DOI
https://doi.org/10.25772/RYCZ-PA73
Defense Date
2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Pharmaceutical Sciences
First Advisor
Phillip M. Gerk
Second Advisor
Christian Bergman
Third Advisor
Matthew Halquist
Fourth Advisor
Audrey Lafrenaye
Fifth Advisor
Masahiro Sakagami
Abstract
The work presented in this dissertation aimed to determine the preclinical pharmacokinetics of Ketone ester (KE) and its metabolites 1,3-BD, BHB, and AcAc, using in vivo and in vitro techniques. KE has gained popularity as an exogenous means to achieve ketosis, be it for therapeutic uses or athletic sports enhancement. Two novel bioanalytical methods were developed and partially validated for quantifying KE and its metabolites in human and rat plasma using LC-MS. The first assay used a reverse-phase C18 column to quantitate ketone ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) (henceforth referred to as KE), (R)-beta-hydroxybutyrate (henceforth referred to as BHB), and (R)-1,3-butanediol (henceforth referred to as 1,3-BD), and the second assay used a HILIC (Hydrophilic Interaction Liquid Chromatography) column for the quantitation of acetoacetate (AcAc). Both assays were found to be selective and sensitive for the respective molecules and showed accuracy precision within the acceptable range, as specified by the ICH M10 guidelines. The assays had a wide range of quantification- 1-500 µM for KE, 50-4000 µM for 1,3-BD, 50- 5000 µM for BHB, and 50-4000 µM for AcAc. Together, these assays were used to track the kinetics of ketone ester and its proximal metabolites in rats after IV and oral dosing and in one human subject after oral dosing. They were also used for quantification of KE and 1,3-BD concentrations in human and rat liver subcellular fractions. A fit-for-purpose HPLC-UV assay was developed for (±)aldol quantification in human liver subcellular fraction.
The metabolism of ketone ester was studied in human serum albumin, plasma, and S9 liver fraction. It was observed that HSA showed esterase activity and contributed to the metabolism of KE. KE showed rapid and non-saturable (up to 15 mM) metabolism in human plasma and human and rat liver S9 fractions with a high predicted extraction hepatic ratio of > 90%. Hepatic clearance was estimated to be around 606 mL/min/kg BW in humans and 700 and 1300 mL/min/kg BW in male and female rats, respectively. 1,3-BD showed saturable metabolism in rat and human liver subcellular fractions with Km of about 8.0 mM (humans), 19.3 mM (male), and 11.9 mM (female rats). The extraction ratio of 1,3-BD was estimated to be < 20% in humans and rats. Aldol, the aldehyde intermediate in the conversion of 1,3-BD to BHB, showed non-saturable (up to 50 mM) clearance in human liver cytosolic fraction with a predicted extraction ratio of >90 % and estimated hepatic clearance of 18.7 mL/min/kg BW. 1,3-BD showed low protein binding in human plasma (~20%).
Results from the in vivorat studies of KE showed that KE was very rapidly eliminated with a half-life of about 3 mins. BHB had a half-life of about 15 mins, while 1,3-BD and AcAc had half-lives of around 25 and 30 mins, respectively. 1,3-BD, similar to the results obtained from the in vitro studies, showed slow elimination. The oral bioavailability of KE, 1,3-BD, and BHB was around 3%, 46% and 79%, respectively. It was seen that KE, BHB, and AcAc all reached a steady state after a 3-hour infusion of KE, while 1,3-BD had not yet reached a steady state (again suggesting poor elimination). A steady state concentration of about 2.4 mM was achieved for BHB with a 1000 mM dosing solution of KE infused at 4 mL/hr/kg. It was observed that plasma BHB and blood glucose showed an inverse profile with respect to each other, and this was observed after IV (bolus and infusion) and oral dosing. The hepatic clearance estimated for KE, and 1,3-BD from in vitro experiments was in good agreement (within 2-fold difference) with the total clearance calculated from in vivo rat studies indicating that the liver was the main organ for clearing these molecules. The volume of distribution calculated for KE, 1,3-BD, and BHB from the in vivo rat studies was greater than the blood volume suggesting that these molecules distribute deeper, beyond the blood compartment. A long-term (24-hour) infusion of KE was administered to evaluate its safety profile. No change was observed in the hematocrit and no signs of hemolysis were seen. Blood glucose was significantly reduced at the end of the infusion, which was expected. Blood urea nitrogen also showed a significant reduction after the infusion and will need further investigation. The results obtained from the studies described in this dissertation indicate that an IV infusion of KE can be used as a potential strategy to safely elevate and maintain blood BHB levels. These studies will find use in designing preclinical experiments to assess the pharmacological effects of KE infusion on TBI or heart disease models and also in planning future clinical studies.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-11-2023
Included in
Other Pharmacy and Pharmaceutical Sciences Commons, Pharmaceutics and Drug Design Commons