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. Author manuscript; available in PMC: 2022 Sep 15.
Published in final edited form as: AAPS J. 2021 Sep 15;23(5):106. doi: 10.1208/s12248-021-00631-8

Table I.

Summary of results from calcein-AM and digoxin screening assays

Excipient 1Functional Category Calcein-AM Assay Digoxin Assay
Normalized Accumulation Ratio2 EV Ratio3 MDR1 Ratio4 Classification5 Fold Change in Flux6 Lucifer Yellow Permeability (%)7 Classification8
D&C Red No. 28 Dye 2.60 0.10 0.26 False positive 0.45 186 Fluorescent
Benzalkonium chloride Antimicrobial 2.34 0.07 0.17 False positive 3.87 7.79 Permeabilizer
D&C Red No. 27 Dye 2.30 0.14 0.32 False positive 0.75 211 Fluorescent
Docusate sodium Surfactant 2.16 0.22 0.49 False positive 2.47 7.6 Permeabilizer
D&C Red No. 3 Dye 1.88 0.41 0.77 False positive 1.02 65.6 Fluorescent
D&C Red No. 33 Dye 1.78 0.02 0.04 False positive 1.18 0.11 Inert
D&C Red No. 6 Dye 1.58 0.92 1.47 Inhibitor 1.20 0.14 Inert
D&C Brown No. 1 Dye 1.58 0.94 1.48 Inhibitor 0.68 0.15 Inert
NaHCO3 Buffering agent 1.57 0.88 1.38 Inert 1.55 0.12 Non-specific
D&C Orange No. 4 Dye 1.53 0.06 0.10 False positive 0.96 0.15 Inert
Naphthol blue black Dye 1.38 1.15 1.60 Inert 1.08 0.01 Inert
FD&C Red No. 4 Dye 1.38 0.83 1.15 Inert 1.28 0.12 Inert
Butylparaben Antimicrobial 1.37 1.15 1.58 Inert 1.08 0.38 Inert
Epilactose Other 1.35 0.79 1.07 Inert 0.80 0.08 Inert
Acid blue 9 Dye 1.34 1.56 2.09 Inert 0.67 0.22 Inert
Sodium 1,2-ethanedisulfonate Salt forming 1.29 0.87 1.13 Inert 1.11 0.11 Inert
Light green CF yellowish Dye 1.28 1.44 1.85 Inert 0.55 0.31 Inhibitor
Dextrose Flavoring agent 1.27 0.89 1.15 Inert 0.99 0.11 Inert
FD&C Yellow No. 5 Dye 1.23 0.84 1.04 Inert 1.03 0.57 Inert
Rhodamine B Dye 1.21 0.97 1.17 Inert 0.38 11.3 Permeabilizer
Fumaric acid Flavoring agent 1.18 0.90 1.06 Inert 0.69 0.11 Inert
Sodium salicylate Other 1.17 0.87 1.02 Inert 1.15 1.25 Inert
Dibutyl sebacate Plasticizer 1.16 1.05 1.22 Inert 0.96 0.11 Inert
Ethanol Antimicrobial 1.16 0.91 1.06 Inert 0.86 0.06 Inert
Indigo carmine Dye 1.15 0.85 0.98 Inert 1.06 0.08 Inert
Erythritol Flavoring agent 1.15 0.90 1.03 Inert 0.93 0.05 Inert
Ethyl vanillin Flavoring agent 1.14 1.08 1.23 Inert 1.07 0.19 Inert
Ethyl maltol Flavoring agent 1.12 0.90 1.00 Inert 0.96 0.09 Inert
Tributyl-2-acetyl citrate Plasticizer 1.08 0.91 0.98 Inert 1.10 0.13 Inert
Sucrose Coating agent 1.07 0.82 0.88 Inert 1.03 0.20 Inert
Sodium bitartrate Flavoring agent 1.06 1.06 1.13 Inert 1.28 0.29 Inert
Oil orange SS Dye 1.05 0.87 0.92 Inert 1.89 0.38 Non-specific
L-ascorbic acid Antioxidant 1.05 0.81 0.85 Inert 1.08 0.29 Inert
Butyl alcohol Solubilizer 1.04 1.09 1.15 Inert 1.19 0.13 Inert
FD&C Red No. 40 Dye 1.04 1.09 1.15 Inert 1.25 0.03 Inert
Ammonium glycyrrhizate Flavoring agent 1.04 1.11 1.2 Inert 1.54 0.11 Non-specific
