Long-Acting Nanoformulated Small Molecule Inhibitor Prodrugs for Antiviral Treatment and Prevention

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Long-Acting Nanoformulated Small Molecule Inhibitor Prodrugs for Antiviral Treatment and Prevention University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-4-2019 Long-Acting Nanoformulated Small Molecule Inhibitor Prodrugs for Antiviral Treatment and Prevention Nathan Smith University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Nanomedicine Commons Recommended Citation Smith, Nathan, "Long-Acting Nanoformulated Small Molecule Inhibitor Prodrugs for Antiviral Treatment and Prevention" (2019). Theses & Dissertations. 354. https://digitalcommons.unmc.edu/etd/354 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. Long-Acting Nanoformulated Small Molecule Inhibitor Prodrugs for Antiviral Treatment and Prevention by Nathan Smith A Dissertation Presented to the Faculty of the University of Nebraska Medical Center Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Medical Sciences Interdepartmental Area Specialization: Pharmacology and Experimental Neuroscience Under the Supervision of Dr. Howard E. Gendelman University of Nebraska Medical Center Omaha, Nebraska April, 2019 Supervisory Committee: Dr. Howard E. Gendelman, M.D. Dr. Larisa Poluektova, M.D., Ph.D. Dr. JoEllyn McMillan, Ph.D Dr. David Oupicky, Ph.D. Dr. Laura Bilek, PT, Ph.D. Long-Acting Nanoformulated Small Molecule Inhibitor Prodrugs for Antiviral Treatment and Prevention Nathan Smith University of Nebraska Medical Center, 2019 Supervisor: Howard E. Gendelman, M.D. Abstract Over 30 years of antiretroviral therapy (ART) has transformed human immunodeficiency virus type one (HIV-1) outcomes from certain death to a nearly complete and normal life [1]. Though disease growth has slowed, the virus still creates substantial co-morbid diseases as well as social and economic burdens worldwide [2]. Lack of success with curative efforts combined with shortcomings in global treatment remain to be properly addressed. The requirement for daily use of combination antiretroviral drugs (ARVs) in low to middle income countries - where the majority of the HIV-1 positive population resides - can lead to breaks in regimen adherence due to a lack of access to ARV, high cost, social stigma, and lack of disease awareness [3]. Treatment breaks speed viral drug resistance and longer-term adverse events, ultimately shortening patient life-span. Hospital feedback in low to middle income countries indicates patient preference for a variety of treatment options, including long-acting (LA) strategies [4]. The need for treatment and by extension curative options is paramount in HIV-1 research. With an eye toward the future of HIV treatment, our laboratories generated a platform for small molecule antiretroviral drug nanoformulations by creating potent hydrophobic lipophilic ARV prodrugs with stable long-term physicochemical properties [5, 6]. Herein, this platform is extended to multiple small molecule inhibitors intended for antiviral therapy and HIV-1 eradication. A small molecule inhibitor used in cancer treatment was chemically modified to prolong drug activity, then tested for anti-HIV activities in the context of immune therapy in curative efforts. Additionally, two nucleoside reverse transcriptase inhibitors (NRTI) were chemically modified to produce long-acting drug iterations. Prodrug nuceloTide (ProTide) technology was used to chemically alter the NRTIs. A tenofovir ProTide was constructed and formulated, increasing prodrug accumulation. Following the same strategy, a phosphoroamidate prodrug nucleotide (ProTide) of lamivudine (3TC), called (M23TC) was synthesized and nanoformulated (NM23TC). A single treatment of NM23TC in human monocyte-derived macrophages (MDM) with NM23TC showed increased drug uptake, retention, intracellular 3TC triphosphate (3TC-TP) and sustained antiretroviral activities for 30 days. Pharmacokinetic analysis of NM23TC administered to Sprague Dawley rats showed sustained prodrug levels in blood and tissues. Further, in vitro studies showed intact prodrug was released over a month from MDMs. When tested in CEM CD4+ T cells, the prodrug was shown to rapidly convert to its 3TC-TP. Taken together, a putative mechanism for extended delivery of active compound to viral cell targets was constructed. This mechanism was strongly supported when tested as a broad- spectrum agent in model for hepatitis B virus infection in humanized mice. A single intramuscular injection produced sustained antiviral activities for one month. Taken together, the approaches give validation to potential value of the platform of transforming daily medications into nanoformulated prodrugs for long-acting antiviral activities. Table of Contents List of Figures and Tables .................................................................................. i List of Abbreviations .......................................................................................... ii Acknowledgements ............................................................................................ v Chapter 1: Background and Significance........................................................ 1 1.1. Global Impact and Challenges of the Human Immunodeficiency Virus..................... 2 1.2. HIV-1 Structure and Life Cycle ................................................................................ 2 1.3. Disease Timeline ..................................................................................................... 4 1.4. Host Immune Response .......................................................................................... 5 1.5. Reservoir Characterization ...................................................................................... 7 1.6. HIV-1 Eradication .................................................................................................... 8 1.7. HIV treatment ........................................................................................................ 10 1.8. Long Acting Nanomedicine for Antiretroviral Therapy ............................................ 15 1.8.1. Liposomes ..........................................................................................................17 1.8.2. Micelles ..............................................................................................................17 1.8.3. Polymer-Chemo Conjugates ...............................................................................18 1.8.4. Solid Lipid Nanoparticles (SLN) ..........................................................................18 1.8.5. Niosomes ...........................................................................................................19 1.8.6. Nanosuspensions ...............................................................................................19 1.8.7. Nanoparticle Systems and Characterization .......................................................19 1.9. Nanoparticle Preparation ....................................................................................... 21 1.10. Studying Antiviral Activity in Murine Mouse Models ............................................. 22 1.11. Scope and Significance of the Works Presented ................................................. 23 Chapter 2: Project Hypotheses and Goals .................................................... 34 2.1. Hypotheses and Aims ............................................................................................ 35 Project 1: IDO Inhibitor (Chapter 3) ...............................................................................35 Projects 2 and 3: Long-Acting NRTI ProTides (Chapters 4 and 5) ................................36 Quaternary aim (Chapter 6): NM23TC Testing Against Hepatitis B in Humanized Mice38 Chapter 3: Development and testing of a nanoformulated IDO inhibitor for HIV Eradication. ................................................................................................ 39 3.1. Abstract. ............................................................................................................... 40 3.2. Introduction.......................................................................................................... 40 3.3. Materials and Methods ........................................................................................ 44 3.3.1. Chemicals and Reagents ....................................................................................44 3.3.2. Synthesis and Characterization of Epacadostat Prodrugs...................................44 3.3.2.1. Synthesis of M1-Epacadostat ..........................................................................44 3.3.2.2. Synthesis of M2-Epacadostat ..........................................................................45 3.3.2.3. Synthesis of M3-Epacadostat ..........................................................................45 3.3.2.4. Synthesis of M4-Epacadostat ..........................................................................46 3.3.2.5. Prodrug Physicochemical Characterization ......................................................46 3.3.3. Folic Acid-P407 Conjugation ...............................................................................47 3.3.4. Epacadostat and M1-Epacadostat Formulation .................................................. 48 3.3.4.1. High-pressure
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