Imperatorin is Transported through Blood-Brain Barrier by Carrier

Original Article

Biomol Ther 25(4), 441-451 (2017)

Imperatorin is Transported through Blood-Brain Barrier by Carrier-Mediated Transporters Temdara Tun1 and Young-Sook Kang1,* College of Pharmacy, Drug Information Research Institute and Research Center for Cell Fate Control, Sookmyung Women’s University, Seoul 04310, Republic of Korea

1

Abstract Imperatorin, a major bioactive furanocoumarin with multifunctions, can be used for treating neurodegenerative diseases. In this study, we investigated the characteristics of imperatorin transport in the brain. Experiments of the present study were designed to study imperatorin transport across the blood-brain barrier both in vivo and in vitro. In vivo study was performed in rats using single intravenous injection and in situ carotid artery perfusion technique. Conditionally immortalized rat brain capillary endothelial cells were as an in vitro model of blood-brain barrier to examine the transport mechanism of imperatorin. Brain distribution volume of imperatorin was about 6 fold greater than that of sucrose, suggesting that the transport of imperatorin was through the blood-brain barrier in physiological state. Both in vivo and in vitro imperatorin transport studies demonstrated that imperatorin could be transported in a concentration-dependent manner with high affinity. Imperatorin uptake was dependent on proton gradient in an opposite direction. It was significantly reduced by pretreatment with sodium azide. However, its uptake was not inhibited by replacing extracellular sodium with potassium or N-methylglucamine. The uptake of imperatorin was inhibited by various cationic compounds, but not inhibited by TEA, choline and organic anion substances. Transfection of plasma membrane monoamine transporter, organic cation transporter 2 and organic cation/carnitine transporter 2/1 siRNA failed to alter imperatorin transport in brain capillary endothelial cells. Especially, tramadol, clonidine and pyrilamine inhibited the uptake of [3H]imperatorin competitively. Therefore, imperatorin is actively transported from blood to brain across the blood-brain barrier by passive and carrier-mediated transporter. Key Words: Imperatorin, Alzheimer’s disease, Blood-brain barrier, Proton coupled antiporter

INTRODUCTION

PD (Kim et al., 2002; Sigurdsson and Gudbjarnason, 2007). It has a small molecular weight (270 g/mol) and a large value of log P (3.65). Recently, it has been reported that imperatorin is highly passed through the blood-brain barrier (BBB) according to in vivo (oral administration) and in vitro permeability data using LC-MS/MS analysis method (Lili et al., 2013). However, the characteristics of imperatorin transport through the BBB remains unknown. Thus, it is important to investigate the transport characteristics of imperatorin to predict the effect of imperatorin on AD and PD. BBB is formed by three cellular elements (astrocytes, pericytes and endothelial cells) at the lining of the tight junction. It expresses multiple transporters, which can influence the BBB permeability of their substrates (Ohtsuki and Terasaki, 2007). These transporters can mediate the blood-to-brain influx for nutrient and other essential molecules as well as the

Imperatorin is isolated from the root of Angelica dahurica. It is a major bioactive furanocoumarin (Baek et al., 2000). It has long been recognized that imperaotrin exhibits many biological properties such as anticancer (Kozioł and SkalickaWoźniak, 2016), antibacterial (Stavri and Gibbons, 2005) antiinflammatory (Abad et al., 2001) and HIV replication-inhibiting activities (Sancho et al., 2004). It is also therapeutically helpful for disorders with high anxiety level and memory impairment (Budzynska et al., 2012). Alzheimer’s disease (AD) and Parkinson’s disease (PD) are neurodegenerative diseases characterized by cholinergic dysfunction with cholinergic deficiency in the brain (Kozioł and Skalicka-Woźniak, 2016). Imperatorin as an inhibitor of acetylcholinesterase (AChE) might be useful for treating AD and

Received Apr 4, 2017 Revised Apr 12, 2017 Accepted Apr 13, 2017 Published Online May 30, 2017

Open Access https://doi.org/10.4062/biomolther.2017.082 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Corresponding Author

