a Department of Pharmaceutical Sciences, Ferrara University, via Fossato di Mortara 19, 44100 Ferrara, Italyb Department of Biology, General Physiology Section, via Borsari 46, 44100 Ferrara, Italy
c Department of Experimental Clinical Medicine, Pharmacology Section, via Fossato di Mortara 19, 44100 Ferrara, Italyd Department of Obstetrics and Gynecology, Medical College of Georgia, Augusta, GA, USA
Received 14 April 2004; received in revised form 15 October 2004; accepted 25 October 2004Available online 19 December 2004
Ascorbic acid (AA) or 6-Br-ascorbate (BrAA) conjugation has been investigated as a tool to improve brain drug delivery by the Vransporter SVCT2. To this aim, the effects of AA- or BrAA-conjugation on drug affinity and uptake have been assessed in vitro,uman retinal pigment epithelium (HRPE) cells, and compared in vivo on mice. Nipecotic, kynurenic and diclofenamic acids wes model drugs. Kinetic and inhibition experiments referred to [14C]AA uptake into HRPE cells showed that nipecotic and kynurenic aecame able to interact with SVCT2, as competitive inhibitors, only when conjugated to AA or BrAA. Surprisingly, diclofenamic acppeared able to interact with SVCT2, with an affinity that was significantly increased or decreased by AA or BrAA conjugation, resPLC analysis, performed on HRPE cells, confirmed the SVCT2 mediated transport for the BrAA-conjugate of nipecotic acidynurenic acids conjugates although interacting with the transporter did not enter the cells. In accordance, only the nipecotic acidhowed anticonvulsant activity after systemic injection in mice.2004 Elsevier B.V. All rights reserved.
eywords:Brain delivery; Drug targeting; HRPE cells; SVCT2 transporter; Vitamin C
. Introduction
Drug delivery into the central nervous system (CNS) ap-ears currently as one of the most important aspects in devel-pment of therapeutic approaches for brain diseases. Manyifferent strategies have been proposed as a way to delivercompound from the blood to the brain, ranging from inva-
ive approaches to mildest methods involving the modifica-ion of the physicochemical properties of drugs (Scherrmann,002). Recently, the progress of molecular cloning and ex-ression of transporter genes suggests the synthesis of pro-
drugs able to interact with specific transporters of native cpounds (Padridge, 2002). Several specific transporters habeen identified in boundary tissues between blood andcompartments. Some of them are involved in the activeply of nutrients (i.e. glucose, amino acids and vitamins)have been used to explore pro-drugs with improved brailivery (Bonina et al., 2000; Ohnishi et al., 2000; TamaiTsuji, 2000).
An essential nutrient for all mammalians is ascorbic(AA; Fig. 1). The highest concentrations of this vitaminbe found in the eye, spinal cord and brain, where it isessary as antioxidant, as neuromodulator for acetylchand noradrenaline release or it contributes for myelinmation (Rose and Bode, 1991; Friedman and Zeidel, 1
260 A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269
Fig. 1. Structural formulae of ascorbic acid (AA), 6-Br-ascorbate (BrAA), nipecotic, kynurenic and diclofenamic acids. The conjugates of these three last drugswith AA (AA–Nipec, AA–Kynur and AA–Diclo) and BrAA (BrAA–Nipec, BrAA–Kynur and BrAA–Diclo) are also shown.
Gilchrest, 1999; Rice, 2000). A new class of Na+ dependentVitamin C transporters (SVCT) has been recently character-ized (Daruwala et al., 1999; Rajan et al., 1999; Tsukaguchiet al., 1999) and found to be of fundamental significance tomaintain the necessary AA intake for humans (Sotiriou etal., 2002) due to their inability in synthesizing this vitamin(Nishikimi et al., 1994): SVCT1 is responsible for ascor-bic acid absorption from intestine and to recover it by kid-ney, whereas SVCT2, expressed by neuroepithelial cells ofthe choroids plexus and the retinal pigmented epithelium, al-lows to accumulate the vitamin in the brain and eye, respec-tively (Friedman and Zeidel, 1999; Gilchrest, 1999; Rice,2000).
