Goldenseal (Hydrastis canadensis) 43525850
An ex vivo approach to botanical-drug interactions: A proof of concept study CSV JSON

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 methodoloy 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 silybinA, 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.

1 . 3A4/5 Inhibition by Berberine (Plasma) (id=NPDI-KGE4lw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

midazolam 708298

Berberine 19012197

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

Fig 6 (Plasma) 1000 μM
Figure 4b

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

0.2 mg/ml

80 μl

10 min

NADPH regenerating system

10 μM

10 min

30, 100, 300, 600, 1000 μM

2 . 3A4/5 Inhibition by Berberine (PB) (id=NPDI-PaRJNQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

midazolam 708298

Berberine 19012197

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

Figure 4b (in parentheses)
Inhibition from Fig 6 (1000 μM)

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

0.2 mg/ml

80 μl

10 min

NADPH regenerating system

10 μM

10 min

30, 100, 300, 600, 1000 μM

3 . 3A4/5 Inhibition by Goldenseal (id=NPDI-hYXlNQ)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

midazolam 708298

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

Figure 2.

After incubation with the serum samples G1, G2, G3, G4,G5 (30, 100, 300, 600, 1000 μM) the activity of CYP3A on midazolam 1’-hydroxylation was determined to be 100.8±2.7%, 103.0±3.1%, 90.0±3.1%, 84.8±1.7%, 87.5%±2.3% of the control, respectively.

Compared to the control, G3, G4 and G5 significantly inhibited CYP3A4/5 activity (Figure 2). Interestingly, these three samples contained relatively higher concentrations of hydrastine (2.19-22.90-fold) than those in the samples G1 and G2 while berberine concentrations in the samples G3, G4, and G5 were lower (0.08-0.17 folds) than those in samples G1 and G2. In general, subjects administered GS exhibited higher hydrastine serum concentrations than berberine

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

0.2 mg/ml

80 μl

10 min

NADPH regenerating system

10 μM

10 min

30, 100, 300, 600, 1000 μM

4 . 3A4/5 Inhibition by Hydrastine (Plasma) (id=NPDI-gAs6gw)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

midazolam 708298

hydrastine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

300 μM estimate from Fig 6
Fig 4a in parentheses

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

0.2 mg/ml

80 μl

10 min

NADPH regenerating system

10 μM

10 min

3, 10, 30, 100, 300 μM

5 . 3A4/5 Inhibition by Hydrastine (PB) (id=NPDI-6AJ-fg)

In Vitro Enzyme Inhibition Experiment

Inhibition was detected.  Cutoff used — not specified

midazolam 708298

hydrastine

1'-hydroxymidazolam

  • CYP3A

Cell fraction Pooled human liver microsomes -7999662

Results

Inhibition from Fig 6 (300 μM)
Fig 4a. (in parentheses)

Compound measured Measurement Value Study sequence Additional information N replicates

Experimental Conditions

0.2 mg/ml

80 μl

10 min

NADPH regenerating system

10 μM

10 min

3, 10, 30, 100, 300 μM