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Copyright © 2017 The Korean Society of Applied Pharmacology

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Biomol Ther 25(4), 441-451 (2017)

lowing administration, blood samples (0.3 mL) were collected via polyethylene 50 (PE 50) tube implanted in the left femoral artery at 0.25-60 min. At 60 min after injection, brain and other organs were collected. Organ samples were solubilized (Tri-Carb 2810TR; PerkinElmer, USA) with ultima gold (PerkinElmer, Packard Co with solunene-350 (PerkinElmer, Waltham, MA, USA) and radioactivity was counted by using Tri-Carb liquid Plasma radioactivity (dpm/ml) wasa converted to the scintillapercentage of injected dos tion counter2810TR; (Tri-Carb 2810TR; PerkinElmer) withgold ultima gold (Tri-Carb PerkinElmer, USA) with ultima (PerkinElmer, Packard C (PerkinElmer). milliliter (ml). The %ID/ml was fit to a bi-exponential equation (1): Plasma (dpm/ml) was wasconverted converted to the per- of injected do Plasma radioactivity radioactivity (dpm/ml) to the percentage −𝐾𝐾2per 𝑡𝑡 + 𝐴𝐴 (ID) 𝑡𝑡 (1) centage of injected milliliter (ml). The %ID/ml %ID/ml = 𝐴𝐴1 𝑒𝑒 −𝐾𝐾1dose 2 𝑒𝑒 milliliter The %ID/mlequation was fit to(1): a bi-exponential equation (1): was fit to a(ml). bi-exponential The intercepts (A1 and A2) and the slopes (k1 and k2) were used to co (1) %ID/ml = 𝐴𝐴1 𝑒𝑒 −𝑘𝑘1 𝑡𝑡 + 𝐴𝐴2 𝑒𝑒 −𝑘𝑘2 𝑡𝑡 (1) pharmacokinetic parameters, The and AA2) and the slopes (k1 and k2) were used to c The intercepts intercepts (A1 (A1 and 2) and the slopes (k1 and k2) were Pharmacokinetic parameters were computed as described previously (Lee et a used to compute the pharmacokinetic parameters. pharmacokinetic parameters, Pharmacokinetic parameters were computed as described obtain the area under the plasma concentration curve (AUC) at 60 min. previously (Lee et al., 2014) to obtain the area under the plasPharmacokinetic parameters were computed as described previously (Lee et ma concentration curve (AUC) at 60area min.(PS) product or organ clearance (μl/m The BBB permeability-surface The BBB permeability-surface area (PS) product or organ obtain the area under the plasma concentration curve (AUC) at 60 min. clearance (μl/min/g) determined using determined using thewas following equation (2): the following equation (2): The BBB permeability-surface area (PS) product or organ clearance (μl/

brain-to-blood efflux to eliminate metabolites and neurotoxic compounds from brain (Ohtsuki and Terasaki, 2007). Several influx and efflux drug transporter are expressed at the BBB, including sodium-independent glucose transporter (GLUT1/Slc2a1), monocarboxylate transporter 1 (MCT1/Slc16a1), amino acid transporter, organic anion transporter 3 (Oat3/Slc22a8), organic anion-transporting polypeptides (Oatps/Slco) and multidrug resistance-associated protein (Mrps/ ABCC) (Ohtsuki and Terasaki, 2007). Organic cation transporters (Oct1-3/ Slc22a1-3), high-affinity choline transporter (ChT/Slc5a7), organic cation/carinitine transporters 1-2 (Octn1-2/Slc22a4-5), plasma membrane monoamine transporter (Pmat/Slc29a4), and multidrug and toxin extrusion protein (Mate/Slc47a) are involved in the influx and efflux transport of various cationic drugs (Okura et al., 2008; Roth et al., 2012). Organic cation drugs and opioids such as pyrilamine (Okura et al., 2008), oxycodone (Okura et al., 2008), diphenhydramine (Sadiq et al., 2011), tramadol (Kitamura et al., 2014), nicotine (Cisternino et al., 2013) and clonidine (André et al., 2009) are transported by the proton coupled antiporter. However, the molecular nature of this antiporter remains unknown. It has been reported that this transporter is dependent on energy and oppositely directed proton gradient. However, it is independent on membrane potential or sodium (Shimomura et al., 2013). The objective of the present study was to investigate the transport mechanism of imperatorin across the BBB using in vivo intravenous injection (IV) and in situ internal carotid artery perfusion (ICAP) techniques. To clarify the functional properties of imperatorin influx at the BBB and its interaction with several transporters of substrates, in vitro uptake studies, Real-Time PCR and siRNA transfection were performed using conditionally immortalized rat brain capillary endothelial cells (TR-BBB cells).

