University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2011 MECHANISM AND KINETICS OF COVALENT AMIDE-LINKED ADDUCT FORMATION IN SOLUTION AND SOLID PEPTIDE FORMULATIONS Michael Paul DeHart University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation DeHart, Michael Paul, "MECHANISM AND KINETICS OF COVALENT AMIDE-LINKED ADDUCT FORMATION IN SOLUTION AND SOLID PEPTIDE FORMULATIONS" (2011). Theses and Dissertations--Pharmacy. 119. https://uknowledge.uky.edu/pharmacy_etds/119 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Michael Paul DeHart, Student Dr. Bradley D. Anderson, Major Professor Dr. Jim Pauly, Director of Graduate Studies MECHANISM AND KINETICS OF COVALENT AMIDE-LINKED ADDUCT FORMATION IN SOLUTION AND SOLID PEPTIDE FORMULATIONS DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Michael Paul DeHart Lexington, KY Director: Dr. Bradley D. Anderson, the H. B. Kostenbauder Professor of Pharmaceutical Sciences Lexington, Kentucky 2011 Copyright © Michael Paul DeHart 2011 ABSTRACT OF DISSERTATION MECHANISM AND KINETICS OF COVALENT AMIDE-LINKED ADDUCT FORMATION IN SOLUTION AND SOLID-PEPTIDE FORMULATIONS Peptides and proteins are prone to chemical and physical degradation in solutions and solids. One of the most common chemical degradation pathways involves deamidation of asparagine residues through a reactive succinimide intermediate resulting in the formation of aspartyl and isoaspartyl degradation products. In this work, kinetic studies in aqueous solutions demonstrated that the succinimide intermediate is susceptible to attack by nucleophiles other than water. This may lead to a variety of alternative degradants. In solutions containing high concentrations of ammonia, the succinimide generated in the deamidation of the model peptides Phe-Asn-Gly and Phe-isoAsn-Gly underwent back-reaction with ammonia leading to peptide isomerization. Intramolecular cyclization involving the N-terminus of Phe-Asn-Gly to form a cyclic diketopiperazine was also observed. Experiments employing the succinimide as the starting reactant and kinetic models confirmed that the above pathways proceed exclusively through an imide intermediate. To maximize their stability, proteins and peptides are formulated as lyophilized solids. However, succinimide-mediated deamidation remains important in solidTstate degradation. Accelerated stability studies of the model peptide Gly-Phe-Asn-Gly in the presence of excess Gly-Val in amorphous lyophiles revealed the formation of four deamidation products from the reaction of the succinimide intermediate with water as well as four covalent amide-linked adducts. The amide-linked adducts were identified as diastereomers formed by nucleophilic attack of the Gly-Val N-terminus at either the ■ alpha or beta-carbonyl of the succinimide. A comprehensive kinetic model confirmed that the formation of all four hydrolysis products and the four covalent-adducts proceed exclusively through the succinimide. The relative ratio of formation of hydrolysis products to covalent-adducts reflects a competition between water and the N-terminus of neighboring peptides for reaction with the succinimide intermediate. This ratio varies with formulation. The sensitivity of the individual reaction steps involved in deamidation and covalent adduct formation to formulation changes (e.g., variation in water content and dilution of reactants by increasing excipient content) were explored. Increasing water content accelerated adduct and hydrolysis degradant formation while increases in concentration of a polymeric excipient (HPMC) appeared to haye no significant effect. The novel mechanism described offers an alternative pathway for peptide and protein aggregation, particularly in amorphous solid formulations. KEYWORDS: Asparagine deamidation, aggregation, covalent adduct, amorphous lyophiles, reaction kinetics Students Signature MECHANISM AND KINETICS OF COVALENT AMIDE-LINKED . ADDUCT FORMATION IN SOLUTION AND SOLID PEPTIDE FORMULATIONS By Michael Paul DeHart To my wife, Wendie, who has always loved me, encouraged me, believed in me, and never doubted me. To Jack, the miracle that he is, and for what our future holds together with his funny daddy and to the little one lost. ACKNOWLEDGMENTS I would be remised if I did not acknowledge certain people who helped make this possible. First and foremost to Dr. Brad Anderson for the guidance he has provided throughout my graduate career. Sharing his attention to detail, quality of work, and knowledge about science is something that I know will help me in my future. I always said “what doesn’t kill you, makes you stronger” in graduate school. Dr Anderson did not kill me, and for that, I am stronger. To my committee members - Dr. Bummer, Dr. Hilt, Dr. Munson, and Dr. Plattner - for their advice and help to see this to completion. I would also like to thank those that work behind the scene to help get things accomplished. Thanks to Larry Farmer for all his help in the ASTeCC building. Thanks to all the College of Pharmacy staff - Catina, Rodney, Kendra, Linda, and Tammi (even as far back as Mandy and Belinda) who have helped me with the paperwork and ordering. There is no doubt that you will learn about science in graduate school, but what you don’t realize is that you learn about other people, their cultures, and yourself 1 will be forever grateful for those fellow graduate students over the years who have taught me about different cultures, people, and religions. And all this would not have been possible if it wasn’t for my family. Thanks to my parents for encouraging and believing in me all along the way. Ill TABLE OF CONTENTS Acknowledgments................................................................................................................ iii Lists of Tables...................................................................................................................... vi Lists of Figures................................................................................................................... viii Chapter 1 - Statement of the Problem and Hypotheses............................................................ 1 Chapter 2 - Background and Literature Review........................................................................5 Introduction....................................................................................................................... 5 Deamidation Review...........................................................................................................7 Asparagine Involvement in Alternate Degradant Formation................................................. 12 Succinimide Involvement in Aggregation........................................................................... 14 Glass Transition Temperature.............................................................................................16 Describing Reaction Kinetics in Amorphous Solids.............................................................17 Factors Influencing Reactivity in Amorphous Solids...........................................................19 References...................................................... 24 Chapter 3 - The Role of the Cyclic Imide in Alternate Degradation Pathways for Asparagine- Containing Peptides and Proteins........................................................................................... 38 Introduction..................................................................................................................... 38 Materials and Methods......................................................................................................42