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Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier

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Abstract

Purpose

To investigate the blood-to-retina verapamil transport at the blood-retinal barrier (BRB).

Methods

EverFluor FL Verapamil (EFV) was adopted as the fluorescent probe of verapamil, and its transport across the BRB was investigated with common carotid artery infusion in rats. EFV transport at the inner and outer BRB was investigated with TR-iBRB2 cells and RPE-J cells, respectively.

Results

The signal of EFV was detected in the retinal tissue during the weak signal of cell impermeable compound. In TR-iBRB2 cells, the localization of EFV differed from that of LysoTracker® Red, a lysosomotropic agent, and was not altered by acute treatment with NH4Cl. In RPE-J cells, the punctate distribution of EFV was partially observed, and this was reduced by acute treatment with NH4Cl. EFV uptake by TR-iBRB2 cells was temperature-dependent and membrane potential- and pH-independent, and was significantly reduced by NH4Cl treatment during no significant effect obtained by different extracellular pH and V-ATPase inhibitor. The EFV uptake by TR-iBRB2 cells was inhibited by cationic drugs, and inhibited by verapamil in a concentration-dependent manner with an IC50 of 98.0 μM.

Conclusions

Our findings provide visual evidence to support the significance of carrier-mediated transport in the blood-to-retina verapamil transport at the BRB.

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Abbreviations

ABCB1:

ATP-biding cassette transporter subfamily B member 1

ABCC1:

ATP-biding cassette transporter subfamily C member 1

ABCC4:

ATP-biding cassette transporter subfamily C member 4

ABCG2:

ATP-biding cassette transporter subfamily G member 2

ARVO:

Association for Research in Vision and Ophthalmology

BCRP:

Breast cancer resistance protein

BRB:

Blood-retinal barrier

CH:

Choroid

C/M ratio:

Cell-to-medium ratio

CNG:

Cyclic nucleotide-gated

DAPI:

4′,6-Diamidino-2-phenylindole

ECF:

Extracellular fluid

EFV:

EverFluor FL Verapamil (BODIPY® FL Verapamil)

FCCP:

Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone

FI ratio:

Fluorescence intensity ratio

GCL:

Ganglion cell layer

IC50 :

50% Inhibitory concentration

ILM:

Inner limiting membrane

INL:

Inner nuclear layer

IPL:

Inner plexiform layer

Km :

Michaelis constant

LTR:

LysoTracker® Red

MDR1:

Multi drug resistance 1

MPP+ :

1-Methyl-4-phenylpyridinium

MRP1:

Multi drug resistance-related protein 1

MRP4:

Multi drug resistance-related protein 4

Oat3:

Organic anion transporter 3

Oatp1a4:

Organic anion transporting polypeptide 1a4

OLM:

Outer limiting membrane

ONL:

Outer nuclear layer

OPL:

Outer plexiform layer

PAH:

p-Aminohippuric acid

PBS:

Phosphate-buffered saline

P-gp:

P-glycoprotein

POS:

Photoreceptor outer segment

Rho-D:

Rhodamine B isothiocyanate-dextran

RPE:

Retinal pigment epithelium

RUI:

Retinal uptake index

SLC:

Solute carrier

SLCO:

Solute carrier organic anion

TEA:

Tetraethylammonium

V-ATPase:

Vacuolar-type H+-ATPase

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Acknowledgments and Disclosures

The present study was financially supported in part by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number JP16H05110 and JP17K08409), JSPS Core-to-Core Program (B. Asia-Africa Science Platforms), and Research Grants from the Smoking Research Foundation and the Takeda Science Foundation. The authors thank Miss Reina Makino for her technical support involving confocal microscopy. The authors declare no conflict of interest.

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Author notes

  1. Na Y. Kubo and A. Nakazawa contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630, Sugitani, Toyama, 930-0194, Japan

    Yoshiyuki Kubo, Ayumi Nakazawa, Shin-ichi Akanuma & Ken-ichi Hosoya

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  1. Yoshiyuki Kubo
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  2. Ayumi Nakazawa
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  3. Shin-ichi Akanuma
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  4. Ken-ichi Hosoya
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Correspondence to Ken-ichi Hosoya.

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Kubo, Y., Nakazawa, A., Akanuma, Si. et al. Blood-to-Retina Transport of Fluorescence-Labeled Verapamil at the Blood-Retinal Barrier. Pharm Res 35, 93 (2018). https://doi.org/10.1007/s11095-018-2384-7

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  • DOI: https://doi.org/10.1007/s11095-018-2384-7

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