Taking all this into account, we have therefore hypothe-sized that drugs able to interact with the SVCT2 transporterisoform can potentially be transported into the brain. Conse-quently, we started a preliminary investigation, aimed to ex-plore the conjugation with ascorbic acid as a possible meanto improve the entry of drugs that are not effectively deliv-ered into the brain (Manfredini et al., 2002). During this study,we have demonstrated that human retinal pigment epitheliumcells (HRPE) selectively express the SVCT2 transporter. Thiscell line has been therefore employed by us to perform invitro analysis about the interaction of several drugs and theirascorbyl-conjugates toward SVCT2 (Manfredini et al., 2002).I nt , but
characterized by potential anticonvulsant activity and con-trol of neurodegenerative disorders, respectively (Hodgkinsand Schwarcz, 1988; Krogsgaard-Larsen et al., 2000; Lam-bert, 2000). According to our preliminary results, these drugsdo not interact with SVCT2, but their interaction can be ob-tained upon conjugation at position 6 of AA (AA–Nipec andAA–Kynur; Fig. 1). Similarly, nipecotic acid becomes ableto show significant in vivo anticonvulsant effects only whenconjugated to AA (Manfredini et al., 2002). We have alsodemonstrated that Diclofenac (Fig. 1), proposed for poten-tial application in Alzheimer’s diseases (Scharf et al., 1999;Halliday et al., 2000; Hull et al., 2000), has a better affinityfor SVCT2 than AA-itself and that the Diclofenac conjuga-tion at position 6 of AA (AA–Diclo;Fig. 1) allows to furtherimprove the affinity of this drug (Manfredini et al., 2002;Dalpiaz et al., 2004). Finally, we have also observed that thepresence of a Br substituent in position 6 of ascorbic acid in-creases the affinity of AA for SVCT2 transporter (Manfrediniet al., 2002).
According to these information, and considering the needsof further studies on the SVCT2 mediated transport intothe brain, we have undertaken the present experimentalwork in order to investigate the profile of interaction to-ward the SVCT2 transporters of the selected drugs (nipecotic,kynurenic and diclofenamic acids) and their related conju-gb een
n particular, nipecotic and kynurenic acids (Fig. 1) have beeaken into account as drugs unable to reach the brain
ates with the 6-Br-ascorbate (BrAA) derivative (Fig. 1). Theehaviour of this new family of BrAA-conjugates has b
A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269 261
described and compared with the properties of AA, BrAA andAA-conjugates, by us anticipated (Manfredini et al., 2002)and here described in detail. In particular, the affinity, the inhi-bition properties of ascorbate SVCT2 mediated transport andthe uptake mechanisms into HRPE cells have been analysed.Finally, the in vivo central effects of some BrAA-conjugateshave been investigated and compared with those referred toparent AA-conjugates (Manfredini et al., 2002; Dalpiaz etal., 2004).
2. Materials and methods
2.1. Preparation of study compounds
The study molecules were prepared by a few step syntheticprocedure. Condensation between 2,3-di-benzyl ascorbicacid (Kato et al., 1988) or 6-bromo-2,3-dibenzyl ascor-bic acid (Raic-Malic et al., 2000) and the correspondingdrugs was obtained in standard conditions, underN,N′-dicyclohexylcarbodimide catalysis. Nipecotic and kynurenicacids needed previous protection at the amino and hydroxylfunctions, respectively. Thus, nipecotic acid was protected astert-butoxycarbonyl derivative (BOC) as reported in literature(Kim et al., 2000) and kynurenic acid as benzyl derivative.T thyle
erefi cotica cid( .
2nely
m gelp 4 nmU er-msD osi-t -e tants( aryt s per-f er-a edo naly-s val-u
his required prior conversion to the kynurenic acid mester (Spath, 1921).
The 6-O- and 6-bromo-5-O-ascorbates thus obtained wnally deprotected by hydrogenolysis. In the case of nipecid derivatives, prior treatment with trifluoroacetic aTFA) was required to remove the BOC-protective group
.1.1. ChemistryReaction courses and product mixtures were routi
onitored by thin-layer chromatography (TLC) on silicarecoated F254 Merck plates with detection under 25V lamp and/or by spraying with a diluted potassium panganate solution. Nuclear magnetic resonance (1H NMR)
pectra were determined for solution in CDCl3-CD3OD-MSO-d6 on a Bruker AC-200 spectrometer and peak p
ions are given in parts per million (δ) downfield from tetramthylsilane as internal standard, whereas coupling consJ) in Hertz. Melting points were obtained in open capillubes and are uncorrected. Column chromatography waormed with Merck 60–200 mesh silica gel. Ambient tempture was 22–25◦C. All drying operations were performver anhydrous magnesium or sodium sulphate. Microais, unless indicated, were in agreement with calculatedes within±0.4%.
