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Investigation of Fluorescent Substrates and Substrate-Dependent Interactions of a Drug Transporter Organic Anion Transporting Polypeptide 2B1 (OATP2B1)

Abstract

Purpose

In this study, we investigated organic anion transporting polypeptide 2B1 (OATP2B1)-mediated uptake of fluorescent anions to better identify fluorescent substrates for in vitro OATP2B1 assays. The OATP2B1 is involved in the intestinal absorption and one of the pharmacokinetic determinants of orally administered drugs.

Methods

A microplate reader was used to determine the cellular accumulation of the fluorescent compounds into the OATP2B1 or the empty vector-transfected HEK293 cells.

Results

Two types of derivatives were found to be OATP2B1 substrates: heavy halogenated derivatives, such as 4′,5′-dibromofluorescein (DBF), and carboxylated derivatives, such as 5-carboxyfluorescein (5-CF). The DBF and 5-CF were transported in a time and concentration-dependent manner. The DBF was transported at a broad pH (pH 6.5–8.0) while 5-CF was transported at an acidic pH (pH 5.5–6.5). The Km values were 0.818 ± 0.067 μM at pH 7.4 for DBF and 8.56 ± 0.41 μM at pH 5.5 for 5-CF. The OATP2B1 inhibitors, including atorvastatin, bromosulfophthalein, glibenclamide, sulfasalazine, talinolol, and estrone 3-sulfate, inhibited the DBF and the 5-CF transport. Contrastively, testosterone, dehydroepiandrosterone sulfate, and progesterone inhibited the DBF transport but stimulated the 5-CF transport. Natural flavonoid aglycones, such as naringenin and baicalein, also exhibited substrate-dependent effects in this manner.

Conclusion

We found two fluorescein analogs, DBF and 5-CF as the OATP2B1 substrates that exhibited substrate-dependent interactions.

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Abbreviations

5-AF:

5-aminofluorescein

5-CF:

5-carboxyfluorescein

6-CF:

6-carboxyfluorescein

AUC:

Area under the curve

BSP:

bromosulfophthalein

CDCF:

5-carboxy-2′,7′-dichlorofluorescein

CDCSF:

5-carboxy-2′,7′-dichlorosulfonfluorescein

COR:

Corticosterone

E1S:

Estrone 3-sulfate

E2G:

Estradiol-17β-glucuronide

DBF:

2′,7′-dibromofluorescein

DCF:

2′,7′-dichlorofluorescein

DDI:

Drug-drug interaction

DEX:

Dexamethasone

DHEAS:

Dehydroepiandrosterone sulfate

EY:

Eosin Y

FDI:

Food-drug interaction

FL:

Fluorescein

OG:

Oregon Green

OATP:

Organic anion transporting polypeptide

PRO:

Progesterone

RB:

Rose bengal

Rho123:

Rhodamine123

SF:

sulfonfluorescein

TET:

Testosterone

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Correspondence to Tomohiro Nabekura.

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Kawasaki, T., Shiozaki, Y., Nomura, N. et al. Investigation of Fluorescent Substrates and Substrate-Dependent Interactions of a Drug Transporter Organic Anion Transporting Polypeptide 2B1 (OATP2B1). Pharm Res 37, 115 (2020). https://doi.org/10.1007/s11095-020-02831-x

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Key Words

  • drug−drug interaction (DDI)
  • fluorescent substrate
  • food–drug interaction (FDI)
  • inhibition assay
  • organic anion transporting polypeptide 2B1 (OATP2B1)