The Development of a High-Throughput Enzymatic Method for the Evaluation of CYP3A4 Drug-Drug Interactions using Fluorescence-Based Assays

Author

• Jaelyn Jenifer, Virginia Commonwealth UniversityFollow

Defense Date

2026

Document Type

Directed Research Project

First Advisor

Christopher Ehrhardt, Ph.D.

Second Advisor

Emanuele Alves, Ph.D.

Abstract

The opioid epidemic in the United States has raised concern about overdose deaths and the possibility of drug interactions between opioids and medications used to treat chronic diseases such as Human Immunodeficiency Virus (HIV). Limited access to treatment has led many individuals to manage opioid withdrawal symptoms on their own. The most popular option adopted was the use of kratom. Kratom is an herbal product obtained from the plant Mitragyna speciosa and is an emerging substance with a potential for abuse and addiction. Kratom’s main alkaloids, mitragynine (MG) and 7-hydroxymitragynine (7-HMG), act on the µ-opioid receptor (MOR) as agonists. In recent years, the use of kratom for pain relief, particularly as a substitute for opioids, has increased in the U.S. Between 2014 and 2019, 3,484 kratom-related exposures were reported to U.S. poison centers. Among them, 13% resulted in major outcomes, with 37% of these severe cases involving kratom associated with other drugs, including antiretrovirals. Therefore, kratom-related toxicity appears to be more likely when in combination with other drugs. Antiretroviral drugs used to treat HIV positive patients can modulate the metabolism of several drugs through induction or inhibition of the CYP450 enzymes. This project aimed to evaluate the capacity of kratom alkaloids to modify the activity of antiretroviral drugs used to treat HIV infections. To achieve this goal, a high-throughput method using CYP3A4 was developed. The CYP3A4 enzyme was chosen because both kratom’s major alkaloids and HIV antiretrovirals are metabolized using this pathway. The drug concentration range tested was chosen to reflect the maximum plasma concentration of drug seen in users. Ketoconazole served as the model inhibitor, and enzyme activity in its presence was compared with activity measured with kratom alkaloids and antiretroviral drugs. Recombinant CYP3A4 was incubated with proper reagents in the presence or absence of ketoconazole, kratom alkaloids, and antiretroviral drugs. Key assay conditions, including buffer, cofactors, enzyme and drug concentrations, incubation time, temperature, and fluorescence detection, were evaluated. Fluorescence measurements were used to quantify E_max and EC₅₀ and to assess statistical significance. The in vitro evaluation of MG and 7‑HMG showed that, within the concentration ranges tested, MG did not interfere with the metabolism of the HIV antiretrovirals examined. Alternatively, 7-HMG had a nuanced attenuating effect on ritonavir’s inhibitory capacity. As deaths associated with polydrug use continue to rise, rapid and cost-efficient methods for assessing potential drug interactions remain essential.

Rights

© The Author(s)

Is Part Of

VCU Master of Science in Forensic Science Directed Research Projects

Date of Submission

4-28-2026