Effects of Antioxidants in Drugs Products

on Intestinal Drug Transporters

Sook Wah Yee
University of California, San Francisco
 Motivation

Digoxin, Rosuvastatin, Rosuvastatin

Fexofenadine, Sulfasalazine
Dabigatran
• Inhibitors of P-gp and BCRP can increase drug bioavailability and hence
increase drug level.
• Inhibitors of OATP2B1 can reduce drug bioavailability and reduce drug level.
 Motivation

Nitrosamine Ranitidine Ranitidine Valsartan

contamination Losartan Losartan
drugs:
• Nitrosamine contamination in drug products: Valsartan, Losartan,
Ranitidine. These are substrates of these transporters.
• Can anti-oxidants used in drug products inhibit these transporters?
 Approach

1. Select a list of antioxidants

• 22 Antioxidants in FDA Inactive Ingredient Database
• 8 Antioxidants from natural product
2. Purchase and plate them in 96-well plate for screening at 200 µM
Approach

3. Screen compounds using membrane vesicles overexpressing P-gp or BCRP.

• Substrates: 3H-N-methylquinidine (P-gp), 3H-CCK (BCRP)
4. Screen compounds using HEK293 cells overexpressing OATP2B1.
• Substrate: 3H-estrone sulfate
5. Determine % uptake of each compound.
Results: % Uptake
Antioxidants OATP2B1 BCRP
1 103.6 92.7
P-gp
113.6
Lower % Uptake, stronger inhibition by the compounds
2 28.3 50.9 86.9
3 137.1 102.8 98.1
Ascorbyl palmitate (AP)
4 106.5 90 134.8
5 79.3 86.5 149.1
6 83.5 81 67.9
7 85.4 78.5 118.9
8 128.8 93.1 92
9
10
19.8
119.8
46.7
75.8
42.1
133.8
Butylated hydroxyanisole (BHA)
11 18.8 4.5 0
12 119.3 99.7 82.6
Carnosic acid
13 116.1 82.7 90.3
14 112.8 110.4 69.3
15 118.2 112.5 58.2
16 123.3 88.2 81.8
17 136.4 91 100.4
18 107.9 95.9 79.8
19 125.6 99.7 76.9
20 108.9 92.4 87.6
21 120.3 109 32.5
22 113.2 94.8 86.7
23 97.8 83 83.1
24 98.8 104.8 103.5
25 103.1 90.3 121.3
26 90.1 110.4 96.1
27 118.9 94.5 78.8
28 92.9 99.7 77.7
29 104.4 114.2 77.4
• Cyclosporin A – BCRP, P-gp
30 101.1 96.9 108
Known Inhibitor 83.3 8.3 0
Known inhibitor for one or more of these • Verapamil – P-gp
Known Inhibitor 194.2 50.5 0 transporters: OATP2B1, BCRP and P-gp • Bromosulfophthalein – All three transporters
Known Inhibitor 10.9 0.3 0 • Erlotinib – OATP2B1
Known Inhibitor 10.5 58.5 63
Known Inhibitor 8.2 0 0
• Curcumin – All three transporters
 Antioxidant inhibits intestinal transporters in the medium to
high µM range
AP IC50 : 23 ± 10 µM
BHA IC50: 206 ± 14 µM BHA IC50 : 182 ± 49 µM
BHA IC50 : 72 ± 19 µM
221010_BCRP_norm
20929_preinc
OATP2B1 125
BCRP 150 P-gp
P-gp
% Uptake of 3H-Esterone sulfate

150 AP
Ascorbyl Palmitate

% Uptake of 3H-N-methylquinidine
BH 125
125 Butylated Hydroxyanisole 100

% Uptake of 3H-CCK8
100
100 75

75
75
50
50
50
25
25 25

0
0 0
1 10 1 100 101000 100 1000 1 10 100 1000 10 100 1000

hibitor conc (uM) Butylated Hydroxyanisole conc (µM)

(uM) Butylated hydroxyanisole
[Butylated conc(µM)
Hydroxyanisole] (µM)
Inhibitor conc (µM)
[Antioxidant] (µM) [Butylated Hydroxyanisole]

