Purpose: There are 6.3 million fractures each year in the USA, costing the healthcare system ~$28 billion/year (data source: National Ambulatory Medical Care Survey and the American Academy of Orthopaedic Surgeons). More importantly, greater than 300,000 hip fractures occur every year in the US, and one in four of these patients will die of associated complication (Bentler et al., 2009; Braithwaite et al., 2003). Clearly, a major need exists for a systemically administered drug that can accelerate fracture repair without imposing unwanted side effects. From our perspective, a bone anabolic agent that would concentrate at the fracture site and clear rapidly from unaffected sites should satisfy these requirements.
To address this need, we have designed a fracture-targeted conjugate that contains a bone-homing aspartic acid decapeptide moiety linked to dasatinib, a potent Src inhibitor, via a self-immolative linker. Upon localization to the fracture surface, free dasatinib is released from the conjugate promoting enhanced fracture repair (Fig.1).
Methods: • Synthesis: The Asp10 moiety of dasatinib-Asp10 conjugate (DAC) was synthesized via standard solid phase peptide chemistry using a 2-chlorotrityl chloride resin. Dasatinib was attached to the Asp10 by esterifying it to maleimidylpropionic acid which in turn was conjugated to the cysteine-containing Asp10 via Michael addition.
• Animal studies: a stabilized fracture was introduced into the femoral midshaft of anesthetized ND4 Swiss Webster mice. Fracture-bearing mice were dosed subcutaneously with vehicle, dasatinib (10 umol/kg) or DAC (10 umol/kg) at the indicated frequency. At three weeks post-fracture, healed femurs were collected and subjected to microCT analyses.
Results: As shown in Fig. 1, the DAC contains a dasatinib warhead, releasable ester linker, and fracture-homing aspartic acid decapeptide. Fig. 2A demonstrates that treatment with DAC essentially doubles the bone density (BV/TV) relative to vehicle-treated controls by 3 weeks post fracture induction. While non-targeted dasatinib also enhances mineralization in the fractured region, its impact was less than half that of its targeted counterpart. The microCT images in Fig. 2B show significantly denser trabecular bone microstructures in the targeted group compared to vehicle- and free dasatinib-treated controls. Trabecular thickness (Fig. 2C) is also remarkably increased while trabecular spacing (Fig. 2D) is drastically decreased relative to mice receiving non-targeted drug or vehicle alone.
As seen in Fig. 3A, treatment with DAC every other day for 3 weeks was equally effective as daily injections of DAC, yielding a 114% increase in bone density over PBS-treated controls. Analysis of microCT scans of cross-sections at the fracture calluses (Fig. 3B) confirmed that a q.a.d. dosing schedule is as effective q.d.. Reducing this dosing interval to every four days, however, resulted in a measurable decline in potency. Importantly, even the weekly dosing of DAC resulted in a trabecular density that was comparable to that seen in mice treated every day with the same concentration of free dasatinib. In terms of the body weight changes, the daily dosed cohort displayed the slowest recovery to normal weight (Fig. 3C), suggesting mild toxicity. In contrast, mice receiving q.a.d. dosing of DAC ended up gaining ~5% body weight on day 21 compared to day 1 (Fig. 3D). Simply reducing the dosing frequency to every other day allowed a return to normal weight that was at least as rapid at PBS treated controls, suggesting that the slightly reduced dosing frequency not only maintains the accelerated rate of fracture repair but also avoids any apparent toxicity associated with daily dosing.
Conclusion: We have shown that DAC facilitates femur fracture repair in vivo by increasing bone density at the fracture site. Although nontargeted dasatinib caused a modest improvement in healing rate, targeted dasatinib was dramatically better than free drug, enabling faster bone deposition with lower toxicity. We conclude that alternated day dosing of DAC constitutes a promising new therapy for acceleration of bone fracture repair without imposition of unwanted side effects.
Bentler, S.E., Liu, L., Obrizan, M., Cook, E.A., Wright, K.B., Geweke, J.F., Chrischilles, E.A., Pavlik, C.E., Wallace, R.B., Ohsfeldt, R.L., et al. (2009). The aftermath of hip fracture: discharge placement, functional status change, and mortality. Am. J. Epidemiol. 170, 1290–1299.
Braithwaite, R.S., Col, N.F., and Wong, J.B. (2003). Estimating hip fracture morbidity, mortality and costs. J. Am. Geriatr. Soc. 51, 364–370.
Philip Low– Presidential Scholar for Drug Discovery, Purdue Institute for Drug Discovery, West Lafayette, Indiana