Dmd066266 1956..1959

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 bloodretinal 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 [H]pravastatin were lower than those expected based on its lipophilicity, suggesting that the influx transport across the BRB and BBB was less than the reversedirection transport. The RUI and BUI values for [H]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, [H]pravastatin and the bulk flow marker [C]D-mannitol were found to be eliminated biexponentially from the vitreous humor. The elimination rate constant of [H]pravastatin during the terminal phase was 1.66-fold greater than that of [C]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-b glucuronide (E17bG) influx was evaluated as a model substrate of oatp1a4 and oat3 because the efflux mechanism of E17bG has already been clarified; that is, E17bG 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 dx.doi.org/10.1124/dmd.115.066266. s 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; E17bG, estradiol 17-b glucuronide; Mrp, multidrug resistance–associated protein; oat, organic anion transporter; oatp, organic anion transporting polypeptides; RPE, retinal pigment epithelium; RUI, retinal uptake index. 1956 http://dmd.aspetjournals.org/content/suppl/2015/10/02/dmd.115.066266.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from