An ex vivo approach to botanical-drug interactions: a proof of concept study

Abstract

Ethnopharmacological relevance: Botanical medicines are frequently used in combination with therapeutic drugs, imposing a risk for harmful botanical-drug interactions (BDIs). Among the existing BDI evaluation methods, clinical studies are the most desirable, but due to their expense and protracted time-line for completion, conventional in vitro methodologies remain the most frequently used BDI assessment tools. However, many predictions generated from in vitro studies are inconsistent with clinical findings. Accordingly, the present study aimed to develop a novel ex vivo approach for BDI assessment and expand the safety evaluation methodology in applied ethnopharmacological research.

Materials and methods: This approach differs from conventional in vitro methods in that rather than botanical extracts or individual phytochemicals being prepared in artificial buffers, human plasma/serum collected from a limited number of subjects administered botanical supplements was utilized to assess BDIs. To validate the methodology, human plasma/serum samples collected from healthy subjects administered either milk thistle or goldenseal extracts were utilized in incubation studies to determine their potential inhibitory effects on CYP2C9 and CYP3A4/5, respectively. Silybin A and B, two principal milk thistle phytochemicals, and hydrastine and berberine, the purported active constituents in goldenseal, were evaluated in both phosphate buffer and human plasma based in vitro incubation systems.

Results: Ex vivo study results were consistent with formal clinical study findings for the effect of milk thistle on the disposition of tolbutamide, a CYP2C9 substrate, and for goldenseal׳s influence on the pharmacokinetics of midazolam, a widely accepted CYP3A4/5 substrate. Compared to conventional in vitro BDI methodologies of assessment, the introduction of human plasma into the in vitro study model changed the observed inhibitory effect of silybin A, silybin B and hydrastine and berberine on CYP2C9 and CYP3A4/5, respectively, results which more closely mirrored those generated in clinical study.

Conclusions: Data from conventional buffer-based in vitro studies were less predictive than the ex vivo assessments. Thus, this novel ex vivo approach may be more effective at predicting clinically relevant BDIs than conventional in vitro methods.

Keywords: Botanical–drug interactions; Cytochrome P450s; Ex-vivo model; Goldenseal; Milk thistle.

Figure 1

The potential for inhibition of CYP2C9 activity by one or more components of milk thistle in 5 individuals’ plasma was assessed with an ex vivo drug interaction model. The relative CYP2C9 activity in the control group was defined as 100%. Five different milk thistle-containing plasma samples are represented as M1-M5. Data represent means ± S.D for 3 independent experiments.

Figure 2

The potential for inhibition of CYP3A4/5 activity by one or more components of goldenseal in 5 individuals’ serum was assessed with an ex vivo drug interaction model. The relative CYP3A4/5 activity in control group was defined as 100%. Five different goldenseal containing serum samples are represented as G1-G5. Data represent means ± S.D. for 3 independent experiments. *P<0.05, **P<0.01 versus control.

Figure 3

The inhibitory effect of silybin A (A) and silybin B (B) on TOLB 4-hydroxylation catalyzed by CYP2C9 was assessed by determining the formation of 4-hydroxy-TOLB. IC50 values (μM) for silybin A and silybin B in phosphate buffer (PB) and plasma are presented in parentheses. Data are means ± SD of 3 independent experiments.

Figure 4

The inhibitory effect of berberine (A) and hydrastine (B) on midazolam 1’-hydroxylation catalyzed by CYP3A4/5 was assessed by determining the formation of 1’-hydroxy-midazolam. IC50 values (μM) for berberine and hydrastine in phosphate buffer (PB) and plasma are presented in parentheses. Data are means ± SD of 3 independent experiments.

Figure 5

Inhibition of CYP2C9–mediated TOLB 4-hydroxylation by silybin A (1-100 μM) (A) and silybin B (1-100 μM) (B) in phosphate buffer compared to blank human plasma. The relative CYP2C9 activity in the control group was defined as 100%. Data represent means ± S.D. from 3 independent experiments. **p<0.01 versus control.

Figure 6

Inhibition of CYP3A4/5 –mediated midazolam 1’-hydroxylation by hydrastine (3-300 μM) (A) or berberine (100-1000 μM) (B) in phosphate buffer compared to blank human plasma. The relative CYP3A4/5 activity in the control group was defined as 100%. Data represent means ± S.D. from 3 independent experiments. **p<0.01 versus control.