HRPE cell line was originally developed by Del MonDel Monte and Maumenee, 1981) and characterized in dail by Dutt et al. (1989). More recently, this HRPE cell linas found to express endogenously an ascorbate tranystem kinetically similar to the transport system mediy SVCT2 (Rajan et al., 1999) whose mRNA was selectiveetected by RT-PCR analysis (Manfredini et al., 2002). Theells were routinely grown in 1:1 mixture of Dulbecco’s mfied Eagle’s and Ham’s F12 media, supplemented withBS, 50�g/mL streptomycin and 50 IU/mL penicillin (Gibcaboratories, Invitrogen Italia, Milan, Italy) at 37◦C in 5%O2. Cells employed for uptake measurements were se
n 24-well tissue culture plates and grown to confluenceays).
262 A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269
2.3. SVCT2 transporter interactions
Transport assays were performed following the methoddescribed byRajan et al. (1999)andWashko et al. (1989)forthe determination of intracellular amounts of radiolabelledascorbic acid. Briefly, the uptake buffer was freshly preparedeach time, the composition was: 25 mM Hepes/Tris (pH 7.5),140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4,5 mM glucose. One millimolar dithiothreitol (DTT; Sigma,St. Louis, MO, USA) was also added to the uptake bufferto prevent the oxidation of AA. At this concentration DTThad no effect on the transport process. Incubation time forthe transport measurements was 60 min (according to pre-vious time course experiments) at 37◦C; after this time theuptake buffer containing the radioactive substrate was aspi-rated and cells were washed with 2× 2 mL of ice-cold uptakebuffer. Cells were then solubilized in 250�L of 0.2 M NaOHsolution containing 0.5% CHAPS (Sigma), transferred tovials containing 3 mL of scintillation cocktail (Ultima Gold,Packard, Milan, Italy) and radioactivity associated with thecells was evaluated by scintillation spectrometry (�-counterBeckmann LS 6000).
The kinetics of [14C]AA (6 mCi/mmol; NEN Life Science,Boston, MA, USA) uptake, mediated by SVCT2, was anal-ysed using concentration of AA ranging from 2.5 to 1000�M.T 14
cells were washed with the uptake buffer and then lysedby adding deionized water (0.3 mL per well of a 24-wellplate) and freezing the cells at−80◦C for 30 min. The cellswere then thawed on ice, and the lysate was centrifuged(12,500×g, 10 min) to remove cellular membranes. The su-pernatant (40�L) was used to measure the levels of the testsubstrate by HPLC.
2.5. HPLC analysis
HPLC apparatus consisted of a modular chromatographicsystem (Model 1100 series pump and diode array detector;Agilent, Waldbronn, Germany) linked to an injection valvewith 50�L sample loop (Model 9125; Rheodyne, Cotati,CA, USA). Analyses were all performed at room temper-ature. Data acquisition and processing were accomplishedwith a personal computer using Chem Station software (Agi-lent). The identity peaks was assigned by co-chromatographywith the authentic standards. Quantification was performedby integration of peak areas using external standardiz-ation.
2.5.1. BrAA HPLC analysisChromatography was performed on a reversed-phase
RAD-PAK Nova Pak column (Waters, Minneapolis, MN,U hasec andm of9 md om tiont
2hase
c n,1 A).T con-s andm M( ow-r di-a timef
2hase
c n,1 ag h).T sistedo nolw tot ten-t
he concentration of [C]AA ranged from 2.5 to 50�M andas kept constant at 50�M. Data were analysed by nonliar regression of Michaelis–Menten equation and confiy Eadie–Hofstee linear regression. The aspecific uptaketected in the presence of 10 mM AA and was found t
ower than 2% with respect to the total uptake at all [14C]AAoncentrations investigated.
Inhibition of AA transport was determined by addingndicated concentrations of unlabelled compounds to pells along with either [14C]AA at fixed concentration 50�M,r [14C]AA ranging from 2.5 to 100�M. The unlabelled inibitor concentrations displacing 50% of [14C]AA (IC50 val-es) were obtained by computer analysis of displaceurves. Inhibitory constants (Ki values) were derived frohe IC50 values according to the Cheng and Prusoff equai = IC50/(1+[C* ]/Kt
* ), where [C* ] is the concentration of th14C]AA andK∗
t its Michaelis–Menten constant (Cheng andrusoff, 1973). Competition studies were performed acco
ng to the Lineweaver–Burk analysis. All calculations werformed using the computer program Graph Pad PGraphPad, San Diego, USA).
Statistical analysis was performed by ANOVA followy Dunnett’st-test. Difference was considered statisticignificant atp-values less than 0.05.