[𝑉𝑉 −𝑉𝑉 ]𝐶𝐶 (t)

𝑡𝑡

𝐷𝐷 0 𝑝𝑝 (𝑡𝑡) = ∫(2): determined equation , 𝐴𝐴𝐴𝐴𝐴𝐴 𝐶𝐶 (𝑡𝑡)𝑑𝑑𝑑𝑑 (2) (2) PS productusing = 𝑡𝑡the following 0 𝑝𝑝

∫0 𝐶𝐶𝑝𝑝 (𝑡𝑡)𝑑𝑑𝑑𝑑

where VD is the brain/plasma ratio brain volume of distribution, an [𝑉𝑉𝐷𝐷terminal −𝑉𝑉0 ]𝐶𝐶𝑝𝑝 (t) 𝑡𝑡 or the brain/plasma the brain volume where VD is the , 𝐴𝐴𝐴𝐴𝐴𝐴 (𝑡𝑡) =ratio PS product = terminal ∫0 𝐶𝐶or 𝑡𝑡 𝑝𝑝 (𝑡𝑡)𝑑𝑑𝑑𝑑 (2) (𝑡𝑡)𝑑𝑑𝑑𝑑 𝐶𝐶 ∫ 𝑝𝑝 of distribution, and0 for V0 is plasma volumes forCthe(t)respective is the terminal plasma con plasma volumes thethe respective organs and p organs and Cp (t) is the terminal plasma concentration (%ID/ is the terminal brain/plasma ratio or the brain volume distribution, a where V D ml). The terminal brain uptake, expressed as %ID/g brain, wasof (%ID/ml). The terminal brain uptake, expressed as %ID/g brain, was calculated fr calculated from the PS (μl/min/g) and the 60-min plasma AUC plasma volumes forthe thefollowing respective organs and Cp (t) is the terminal plasma co (%ID min/ml) using (μl/min/g) and the 60-min plasma equation AUC (%ID(3): min/ml) using the following equation

(%ID/ml). The terminal brain uptake, expressed as %ID/g brain, was calculated f %ID/g (t) = PS product × AUC (t) (3) %ID/g (t) = PS product × AUC (t) (3) (μl/min/g) and the 60-min plasma AUC (%ID min/ml) using the following equatio Brain uptake index method (BUI): BUI technique was performed as reported previously (Suzuki et al., 2002). After Brain uptake index method (BUI) the common carotid arrat%ID/g was anesthetized ketamine, (t) = PS productwith × AUC (t) (3) tery was injected with 200 ml Ringer-HEPES buffer containBUI technique was performed as reported previously (Suzuki et al., 2002). Aft ing [3H]imperatorin (2.5 mCi) with or without unlabeled inhibitor 14 compound andindex [ C]n-butanol (0.5 mCi) used an internal Brain uptake method (BUI) anesthetized with ketamine, the common carotidasartery was injected with 200 µ reference compound. Rat was decapitated 15 s after injection radioactivity was performed using Tri-Carb Liquid Scintillation Counters. distri BUI technique was performed as reported(2.5 previously (Suzuki et al., The 2002). Af andHEPES cerebrum were dissolved in solunene-350. Their radioacµCi)with or without unlabeled buffer containing [3H]imperatorin tivity was performed using Tri-Carb Liquid Scintillation Coun3 3 characteristic ofwith [ H]imperatorinthe were expressed using the percentage of with [ H]impe 3 carotid anesthetized common was injected 200 ters. The distribution characteristic of [used H]imperatorin were C]n-butanol (0.5 µCi) as anartery internal reference compound compound and [14ketamine, 3 14the percentage of [ H]imperatorin uptake expressed using uptake relative to [ containing C]n-butanol that was expressed byµCi)with Eq (4): or without unlabele 14 H]imperatorin (2.5 HEPES [3was decapitated 15s after that injection and cerebrum were relative to [buffer C]n-butanol expressed by Eq (4):dissolved in solunene-3

