Abstract
Systemically administered pravastatin effectively treats diabetic retinopathy without central nervous system side effects. The efflux transport mechanism of pravastatin from the brain has already been clarified. In this study, the influx of pravastatin across the blood-retinal and blood-brain barriers (BRB and BBB) and the efflux of pravastatin from the retina were investigated using rats. Pravastatin influx (blood-to-tissues) was assessed using the retinal and brain uptake index (RUI and BUI) methods, and microdialysis was performed to investigate the efflux (retina-to-blood) transport of pravastatin. The RUI and BUI values for [3H]pravastatin were lower than those expected based on its lipophilicity, suggesting that the influx transport across the BRB and BBB was less than the reverse-direction transport. The RUI and BUI values for [3H]pravastatin were significantly decreased by pravastatin, digoxin, and probenecid, indicating that pravastatin undergoes carrier-mediated influx transport in the blood-to-tissues direction across the BRB and BBB. After intravitreal injection, [3H]pravastatin and the bulk flow marker [14C]d-mannitol were found to be eliminated biexponentially from the vitreous humor. The elimination rate constant of [3H]pravastatin during the terminal phase was 1.66-fold greater than that of [14C]d-mannitol. Efflux transport was reduced in the retinal presence of pravastatin, digoxin, and benzylpenicillin, suggesting that pravastatin is transported via efflux transporters. In conclusion, pravastatin is transported across the BRB via uptake and efflux transporters in both the blood-to-retina and retina-to-blood directions, and the retina-to-blood transporters are dominant, based on the lower values of the RUI compared with the values expected from the lipophilicity
Introduction
Although some statins have pharamacologic effects on diabetic retinopathy, as well as central nervous system (CNS) side effects, systemically administrated pravastatin reduces signs of diabetic retinopathy in diabetic patients and does not produce CNS side effects such as sleep disturbances (Gordon et al., 1991; Saheki et al., 1994).
Generally, the permeability of drugs into the retina from the blood is strictly regulated by the blood-retinal barrier (BRB), which is analogous to the blood-brain barrier (BBB). The BRB consists of both retinal capillary endothelial cells (inner BRB) and retinal pigment epithelial (RPE) cells (outer BRB), which together form the complex tight junctions that restrict paracellular solute transport. The control of the intraocular environment and the maintenance of neuroretinal homeostasis are mediated, in part, by the various transporters expressed at the BRB (Hosoya and Tomi, 2005).
Pravastatin is a substrate of the organic anion transporting polypeptides1a4 (oatp2; slco1a4) and the organic anion transporter 3 (oat3; slc22a8) (Kikuchi et al., 2004). Oatp1a4 is localized to both the luminal and abluminal membranes of the inner BRB and on the apical membrane of RPE cells of rats (Akanuma et al., 2013), whereas oat3 is expressed on only the abluminal side of the rat inner BRB and is not found on RPE cells (Hosoya et al., 2009). The expression pattern of these transporters at the BBB is similar to that at the BRB; specifically, oatp1a4 is found on both the luminal and abluminal sides of the rat brain capillary endothelium, whereas oat3 is found on only the abluminal side. Previously, transporters, including oatp1a4 and oat3, have been implicated in the brain-to-blood transport of pravastatin, and the efflux of pravastatin across the BBB has been found to be more than 3-fold greater than its uptake (Kikuchi et al., 2004). The transport mechanism of pravastatin across the BRB, however, remains unclear.
The aim of the present study was to clarify the characteristics of pravastatin transport across the BRB in both the blood-to-retina and retina-to-blood directions and to obtain further evidence to support the clinical effects of pravastatin on diabetic retinopathy. Pravastatin influx (blood-to-tissues) was assessed using the retinal uptake index (RUI) and compared with that of the BBB, and microdialysis was performed to investigate efflux (retina-to-blood) transport of pravastatin. In addition, estradiol 17-β glucuronide (E17βG) influx was evaluated as a model substrate of oatp1a4 and oat3 because the efflux mechanism of E17βG has already been clarified; that is, E17βG is transported from the vitreous humor to the blood via at least oatp1a4 (Katayama et al., 2006) and from the brain to blood via the oat family (mainly oat3) and oatp1a4 (Sugiyama et al., 2001).
Materials and Methods
Animals and Reagents.