Na3PO4 Buffering agent 1.04 1.35 1.40 Inert 1.48 0.09 Non-specific
Succinic acid Flavoring agent 1.03 0.95 0.98 Inert 0.86 0.13 Inert
Ponceau 3R Dye 1.03 0.99 1.02 Inert 0.97 0.05 Inert
2-Ethoxyethanol Solvent 1.02 0.93 0.95 Inert 1.37 0.08 Inert
Pigment yellow 62 Dye 1.02 1.18 1.21 Inert 0.99 0.05 Inert
Carmine Dye 1.02 1.25 1.28 Inert 1.26 0.16 Inert
Ponceau xylidine Dye 1.01 1.06 1.08 Inert 1.05 0.09 Inert
Propyl gallate Antimicrobial 1.00 1.22 1.23 Inert 1.54 0.31 Non-specific
Cetyl alcohol Coating agent 1.00 1.18 1.18 Inert 0.82 0.09 Inert
Sodium benzoate Antimicrobial 0.99 0.84 0.84 Inert 0.99 0.18 Inert
Triethyl citrate Plasticizer 0.99 0.86 0.86 Inert 1.04 0.33 Inert
EDTA Chelator 0.99 1.01 1.01 Inert 0.93 0.09 Inert
L-arginine Nutrient suppl. 0.99 1.14 1.13 Inert 1.33 0.07 Inert
D-tagatose Flavoring agent 0.98 1.03 1.01 Inert 1.08 0.06 Inert
Acesulfame potassium Flavoring agent 0.98 0.86 0.85 Inert 1.04 2.08 Inert
Neotame Flavoring agent 0.98 1.08 1.07 Inert 0.97 0.11 Inert
Vanillin Flavoring agent 0.98 0.97 0.95 Inert 1.33 0.04 Inert
Propylparaben Antimicrobial 0.97 1.19 1.15 Inert 1.43 0.20 Inert
Xylitol Flavoring agent 0.97 1.10 1.07 Inert 1.37 0.06 Inert
Ext D&C yellow No. 7 Dye 0.97 1.20 1.16 Inert 1.23 0.14 Inert
Potassium sorbate Antimicrobial 0.96 1.19 1.15 Inert 1.45 0.10 Non-specific
Sorbitol Diluent 0.96 0.87 0.85 Inert 0.97 0.13 Inert
Glycerin Antimicrobial 0.96 0.85 0.82 Inert 0.88 0.28 Inert
Sodium gluconate Nutrient suppl. 0.96 1.10 1.06 Inert 1.10 0.18 Inert
Propylene glycol Antimicrobial 0.96 0.85 0.81 Inert 1.32 0.27 Inert
Sodium acetate Antimicrobial 0.95 1.20 1.15 Inert 1.16 0.11 Inert
Butylated hydroxytoluene Antioxidant 0.95 0.99 0.95 Inert 1.45 2.57 Non-specific
Calcium pyrophosphate Nutrient suppl. 0.95 1.07 1.02 Inert 1.05 0.05 Inert
Transanethol Flavoring agent 0.94 1.20 1.14 Inert 1.28 0.31 Inert
Maltitol Flavoring agent 0.94 1.05 0.98 Inert 1.01 0.09 Inert
Lactose monohydrate Diluent 0.93 0.99 0.93 Inert 1.25 0.09 Inert
Isopropanol Antimicrobial 0.93 1.04 0.97 Inert 1.01 0.10 Inert
Sodium lauryl sulfate Surfactant 0.93 0.92 0.86 Inert 2.23 7.98 Permeabilizer
Glycine Buffering agent 0.92 1.06 0.97 Inert 1.36 0.11 Inert
L-Malic acid Flavoring agent 0.92 1.17 1.08 Inert 1.21 0.12 Inert
Acetophenone Solubilizer 0.92 1.14 1.05 Inert 1.20 0.04 Inert
Mannose Flavoring agent 0.91 1.14 1.04 Inert 1.00 0.06 Inert
Methanolamine Buffering agent 0.91 1.13 1.03 Inert 1.11 0.11 Inert
Sodium thiosulfate Antioxidant 0.91 0.97 0.88 Inert 0.99 0.11 Inert
Na2HPO4 Buffering agent 0.91 1.04 0.94 Inert 1.34 0.06 Inert
Glyceryl caprylate Surfactant 0.90 1.08 0.97 Inert 1.05 0.13 Inert
Methylparaben sodium Antimicrobial 0.90 1.10 0.99 Inert 1.59 0.07 Non-specific
Sodium citrate Buffering agent 0.89 1.11 1.00 Inert 1.13 0.18 Inert
Mannitol Diluent 0.89 1.22 1.08 Inert 1.24 0.13 Inert