MATERIALS AND METHODS Radioisotope and reagents

Radiolabeled compound [3H]imperatorin (3.7 Ci/mmol) was purchased from American Radiolabeled Chemical, Inc (St. Louis, MO, USA). Unlabeled compounds such as imperatorin, tramadol hydrochloride, pyrilamine maleate salt, verapamil hydrochloride, quinidine, nicotine, clonidine hydrochloride, 1-Methyl-4-phenylpyridinium ion (MPP+) and other compounds were purchased from Sigma Aldrich (St. Louis, MO, USA).

Animals

Male Sprague-Dawley rats (SD rats, 7 weeks, 250-350 g) were purchased from Koatech Inc (Pyeongtaek, Korea). All animal experiments were approved by the Committee of the Ethics of Animal Experimentation of Sookmyung Women’s University (Seoul, Korea; Approval No.: SMWUIACUC-16017-014).

C]n-butanol (0.5 µCi) used as an internal reference compoun compound and [14[3H] {[14C](dpm in the brain)} 페이지 6 / 40 BUI (%) = [3H] X 100 (4) (4) in the injectate solution)} decapitated {[14C] 15s(dpm after injection and cerebrum were dissolved in solunene-3

Internal carotid artery perfusion technique: 페이지 6 / 40ICAP technique was performed as reported previously (Takasato et al., 1984; Lee and artery Kang, perfusion 2016). SD rats were anesthetized with Internal carotid technique ketamine/xylazine (100 mg/kg and 2 mg/kg). [3H]Imperatorin (270 nM) with or without unlabeled imperatorin and inhibitor ICAP technique was performed as reported previously and Kang, 2016; Tak compounds were diluted in KHB and perfused into the(Lee internal

In vivo brain uptake study

Intravenous injection technique (Pharmacokinetic): Pharmacokinetic parameters and brain uptake of [3H]imperatorin were investigated in rats following a single IV injections according to previous reports (Pardridge et al., 1994; Lee et al., 2014). SD rats were anesthetized ketamine/xylazine (100 mg/kg and 2 mg/kg; Yuhan, Seoul, Korea). [3H]Imperatorin (1.35 μM) was injected to the left femoral vein of SD rat. Fol-

carotid artery of rat at a flow rate of 4mL/min for 15 sec using al., 1984). SD rats were anesthetized with ketamine/xylazine (100 mg/kg and 2 m micro-syringe pump. To examine [3H]imperatorin transport on pH alteration, HCl or NaOH was added to KHB in some experi[3H]Imperatorin (270 nM) with or without unlabeled imperatorin and inhibitor com ments after gassing to bring the pH to 6.40, 7.40 or 8.40.

were diluted in KHB and perfused into the internal carotid artery of rat at a flow https://doi.org/10.4062/biomolther.2017.082

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4mL/min for 15 sec using micro-syringe pump. To examine [3H]imperatorin trans

pH alteration, HCl or NaOH was added to KHB in some experiments after gassing t the pH to 6.40, 7.40 or 8.40.

pH pH alteration, alteration, HCl HCl or or NaOH NaOH was was added added to to KHB KHB in in some some experiments experiments after after gassing gassing to to bring bring the the pH pH to to 6.40, 6.40, 7.40 7.40 or or 8.40. 8.40.

