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

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.

     

Propranolol Transport Across the Inner Blood–Retinal Barrier: Potential Involvement of a Novel Organic Cation Transporter

The influx transport of propranolol across the inner blood–retinal barrier (BRB) was investigated. In the in vivo analysis of carotid artery single-injection method, [³H]propranolol uptake by the retina was greater than that of an internal reference compound, and was reduced by several organic cations. In the in vitro uptake study, TR-iBRB2 cells, an in vitro model of the inner BRB, showed a time-, concentration-, pH- and temperature-dependent [³H]propranolol uptake, suggesting the involvement of a carrier-mediated transport process in the influx of propranolol across the inner BRB. In the inhibition study, various organic cations, including drugs and candidates for the treatment of the retinal diseases, inhibited the [³H]propranolol uptake by TR-iBRB2 cells with no significant effects by the substrates and inhibitors of well-characterized organic cation transporters, suggesting that the influx transport of propranolol is performed by a novel transporter at the inner BRB. An analysis of the relationship between the inhibitory effect and the lipophilicity of inhibitors suggests a lipophilicity-dependent inhibitory effect of amines on the [³H]propranolol uptake by TR-iBRB2 cells. These results showed that influx transport of propranolol across the inner BRB is performed by a carrier-mediated transport process, suggesting the involvement of a novel organic cation transporter.     

Blood-to-retina transport of riboflavin via RFVTs at the inner blood-retinal barrier

Riboflavin (vitamin B₂) supply to the retina across the inner blood-retinal barrier (BRB) was investigated. In rats, the apparent influx permeability clearance of [³H]riboflavin (62.8 μL/(min·g retina)) was much higher than that of a non-permeable paracellular marker, suggesting the facilitative influx transport of riboflavin across the BRB. The retinal uptake index (RUI) of [³H]riboflavin was 59.0%, and significantly reduced by flavin adenine dinucleotide (FAD), but not by l-ascorbic acid, suggesting the substrate specificity of riboflavin transport. TR-iBRB2 cells, an in vitro model of the inner BRB, showed a temperature- and concentration-dependent [³H]riboflavin uptake with a K_m of 113 nM, suggesting that the influx transport of riboflavin across the inner BRB involves a carrier-mediated process. [³H]Riboflavin uptake by TR-iBRB2 cells was slightly altered by Na⁺- and Cl⁻-free buffers, suggesting that riboflavin transport at the inner BRB is preferentially Na⁺- and Cl⁻-independent. [³H]Riboflavin uptake by TR-iBRB2 cells was significantly reduced by riboflavin analogues while the uptake remained unchanged by other vitamins. The function and inhibition profile suggested the involvement of riboflavin transporters (SLC52A/RFVT) in riboflavin transport at the inner BRB, and this is supported by expression and knockdown analysis of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3) in TR-iBRB2 cells.     

Blood-to-Retina Transport of Imperatorin Involves the Carrier-Mediated Transporter System at the Inner Blood-Retinal Barrier

This study investigated the mechanism of transporting imperatorin across the inner blood-retinal barrier (iBRB). The carotid artery single injection method was used to calculate the retinal uptake index (RUI) of [³H]imperatorin in vivo, whereas the retinal capillary endothelial cell lines were used for the in vitro uptake and mRNA expression assays. RUI value of [³H]imperatorin was greater than that of the reference compound ([¹⁴C]n-butanol). [³H]Imperatorin significantly reduced the RUI in the presence of neuroprotective organic cationic drugs at 10 mM. However, tetraethylammonium and p-aminohippuric acid showed no significant effects. [³H]Imperatorin uptake by TR-iBRB2 cells was time-, pH-, energy-, and concentration-dependent with a K_m value of 679 ± 130 μM. In addition, the uptake study showed insensitivity to sodium and membrane potential. Various organic cations including pyrilamine, nicotine, and clonidine significantly reduced the uptake of [³H]imperatorin, whereas organic anions and monocarboxylic acids did not. Furthermore, the mRNA expression level dropped markedly with rOCTN1, rOCTN2, rPMAT, and rMATE1 small interfering RNAs in the transfection study. Moreover, [³H]imperatorin uptake remained neutral with small interfering RNA transfections. Our results indicate that imperatorin transport across the iBRB involves carrier-mediated transporter system.     