.4. Uptake in HRPE cells
Intracellular accumulation of BrAA, BrAA–Nipec anrAA–Kynur were measured incubating HRPE cells w
ncreasing concentrations of unlabelled analogues inake buffer for 60 min at 37◦C. Following the incubation
SA). The detector was set at 250 nm. The mobile ponsisted of a mixture of 50 mM acetate buffer (pH 4.7)ethanol (Carlo Erba Reagenti, Milan, Italy) with a ratio5:5 (v/v). 0.2 mM EDTA and 1 mM tetrabutylammoniuihydrogenphosphate [(But)4NH2PO4] were also added tobile phase. The flow-rate was 0.8 mL/min and reten
ime was 9.8 min.
.5.2. BrAA–Nipec HPLC analysisChromatography was performed on a reversed-p
olumn (Synergi 4u Polar-RP 80A cartridge colum50 mm× 4.6 mm i.d.; Phenomenex, Torrance, CA, UShe detector was set at 268 nm. The mobile phaseisted of a mixture of 50 mM acetate buffer (pH 4.7)ethanol with a ratio of 85:15 (v/v). 0.2 mM EDTA and 1 m
But)4NH2PO4 were also added to mobile phase. The flate was 0.8 mL/min and retention time of BrAA–Nipecsteroisomers were 5.80 and 7.07 min and the retention
or BrAA was 10.8 min.
.5.3. BrAA–Kynur HPLC analysisChromatography was performed on a reversed-p
olumn (Hypersil BDS C-18 5U cartridge colum50 mm× 4.6 mm i.d.; Alltech, Milan, Italy) equipped withuard column packed with Hypersil C-18 material (Allteche detector was set at 348 nm. The mobile phase conf a mixture of 50 mM acetate buffer (pH 4.7) and methaith a ratio of 64/36 (v/v). 0.2 mM EDTA was also added
he mobile phase. The flow rate was 0.8 mL/min. The reion time was 5.95 min.
A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269 263
2.6. Effects of the drugs on pentylenetetrazol-inducedseizures
Swiss albino male mice (25–30 g body weight) were usedfor the in vivo experiments. In any experimental sessiona maximum of 20 animals were acutely injected i.p. withsaline (control), nipecotic acid (0.75 mmol/kg), AA–Nipec(0.75 mmol/kg), BrAA–Nipec (0.75 mmol/kg), kynurenicacid (0.50 mmol/kg) and BrAA–Kynur (0.50 mmol/kg). Di-clofenamic acid (3.75 mmol/kg) was injected 15 min beforeBrAA–Nipec. Twenty-five minutes after the treatment, allmice were subcutaneously injected with pentylenetetrazol(80 mg/kg) and the animals were observed for the follow-ing 30 min. The latency to appearance of generalized tonicconvulsions was measured to evaluate the effects of the treat-ments on pentylenetetrazol-induced convulsion (Manfrediniet al., 2002; Dalpiaz et al., 2004).
Statistical analysis was performed by ANOVA followedby the Newman–Keuls test for multiple comparisons.
3. Results
3.1. SVCT2 transporter interactions
2i er-b t-u fsteel Thispr is oft r thantV4
F AAw swc ta. V,Ai
Fig. 3 displays the inhibition curves of [14C]AA up-take referred to the newly synthesized BrAA conjugates ofnipecotic, kynurenic and diclofenamic acids. These data arereported in comparison with the inhibition curves of AA,BrAA, the free acids and their AA-conjugates previously de-scribed by us (Manfredini et al., 2002).
Fig. 3A reports the behaviour in the presence of nipecoticacid and its conjugates with AA and BrAA (AA–Nipec andBrAA–Nipec), whereasFig. 3B and C report the behaviourobtained with kynurenic and diclofenamic acids, respec-tively, and their conjugates with AA and BrAA (AA–Kynur,BrAA–Kynur, AA–Diclo and BrAA–Diclo).
The inhibition constant values (Ki ) obtained by the anal-ysis of curves referred to AA and BrAA are 20.1± 1.6 and2.69± 0.13, respectively (Table 1).
As it can be observed, nipecotic and kynurenic acids werenot able to inhibit the [14C]AA uptake in the concentrationrange investigated (0.5–5000�M). On the other hand, afterconjugation with AA these acids showed the ability to inter-act with SVCT2 transporter (Fig. 4A and B): theKi values of
Fig. 3. Inhibition of 50�M [14C]AA uptake by ascorbic acid (AA), 6-bromoascorbate (BrAA), nipecotic (A), kynurenic (B), diclofenamic (C)acids and their conjugates with ascorbate and 6-bromoascorbate (A–C, re-spectively). [14C]AA uptake in the presence of inhibitors was measured at37◦C. These are single representative experiments performed in duplicate.