Tun and Kang. Imperatorin Transport across Blood-Brain Barrier

The The ionic ionic composition composition of of the the KHB KHB perfusate perfusate was was changed changed by by 256 256 mM mM of of mannitol mannitol +

-

instead and Cl Cl-.. Rat Rat was was also also perfused perfused with with carbonate-free carbonate-free HEPES-buffered HEPES-buffered saline. saline. instead of of Na Na+ and and Kd was the first order constant for non-saturable compoThe ionic composition of the KHB perfusate was changed 3 by 256 mM mannitol instead of Na+ andfrom nent respectively. Cl−. the Rat wasactivity also of V the was per VDD of of theof[[3H]imperatorin H]imperatorin was determined determined from the ratio ratio activity of disintegrations disintegrations per Vmax/Km (mL/min/mg protein) value was calculated as the perfused with carbonate-free HEPES-buffered saline. 3 minute (dpm/g) VD of per uptake clearance for saturable transport compound. The satuthe [gram H]imperatorin was determined from the ratio acminute per gram (dpm/g) of of brain. brain. rable component of imperatorin was plotted by non saturable tivity of disintegrations per minute per gram (dpm/g) of brain. uptake from total uptake in Eadie-Hofstee plot. [brain(dpm)/brain(g)] [brain(dpm)/brain(g)] V The inhibitory constant (Ki) was in the presence of 1 mM VDD(μℓ/g) (μℓ/g) = = [perfusate(dpm)/perfusate(µℓ)] Where VD was[perfusate(dpm)/perfusate(µℓ)] the brain volume of the [3H] compound and t was the perfusion timeclonidine (15 Sec). or pyrilamine. It was calculated from the tramadol, following equation The permeability surface was using the following equation(9): The BBB BBB surface area area (PS) (PS) product product was calculated calculated following For permeability the concentration-dependency experiment (André etusing al., the 2009) , theequation flux of The BBB permeability surface area (PS) product was calcu(5): lated using the following equation (5):the flux (J ; nmol/min/g of brain) isV=V (9) max×C/[Km×(1+I/Ki)+C]+Kd×C (5): [3H]imperatorin was calculated from given by equation in PS(μℓ/min/g)= (5) (µℓ/g)/ tt (min) (min) (5) (5) PS(μℓ/min/g)= V VDD(µℓ/g)/ (6):