Involvement of reduced folate carrier 1 in the inner blood-retinal barrier transport of methyltetrahydrofolate

The purpose of this study was to elucidate the mechanism of methyltetrahydrofolate (MTF) transport at the inner blood-retinal barrier (inner BRB). The characteristics and function of MTF transport at the inner BRB were examined using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model of the inner BRB. The [3H]MTF uptake by TR-iBRB2 cells increased with lowering extracellular pH and was Na+- and Cl--independent. The [3H]MTF uptake was concentration-dependent with a K(m) of 5.1 microM. This process was inhibited by reduced folate carrier 1 (RFC1) substrates, such as methotrexate and formyltetrahydrofolate, in a concentration-dependent manner with an IC50 of 8.7 and 2.8 microM, respectively, suggesting that RFC1 mediates MTF uptake in TR-iBRB2 cells. Although both RFC1 and proton-coupled folate transporter (PCFT) mRNA, which are pH-sensitive folate transporters, are expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells, the expression level of RFC1 mRNA was 83- and 49-fold greater than that of PCFT, respectively. Taken together, the above findings are consistent with the involvement of RFC1 in the inner BRB transport of MTF.     

Inner blood-retinal barrier mediates l-isomer-predominant transport of serine

D-serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the blood-to-retina transport of [(3) H]L-serine with an influx clearance of 49.9 μL/(min·g retina), which is greater than that of [(3) H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [(3) H]L-Serine and [(3) H]D-serine uptake by TR-iBRB2 cells took place in an Na(+)-dependent and a concentration-dependent manner with Michaelis constant values of 97.5 μM and 9.63 mM, respectively. The uptake process of [(3) H]L-serine and [(3) H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina.     

Altered Brain Uptake of Therapeutics in a Triple Transgenic Mouse Model of Alzheimer’s Disease

Purpose

The purpose of this study was to systematically assess the impact of Alzheimer’s disease (AD)-associated blood–brain barrier (BBB) alterations on the uptake of therapeutics into the brain.

Methods

The brain uptake of probe compounds was measured in 18–20 month old wild type (WT) and triple transgenic (3×TG) AD mice using an in situ transcardiac perfusion technique. These results were mechanistically correlated with immunohistochemical and molecular studies.

Results

The brain uptake of the paracellular marker, [¹⁴C] sucrose, did not differ between WT and 3×TG mice. The brain uptake of passively diffusing markers, [³H] diazepam and [³H] propranolol, decreased 54–60% in 3×TG mice, consistent with a 33.5% increase in the thickness of the cerebrovascular basement membrane in 3×TG mice. Despite a 42.4% reduction in P-gp expression in isolated brain microvessels from a sub-population of 3×TG mice (relative to WT mice), the brain uptake of P-gp substrates ([³H] digoxin, [³H] loperamide and [³H] verapamil) was not different between genotypes, likely due to a compensatory thickening in the cerebrovascular basement membrane counteracting any reduced efflux of these lipophilic substrates.

Conclusion

These studies systematically assessed the impact of AD on BBB drug transport in a relevant animal model, and have demonstrated a reduction in the brain uptake of passively-absorbed molecules in this mouse model of AD.     

Effects of Bisphosphonates on Glucose Transport in a Conditionally Immortalized Rat Retinal Capillary Endothelial Cell Line (TR-iBRB Cells)

The objective of the present study was to elucidate the effect of bisphosphonates, anti-osteoporosis agents, on glucose uptake in retinal capillary endothelial cells under normal and high glucose conditions. The change of glucose uptake by pre-treatment of bisphosphonates at the inner blood-retinal barrier (iBRB) was determined by measuring cellular uptake of [³H]3-O-methyl glucose (3-OMG) using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB cells) under normal and high glucose conditions. [³H]3-OMG uptake was inhibited by simultaneous treatment of unlabeled D-glucose and 3-OMG as well as glucose transport inhibitor, cytochalasin B. On the other hand, simultaneous treatment of alendronate or pamidronate had no significant inhibitory effect on [³H]3-OMG uptake by TR-iBRB cells. Under high glucose condition of TR-iBRB cells, [³H]3-OMG uptake was increased at 48 h. However, [³H]3-OMG uptake was decreased significantly by pre-treatment of alendronate or pamidronate compared with the values for normal and high glucose conditions. Moreover, geranylgeraniol (GGOH), a mevalonate pathway intermediate, increased the uptake of [³H]3-OMG reduced by bisphosphonates pre-treatment. But, pre-treatment of histamine did not show significant inhibition of [³H]3-OMG uptake. The glucose uptake may be down regulated by inhibiting the mevalonate pathway with pre-treatment of bisphosphonates in TR-iBRB cells at high glucose condition.     