The kinetics of the [14C]AA uptake mediated by SVCTs represented inFig. 2. The rate was found to be hypolically related to [14C]AA concentration, indicating sarability of the uptake process. The related Eadie–Ho
inear regression of data is represented in the inset.lot was linear in the concentration range investigated (n= 8,= 0.991,p< 0.0001) and, analogously, computer analys
he saturation experiment suggested a one-site, rathewo sites model. The Michaelis–Menten constant (Kt) andmax values for the transport process are 36± 3�M and.3± 0.3 nmol/(106 cells per 60 min), respectively.
ig. 2. Kinetics of ascorbate (AA) uptake in HRPE cells. Uptake ofas measured by 60 min incubation at 37◦C, pH 7.5. AA concentrationere in the range 2.5–1000�M. The concentration of [14C]AA was keptonstant at 50�M. Inset: Eadie–Hofstee transformation of the same daA transport (nmol/(106cells per 60 min)); S, AA concentration (�M). This
s a single representative experiment performed in duplicate.
264 A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269
Table 1Inhibition constant values (Ki ) of AA, BrAA, nipecotic, kynurenic, diclofe-namic acids and their conjugates with AA and BrAA, obtained by inhibitionof 50�M [14C]AA uptake on HRPE cells
Values± S.E.M. are from three independent experiments performed in du-plicate.
AA–Nipec and AA–Kynur are 1187± 78 and 39.8± 3.2�M,respectively (Table 1). It is interesting to observe that con-jugation with BrAA increases the affinity for SVCT2 ofnipecotic and kynurenic acids with respect to AA. In fact, theKi values of BrAA–Nipec (193± 14�M) and BrAA–Kynur(7.4± 0.8�M) appear about five times lower thanKi valuesof AA-conjugates (Table 1).
As reported inFig. 3C, diclofenamic acid was ableto strongly inhibit the [14C]AA uptake, showing aKivalue (3.35± 0.16�M) similar to that observed for BrAA(2.69± 0.13�M). Moreover, the conjugation with AA in-creases the diclofenamic acid affinity for SVCT2. TheKi
F e( cotica -c RPEc -p ofa
value of the conjugate AA–Diclo is, indeed, 0.19± 0.01�M.In contrast to that observed for nipecotic and kynurenic acids,the conjugation of diclofenamic acid with BrAA decreasesthe inhibition potency with respect to AA. In fact, theKivalue of BrAA–Diclo (21.4± 1.8�M) is about hundred timeshigher than theKi value of AA–Diclo (Table 1). Based onthese data, we examined inhibition kinetics of the AA andBrAA analogues in greater detail.
Table 2reports theKt andVmax values obtained by ki-netic experiments of [14C]AA uptake performed in the ab-sence and in the presence of different concentrations of theBrAA-conjugates together with the values of other inhibitors(AA, its conjugates with nipecotic and kynurenic acids andBrAA) by us anticipated (Manfredini et al., 2002). We cannote that in the presence of increasing concentrations,Kt val-ues increase, whereas the correspondingVmax values are notaffected by the presence of inhibitors. These data suggest acompetitive inhibition of [14C]AA uptake as shown by theLineweaver–Burk analysis, reported inFig. 4A and B.
3.2. Uptake into HRPE cells
The entering of unlabelled compounds into HRPE cellsby SVCT2 mediated uptake was firstly investigated onBrAA by HPLC analysis. The kinetics, reported inFig. 5,s y ofE -p thep AAd ssa c-t er withr
nots bitorb the
TK int itors
C
[B[[[[[[[[[
V n du-p
ig. 4. Inhibition of [14C]AA transport by (A) 75�M 6-bromoascorbatBrAA) and increasing concentrations of the AA-conjugates of nipend kynurenic acids. Inhibition of [14C]AA transport by (B) increasing conentrations of the BrAA-conjugates of nipecotic and kynurenic acids. Hells were incubated for 60 min at 37◦C. The compounds inhibit in a cometitive manner the uptake of [14C]AA in HRPE. Double reciprocal plots
representative determination are shown.
how the same pattern registered for AA. The linearitadie–Hofstee plot (n= 8, r = 0.994,p< 0.0001) and comuter analysis of the saturation experiment confirmedresence of a single transporter population also for thiserivative. TheKt andVmax values for the transport procere 5.1± 0.4�M and 4.3 nmol/(106 cells per 60 min), respe
ively, validating that the affinity of BrAA for SVCT2 and thelated uptake rate are one order of magnitude higherespect to AA.
