Shyh-Dar Li

Chronic pain affects approximately 20% of Canadians and is a global healthcare burden with limited treatment options. The high incidence of comorbid chronic pain and depression additionally worsens patient’s quality of life and treatment outcomes. Antidepressants are used for the treatment of persistent pain but show limited efficacy and often induce serious side effects. Opioids are highly potent analgesics but are associated with severe adverse effects frequently limiting their use in chronic pain management. Endogenous opioids act on our nervous system to regulate pain and mood. The endogenous opioid peptide Leucine-enkephalin (Leu-ENK) produces analgesia with fewer adverse effects compared to conventional opioids. However, its poor stability and low membrane permeability make Leu-ENK ineffective when administered peripherally. We developed the N-pivaloyl Leu-ENK analog KK-103, which showed a high relative binding affinity for the delta opioid receptor (DOR) and was stable in plasma for 5 h. In the hotplate model, subcutaneous (s.c.) KK-103 showed 10-fold improved antinociception compared to Leu-ENK and produced a longer-lasting antinociceptive activity than morphine. In the formalin model, KK-103 reduced the licking and biting time of mice by ~60% relative to the vehicle group. We demonstrated a similar dose to maximum antinociceptive-effect relationship of KK-103 in the hotplate and formalin foot models. Contrasting morphine, KK-103 did not induce breathing depression, physical dependence, and tolerance in the models and experimental timelines used in this study. Additionally, KK-103’s minimal gastrointestinal inhibition and absence of sedation demonstrated its potential as a safe and effective analgesic. We also demonstrated KK-103’s increased systemic adsorption and plasma exposure compared to Leu-ENK and observed brain uptake of radiolabeled KK-103 after s.c. administration. The antinociceptive effect of KK-103 was primarily mediated by opioid receptors in the central nervous system and specifically the activation of the DOR. We detected the conversion of KK-103 to Leu-ENK in vitro which may induce the observed effects of KK-103 in mice. Finally, we determined an antidepressant-like activity of KK-103, which was comparable to that of the antidepressant desipramine. This work showed the potential application of KK-103 for pain and depression and provides the theoretical framework for future R&D of ENK therapeutics.

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Many small molecule drugs face challenges with aqueous solubility during their development, which can lead to issues with drug delivery. A commonly employed method to improve the solubility and delivery of drug-like molecules is to modify the compounds with hydrophilic groups. However, it can be very challenging to incorporate these groups without sacrificing the activity of the molecule. Alternative approaches include encapsulating the hydrophobic drug into a delivery vessel, such as a nanoparticle (NP), or introducing functional groups that help with solubility and delivery of the drug-like molecule but are cleaved in biological settings to release the original drug molecule (for example, a prodrug approach). Combining these approaches, a hydrophilic group such as water-soluble polymer poly(ethylene glycol) (PEG) can be added to a hydrophobic drug molecule to produce an “amphiphile”. These amphiphiles can self-assemble in aqueous media to form NPs where the hydrophobic, poorly water-soluble drug is protected in the core and the water-soluble polymer forms the outer shell. These NPs can have prolonged circulation, reduced clearance, and may therefore enhance efficacy. These PEG groups are appended to the molecule via a functional group that can be cleaved under biological conditions to release the active drug molecule. The physicochemical and pharmacological properties of these NPs can be modulated by altering different aspects of the amphiphilic drug-PEG conjugate. To optimize these conjugates and maximize their therapeutic potential, it is important to take a structure-activity relationship (SAR)-based approach when undertaking such an investigation. To facilitate the rapid and facile generation of drug conjugates, this thesis focused on developing a new platform technology using azide-alkyne click chemistry. Small molecule drugs with poor aqueous solubility were “clicked” to linear short-chain PEG. The resulting PEG conjugates then self-assembled into NPs. Multiple conjugates were generated to demonstrate the robustness and versatility of this technology. This thesis demonstrated that the developed click chemistry platform is a robust synthetic technology that has potential application to improve the delivery of poorly soluble drugs.

