effect of transporter inhibition on the distribution of cefadroxil in...

FLUIDS AND BARRIERSOF THE CNS

Chen et al. Fluids and Barriers of the CNS 2014, 11:25http://www.fluidsbarrierscns.com/content/11/1/25

RESEARCH Open Access

Effect of transporter inhibition on the distributionof cefadroxil in rat brainXiaomei Chen1,2, Irena Loryan2, Maryam Payan2,3, Richard F Keep4, David E Smith1*

and Margareta Hammarlund-Udenaes2

Abstract

Background: Cefadroxil, a cephalosporin antibiotic, is a substrate for several membrane transporters includingpeptide transporter 2 (PEPT2), organic anion transporters (OATs), multidrug resistance-associated proteins (MRPs),and organic anion transporting polypeptides (OATPs). These transporters are expressed at the blood–brain barrier(BBB), blood-cerebrospinal fluid barrier (BCSFB), and/or brain cells. The effect of these transporters on cefadroxildistribution in brain is unknown, especially in the extracellular and intracellular fluids within brain.

Methods: Intracerebral microdialysis was used to measure unbound concentrations of cefadroxil in rat blood,striatum extracellular fluid (ECF) and lateral ventricle cerebrospinal fluid (CSF). The distribution of cefadroxil in brainwas compared in the absence and presence of probenecid, an inhibitor of OATs, MRPs and OATPs, where bothdrugs were administered intravenously. The effect of PEPT2 inhibition by intracerebroventricular (icv) infusion ofAla-Ala, a substrate of PEPT2, on cefadroxil levels in brain was also evaluated. In addition, using an in vitro brain slicemethod, the distribution of cefadroxil in brain intracellular fluid (ICF) was studied in the absence and presence oftransport inhibitors (probenecid for OATs, MRPs and OATPs; Ala-Ala and glycylsarcosine for PEPT2).

Results: The ratio of unbound cefadroxil AUC in brain ECF to blood (Kp,uu,ECF) was ~2.5-fold greater duringprobenecid treatment. In contrast, the ratio of cefadroxil AUC in CSF to blood (Kp,uu,CSF) did not change significantlyduring probenecid infusion. Icv infusion of Ala-Ala did not change cefadroxil levels in brain ECF, CSF or blood.In the brain slice study, Ala-Ala and glycylsarcosine decreased the unbound volume of distribution of cefadroxil inbrain (Vu,brain), indicating a reduction in cefadroxil accumulation in brain cells. In contrast, probenecid increasedcefadroxil accumulation in brain cells, as indicated by a greater value for Vu,brain.

Conclusions: Transporters (OATs, MRPs, and perhaps OATPs) that can be inhibited by probenecid play an importantrole in mediating the brain-to-blood efflux of cefadroxil at the BBB. The uptake of cefadroxil in brain cells involves boththe influx transporter PEPT2 and efflux transporters (probenecid-inhibitable). These findings demonstrate that drug-druginteractions via relevant transporters may affect the distribution of cephalosporins in both brain ECF and ICF.

Keywords: Blood–brain barrier, Blood-cerebrospinal fluid barrier, Intracerebral microdialysis, Brain slice, Cefadroxil,Multidrug resistance-associated proteins, Organic anion transporters, Organic anion transporting polypeptides, Peptidetransporter 2, Probenecid

* Correspondence: [email protected] of Pharmaceutical Sciences, College of Pharmacy, University ofMichigan, Ann Arbor, Mi 48109, USAFull list of author information is available at the end of the article

© 2014 Chen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated.

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Chen et al. Fluids and Barriers of the CNS 2014, 11:25 of 12http://www.fluidsbarrierscns.com/content/11/1/25

BackgroundCephalosporins, a class of beta-lactam antibiotics, havebeen widely used for the prophylaxis and treatment of avariety of infections [1]. In addition to their antibacterialactivity, the therapeutic effects of different cephalosporinsdepend on their pharmacokinetics and tissue distribution,which are affected by multiple membrane transporters.Some cephalosporins are substrates of proton-coupledoligopeptide transporters (POTs) [2], organic anion trans-porters (OATs) [3], organic anion transporting polypep-tides (OATPs) [4,5], and multidrug resistance-associatedproteins (MRPs) [6,7]. These transporters are widely dis-tributed in several tissues including the kidney, liver, intes-tine, and brain [8], influencing cephalosporin absorption,distribution, and elimination.Among all tissues, drug delivery to brain is the most

challenging because of the blood–brain barrier (BBB),situated at the cerebral endothelium, and blood-CSFbarrier (BCSFB) at the choroid plexus epithelium [9]. Inaddition to tight junctions limiting paracellular diffusion[10,11], the BBB and BCSFB express many transportersresponsible for chemical exchange between brain andblood including efflux transporters important for pro-tecting the brain from waste products and potentialtoxins [12]. Among the cephalosporin transporters, theMRPs and OATs at the BBB and BCSFB are believed totransport substrates from brain (and CSF) to blood as ef-flux transporters [13-15]. Peptide transporter 2 (PEPT2,a member of POTs) at the apical side (CSF facing) of theBCSFB is able to transport substrates from the CSF sidetowards blood [16]. The OATPs are expressed bothat the BBB and BCSFB as bidirectional transporters[17,18]. The above mentioned transporters are alsoexpressed on the cell membrane of brain cells (neurons,astrocytes, and microglia) [13,14,18,19], potentially af-fecting cephalosporin distribution after their entry intobrain. Thus, membrane transporters may influence thebrain distribution of cephalosporins and influence theireffectiveness for treating central nervous system (CNS)infections.Cefadroxil is a first-generation cephalosporin and used