On the other hand, the BrAA–Kynur conjugate doeseem transported by SVCT2, despite its competitive inhiehaviour: in fact, the presence of this conjugate in
able 2
t andVmax values obtained by [14C]AA kinetic experiments performedhe absence and in the presence of increasing concentrations of inhib
alues± S.E.M. are from three independent experiments performed ilicate.a p< 0.001 vs. [14C] ascorbate alone.b p< 0.05 vs. [14C] ascorbate alone.
A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269 265
Fig. 5. Kinetics of 6-bromoascorbate (BrAA) uptake in HRPE cells, ob-tained by HPLC analysis. Uptake of BrAA was measured by 60 min incuba-tion at 37◦C, pH 7.5. BrAA concentrations were in the range 0.25–100�M.Inset: Eadie–Hofstee transformation of the same data. V, BrAA transport(nmol/(106cells per 60 min)); S, BrAA concentration (�M). This is a singlerepresentative experiment performed in duplicate.
extracellular fluid (500�M, a concentration two order ofmagnitude higher than itsKi value) has not allowed its uptakeinto HRPE cells.
Fig. 6reports the HPLC chromatogram referred to 500�MBrAA–Kynur (peak at 6 min) in the extracellular fluid (A),and the related chromatogram referred to the intracellu-lar fluid after incubation (60 min) (B). It can be observedthat BrAA–Kynur was not transported into HRPE cell (noBrAA–Kynur intracellular amounts detected).
The SVCT2 transporter seems instead to mediate the up-take into HRPE cells of the BrAA–Nipec conjugate. In fact,the analysis performed with 5 mM BrAA–Nipec (a concen-tration one order of magnitude higher than itsKi value) al-lowed to observe a consistent uptake into HRPE cells with arate of 0.9± 0.1 nmol/(106 cells per 60 min), as we can ob-serve inFig. 7. Similar amounts of both diasteroisomers ofthis conjugate (peaks at 5.8 and 7.1 min;Fig. 7A) were foundinto the cells (Fig. 7B). The presence of BrAA in intracellularfluids indicates that the BrAA–Nipec conjugate show sus-ceptibility to undergoing enzymatic hydrolysis. Moreover,the uptake was totally inhibited by the presence of 10 mMAA (no BrAA–Nipec intracellular amounts detected), con-firming that BrAA–Nipec is accumulated into HRPE cellsby SVCT2 mediated uptake. No significant degradation ofthe conjugates has been detected during the incubation withHRPE cells, being their peaks area the same before and afterthe incubation.
3.3. Effects of the drugs on pentylenetetrazol-inducedseizures
Table 3reports the effects of i.p. injection of BrAA–Nipecon pentylenetetrazole (PTZ)-induced convulsion in mice,in comparison with nipecotic acid and AA–Nipec effects( n-j on-v tica theirg
F uid of H t 37C
ig. 6. HPLC chromatograms for 500�M BrAA–Kynur in extracellular flfor 60 min (B). Any uptake of BrAA–Kynur has been registered.
Manfredini et al., 2002). In the saline group the s.c. iection of pentylenetetrazol (80 mg/kg) induced tonic culsions with a latency of 621± 27 s, whereas nipecocid-treated mice showed no apparent abnormality ineneral behaviour (Manfredini et al., 2002). The i.p injection
RPE cells (A) and for the related intracellular fluid after incubation a◦
266 A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269
Fig. 7. HPLC chromatograms for 200 nM BrAA–Nipec (A) and for the difference between chromatogram of the intracellular fluids of HRPE cells incubatedwith 5 mM BrAA–Nipec and buffer solution (B). The peak at 10.8 min is referred to BrAA. The difference has been performed using the Chem Station software(Agilent).
Table 3Effect of i.p. injection of nipecotic acid (0.75 mmol/kg), AA–Nipec(0.75 mmol/kg) and BrAA–Nipec (0.75 mmol/kg) in the absence and in thepresence of diclofenamic acid (3.75 mmol/kg) on pentylenetetrazole (PTZ)-induced convulsion in mice
BrAA–Nipec + diclofenamic acid + PTZ 691± 25a p< 0.05, significantly different from control and nipecotic acid groups,
according to ANOVA followed by the Newman–Keuls test for multiple com-parisons.
of AA–Nipec and BrAA–Nipec (0.75 mmol/kg) significantlyincreased the latency to appearance of PTZ-induced tonicconvulsions, while nipecotic acid (0.75 mmol/kg) was in-effective. It is important to underline that BrAA–Nipecbecomes ineffective in the presence of diclofenamic acid(3.75 mmol/kg), by itself ineffective on PTZ-induced con-vulsions (Dalpiaz et al., 2004). In addition neither kynurenicacid (0.50 mmol/kg) nor BrAA–Kynur (0.50 mmol/kg) sig-nificantly affect PTZ-induced tonic convulsions (data notshown). In all groups of animals no lethality was observed.