Methanol Solubilizer 0.89 1.02 0.92 Inert 1.05 0.10 Inert
L-Histidine Nutrient suppl. 0.89 1.09 0.98 Inert 1.18 0.22 Inert
Piperazine Flavoring agent 0.88 1.05 0.94 Inert 1.58 0.07 Non-specific
Tromethamine Buffering agent 0.87 1.28 1.12 Inert 1.29 0.07 Inert
Cinnamaldehyde Flavoring agent 0.87 1.10 0.96 Inert 3.14 103 Fluorescent
DL-α-tocopherol acetate Antioxidant 0.87 1.19 1.05 Inert 1.50 0.09 Non-specific
Meglumine Buffering agent 0.87 1.24 1.07 Inert 1.02 0.11 Inert
Maleic acid Buffering agent 0.87 1.06 0.92 Inert 1.13 0.09 Inert
Ethylparaben Antimicrobial 0.87 1.27 1.11 Inert 1.10 0.14 Inert
Galactose Flavoring agent 0.86 1.09 0.94 Inert 1.34 0.06 Inert
Malic acid Flavoring agent 0.86 1.12 0.97 Inert 1.14 0.08 Inert
Tartaric acid Flavoring agent 0.86 1.14 0.98 Inert 1.47 0.08 Non-specific
Glutamic acid Nutrient suppl. 0.86 1.18 1.02 Inert 0.72 0.12 Inert
Lactitol Diluent 0.86 1.03 0.89 Inert 1.17 0.09 Inert
Isomalt Coating agent 0.86 1.06 0.91 Inert 1.52 0.08 Non-specific
Amarnath Dye 0.86 1.22 1.05 Inert 1.12 0.02 Inert
NaH2PO4 Buffering agent 0.85 1.04 0.89 Inert 1.31 0.06 Inert
Maltose Flavoring agent 0.85 1.11 0.95 Inert 1.61 0.10 Non-specific
Urea Surfactant 0.85 1.28 1.08 Inert 1.21 0.07 Inert
Neohesperidin dihyhrochalone Flavoring agent 0.84 1.13 0.96 Inert 1.61 0.18 Non-specific
Sodium sulfite Antimicrobial 0.84 1.04 0.87 Inert 0.95 0.09 Inert
Myristyl alcohol Emollient 0.83 1.24 1.03 Inert 0.90 0.08 Inert
FD&C yellow No. 6 Dye 0.82 1.25 1.02 Inert 1.03 0.19 Inert
Fructose Flavoring agent 0.82 1.06 0.87 Inert 1.36 0.14 Inert
Methacrylic acid Other 0.81 1.38 1.13 Inert 1.10 0.08 Inert
Acetone Solubilizer 0.81 1.14 0.93 Inert 1.08 0.06 Inert
DL-Aspartic acid Nutrient suppl. 0.81 1.30 1.06 Inert 1.14 0.07 Inert
Sodium 4-aminobenzoate Other 0.79 1.12 0.89 Inert 1.03 0.13 Inert
Sucrose palmitate Surfactant 0.79 1.30 1.04 Inert 1.48 2.60 Non-specific
Aspartame Flavoring agent 0.79 1.29 1.03 Inert 1.18 0.10 Inert
Chloromethane Other 0.79 1.22 0.97 Inert 1.38 0.04 Inert
β-Cyclodextrin Solubilizer 0.79 1.18 0.94 Inert 0.25 0.14 Inhibitor
Maltol Flavoring agent 0.78 1.04 0.82 Inert 0.96 0.09 Inert
MgSO4 Nutrient suppl. 0.77 1.22 0.94 Inert 0.84 0.04 Inert
Sucralose Flavoring agent 0.75 0.98 0.74 Inert 1.11 0.16 Inert
Sodium carbonate Buffering agent 0.72 1.24 0.90 Inert 1.39 0.19 Inert
Ethyl acetate Flavoring agent 0.71 1.29 0.91 Inert 1.07 0.06 Inert
Leucine Flavoring agent 0.69 1.07 0.75 Inert 1.40 0.06 Inert
DL-Lactic acid Flavoring agent 0.67 1.31 0.88 Inert 1.15 0.07 Inert
Cysteine hydrochloride Nutrient suppl. 0.66 1.33 0.87 Inert 1.05 0.06 Inert
Sucrose monolaurate Surfactant 0.53 1.28 0.68 Non-specific 1.54 2.24 Non-specific
Yellow AB Dye 0.44 0.38 0.17 Non-specific 1.81 0.14 Non-specific
Cetylpyridinium chloride Antimicrobial 0.18 0.34 0.06 Non-specific 3.35 6.22 Permeabilizer
1