Male Sprague-Dawley Mdr1a knockout rats (6–7 weeks old) and wild-type Sprague-Dawley rats (6–8 weeks old) were obtained from Sage Laboratories (St. Louis, MO) and Charles River Laboratories (Yokohama, Japan), respectively. All experiments were performed according to the Ethical Guidelines for Animal Experiments of Santen Pharmaceutical Company (Ikoma, Japan). Pravastatin [3H(G)] sodium salt ([3H] pravastatin, 5 Ci/mmol) and n-[1-14C]butanol ([14C]n-butanol, 2 mCi/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). [Estradiol-6,7-3H(N)]-estradiol 17β-D-glucuronide ([3H]E17βG, 36.6 Ci/mmol) and d-[1-14C]mannitol ([14C]d-mannitol, 55 mCi/mmol) were purchased from PerkinElmer (Waltham, MA). All other chemicals were commercially available and of reagent grade.
Uptake Index Method and Microdialysis Study.
The RUI and brain uptake index (BUI) were calculated according to previously reported uptake index methods. A microdialysis study was performed as previously described. Each experiment was performed once. The details are provided in the Supplemental Material.
Data Analysis.
All data, which were obtained from independent biologic samples, are expressed as the mean ± S.E. (n = 3 or 4 rats). An unpaired, two-tailed Student’s t test was used to assess the significance of the differences between the means of the two groups. The statistical significance of the differences in the means of the groups was determined using Bartlett’s test followed by Dunnett’s multiple comparison test. P < 0.05 was considered statistically significant.
Results and Discussion
The RUI and BUI of [3H]pravastatin and [3H]E17βG in wild-type rats were almost the same as those in Mdr1a knockout rats and were lower than the lipophilicity trend line (Fujii et al., 2014) (Fig. 1, A and B). The results indicate that neither of these compounds is recognized by P-glycoprotein and that influx transport into tissues across the BRB and BBB is less than efflux transport. The RUI and BUI of [3H]pravastatin were significantly reduced by pravastatin (40 mM), digoxin (0.01 mM)—which has a very high affinity for oatp1a4—and probenecid (1 mM), an inhibitor of both the oatp and oat families (Table 1). Also, [3H]E17βG influx across the BRB was significantly reduced by administration of digoxin and probenecid, although the BUI values were unchanged (Supplemental Fig. 1). These results suggested that oatp1a4 plays a role in the blood-to-retina transport of organic anions and that oatp1a4 is involved in both BRB- and BBB-mediated pravastatin influx. Further study is needed to elucidate the reason why no change was observed in the BUI for E17βG.
(A) Correlation of the RUI and (B) BUI with the log D7.4 of pravastatin (log D7.4: −0.67) and E17βG (log D7.4: −2.03) in wild-type (closed symbols) and Mdr1a knockout (open symbols) rats. The log D7.4 value of each compound was calculated using ACD/Percepta [Advanced Chemistry Development Inc. (Toronto, Canada)]. The line represents the lipophilicity trend line in wild-type rats using the data of d-mannitol, thiourea, and progesterone, which are expected to permeate by passive diffusion. The line represents the linear regression curve according to the linear least-squares methods for the three compounds in wild-type rats. RUI = 69.4 × exp (0.493 × log D7.4) (r2 = 0.993) (A) and BUI = 16.9 × exp (0.496 × log D7.4) (r2 = 0.998) (B) (Fujii et al., 2014). (C) Time-profile of [3H]pravastatin and [14C]d-mannitol in the vitreous humor after their intravitreous injection into rats. Closed squares and open squares represent the concentrations of [3H]pravastatin and [14C]d-mannitol in the dialysate, respectively. (D) Inhibitory effect on the difference in the elimination rate constants (β) between [3H]pravastatin and [14C]d-mannitol during the terminal phase. Percentage of control was calculated as follows: {β value of [3H]pravastatin − β value of [14C]d-mannitol in the presence of inhibitor}/{β value of [3H]pravastatin − β value of [14C]d-mannitol in the absence of inhibitor} × 100. The statistical significance of the differences in the means of the groups was determined using Bartlett’s test followed by Dunnett’s multiple comparison test. *P < 0.05 and **P < 0.01, significantly different from control. Each column or symbol represents the mean ± S.E. (n = 3 or 4 rats).
The effect of compounds on the retinal and brain uptake index for [3H]pravastatin in wild-type rats
[3H]Pravastatin (10 µCi/rat), with [14C]n-butanol (0.1 µCi/rat) as the highly diffusible reference, injected into the carotid artery in the absence (control) and presence of inhibitors. Values represent means ± S.E. (n = 3 or 4 rats).
