berberine
Inhibitory Mechanisms of Human CYPs by Three Alkaloids Isolated from Traditional Chinese Herbs
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The three purified herbal compounds tetrahydropalmatine (Tet), neferine and berberine (Ber) were exploredin vitro for basic inhibition mechanisms towards recombinant human CYP1A2, CYP2D6 and CYP3A4 meta- bolic activities. Phenacetin, dextromethorphan and testosterone, respectively, were used as CYP1A2, CYP2D6 and CYP3A4 substrates, and their metabolites were determined by validated HPLC methodologies. Positive inhibition controls were used. Mechanism-based (irreversible) inhibition was assessed by time-dependent and nicotinamide adenine dinucleotide phosphate-dependent and reversible inhibition by Lineweaver–Burk plot assessments. Inhibition mechanisms were also assessed by computerized interaction prediction by using the Discovery Studio CDOCKER software (Accelrys, San Diego, CA, USA). Tetrahydropalmatine showed a mechanism-based inhibition of both CYP1A2 and CYP2D6, and Ber of CYP2D6. Neferine and Ber both showed a nonmechanistic inhibition of CYP1A2. All compounds showed a similar and significant mechanism-based inhibition of CYP3A4. Tetrahydropalmatine and Ber demonstrated both reversible and irreversible inhi- bition of CYP2D6 and CYP3A4. Tetrahydropalmatine and Ber displayed H-bond and several Pi-bond connec- tions with specific amino acid residues of CYP1A2, CYP2D6 and CYP3A4, giving further knowledge to the identified reversible and irreversible herb–drug interactions. Tetrahydropalmatine and Ber should be considered for herb–drug interactions in clinical therapy until relevant clinical studies are available.
1 . 2D6 pre-incubation with NADPH (id=NPDI-ykfGxQ)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — not specified
- CYP2D6 4173631
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Estimated from fig 2 at 30 mins
Table 2
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
100-pmol CYP/mg
Commercially available
73755
400 μL
25 min
NADPH
NADPH regenerating system
P450 reductase
100 μM
0,5,15,30, and 45 min
NADPH with precipitant
7.43-μM
2 . 1A2 preincubation with NADPH (id=NPDI-EjO7dA)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — not specified
- CYP1A2 4312402
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
estimated from fig 2
table 2
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
110-pmol CYP/mg
Commercially available
41273
400 μL
25 min
NADPH
NADPH regenerating system
P450 reductase
100 μM
0,5,15,30, and 45 min
NADPH with precipitant
75 μM
3 . 3A4 preincubation without NADPH (id=NPDI-iBWK6Q)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — not specifed
- CYP3A4 4306811
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Results
Results estimated above from 30 min of preincubation (Fig 2)
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
141-pmol and 149-pmol
Commercially available
82636 and 38275
400 μL
10 min
NADPH
NADPH regenerating system
P450 reductase
100 μM
0,5,15,30, and 45 min
No NADPH with precipitant
50.5 μM
4 . 3A4 preincubation with NADPH (id=NPDI-q61pww)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — not specified
- CYP3A4 4306811
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 Co-expressed
Results
Results above from 30 min preincubation with NADPH (fig 2)
Table 2
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
141-pmol and 149-pmol
Commercially available
82636 and 38275
400 μL
10 min
NADPH
NADPH regenerating system
P450 reductase
100 μM
0,5,15,30, and 45 min
NADPH with precipitant
50.5 μM
5 . 2D6 preincubation without NADPH (id=NPDI-wjO31A)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — not specified
- CYP2D6 4173631
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Estimate from Fig 2 at 30 mins
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
100-pmol CYP/mg
Commercially available
73755
400 μL
25 min
NADPH
NADPH regenerating system
P450 reductase
100 μM
0,5,15,30, and 45 min
No NADPH with precipitant
7.43-μM
6 . 1A2 preincubation without NADPH (id=NPDI-MsX4aw)
In Vitro Enzyme Inhibition Experiment
Inhibition was detected. Cutoff used — not specified
- CYP1A2 4312402
Recombinant expression system
Baculovirus-insect cells
Cytochrome B5 No
Results
Estimated from fig 2 at 30 mins
| Sample | Compound measured | Value | Measurement | Study sequence | Additional information | N replicates |
|---|
Experimental Conditions
110-pmol CYP/mg
Commercially available
41273
400 μL
25 min
NADPH
NADPH regenerating system
P450 reductase
100 μM
0,5,15,30, and 45 min
No NADPH with precipitant
75 μM