(A-3) Methods for synthesis of an alendronate containing polymer for future bone coating applications

Osteoclasts are important cells in the human body and are responsible for degrading bone during the normal bone remodeling process [1]. In certain circumstances the activity of osteoclasts can have undesired or even harmful impacts such as in osteoporosis, bone cancer, or with premature resorption of autologous bone grafts. In particular, pediatric patients who receive a decompressive craniectomy and have their autologous bone reinserted into the defect have extremely high graft failure rates. In one clinical study, out of 54 pediatric patients, 50% experienced resorption issues after the bone was replaced [2]. One potential solution to this problem would be to inhibit osteoclast activity around the bone graft. For clinical indications like osteoporosis with overactive osteoclasts, a class of drugs called bisphosphonates are often systemically administered to selectively inhibit these cells. To adapt this systemic drug system for a localized therapy, it is hypothesized that coating a pediatric patient’s autologous bone graft with a polymer containing a bisphosphonate drug will locally inhibit osteoclasts in the surrounding area, preventing overactive bone resorption. The aim of this study is to create a new polymer containing alendronate, a type of bisphosphonate, for construction into a layer-by-layer film that will coat a bone graft.   

Before synthesizing a cationic copolymer containing the alendronate therapeutic, the solubility of the proposed polymer constituents, Alendronate Sodium Trihydrate, N,N’-Ethylenebisacrylamide, and 1,3-Di-4-piperidylpropane, were studied. Studies involved adding different solvents and solvent combinations to small quantities of constituents and qualitatively examining the reagent solubility. Different conditions such as changing the temperature were also examined, and observations were recorded. Based on the solubility studies, appropriate solvents were selected and trials using different methods to synthesize the polymer were conducted. Factors that were altered between trials included the time and temperature that the reaction was run at, the concentration of alendronate, and the addition of a base as a reaction catalyst. The crude products that were obtained from these syntheses were examined using NMR spectroscopy. Both proton nuclear magnetic resonance (H NMR) and phosphorus nuclear magnetic resonance (P NMR) were used. P NMR was used to confirm the presence of alendronate in products since alendronate contains phosphorus (see Figure 1 A for chemical structure).  

Results and Discussion   

A combination of deionized water and reagent alcohol was found to be the best solvent combination to dissolve all of the polymer precursors, though deionized water with methanol was also suitable. For the synthesis it was found that solubility was increased when reagent alcohol was first added to N,N’-Ethylenebisacrylamide and 1,3-Di-4-piperidylpropane until dissolved and in a separate container deionized water was added to alendronate until dissolved before combining the two. Approximately 0.11 mL of reagent alcohol was needed per milligram of N,N’-Ethylenebisacrylamide and approximately 0.037 mL of reagent alcohol was needed per milligram of 1,3-Di-4-piperidylpropane for it to dissolve. Heat was not found to have a significant impact on the solubility of the constituents based on a small trial with samples in a bath of 61 degrees Celsius. In the presence of a catalytic base, the reaction solubility improved. The NMR results in parts B and C of Figure 1 are from one synthesis run for 96 hours at room temperature. Figure 1 B shows the H NMR spectrum of the product including the aligning chemical structure. P NMR spectrum results (data not included) showed that the peak from the produced synthesis was shifted from the peak of alendronate alone. The combination of these spectra indicates that the alendronate reacted to form a new product.

Conclusions

Initial formulations of cationic polymers containing the bisphosphonate drug alendronate were successfully synthesized. Future work for this study will involve experimenting with catalytic bases other than sodium hydroxide to increase reaction yield. Furthermore, the steps of the synthesis will be refined and steps for purification will be tested and evaluated using NMR. After optimizing the polymer synthesis, the focus of this study will shift to creating a layer-by-layer coating using these created materials to coat a bone graft for in vitro and in vivo testing.

[1]          B. F. Boyce, Z. Yao, and L. Xing, “Osteoclasts have Multiple Roles in Bone in Addition to Bone Resorption,” Crit Rev Eukaryot Gene Expr, vol. 19, no. 3, pp. 171–180, 2009.

[2]          C. A. Bowers, J. Riva-Cambrin, D. A. Hertzler, and M. L. Walker, “Risk factors and rates of bone flap resorption in pediatric patients after decompressive craniectomy for traumatic brain injury: Clinical article,” Journal of Neurosurgery: Pediatrics, vol. 11, no. 5, pp. 526–532, May 2013, doi: 10.3171/2013.1.PEDS12483.