A Dissertation Entitled: Pharmaceutical applications and toxicity of extracted alkenones from marine Isochrysis algae by Kyle McIntosh Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Pharmacology and Experimental Therapeutics Degree in College of Pharmacy and Pharmaceutical Sciences The University of Toledo ___________________________________________ Dr. Amit K. Tiwari, Committee Chair ___________________________________________ Dr. Frederick E. Williams, Committee Member ___________________________________________ Dr. Gabriella Baki, Committee Member ___________________________________________ Dr. Jeffrey Sarver, Committee Member ___________________________________________ Dr. Cyndee Gruden, Interim Dean College of Graduate Studies The University of Toledo August 2019 Copyright 2019, Kyle McIntosh This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Pharmaceutical applications and toxicity of extracted alkenones from marine Isochrysis algae by Kyle McIntosh Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Pharmacology and Experimental Therapeutics Degree in Pharmaceutical Sciences The University of Toledo August 2019 Isochrysis is a commercially available marine algae used for animal feed, human nutrient supplements, and biodiesel. The Isochrysis galbana species is one of four genera of haptophytes that produces unique, long-chain lipids known as alkenones. However, there is a lack of physical characteristics and toxicity data for alkenones in animals, thus, limiting their use in humans. In the first aim of this study, alkenones derived from Isochrysis sp. were evaluated for their chemical structure characteristics as an alternative for waxes used in personal care products. The melting point of the alkenone was determined (71.1–77.4°C), and their thickening capability in five emollients was evaluated and compared to microcrystalline wax and ozokerite. Alkenones showed compatibility with three emollients, isopropyl isostearate, C12-C15 alkyl benzoate, and ethylhexyl methyxycinnamate, and they thickened the emollients similar to the other tested waxes. Lipsticks and lip balms were formulated with and without alkenones. All products remained stable at room temperature for 10 weeks. Lipstick formulated with alkenones was the most resistant to high temperature. Finally, alkenones were compared to three cosmetic thickening waxes in creams. Viscosity, rheology and stability of the iii creams were evaluated. All creams had a gel-like behavior. Overall, the alkenones in these formulas were comparable to the other three waxes. Thus, alkenones can offer a potential green choice as a new personal care structuring compound. For the second aim of this study, we performed acute oral, acute dermal and repeated 28-day dermal toxicity studies using female SAS (an acronym for the company SASCO where the colony was bred) Sprague Dawley rats. Our behavioral studies (level of grooming, eye opening, walking, exploring, body posture, breathing, scratching, and nose flattening) indicated that the specific alkenones had no visible behavioral effects at oral doses up to 4000 mg/kg. In addition, there were no significant changes in food consumption or body weight, and there was no significant histopathological changes in the liver, kidneys, spleen, heart or skin compared to animals treated with vehicle. In the acute and chronic dermal toxicity studies, the alkenones produced less irritation and did not significantly damage the skin based on the Draize skin reaction scale and transepidermal water loss readings compared to the positive control, 1% sodium lauryl sulfate. Overall, our results indicated that alkenones are safe in female Sprague Dawley rats, suggesting that they could be used for both oral and dermal formulations, although additional studies would be required before these alkenones could be applied to human personal care products. iv Acknowledgements I want to thank my parents, Darryl and Cara, my family, and my friends for their endless support, love, and encouragement. I thank Hope Lutheran Church and its members for their spiritual support. I thank God for paving this journey for me and for placing marvelous individuals in my life. My biggest thanks go towards Dr. Amit K. Tiwari for being my advisor for these past four years. He has taught me so much when it comes to research; learning and moving past mistakes in experiments, prioritizing daily tasks, and to become passionate in the work you accomplish. He has also taught me valuable life-long skills which I will practice and carry with me throughout my future careers. I want to thank Dr. Baki for her knowledge of testing cosmetic ingredients, for allowing me to run her devices for this project, for the support of her students who assisted me, and her assistance in the planning of work needed with the alkenones. Dr. Sarver for opening his door to me when I had concerns with software programs to use and for his help with the statistical data. Dr. Williams for keeping me on track, his feedback and support for not just me, but all graduates in the program. Lastly, I want to thank my lab colleagues, Angelique Nyinawabera, Noor Hussein, Haneen Amawi, Saloni Malla, Mariah Pasternak, and Dr. Diwakar Bastihalli Tukaramrao and past lab colleagues. They have challenged me, helped see the value in myself, and know what I have to offer when working in a team setting. These people and others have made my time at Toledo unique and rich with a cultural experience and have given me memories I will treasure. v Table of Contents Abstract .......................................................................................................................... iii Acknowledgements ..............................................................................................................v Table of Contents ............................................................................................................... vi List of Tables ..................................................................................................................... ix List of Figures ......................................................................................................................x List of Abbreviations ........................................................................................................ xii List of Symbols ..................................................................................................................xv I. Introduction ........................................................................................................1 A. Marine Microalgae ........................................................................................1 B. Extractions Obtained from Marine Microalgae ............................................2 C. Alkenones ......................................................................................................3 D. Current Uses of Alkenones ...........................................................................5 a. Paleoclimatology .......................................................................................5 b. Biofuel.......................................................................................................6 c. Cosmetics ................................................................................................. 7 d. Medicine ...................................................................................................7 II. Materials and Methods .....................................................................................10 A. Test Materials ..............................................................................................10 B. Animals .......................................................................................................11 C. Chemical Characterization Methods ...........................................................12 a. Establishment of Solubility .....................................................................12 b. Determination of Melting Point ..............................................................12 vi c. Thickening Capability Test .....................................................................13 d. Developing the Lipstick and Lip Balm Formulation ..............................14 e. Cream Formulation .................................................................................15 f. Determination of Viscosity, Thixotropy, and Rheology .........................17 g. Observing the Liquid Crystalline Structure ............................................18 h. Stability Testing ......................................................................................19 D. Animal Study Design ..................................................................................19 E. Suspension Formulation ..............................................................................21 F. Acute Oral Gavage Administration of the Alkenones .................................22 a. Quantification of White Blood Cells (WBCs), Red Blood Cells (RBCs), Platelets (PLTs), Monocytes and Hemoglobin (Hb) .........................23 b. Determination of Blood Parameters........................................................23 c. Histology .................................................................................................24 G. Single and Repeated Dermal