Background/Purpose: Arthritic diseases affect more than 50 million Americans and, although there are many etiologies, the common manifestation of the disease is pain and limited mobility (1). Delivery of therapeutics to alleviate symptoms and perturb disease progression is limited by the risk of systemic toxicity (2). Intra-articular therapeutic administration is limited to accessible joints and cannot address disseminated disease (3). We have identified a panel of cysteine-knot peptides that target to cartilage in rodents following systemic administration. This work characterizes the in vivopharmacokinetics of cartilage-targeting knottins and assesses the potential for translation to arthritis therapeutics in the clinic.

Methods: Whole body autoradiography (WBA) was used to evaluate biodistribution of knottin peptides in rodents. Radioactive signal of ¹⁴C-KNT-17R (100 nmol, IV) was quantified in the knee and intervertebral disc (IVD) between 5 min and 96hr. Delivery of a cargo was assessed after KNT-17R conjugation to the fluorophore Cy5.5 (10nmol IV, 3hr) or Dexamethasone (100nmol IV, 3 and 24hr). Accumulation in knee and IVD were assessed by fluorescent microscopy and WBA. Binding to human cartilage explants was assessed by microscopy or liquid scintillation counting after overnight incubation with 10mM ¹⁴C or Cy5.5 labeled peptide.

Results: Two independent time course experiments show that peptide KNT-17R distributes rapidly and persists in all types of cartilage in the mouse. The peak signal intensity of 60+/-14 % injected dose/g (knee) and 34+/- 8 % injected dose/g (IVD) was observed 30 min after administration. Peak signal did not significantly decline until after 3 hours (knee) or 8 hours (IVD). Fluorescent microscopy further demonstrated that Cy5.5-KNT-17R accumulated in cartilage of the knee and IVD. Dexamethasone-KNT-17R accumulated in the knee at 41+/-14 % injected dose/g and in the IVD at 20+/-3 % injected dose/g after 3 hours. Signal intensity in cartilage was too low to measure in mice treated with ¹⁴C-Dexamethasone. KNT-17R was found to bind to human cartilage with significantly greater intensity than negative control peptide KNT-15R by fluorescence microscopy and liquid scintillation counting.

Conclusion: This work demonstrates cartilage accumulation by a systemically administered knottin peptide. The peptide is able to deliver a steroid payload to cartilage in multiple clinically relevant joints. Binding to human cartilage explants establishes evidence that a knottin peptide may have promise in the treatment of arthritis in humans.

1. Hootman, Arthritis Rheumatol 2016. 68, 1582-1587

2. Evans, Nat Rev Rheumatol 2014 10, 11-22

3. Larsen, J Pharm Sci 2008 97, 4622-4654

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeted-therapeutic-delivery-to-cartilage-with-knottin-proteins/