Preclinical formulation development and mechanistic studies of pluronic P123 JS-K

The work in this dissertation summarizes the development of a nitric oxide producing prodrug, JS-K in a polymeric Pluronic® P123 micelle formulation. This work is an attempt to advance the formulation development and provide the preclinical rationale by studying: a) formulation stability and toxicity in vitro and in vivo and plasma protein binding of the drug and the formulation in vitro; b) cell distribution of the free drug (JSK) and the formulation (P123/JS-K) in vitro; c) mechanistic pathways triggered by JS-K and P123/JS-K leading to apoptosis and finally; d) development and validation of an analytical method for detection of the drug or formulation and biological metabolites when used in clinical settings. Except for the mechanistic studies performed where the method employed was Western blot or fluorescence analysis, all the other studies were conducted using analytical methods developed on high performance liquid chromatography (HPLC) equipped with ultra violet (UV) detector. The available method lacked metabolite detection and sensitivity with low drug concentration. Therefore, a more robust, sensitive and elaborate method was developed using ultra performance liquid chromatography (UPLC) equipped with a UV detector. The method was also validated as per United States Pharmacopoeia (USP). The validated method could be used for studying biodistribution, pharmacokinetics and potential toxicity in clinical settings. iv The major findings of the dissertation could be succinctly described by stating JS-K in the Pluronic P123 formulation was comparatively more stable than in the free-JS-K formulation. There was no major toxicity observed due to the designed formulation when tested both in vitro and in vivo. P123/JS-K showed preferential distribution in the cell nucleus, indicating some effect produced by the Pluronic® micelle. Mechanistically, not much difference was observed between JS-K and P123/JS-K and both were shown to produce stress response in leukemia cells. Both JS-K and P123/JS-K were nontoxic to CD34+ hematopoietic cells. The work described in this dissertation is an attempt to showcase "Bench to Bedside" research in laboratory settings. An attempt has been made to incorporate all the studies required by the regulatory agencies such as US Food and Drug Administration (FDA). The studies performed and the method developed will lay the platform for future studies that are required for filing an Investigational New Drug (IND) application following current Good Manufacturing Practices (cGMP).