 pathway. In the spleen, treatment with APT001 5 mg/kg compared with untreated rats resulted in only 32 upregulated and 33 downregulated DEGs.
In the biodistribution study APT001 was primarily found in synovium and kidneys after 6 hours and in the spleen after 48 hours. At termination point after 7 weeks, no peptide was detected in any organ indicating complete clearance. Citrullinated peptide without nanoparticle distributed differently, targeting mostly liver and kidney at both 6-hour and 48-hour time points. No histology changes were seen in organs from rats treated with APT001 5 mg/kg compared with untreated rats.
Conclusion: We here confirm previous observations that APT001 attenuates CIA in rats. This study highlights a sustained efficacy over the extended observation period. This study further adds mechanistic insights revealing changes in gene expression in the joints but not in the spleen. This is in line with the proposed mechanism of action being uptake by neutrophils and monocytes and transport and release of APT001 in the joints and modulation of the immune imbalance caused by break of tolerance to citrullinated peptides.
REFERENCES: [1] S. Khatri, J. Hansen, K. Astakhova, Antibodies to synthetic citrullinated peptide epitope correlate with disease activity and flares in rheumatoid arthritis, PLoS One. 15 (2020).
[2] S. Khatri, J. Hansen, A.C. Mendes, I.S. Chronakis, S.C. Hung, E.D. Mellins, K. Astakhova, Citrullinated Peptide Epitope Targets Therapeutic Nanoparticles to Human Neutrophils, Bioconjug. Chem. 30 (2019) 2584–2593.
[3] S. Khatri, J. Hansen, N.B. Pedersen, I.P. Brandt-Clausen, S. Gram-Nielsen, A.C. Mendes, I.S. Chronakis, U.B. Keiding, A.I. Catrina, B. Rethi, M.H. Clausen, T. Kragstrup, K. Astakhova, Cyclic Citrullinated Peptide Aptamer Treatment Attenuates Collagen-Induced Arthritis, Biomacromolecules. 23 (2022).
Acknowledgements: We express our gratitude to Susanne Primdahl for their outstanding contributions with histology part of the study.
Disclosure of Interests: Kira Astakhova Stock ownership and affiliation with the biotech company developing the studied compound (Aptol Pharma)., Tue Wenzel Kragstrup Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, UCB, and Abbvie., Stock ownership and affiliation with the biotech company developing the studied compound (Aptol Pharma)., Bristol-Myers Squibb, UCB, Gilead, and Eli-Lilly., Gilead., Adrian Hernández Bustos: None declared, Nadia Bom Pedersen: None declared, Christopher Mahony: None declared, Adam P. Croft: None declared, Mads Hartvig Clausen Stock ownership and affiliation with the biotech company developing the studied compound (Aptol Pharma).