The most common use is noted for most excipients. For those marked ‘Other’ no clear indication was available.

2

Normalized accumulation ratio =  P−gp+IP−gp−IEV+IEV−I where (P-gp + I) and (P-gp − I) represent the fluorescence of accumulated calcein in HEK-MDR1 cells in the presence and absence of the test compound, respectively and (EV + I) and (EV − I) represent the fluorescence of accumulated calcein in the HEK-EV cells in the presence and absence of the test compound, respectively.

3

EV ratio = EV+IEV−I where (EV + I) and (EV − I) represent the fluorescence of accumulated calcein in the HEK-EV cells in the presence and absence of the test compound, respectively.

4

MDR1 ratio = P−gp+IP−gp−I where (P-gp + I) and (P-gp − I) represent the fluorescence of accumulated calcein in the HEK-MDR1 cells in the presence and absence of the test compound, respectively.

5

Excipients were classified as inhibitors if the normalized accumulation ratio was ≥1.40 and the MDR1 ratio was ≥1.40. Excipients with a normalized accumulation ratio ≥1.40 with decreased ratios in both the EV and MDR1 cells were classified as false positives. Excipients with a normalized accumulation ratio ≤0.60 were classified as non-specific activators. All other excipients were considered inert.

6

Fold change in flux is the ratio of digoxin flux in the basal to apical direction in the presence and absence of the indicated excipient.

7

Lucifer yellow permeability is the percentage of lucifer yellow that moved from the apical to the basal chamber during the 2 hr flux assay.

8

Excipients were classified as inhibitors if the fold change in digoxin flux was ≤0.60 and lucifer yellow permeability was <3%. Excipients that interfered with the lucifer yellow measurement (permeability >50%) were classified as fluorescent and those that increased lucifer yellow permeability >3% and ≤50% were classified as permeabilizers. Excipients that increased digoxin flux ≥40% were classified as non-specific activators. All other excipients were considered inert.