<Emphasis Type="Italic">In Vivo</Emphasis> and <Emphasis Type="Italic">In Vitro</Emphasis> Evidence for Brain Uptake of 4-Phenylbutyrate by the Monocarboxylate Transporter 1 (MCT1)

Purpose

4-Phenylbutyrate (4-PBA) is expected to be a potential therapeutic for several neurodegenerative diseases. These activities require 4-PBA transport into the brain across the blood-brain barrier (BBB). The objective of the present study was to characterize the brain transport mechanism of 4-PBA through the BBB.

Methods

The brain transport of 4-PBA across the BBB was investigated following intravenous (IV) injection and internal carotid artery perfusion (ICAP) in vivo. The mechanism of transport was examined using TR-BBB cells, an in vitro model of the BBB.

Results

The volume of distribution (V_D) of 4-PBA by rat brain was about 7-fold greater than that of sucrose, a BBB impermeable vascular space marker, suggesting the blood-to-brain transport of 4-PBA through the BBB in the physiological state. [¹⁴C]4-PBA uptake by TR-BBB cells showed time-, pH- and concentration-dependence with a K _m of 13.4 mM at pH 7.4 and 3.22 mM at pH 6.0. The uptake was Na⁺ independent, and was significantly inhibited by alpha-cyano-4-hydroxycinnamate (a typical inhibitor for monocarboxylate transport), endogenous monocarboxylate compounds and monocarboxylic drugs. Lactate and valproate competitively inhibited [¹⁴C]4-PBA uptake with K _i value of 13.5 mM and 7.47 mM, respectively. These results indicate the role of monocarboxylate transporters (MCTs) in 4-PBA transport into the brain at the BBB. TR-BBB cells expressed mRNA of rMCT1, 2, and 4, especially, rMCT1 showed high mRNA expression level. In addition, [¹⁴C]4-PBA uptake was inhibited by rMCT1 specific small interfering RNA.

Conclusion

The transport mechanism of 4-PBA from blood to brain across the BBB likely involves MCT1.

     

Riboflavin transport mediated by riboflavin transporters (RFVTs/SLC52A) at the rat outer blood-retinal barrier

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B₂) across the blood-retinal barrier (BRB). In the present study, the blood-to-retina supply of riboflavin across the outer BRB was assessed in RPE-J cells, a rat-derived in vitro cell model of the outer BRB that is formed by the retinal pigment epithelial cells. In the directional uptake analysis on collagen-coated Transwell® inserts, RPE-J cells showed higher basal-to-cell (B-to-C) uptake (22.8 μL/mg protein) of [³H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [³H]riboflavin with a K_m of 297 nM, suggesting the involvement of carrier-mediated process in the blood-to-retina transport of riboflavin across the outer BRB. In RPE-J cells, [³H]riboflavin uptake was affected under a K⁺-replacement condition while no effect was observed under a choline-replacement condition and at different pH values. Uptake of [³H]riboflavin by RPE-J cells was markedly reduced by riboflavin, flavin adenine dinucleotide (FAD), and lumichrome with no significant effect noted for other vitamins. The obtained results suggested the involvement of riboflavin transporters (SLC52A/RFVT) at the outer BRB, and this is supported by the expression and knockdown analyses of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3).     

Blood–brain and retinal barriers show dissimilar ABC transporter impacts and concealed effect of P‐glycoprotein on a novel verapamil influx carrier

Background and Purpose

The respective impact and interplay between ABC (P‐glycoprotein/P‐gp/Abcb1a, BCRP/ABCG2, MRP/ABCC) and SLC transporter functions at the blood–brain barrier (BBB) and blood–retinal barriers (BRB) are incompletely understood.

Experimental Approach

We measured the initial cerebral and retinal distribution of selected ABC substrates by in situ carotid perfusion using P‐gp/Bcrp knockout mice and chemical ABC/SLC modulation strategies. P‐gp, Bcrp, Mrp1 and Mrp4 were studied by confocal retina imaging.

