Traumatic joint injuries significantly increase synovial fluid levels of pro-inflammatory cytokines that can initiate cartilage degeneration leading to osteoarthritis (OA). Articular cartilage is a highly negatively charged, avascular tissue, which relies on synovial fluid convection and electro-diffusion to transport proteins and therapeutics to tissue chondrocytes. No OA drug has yet passed the safety criteria of clinical trials due to ineffective intra-articular (i.a.) delivery methods, which require very high drug doses that cause systemic toxicity. There is a need to design local delivery mechanisms that can enable drugs or drug carriers to rapidly diffuse into the cartilage extracellular matrix to achieve intratissue therapeutic levels before these drugs are cleared from the joint space via lymphatics and synovium vasculature.
This dissertation investigates the effects of size and charge of solutes on their penetration, binding and retention within negatively charged tissues such as cartilage. Based on this understanding we selected Avidin, a globular protein, as a drug carrier owing to its optimal size and high positive charge (66,000 Da, pI 10.5). Avidin resulted in a six-fold upward Donnan partitioning factor at the synovial fluid-cartilage interface, had a 400-fold higher uptake than its electrically neutral counterpart (Neutravidin), and remained bound within cartilage for at least 15 days. Competitive binding experiments revealed that despite Avidin's weak and reversible ionic binding (dissociation constant, KD~150 pM) to the negatively charged glycosaminoglycans, its long term retention was facilitated by large intratissue binding site density (NT~2,920 pM). Thus, structures like Avidin are ideal candidates for local i.a. drug delivery into cartilage.
In vivo animal studies revealed that Avidin retained inside the joint space for extended time periods resulting half-life of 154h in rabbit cartilage which was 5-6 times longer than that in the thinner rat cartilage. This was confirmed to be consistent with the concept that diffusion-binding kinetics scale as the square of tissue thickness, emphasizing the necessity of using larger animal models for studying joint space transport and pharmacokinetics. Avidin's neutral counterpart (Neutravidin) was completely cleared from the joint space of both rats and rabbits within 24h.
We then conjugated Avidin with the glucocorticoid, dexamethasone, using chemical linkers to enable its sustained release. Avidin delivered dexamethasone into cartilage deep zones where majority of chondrocytes reside thereby successfully inhibiting cytokine-induced catabolic activity in cartilage explants in-vitro. A single i.a. injection of Avidin-conjugated drug can thereby enable sustained drug delivery in low doses and therefore has the potential to replace the current clinical practice of using multiple injections of high dose glucocorticoids in patients. The biological efficacy of this system in rescuing degenerative mechanisms of OA is currently being validated in a well-accepted rabbit model of post-traumatic OA as part of a preclinical study
|2003||Ng L, Grodzinsky AJ, Patwari P, Sandy J, Plaas A, Ortiz C. Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy. J Struct Biol. September 2003;143(3):242-257.|
|1994||Sah RL, Chen AC, Grodzinsky AJ, Trippel SB. Differential effects of bFGF and IGF-I on matrix metabolism in calf and adult bovine cartilage explants. Arch Biochem Biophys. January 1994;308(1):137-147.|
|2013||Little CB, Hunter DJ. Post-traumatic osteoarthritis: from mouse models to clinical trials. Nat Rev Rheumatol. August 2013;9(8):485-497.|
|1983||Hoch DH, Grodzinsky AJ, Koob TJ, Albert ML, Eyre DR. Early changes in material properties of rabbit articular cartilage after meniscectomy. J Orthop Res. 1983;1(1):4-12.|
|2014||Bajpayee AG, Wong CR, Bawendi MG, Frank EH, Grodzinsky AJ. Avidin as a model for charge driven transport into cartilage and drug delivery for treating early stage post-traumatic osteoarthritis. Biomaterials. January 2014;35(1):538-549.|
|1989||Sah RL-Y, Kim Y-J, Doong J-YH, Grodzinsky AJ, Plass AHK, Sandy JD. Biosynthetic response of cartilage explants to dynamic compression. J Orthop Res. 1989;7(5):619-636.|
|2007||Lohmander LS, Englund PM, Dahl LL, Roos EM. The long-term consequence of anterior cruciate ligament and meniscus injuries: osteoarthritis. Am J Sports Med. October 2007;35(10):1756-1769.|
|2014||Evans CH, Kraus VB, Setton LA. Progress in intra-articular therapy. Nat Rev Rheumatol. January 2014;10(1):11-22.|
|1998||Johnson DL, Urban WP Jr, Caborn DNM, Vanarthos WJ, Carlson CS. Articular cartilage changes seen with magnetic resonance imaging-detected bone bruises associated with acute anterior cruciate ligament rupture. Am J Sports Med. May–June 1998;26(3):409-414.|
|1997||Torzilli PA, Arduino JM, Gregory JD, Bansal M. Effect of proteoglycan removal on solute mobility in articular cartilage. J Biomech. September 1997;30(9):895-902.|
|2010||Lotz MK. Posttraumatic osteoarthritis: pathogenesis and pharmacological treatment options. Arthritis Res Ther. 2010;12:211.|
|2011||Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier J-P, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol. January 2011;7(1):33-42.|
|1976||Maroudas A. Transport of solutes through cartilage: permeability to large molecules. J Anat. November 1976;122(pt 2):335-347.|
|2004||Leddy HA, Awad HA, Guilak F. Molecular diffusion in tissue-engineered cartilage constructs: effects of scaffold material, time, and culture conditions. J Biomed Mater Res. August 15, 2004;B70(2):397-406.|
|1986||Farndale RW, Buttle DJ, Barrett AJ. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta. September 4, 1986;883(2):173-177.|
|2011||Anderson DD, Chubinskaya S, Guilak F, Martin JA, Oegema TR, Olson SA, Buckwalter JA. Post-traumatic osteoarthritis: improved understanding and opportunities for early intervention. J Orthop Res. June 2011;29(6):802-809.|
|2006||Brown TD, Johnston RC, Saltzman CL, Marsh JL, Buckwalter JA. Posttraumatic osteoarthritis: a first estimate of incidence, prevalence, and burden of disease. J Orthop Trauma. November–December 2006;20(10):739-744.|