To investigate the elimination of pravastatin across the BRB, a microdialysis study was performed. The concentration of [3H]pravastatin and [14C]d-mannitol, coinjected as a bulk flow marker, in the dialysate after intravitreous injection decreased biexponentially (Fig. 1C). The apparent elimination rate constant (β) during the terminal phase of [3H]pravastatin (0.0179 ± 0.001 min−1) was 1.66-fold greater than that of [14C]d-mannitol (0.0107 ± 0.0005 min−1) (Supplemental Fig. 2). The differences in the β value between [3H]pravastatin and [14C]d-mannitol coadministered with digoxin (0.01 mM) and benzylpenicillin (20 mM), an inhibitor of oat3, were both significantly lower than that of the control (Fig. 1D), whereas choline had no effect. These findings suggest that transporters, including oatp1a4 and oat3, were involved in the retinal clearance of pravastatin, in addition to its elimination from the vitreous humor via bulk flow and passive diffusion. Thus, the efflux mechanism of pravastatin across the BRB is similar to that across the BBB (Kikuchi et al., 2004).
Moreover, pravastatin is a substrate of multidrug resistance–associated protein 2 (ABCC2/Mrp2), ABCC4 (Mrp4), and breast cancer resistance protein (ABCG2/Bcrp) (Yamazaki et al., 1997, Hirano et al., 2005; Uchida et al., 2007). Mrp2 protein is expressed in human retina and RPE (Pelis et al., 2009), but Mrp2 cannot be detected in the mouse inner BRB (Tachikawa et al., 2008) or in rat brain (Ohtsuki et al., 2007) at the mRNA level. Mrp4 is found on the luminal side of the mouse inner BRB (Tagami et al., 2009) and both sides of the BBB (Kusuhara and Sugiyama, 2005). Bcrp is localized to the luminal side of the mouse inner BRB (Asashima et al., 2006) as well as the BBB. Taken together, the ATP-binding cassette (ABC) transporters may be involved in the transport of pravastatin across the BRB and BBB. Based on our results and previous reports, asymmetric pravastatin transport across both barriers would be expected (Supplemental Fig. 3). It is important to reveal whether the ABC transporters are involved in the efflux transport of pravastatin across the BRB and BBB in future experiments.
Recently, in a comparison between diabetic and normal rats, increased expression of oatp1a4 mRNA and decreased expression of MRP2 mRNA were found in the liver, as well as a markedly altered pravastatin disposition (Hasegawa et al., 2010). Decreases in renal oat3 function and expression have also been reported in diabetic rats (Phatchawan et al., 2014). It is likely that there are changes in BRB transporters in diabetic rats. The functional and expressional differences in transporters between normal and pathologic conditions might be exploitable when treating diabetic retinopathy.
Oatp1a4 falls into the same subfamily as human OATP1A2 (Hagenbuch and Meier, 2004). OATP1A2 and OAT3 are expressed in human retina/choroid at the mRNA level (Chan et al., 2015), as well as at the BBB (Cha et al., 2001; Gao et al., 2015). In addition, both transporters recognize pravastatin (Takeda et al., 2004; Shirasaka et al., 2010). Therefore, our results support the clinical effects of pravastatin on diabetic retinopathy without the CNS side effects. Pravastatin was transported into the target tissues, retinal endothelial cells, via uptake transporters and showed pharmacologic effects, whereas pravastatin taken into brain endothelial cells was transported via efflux transporters before reaching the CNS.
In conclusion, pravastatin is transported across the BRB and BBB via uptake and efflux transporters in both directions, and the efflux transporters are dominant, based on the lower values of the RUI and BUI than those expected from the lipophilicity. These findings provide useful information for the development of new approaches to the design of precision pharmaceuticals for the systemic delivery of drugs to retinal vessels without CNS side effects.
Acknowledgments
Authorship Contributions
Participated in research design: Fujii, Kawazu, Hosoya.
Conducted experiments: Fujii, Setoguchi.
Contributed new reagents or analytic tools: Fujii.
Performed data analysis: Fujii.
Wrote or contributed to the writing of the manuscript: Fujii, Kawazu, Hosoya.
Footnotes
- Received July 9, 2015.
- Accepted October 1, 2015.
↵
This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- ABC
- ATP-binding cassette
- BBB
- blood-brain barrier
- Bcrp
- breast cancer resistance protein
- BRB
- blood-retinal barrier
- BUI
- brain uptake index
- CNS
- central nervous system
- E17βG
- estradiol 17-β glucuronide
- Mrp
- multidrug resistance–associated protein
- oat
- organic anion transporter
- oatp
- organic anion transporting polypeptides
- RPE
- retinal pigment epithelium
- RUI
- retinal uptake index
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