Key Results

Chemical or physical disruption of P‐gp increased [³H]‐verapamil transport by ~10‐fold at the BBB and ~1.5‐fold at the BRB. [³H]‐Verapamil transport involved influx‐mediated by an organic cation clonidine‐sensitive/diphenhydramine‐sensitive proton antiporter at both barriers; this effect was unmasked when P‐gp was partially or fully inhibited/disrupted at the BBB. Studies of [³H]‐mitoxantrone and [³H]‐zidovudine transport suggested, respectively, that Bcrp efflux was less involved at the BRB than BBB, whereas Mrps were significantly and similarly involved at both barriers. Confocal imaging showed that P‐gp and Bcrp were expressed in intra‐retinal vessels (inner BRB/iBRB) but absent from the blood/basal membrane of cells of the retinal pigment epithelium (outer BRB/oBRB/RPE) where, in contrast, Mrp1 and Mrp4 were localized.

Conclusions and Implications

P‐gp, Bcrp, Mrp1 and Mrp4 are differentially expressed at the outer and inner BRB, resulting in an altered ability to limit substrate distribution at the retina as compared with the BBB. [³H]‐Verapamil distribution is not P‐gp‐specific and involves a proton antiporter at both the BBB and BRB. However, this transport is concealed by P‐gp at the BBB, but not at the BRB, where P‐gp activity is reduced.

Abbreviations

ABC

ATP‐binding cassette

BBB

blood–brain barrier

BRB

blood–retina barrier

CV

choroidal vessels

DPH

diphenhydramine; Elac, elacridar

iBRB

inner blood–retina barrier

KO

knockout

oBRB

outer blood–retina barrier

PD

pharmacodynamics

P‐gp

P‐glycoprotein

pH_e

extracellular/vascular pH

pH_i

intracellular pH

PK

pharmacokinetics

RPE

retinal pigment epithelial cells

SLC

solute carrier

TKO

triple knockout [Abcb1a^−/−, Abcb1b^−/−, Abcg2^−/−] mice

WT

wild type

     

A polyspecific drug/proton antiporter mediates diphenhydramine and clonidine transport at the mouse blood-retinal barrier

Background and Purpose

Transporters at the blood-retinal barrier (BRB), as at the blood–brain barrier (BBB), regulate the distribution of compounds into the neural parenchyma. However, the expression of BRB transporters and their quantitative impact in vivo are still poorly understood.

Experimental Approach

Clonidine and diphenhydramine are substrates of a novel BBB drug/proton-antiporter. We evaluated their transport at the BRB by in situ carotid perfusion in wild-type or knocked-out mice for Oct1-3 (Slc22a1-3).

Key Results

At pharmacological exposure levels, carrier-mediated BRB influx was 2 and 12 times greater than the passive diffusion rate for clonidine and diphenhydramine, respectively. Functional identification demonstrated the involvement of a high-capacity potassium- and sodium-independent proton-antiporter that shared the features of the previously characterized clonidine, diphenhydramine and cocaine BBB transporter. The functional characterization suggests that SLC transporters Oct1-3, Mate1 (Slc47a1) and Octn1-2 (Slc22a4-5) are not involved. Melanin/retinal toxic drugs like antimalarials (amodiaquine, quinine), quinidine and tricyclic antidepressants (imipramine) acted as inhibitors of this proton-antiporter. The endogenous indole derivative tryptamine inhibited the transporter, unlike 5-HT (serotonin), dopamine or L-DOPA. Trans-stimulation experiments with [³H]-clonidine at the BRB indicated that diphenhydramine, nicotine, oxycodone, naloxone, tramadol, 3,4-methylenedioxyamphetamine (MDMA, ecstasy), heroin, methadone and verapamil are common substrates.

Conclusions and Implications

A proton-antiporter is physiologically involved in the transport of clonidine and diphenhydramine and is quantitatively more important than their passive diffusion flux at the mouse BRB. The features of this molecularly unidentified transporter highlight its importance in regulating drug delivery at the retina and suggest that it has the capacity to handle several drugs.     

Hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells

We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [3H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [3H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [3H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.     