7
 Interpreting in vitro study results to determine the potential
for clinical DDIs.
antioxidant
• The investigational drug has the potential to inhibit P-gp, BCRP or OATP2B1 in
vivo if the investigational drug is administered orally, and the Igut / IC50 ≥ 10,
where Igut = dose of inhibitor/250 mL.
antioxidant
an antioxidant
• If in vitro studies indicate that a drug is a P-gp or BCRP or OATP2B1 inhibitor,
the sponsor should consider whether to conduct an in vivo study based on likely
concomitant medications that are known P-gp or BCRP substrates.
FDA IID oral % w/w in 500 mg Max intestinal conc
Antioxidant MW (g/mol)
limit (mg/day) oral unit (µM) in 250 mL fluid
Ascorbyl palmitate
(AP)
414.53 12 2% (10mg) 96.5
Butylated
hydroxyanisole 180.24 8 1.6% (8mg) 178
(BHA)
8
Ref: FDA guidance In Vitro Drug Interaction Studies Jan 2020
 Interpreting in vitro study results to determine the potential
for clinical DDIs.

Max intestinal conc Igut / IC50

Antioxidant (µM) in 250 mL fluid
AP BHA
Ascorbyl palmitate (AP) 96.5 OATP2B1 96.5 / 23 = 4.2 178 / 72 = 2.5
Butylated hydroxyanisole BCRP NA 178 / 206 = 0.9
(BHA) 178 P-gp NA 178 / 182 = 1.0

Igut / IC50 < 10, AP and BHA are antioxidants that are unlikely
9
Ref: FDA guidance In Vitro Drug Interaction Studies Jan 2020 to inhibit OATP2B1, BCRP or P-gp.
Inhibition of OATP2B1-mediated valsartan uptake by two antioxidants

220929_preinc OATP2B1
120 AP
Ascorbyl Palmitate
BHA
100 Butylated Hydroxyanisole
% Uptake of Valsartan

80

60

40

20 AP IC50 : 15 ± 8 µM
10
BHAmin
ICpre-incubation
50 : 22 ± 3 µM
Drug: Valsartan
0
0.1 1 10 1 100 101000 100 1000
Inhibitor conc (uM) Concentrations, µM
[Antioxidant] (µM)

Max intestinal conc Igut / IC50

(µM) in 250 mL fluid
Ascorbyl palmitate AP BHA
(AP) 96.5
OATP2B1 96.5 / 15 = 6.4 178 / 22 = 8.1
Butylated
hydroxyanisole (BHA) 178 Igut / IC50 < 10, AP and BHA are antioxidants that are
unlikely to inhibit OATP2B1. 10
Commonly used antioxidants that have minimal
interactions with intestinal transporters
Antioxidants OATP2B1 BCRP P-gp
Ascorbic acid 103.6 92.7 113.6
Butylated hydroxytoluene (BH) 137.1 102.8 98.1
Cysteine hydrochloride 106.5 90 134.8
Propyl gallate 79.3 86.5 149.1
Vitamin E 83.5 81 67.9
Values shown are % uptake of
Anhydrous citric acid 128.8 93.1 92 transporter substrate when
EDTA (edetate disodium) 112.8 110.4 69.3 antioxidants were tested at 200 µM.
Erythorbic acid 123.3 88.2 81.8
Glycine 125.6 99.7 76.9
Histidine 108.9 92.4 87.6
Methionine 113.2 94.8 86.7
Phosphoric Acid 97.8 83 83.1
Potassium sorbate 98.8 104.8 103.5
Sesamol 103.1 90.3 121.3
Sodium bisulfite 90.1 110.4 96.1
Sodium metabisulfite 118.9 94.5 78.8
Sodium sulfite 92.9 99.7 77.7
Sodium thiosulfate,
104.4 114.2 77.4
pentahydrate
Tartaric Acid 101.1 96.9 108
 Summary
• Initial screening revealed that IC 50 values for inhibition
of intestinal transporters by AP and BHA are close to the
estimated maximal intestinal concentrations for these
antioxidants (97 µM for AP and 178 µM for BHA).

• Future studies are underway to determine the in vitro

inhibition potencies of butylated hydroxyanisole (BHA)
using drugs that are substrates of P-gp and BCRP
(cimetidine).

• If Igut / IC50 ≥ 10, further evaluation to determine

antioxidant-drug interaction potential: e.g. conduct
additional modeling analyses (e.g. PBPK models) or by
conducting in vivo antioxidant-drug interaction study.
Acknowledgements

Megan L. Koleske, Chetan P. Kulkarni, Kathy Giacomini,

Deanna L. Kroetz

1. Office of Generic Drugs, Center for Drug Evaluation

and Research, FDA:
Khondoker Alam, Liang Zhao, Lei Zhang

2. Office of Pharmaceutical Quality, Center for Drug

Evaluation and Research, FDA:
Andre Raw, Bhagwant Rege, Dongmei Lu