(T0930-02-09) Development of Dihydromyricetin for Effective Oral Dosing

Purpose: Dihydromyricetin (DHM) is a bioactive phytochemical flavonoid increasingly recognized in western society for its demonstrated anti-inflammatory and antioxidant activities^1,2. These characteristics endow the compound with a number of therapeutic benefits, which have been demonstrated in nonclinical models to be a potential treatment for various ailments including alcohol-related dyslipidemia, diabetes, anxiety, and Alzheimer's Disease^3-5. However, due to its inherent physicochemical properties, DHM exhibits poor aqueous solubility and absorption, and thus low bioavailability⁶. These limitations pose several translational challenges for clinical application, preventing effective oral dosing and necessitating invasive delivery methods such as oral gavage or injection to achieve therapeutic concentrations. To circumvent these hurdles, our group has developed novel formulations of DHM via crystallization methods that demonstrate markedly improved aqueous solubility, thereby allowing for more effective oral dosing. Therefore, the purpose of this study is to build a pharmacokinetic profile of our lead compounds, accomplished through various methods including physiologically based pharmacokinetic predictive modeling and in vivo studies assessing relative oral bioavailability.

Methods: DHM crystals and salts were formed by facilitating proton transfer from DHM (proton donor) to a base (proton acceptor); thus, DHM extract ( >98% purity) was paired with various chemical bases including triethanolamine (TEA), calcium hydroxide (Ca(OH)₂), and Lysine. Crystallization was achieved using various solvent systems where solid powders were collected following slow evaporation. Crystalline birefringence was assessed using polarized light microscopy, melting points were measured using differential scanning calorimetry (DSC), and crystallinity was confirmed via X-ray Powder Diffraction (XRPD). Finally, single unit crystal structures were obtained using CryoEM Method MicroED. Using physiologically based pharmacokinetic (PBPK) prediction software, GastroPlus™, pharmacokinetic parameters were calculated, and relative bioavailability and percent fraction absorbed (%Fa) were obtained via a parameter sensitivity analysis (PSA) of DHM + TEA co-crystals. Pilot in vivo studies are currently in progress using male and female C57BL/6 mice (n=4/group/sex). Mice will be administered 0.1mg/ml or 1.0 mg/mL of either free DHM, DHM +TEA, DHM + Ca(OH)₂, or DHM + Lysine in their drinking water for a period of 10 days to reach steady state concentrations. Brains will be harvested to assess the ability of DHM to permeate the blood brain barrier, and plasma will be collected to measure levels of circulating DHM. Both assessments will be conducted with liquid chromatography-mass spectrometry/mass spectrometry.

Results: The enhanced solubility of DHM crystals and salts were investigated by physical and structural chemistry-based analyses. DHM + TEA, our lead compound, was confirmed to be a co-crystal and produced suitable diffraction by MicroED, enabling determination of atomic structure. PBPK modeling predicted the C_max for a 5mg oral dose of DHM + TEA co-crystal to be over 100X that of free DHM (2,476.3 ng/ml and 20.73 ng/ml, respectively), and the total AUC, representative of bioavailability, to be ~65X greater (AUC_0-∞ : 10,860 ng/ml·hour versus 166.5 ng/ml·hour, respectively). Through PSA, plots were generated based on twenty separate calculations of %Fa, each derived from different PK simulations conducted at various doses ranging from 0.5-500mg. While the %Fa of free DHM decreases from 0.5-500mg in a sigmoidal manner, the %Fa of DHM + TEA %Fa was demonstrated to not be sensitive to dose from 0.5~100mg (seen as a flat line), predicting that the absorption of DHM remains relatively constant in this range. From 100mg-500mg there is a < 15% change in %Fa with DHM + TEA co-crystal. Additionally, compared to free DHM, the relative oral bioavailability of DHM+ TEA more than doubled between the doses of 0.5-10mg (159% vs 356%, respectively), and increased by over 3000% from 0.5-500mg (159% vs 3223%).

Conclusion: Understanding the ionization macro and microstates of free DHM led to the formation of novel crystal and salt forms of DHM, enabling rapid dissolution in the relevant gastrointestinal pH range for improved oral bioavailability. Our novel formulations are readily soluble in water and are stable at room temperature. Therefore, this discovery allowed for the development of an oral formulation that we used in an animal model, where we demonstrated the utility and palatability of the new formulation in a pre-clinical setting. The predicted PBPK data shows promising evidence that DHM crystals can result in markedly improved absorption and thus, increased bioavailability. Taken together, this data provides initial support for the potential of using a crystal formulation of DHM as a therapeutic intervention for a myriad of inflammation-driven and neurodegenerative diseases, including its current commercial use as a hangover remedy. As a natural product that is readily available, our findings help set the stage for the rapid advancement of DHM into the clinic for improving overall health.

References: 1 Hyun, T., Eom, S., Yu, C. & Roitsch, T. Hovenia dulcis – An Asian Traditional Herb. Planta Medica 76, 943-949 (2010).
2 Liu, D., Mao, Y., Ding, L. & Zeng, X.-A. Dihydromyricetin: A review on identification and quantification methods, biological activities, chemical stability, metabolism and approaches to enhance its bioavailability. Trends in Food Science & Technology 91, 586-597 (2019).
3 Janilkarn-Urena, I. et al. Dihydromyricetin supplementation improves ethanol-induced lipid accumulation and inflammation. Frontiers in Nutrition 10, undefined-undefined (2023).
4 Silva, J. et al. Modulation of hippocampal GABAergic neurotransmission and gephyrin levels by dihydromyricetin improves anxiety. Frontiers in Pharmacology 11, 1008 (2020).
5 Ling, H. et al. Dihydromyricetin improves type 2 diabetes-induced cognitive impairment via suppressing oxidative stress and enhancing brain-derived neurotrophic factor-mediated neuroprotection in mice. Acta Biochimica et Biophysica Sinica 50, 298-306 (2018).
6 Zhang, R. et al. Strategic developments in the drug delivery of natural product dihydromyricetin: applications, prospects, and challenges. Drug Delivery 29, 3052-3070 (2022).
7 Liu, L., Yin, X., Wang, X. & Li, X. Determination of dihydromyricetin in rat plasma by LC-MS/MS and its application to a pharmacokinetic study. Pharmaceutical Biology 55, 657-662 (2016).

Acknowledgements: We would like to thank the members of the Davies and Seidler labs for their support on this project. This project was made possible through the generous funding opportunities provided by Timothy M. Chan Professorship in Complementary Therapeutics (DLD), the USC Institute of Addiction Science (DLD), the BrightFocus Grant (PS) and USC Mann School of Pharmacy and Pharmaceutical Sciences.


Graph shows a comparison of simulated DHM + TEA oral exposure from a 5mg total dose vs observed values, taken from Liu et al.7, of free DHM in rats.


Plots depict percent fraction of the initial oral dose absorbed after gut extraction (%Fa) at 20 simulated doses. While free DHM %Fa decreases from 0.5-500mg in a sigmoidal manner, the DHM + TEA %Fa is not sensitive to dose from 0.5~100mg (seen as a flat line). From 100mg-500mg there is a <15% change in %Fa with DHM + TEA.


Histogram shows the oral bioavailability of the DHM+TEA co-crystals relative to free DHM at a dose range of 0.5-500mg.