Uptake of ANG1005, A Novel Paclitaxel Derivative, Through the Blood-Brain Barrier into Brain and Experimental Brain Metastases of Breast Cancer

Purpose

We evaluated the uptake of angiopep-2 paclitaxel conjugate, ANG1005, into brain and brain metastases of breast cancer in rodents. Most anticancer drugs show poor delivery to brain tumors due to limited transport across the blood-brain barrier (BBB). To overcome this, a 19-amino acid peptide (angiopep-2) was developed that binds to low density lipoprotein receptor-related protein (LRP) receptors at the BBB and has the potential to deliver drugs to brain by receptor-mediated transport.

Methods

The transfer coefficient (K_in) for brain influx was measured by in situ rat brain perfusion. Drug distribution was determined at 30 min after i.v. injection in mice bearing intracerebral MDA-MB-231BR metastases of breast cancer.

Results

The BBB K_in for ¹²⁵I-ANG1005 uptake (7.3?±?0.2?×?10^-3 mL/s/g) exceeded that for ³H-paclitaxel (8.5?±?0.5?×?10^-5) by 86-fold. Over 70% of ¹²⁵I-ANG1005 tracer stayed in brain after capillary depletion or vascular washout. Brain ¹²⁵I-ANG1005 uptake was reduced by unlabeled angiopep-2 vector and by LRP ligands, consistent with receptor transport. In vivo uptake of ¹²⁵I-ANG1005 into vascularly corrected brain and brain metastases exceeded that of ¹⁴C-paclitaxel by 4–54-fold.

Conclusions

The results demonstrate that ANG1005 shows significantly improved delivery to brain and brain metastases of breast cancer compared to free paclitaxel.     

Clobenpropit,a histamine H3 receptor antagonist/inverse agonist,inhibits [3H]-dopamine uptake by human neuroblastoma SH-SY5Y cells and rat brain synaptosomes

Background

Clobenpropit, a potent antagonist/inverse agonist at the histamine H₃ receptor (H₃R), reduced the cytotoxic action of 6-hydroxydopamine (6-OHDA) in neuroblastoma SH-SY5Y cells transfected with the human H₃R. We therefore set out to study whether this effect involved a receptor-independent action on dopamine transport.

Application of membrane permeability evaluated in in vitro analyses to estimate blood-retinal barrier permeability

The relationship between the in vitro membrane permeability and systemic blood-retinal barrier (BRB) permeability of drugs was investigated. To determine membrane permeability trend lines in this relationship, the apparent permeability (P(app)) and initial uptake rate (V) of 23 compounds were evaluated in a parallel artificial membrane permeability assay and the uptake study with a rat retinal endothelial cell line (TR-iBRB2 cells) for comparison with their retinal uptake index (RUI). The RUI values of compounds undergoing passive diffusion across the BRB were correlated with a log of the P(app) [RUI = 7.93 × 10 × exp (0.994 × log P(app)), r(2) = 0.660] and a log of the V [RUI = 26.5 × exp (1.55 × log V), r(2) = 0.581]. The RUI values of compounds undergoing carrier-mediated transport across the BRB were correlated with a log of the V [RUI = 26.5 × exp (0.887 × log V), r(2) = 0.559]. These results showed that the membrane permeability trend lines derived from the RUI and V values reflect the transport of drugs at the BRB, suggesting that an in vitro analysis-based estimation of the BRB permeability can be obtained using TR-iBRB2 cells and membrane permeability trend lines.     

Design and Characterization of a Novel Fluorinated Magnetic Resonance Imaging Agent for Functional Analysis of Bile Acid Transporter Activity

Purpose

To synthesize a trifluorinated bile acid that can be used for ¹⁹F magnetic resonance imaging (MRI) of bile acid enterohepatic circulation, characterize its in vitro transporter affinity, stability, and ¹⁹F-MRI signal, and assess its ability to concentrate in the gallbladder of C57BL/6 mice.

Methods

Target compound CA-lys-TFA was synthesized and tested for affinity toward the apical sodium dependent bile acid transporter (hASBT) and the Na+/taurocholate cotransporting polypeptide (hNTCP). In a pilot study, fasted mice were gavaged with vehicle control, 150 mg/kg or 300 mg/kg CA-lys-TFA. CA-lys-TFA in gallbladder, liver and plasma at t?=?5 h was quantified. Additionally, a 24-h time course (24 mice across eight time points) was studied using 50 mg/kg CA-lys-TFA.