4. Discussion
We have previously discovered that conjugation with AAof model drugs, that do not normally cross the blood–brain
barrier (BBB), gave compounds able to reach the CNS(Manfredini et al., 2002). We have therefore hypothesizedthat this targeting into the brain may be mediated by Vi-tamin C transport systems located in boundary tissues be-tween blood and CNS. The SVCT2 transporter appears agood candidate for uptake studies of AA and its conju-gates in CNS. In fact, this SVCT subtype has not beendetected in the BBB, but is selectively expressed both byneuroepithelial cells of the choroid plexus, allowing the up-take of AA in the brain, in particular the cerebrospinal fluid,and the retinal pigment epithelium, which limits drug en-try into the neuronal retina and vitreous (Rose and Bode,1991; Friedman and Zeidel, 1999; Gilchrest, 1999; Rice,2000; Liang et al., 2001; Angelow et al., 2003). We haveproposed HRPE cells as a cellular model valuable for invitro studies of the endogenous activity of SVCT2 trans-porters. In fact, in HRPE cells an endogenous expressionof ascorbate transport system was detected. This systemshowed kinetic properties similar to the transport processmediated SVCT2 (Rajan et al., 1999). RT-PCR analysis al-lowed us, afterwards, to observe that the ascorbate transporterexpressed in this human cell line is only the SVCT2 iso-form (Manfredini et al., 2002). As previously anticipated,and here described in detail, the kinetic studies for [14C]AAand BrAA (Figs. 2 and 5, respectively) confirmed the exis-t y thelo n atp ns-p
ence of a single class of transporters, as indicated binearity of the Eadie–Hofstee plots. TheKt andVmax valuesbtained for AA and BrAA showed that the Br-substitutioosition 6 of AA enhances the affinity for the SVCT2 traorter (Kt for AA = 36± 3�M; Kt for BrAA = 5.1± 0.4�M)
A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269 267
without impairing the uptake into HRPE cells (Vmax forAA and BrAA are 4.3± 0.3 and 4.0± 0.3 nmol/(106 cellsper 60 min), respectively). These data suggest that modi-fications at position 6 on AA are compatible for the de-sign of analogues that may still be able to interact withthe transporters. Thus, new derivatives were synthesisedas conjugates at position 5 of BrAA of the same modeldrugs (Fig. 1). Inhibition experiments referred to nipecotic(Fig. 3A), kynurenic acids (Fig. 3B) and their conjugatesconfirm, as previously anticipated by us (Manfredini et al.,2002), that these drugs are totally unable to interact withSVCT2 transporters, whereas their conjugates show a sig-nificant inhibition potency on [14C]AA uptake (Table 1).Moreover, as reported inFig. 3A, we have found for the firsttime a remarkable increase in SVCT2 affinity going fromthe nipecotic acid conjugate with AA (Ki = 1187± 78�M)to BrAA conjugate (Ki = 193± 14�M). A similar pro-file has also been found for the kynurenic acid conju-gates (Fig. 3B) with AA (Ki = 39.8± 3.2�M) and BrAA(Ki = 7.4± 0.8�M). Taken together, these data suggest thepossibility to achieve better affinities by conjugation withBrAA rather than AA, but also lead to hypothesise thepresence of accessory interactions that may improve trans-porter recognition. On the other hand, inhibition experi-ments performed on HRPE cells using diclofenamic acida -t nicac T2tt sesiO( n-d thep
so ica urkaV eA the[ imi-l mepT
ndk cellsh acid( weda nt e that acidc
ctso sys-
temic injection of BrAA–Nipec and BrAA–Kynur in com-parison with the previously described effects of kynurenicand nipecotic acids and their AA-conjugates (Manfredini etal., 2002). Neither kynurenic acid nor BrAA–Kynur signifi-cantly affected pentylenetetrazol-induced tonic convulsions,suggesting the inability of the drug and its related BrAA con-jugate to reach the brain. On the other hand, as reported inTable 3, AA–Nipec and BrAA–Nipec significantly increasedthe latency to appearance of pentylenetetrazol-induced tonicconvulsions, while nipecotic acid alone (0.75 mmol/kg) wasineffective. It is to underline that nipecotic acid-treated miceshowed no apparent abnormality in their general behaviour(Manfredini et al., 2002). Moreover, it may be interesting tonote that nipecotic acid does not interact with SVCT2 trans-porters, whereas BrAA–Nipec interacts with SVCT2 and isrecovered into HRPE cells by an SVCT2 mediated uptake.These data may suggest that, upon conjugation with AA orBrAA, nipecotic acid can target the brain. Similarly, it hasbeen reported that esters of nipecotic acid are able to inhibitGABA uptake in the brain (Gokhale et al., 1990) with conse-quent anticonvulsant effects (Frey et al., 1979; Horton et al.,1979), which can be obtained only after enzymatic hydroly-sis of the nipecotic acid esters (Bonina et al., 1999; Gokhaleet al., 1990). The presence of BrAA detected in HRPE cellsafter incubation with the BrAA–Nipec conjugate (Fig. 7B)i ibil-i en-e ularl
sante aved AAS 4A cidi ec,s cellsa ech-a rthera igatet elialt or am ationo d thatS ce ofr st un-ft ecteda picald thet A,e y oft
om-p nica rtant
nd its AA or BrAA conjugates (Fig. 3C) showed a toally different pattern with respect to nipecotic and kynurecids. In fact, as reported by us (Dalpiaz et al., 2004), di-lofenamic acid is able itself to interact with the SVCransporters with an affinity (Ki = 3.35± 0.16�M) similaro that of BrAA; moreover, the AA-conjugation increats affinity by an order of magnitude (Ki = 0.19± 0.01�M).
