| The renin-angiotensin system (RAS) is an intricate endocrine cascade that elicits diverse biological functions. Angiotensin II (Ang II), as a central component of the RAS, mediates inflammatory diseases by binding to the Angiotensin II type 1 receptor (AT1R). In contrast, its binding to Angiotensin II type 2 receptor (AT2R) provides functional antagonism to AT1R axes. Novokinin, a synthetic peptide, activates AT2R and can be considered a potential drug candidate for alternative therapy for RAS-related diseases if its plasma stability and pharmacokinetics could be improved. Hence, we have employed a novel bone-targeting approach where we conjugate bone to polyethylene glycol (PEG) spacer and bone-targeting moiety bisphosphonate (BP) that utilizes the bone as a reservoir and protects it from degradation. We have hypothesized that this delivery approach will improve stability to elicit superior pharmacodynamic characteristics. To fulfill our hypothesis, we first synthesized Novokinin and Novokinin Conjugate (Novo Conj) by conjugation with PEG and BP. They were characterized by HPLC and LC-MS/MS. In addition to this our results indicated that Novo Conj has better stability and higher affinity to the bone. Next, we outlined our second objective to determine and compare the biological activity of conjugated peptide in vitro and in vivo. Our result indicated that Novo Conj showed superior in vitro effects by increasing neurite outgrowth in NG108-15 and prolonging antiproliferative effects in breast cancer cell lines. Similarly, for the in vivo study we observed that Novo Conj alleviated signs and symptoms of experimental arthritis by reducing the plasma nitric oxide level, restoring inflammation-induced weight loss, reducing joint swelling, and restoring the disrupted balance of the RAS. Our final objective was to determine drug pharmacokinetic (PK) parameters in the healthy male Sprague Dawley (SpD) rats. For this study, we developed and validated the quantification of novokinin in the plasma, applied it to PK studies and established its complete drug profile. Overall, we successfully established bone-targeted delivery of novokinin by testing several cell
models and the RA model. We also showed a superior pharmacodynamic effect of conjugated peptide than its native due to its stability and likely improvement in its PK parameters. |