Introduction: Knee osteothritis (OA) is a leading cause of disability in older adults, research into the specific therapies of OA, is a priority in health sciences. In this project a novel peptide-based nanoparticles (NPs) are proposed to satisfy an unmet clinical need: sustained drug delivery to the knee joint. The objective is to develop a delivery system R- 954 from nanogels hyaluronic acid (HA) /chitosan (CH), either by encapsulation ( without covalent bond ), or by grafting to the HA ( with covalent bond). The peptide r954 is an antagonist of the bradykinin receptor (BKB1R) and BQ123 is an antagonist of the endothelin receptor (ETA-R). Kaufman et al. and Moldovan et al[1],[2]. showed that both endotelin-1 (ET-1) and bradykinin play crucial role in vivo in OA pathophysiology, through their catabolic functions in cartilage degradation and inflammation involving metalloproteases MMPs (MMP1-and MMP-13), nitric oxide (NO), and prostaglandins (PGE2) . Hyaluronic acid and chitosan based nanohydrogels were chosen as carriers for the two peptides because they could provide an enhanced protection against biochemical aggression and sustained release.
Materials and Methods: In the first part, the peptide coupling of the r954 petide and hyaluronic acid (HA) was carried out. The conjugation of the peptide to HA was performed using the well established coupling chemistry of pH sensitive linkers. The carboxylic and primary amine groups present on the peptides allow for simple ligation.
To quantify the release and stability of this novel peptide-based drug, a fast and accurate liquid chromatography –tandem mass spectrometry (LC-MS/MS) method was developed. In this method the proteins of the HSF are simply precipitated with acetonitrile, the sample is diluted with mobile phase and injected directly without further purification steps. The method was validated according to the EMA guideline on bioanalytical method validation.
Results and discusssion: Peptide release and degradation kinetics study of the coupled composite in human synovial fluid (HSF) and buffer solutions.
We found that a 23% of r954 was released in hsf from the coupled composite in 77 hrs, it means in 39 days the total release. However the release of the peptide bq123 under the same conditions takes a very long time.
The degradation kinetics of the peptide r954 and bq123 in the physiological matrix human synovial fluid and under various pH conditions were investigated. The stability testing of the peptide r954 was carry out to provide evidence on how the quality of the drug substance varies with time under the influence of the temperature. When r954 was incubated in hsf at 37 °C, was stable during the first 77 hrs.
Conclusion: Results are very encouraging and show that the vectorization strategy is feasible . The main perspective: Synthesize sterile synthetic nanogels containing both peptides (encapsulated or grafted) to assess the cytotoxicity and the effects on human cartilage.
Dr. Pierre Sirois for the supply of R -954 peptide.; Canada Research Chair in Bio-inspired Materials and Interfaces, Faculty of Pharmacy, Université de Montreal
References:
[1] Kaufman et al, Nociceptive tolerance is improved by bradykinin receptor B1 antagonism and joint morphology is protected by both endothelin type A and bradykinin receptor B1 antagonism in a surgical model of osteoarthritis. Arthritis Research & Therapy (2011)
[2] Moldovan et al, Dual antagonism of endothelin type A1 and bradykinin b1 receptors for treating pain and preventing cartilage degradation. Patent 20120202744