Results

CA-lys-TFA was a potent substrate of hASBT (Kt?=?39.4 μM, normalized Vmax?=?0.853) and hNTCP (Kt?=?8.99 μM, normalized Vmax?=?0.281). ¹⁹F MRI phantom imaging showed linear signal-concentration dependence. In vivo studies showed that rapid accumulation of CA-lys-TFA in the gallbladder was maximal within 4–7 h.

Conclusions

These findings suggest that CA-lys-TFA, a fluorinated non-radioactive bile acid analogue, has potential for use in MRI to measure in vivo bile acid transport and diagnose bile acid malabsorption and other conditions associated with impaired bile acid transport.     

Evaluation of an Immortalized Retinal Endothelial Cell Line as an In Vitro Model for Drug Transport Studies Across the Blood-Retinal Barrier

Purpose. To evaluate the growth and barrier properties of an immortalized rat retinal endothelial cell line (TR-iBRB) maintained on permeable membrane for drug transport studies. Methods. TR-iBRB cells were grown on permeable membrane filters. The effect of coating material on cell growth was investigated. Transport of [¹⁴C]-3-O-methyl-D-glucose (3-OMG), AGN 194716, AGN 195127, AGN 197075, acebutolol, alprenolol, atenolol, brimonidine, carbamazepine epoxide (CBZ-E), metoprolol, nadolol, rhodamine 123, and sotalol was measured across the cultured cell layer to determine the apparent permeability coefficients (P_app). Rhodamine 123 uptake into these cells in the presence of these test compounds was evaluated. Western blot was performed to detect the efflux transporter P-glycoprotein (P-gp). Bidirectional transport in MDR1-MDCK cell monolayers overexpressing the human P-gp was measured for AGN 197075. Results. TR-iBRB cells form confluent cell layers when grown on fibronectin-coated membrane and exhibit characteristic spindle-shaped morphology. A good correlation between P_app and cLogD (pH 7.4) of the compounds tested was observed, except for 3-OMG, AGN 197075, and rhodamine 123, which are substrates of carrier-mediated transport systems such as P-gp and a glucose transporter (GLUT1). When grown on permeable membrane, TR-iBRB cells expressed functional P-gp and GLUT1. Conclusions. TR-iBRB cells, when grown on permeable membrane, provide a useful tool for predicting permeability across the BRB. The usefulness of this model for high-throughput screening and rank ordering of drug candidates intended for the back of the eye in treatment of ocular diseases needs further characterization upon correlation with in vivo data.     

Persistent effects of chronic clozapine on the cellular and behavioral responses to LSD in mice

Rationale

In schizophrenia patients, optimal treatment with antipsychotics requires weeks to months of sustained drug therapy. However, single administration of antipsychotic drugs can reverse schizophrenia-like behavioral alterations in rodent models of psychosis. This raises questions about the physiological relevance of such antipsychotic-like activity.

Objective

This study evaluates the effects of chronic treatment with clozapine on the cellular and behavioral responses induced by the hallucinogenic serotonin 5-HT_2A receptor agonist lysergic acid diethylamide (LSD) as a mouse model of psychosis.

Method

Mice were treated chronically (21 days) with 25 mg/kg/day clozapine. Experiments were conducted 1, 7, 14, and 21 days after the last clozapine administration. [³H]Ketanserin binding and 5-HT _2A mRNA expression were determined in mouse somatosensory cortex. Head-twitch behavior, expression of c-fos, which is induced by all 5-HT_2A agonists, and expression of egr-1 and egr-2, which are LSD-like specific, were assayed.

Results

Head-twitch response was decreased and [³H]ketanserin binding was downregulated in 1, 7, and 14 days after chronic clozapine. 5-HT _2A mRNA was reduced 1 day after chronic clozapine. Induction of c-fos, but not egr-1 and egr-2, was rescued 7 days after chronic clozapine. These effects were not observed after short treatment (2 days) with clozapine or chronic haloperidol (1 mg/kg/day).

Conclusion

Our findings provide a murine model of chronic atypical antipsychotic drug action and suggest downregulation of the 5-HT_2A receptor as a potential mechanism involved in these persistent therapeutic-like effects.