n the other hand, the newKi value referred to BrAA–DicloKi = 21.4± 1.8�M) indicates that the BrAA-conjugation iuces a consistent reduction of affinity with respect toarent compound.
In view of the overall information, inhibition kineticf BrAA- and AA-derivatives of nipecotic and kynurencids was next examined in detail. The Lineweaver–Bnalysis reported for the first time inFig. 4A (Kt andmax values are reported onTable 2) indicates that thA-conjugates of nipecotic and kynurenic acids inhibit
14C]AA uptake according to a competitive process. Sarly, we have found that the BrAA-conjugates with the saarent drugs appear as competitive inhibitors (Fig. 4B andable 2).
The uptake of BrAA-conjugates of nipecotic aynurenic acids was investigated: no uptake into HRPEas been observed for the BrAA conjugate of kynurenicFig. 6); on the contrary, the nipecotic acid conjugate shosignificant uptake (Fig. 7), which was totally inhibited i
he presence of 10 mM ascorbate. These results indicathe SVCT2 transporters are involved in the nipecoticonjugate transport into HRPE cells.
To support our in vitro data we evaluated the effen pentylenetetrazol-induced convulsions in mice of
t
ndicate that this Vitamin C derivative shows susceptty to undergoing enzymatic hydrolysis, even if, in gral, the BrAA-conjugates show high stability at extracell
evel.We have also analysed the BrAA–Nipec anticonvul
ffects in the presence of diclofenamic acid, which we hemonstrated to be a non competitive inhibitor of theVCT2-uptake (Manfredini et al., 2002; Dalpiaz et al., 200).s reported inTable 3, the presence of diclofenamic a
n mice abolished the anticonvulsant effect of BrAA–Nipuggesting that the uptake of this conjugate into HRPEnd into the brain may be related by the same transport mnism, although other mechanims could be involved. Funalysis should be performed in order to better invest
his latter aspect. Moreover, the knowledge of trans-epithransport mechanisms involving SVCT2 may help us fore complete comprehension of cell membrane permef the conjugates. In this regard, it has been hypothesizeVCT2 transporters are targeted to the basolateral surfa
etinal pigment epithelia (Liang et al., 2001) which appearo be polarized in two distinct regions: the basolateraloldings and the apical microvilli (Chancy et al., 2000). Theransepithelial transport of ascorbic acid has been detnd, therefore, hypothesized to have a basolateral to airection (Hediger, 2002). These information suggest that
ransport of BrAA-conjugate may be similar to that of Aven if additional work is needed to establish the validithis hypothesis.
In conclusion, we have prepared a new series of counds based on BrAA conjugation of nipecotic, kynurend diclofenamic acids. These derivatives showed impo
268 A. Dalpiaz et al. / European Journal of Pharmaceutical Sciences 24 (2005) 259–269
interaction and transport capability through the SVCT2transporters. We can confirm that nipecotic acid conjugates(AA–Nipec and BrAA–Nipec), differently from the parentcompound, show anticonvulsant effects whereas kynurenicacids derivatives (AA–Kynur and BrAA–Kynur) do not.The therapeutic effects of nipecotic acid conjugates are to-tally inhibited by the presence of diclofenamic acid, a non-competitive inhibitor of SVCT2.
Acknowledgements
This work was supported by University of Ferrara andNational Institutes of Health Grant (HD37150).
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, atdoi:10.1016/j.ejps.2004.10.014.
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