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Life threatening liver diseases such as cirrhosis and viral hepatitis accounts for 2 million global deaths annually. The major limitations of the current pharmacological therapies are the inability to deliver sufficient concentrations of therapeutic drugs to the diseased liver cells and the activation of undesired systemic side effects. The development of lipid-nanoparticles (LNPs) for small molecular drugs delivery has grown rapidly in the past decades. Although some were successful in targeting the liver, not so many were able to target the hepatocytes, where most liver diseases reside. To address this issue this dissertation is focused on the development of a novel hepatocyte-targeting formulation, called Phospholipid-Free Small Unilamellar Vesicles (PFSUVs), to improve the treatment of many liver diseases including liver stage malaria and viral hepatitis. The developed formulation is composed of high cholesterol content (~ 83%) and TWEEN 80, a non-ionic surfactant.The first part of this dissertation explored the effect of PFSUVs’ size on the intrahepatic distribution. We found that small size PFSUVs (60 nm) selectively target the hepatocytes, whereas larger PFSUVs with an average size of 120 nm accumulate in the Kupffer cells. The second and third parts of the dissertation explored two different hepatocyte-related disease applications. First, we examined the ability of PFSUVs to improve the treatment of liver-stage malaria by efficiently delivering primaquine to the hepatocytes and reducing its hemolytic toxicity. The second application focused on enhancing the treatment of hepatitis B virus (HBV). This was achieved by stimulating the immune system to trigger the production of endogenous interferon-α, a proinflammatory cytokine. We evaluated the efficacy of PFSUVs in significantly reducing hepatitis B surface antigen (HBsAg) in a mouse model when delivering an immunomodulatory agent to the hepatocytes. The fourth part of this thesis was to explore the mechanism behind the selectivity of PFSUVs to hepatocytes. In addition to the small size of PFSUVs, we demonstrated the effect of serum proteins, specifically apolipoproteins, on the internalization of PFSUVs into hepatocytes. This work demonstrated the importance of cell-specific targeting and the potential of PFSUVs to improve the treatment in many hepatocytes-associated diseases.

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More than 70% of drugs exhibit poor water solubility, thereby limiting their clinical applications. Formulating these drugs into liposomes is a feasible approach to increase their solubility and improve the therapeutic efficacy. However, encapsulating hydrophobic drugs into the lipid bilayer of liposomes often results in burst drug release and liposomal instability, due to the weak association between the drugs and the lipid bilayer. Additionally, the capacity of the lipid bilayer is limited, leading to inefficient drug loading. To address this issue, this thesis focused on developing a new loading technology, called Solvent-assisted Active Loading Technology (SALT), to allow stable and efficient loading of poorly water-soluble drugs into the aqueous core of liposomes. This technology involved the addition of a certain amount of a water-miscible organic solvent into the mixture of a poorly soluble drug and preformed liposomes incorporated with a trapping agent inside the aqueous core. The solvent was not only used to help drug dissolution, but also to facilitate drug permeation into the liposomal core to form drug complexes with the trapping agent by increasing the membrane permeability during the drug loading. We have generated multiple examples to demonstrate the robustness and potential utilities of this technology. As a proof-of-principle, the first part of the thesis focused on developing the SALT for stable loading of a model drug, staurosporine (STS, insoluble weakly basic drug), into liposomes and optimizing the fabrication of a liposomal STS formulation for in vivo therapy of tumor. The second part of this dissertation was to explore whether the SALT is a flexible platform for formulating other types of poorly soluble drugs such as gambogic acid (GA, insoluble weakly acidic drug) into liposomes. We also examined whether other miscible solvents (other than DMSO) could be utilized in the system and their roles in promoting drug loading. The third part of this thesis was to demonstrate another utility of the SALT for preparing an oral pediatric formulation of mefloquine with bitterness masking. This thesis work demonstrated that SALT was a robust drug loading technology to develop stable liposomal formulations for poorly soluble drugs with practical utilities.

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