where I was the concentration of each mutual inhibitory effects of compound, as the inhibitor concentration. Energy, sodium ion and membrane potential dependency where VD was the brain volume of the [3H] compound and t 페이지 페이지 77 // 40 40 of imperatorin uptake by TR-BBB cells were determined as was the perfusion time (15 Sec). Jin = PS× described previously (Kitamura et al., 2014). The uptake was For the C concentration-dependency experiment (André et tot (6) evaluated under ATP-depleted condition by 20 min pre-incual., 2009), the flux of [3H]imperatorin was calculated from the bation with 0.1% of sodium azide (NaN3) and 25 mM of roteflux (Jin; nmol/min/g of brain) is given by equation (6): none (dissolved in the transport buffer containing 0.2% DMSO) 3 [ H]imperatorin brain flux (Jin) or cellular velocity (µmol/min/g) was described as which were metabolic energy inhibitor. In this experiment, 10 = PS×C JinThe tot (6) mM D-glucose in the ECF buffer was replaced by 10 mM 3-Osaturable (Michaelis-Menten term). A passive unsaturable component has been measured methylglucose to reduce metabolic energy. To assess sodium The [3H]imperatorin brain flux (Jin) or cellular velocity ion dependency, the uptake was measured under sodium ion(mmol/min/g) was with equation (7):described as saturable (Michaelis-Menten free conditon by replacing NaCl in ECF buffer with NMG+. The term). A passive unsaturable component has been measured with equation (7): uptake also performed under membrane-disrupted condition by replacing of sodium ion with KCl followed by treatment with 10 mM valinomycin (transport buffer containing 0.2% DMSO) Jin= + passive Ctot (7) (7) for 10 min. In order to evaluate the effect of proton gradient on imperatorin uptake by TR-BBB cells, cells were simultaneously treated with 10 mM carbonyl cyanide-p-trifluoromewhere Ctot (mM) was the total imperatorin concentration in (FCCP, a protonophore). FCCP was perfusate incubation buffer, Vmax (mmol/min/g) is the maxiconcentration in perfusatethoxyphenylhydrazone or incubation buffer, where Cor tot (mM) was the total imperatorin dissolved in transport buffer containing 0.10% DMSO. For exmal velocity of transport, and Km (mM) of imperatorin was the 3 tracellular pHwas (pHthe concentration at theishaft-maximal carrier of velocity. Kpassive (mL/ the maximal velocity transport, and K imperatorin Vmax (µmol/min/g) e) dependent, [ H]imperatorin uptake at pH m (mM) of min/g) was an unsaturable component representing the rate 6.4 and pH 8.4. To examine the effect of intracellular pH (pHi) concentration at thediffusion. haft-maximal carrierfitted velocity. was an unsaturable transport by passive Data were usingKnonlinear dependent, cells were pre-treated with 30 mM of ammonium passive (µL/min/g) regression analysis. chloride (NH4Cl) for 30 min to reduce pHi and simultaneously 3 representing rate in transport by cells: passiveTR-BBB diffusion. Data werewith fitted usingNH4Cl to increase pHi (Okura et al., 2008). [component treated 30 mM H]Imperatorin uptakethe study TR-BBB cells were cultured according to a previously report (Terasanonlinear regression analysis. ki and Hosoya, 2001; Kang et al., 2002). For in vitro uptake RNA interference analysis study, [3H]imperatorin transport in TR-BBB cells was perFor gene silencing of a set of four siRNAs (GE Healthformed described previously (Kang et al., 2002). Cells were care Dharmacon, Inc., Landsmeer, Netherlands) specific for then[3incubated with 200 mL study transport buffer containing 135 nM rOctn2, rPmat, rOcnt1, rOct2 and negative control were used H]Imperatorin uptake in TR-BBB cells [3H]imperatorin with or without selected compounds at 37°C in TR-BBB cells, including target sequences of Octn2, rPmat, rOcnt1, rOct2 and negative control. TR-BBB cells were seedfor a designed time. Aliquots were collected to count the radioed onto collagen-coated 6- and 24- well plates at a density activity using the Tri-Carb Liquid Scintillation Counter. Cellular 5 2 of 1×10 proteinTR-BBB contentcells waswere determined with a DC assay kit(Terasaki cells/cm . At 24 h after seeding, siRNAs specific for cultured according to aprotein previously report and Hosoya, 2001; using bovine serum albumin (Bio-Rad Laboratories Co, HerOctn2, Pmat, rOcnt1and rOct2 (200 nM) or negative control cules, USA) as For a standard. [³H]Imperatorin uptake was (control) KangCA, et al., 2002). in vitro uptake study, [3H]imperatorin transportsiRNA in TR-BBB cells were was transfected into TR-BBB cells using Liexpressed as cell-to-medium (mL/mg protein) ratio as follows: pofectamine® 2000 transfection reagent (Invitrogen, Carlsbad, performed (dpm/mL) described previously (Kangper et milligram al., 2002). cell Cellsprotein were then incubated with 200 µL to manufacturer’s protocol. Cells were radioactivity in the sample CA, USA) according (dpm/mg protein). used for quantitative real-time PCR and [3H]imperatorin up페이지 8 / 40 for 5 min The initial uptake of imperatorin was measured take was analyzed at 48 h after the initiation of transfection For kinetic studies, the Michaelis-Menten constant (Km) and (Lee and Kang, 2016). the maximum uptake rate (Vmax) of [3H]imperatorin were estimated using the following equation (8): Statistical analysis All data were expressed as means ± standard error of the means (SEM). Statistical analysis of data was performed by V=Vmax·C/(Km+C)+Kd·C (1) (8) one-way ANOVA analysis of variance followed by Dennett’s (post hoc test) for single and multiple comparisons, respecwhere V and C were the initial uptake rate of [3H]imperatorin at tively. Statistically significant was considered at p