clinically mainly to treat urinary tract infections [1]. Thecurrent study employed cefadroxil as a model drug toexamine the potential impact of transporters on thebrain distribution of cephalosporins, as it has been re-ported to be a substrate of POTs, OATs, MRPs, andOATPs [5,6,20-22]. In small intestine, PEPT1, a memberof POTs, mediates peptide/mimetic uptake at the apicalside of enterocytes, leading to a high oral bioavailability[23,24]. Thus, PEPT1 knockout led to a 23-fold reductionin peak plasma concentrations and a 14-fold decrease insystemic exposure of cefadroxil in mice [24]. Also, MRP3and MRP4, at the basolateral side of enterocytes, maycontribute to the further transport of cefadroxil from

enterocyte to blood [22]. The kidney is the main elimin-ation organ for cefadroxil and studies in PEPT2 null miceindicate that this is the principal transporter involved incefadroxil reabsorption [20]. Moreover, the clearance ofcefadroxil is significantly reduced by co-administration ofprobenecid [20,25]. Probenecid is widely known as an in-hibitor of OATs, which mediates renal secretion at thebasolateral membrane of proximal tubule epithelia. How-ever, probenecid can also inhibit the MRPs and OATPsthat transport substrates from blood to urine via thekidney [26,27].Studies on the distribution of cefadroxil in brain have

focused on the function of PEPT2 at the BCSFB andbrain cells. PEPT2 in choroid plexus removes cefadroxilfrom CSF. As a result, the CSF-to-blood concentrationratio of cefadroxil in wild-type mice was markedly lowerthan that in PEPT2 knockout mice [20,28]. In addition,cefadroxil inhibited the uptake of PEPT2 substrates inrodent neonatal astrocytes, demonstrating an uptakefunction of PEPT2 in brain cells [19,29,30]. However,there are no studies on the influence of transporters onthe distribution of cefadroxil in brain extracellular fluid(ECF). A deeper understanding of the effect of trans-porters on CNS cephalosporin distribution is helpful forthe more efficient use of cephalosporins for treatingbrain infections like meningitis.The present study examined the impact of transporters

on cefadroxil distribution in brain ECF and CSF usingprobenecid, an inhibitor of OATs, MRPs and OATPs, aswell as Ala-Ala, a substrate of PEPT2. In vivo microdial-ysis was applied to measure cefadroxil concentrations inrat brain ECF, CSF and blood. In addition, an in vitrobrain slice method was performed to study cefadroxildistribution within the rat brain parenchyma.

MethodsChemicalsCefadroxil, cefadroxil-D4 (deuterated), probenecid, Ala-Ala,glycylsarcosine (GlySar), and amoxicillin were purchasedfrom Sigma-Aldrich (St. Louis, MO, USA), isoflurane fromBaxter Medical AB (Kista, Sweden), and 100 IU/mL hep-arin from Leo Pharma AB (Malmö, Sweden). Acetonitrileand formic acid were obtained from Merck (Darmstadt,Germany). All other chemicals were of analytical grade orbetter. Ringer’s solution was used to perfuse the microdialy-sis probes and consisted of 145 mM NaCl, 0.6 mM KCl,1.0 mM MgCl2, and 1.2 mM CaCl2 in 2 mM phosphatebuffer, pH 7.4. Artificial extracellular fluid (aECF), used tobuffer the brain slices, was comprised of 10 mM glucose,129 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 0.4 mMK2HPO4, 1.4 mM CaCl2, and 25 mM HEPES, pH 7.6, atroom temperature. Normal saline was obtained from BraunMedical AB (Stockholm, Sweden) and the Milli-Q system


(Millipore, Bedford, Massachusetts) was used to purifythe water.

AnimalsMale Sprague–Dawley rats (260–300 g) were obtainedfrom Taconic (Lille Skensved, Denmark). Rats were ac-climated for at least 7 days in a temperature- andhumidity-controlled environment with 12-hour light/dark cycles before study. The protocols in this studywere approved by the Animal Ethics Committee of UppsalaUniversity, Sweden (C351/11 and C328/10).

Microdialysis study of cefadroxil in the absence andpresence of probenecidSurgery was performed one day before microdialysis inorder to implant vessel catheters and microdialysis probesas described previously [31] with modification. Briefly,under isoflurane anesthesia and with body temperaturecontrolled at 38°C (CMA/150 temperature controller,CMA, Stockholm, Sweden), catheters were inserted intothe left femoral vein for cefadroxil infusion, the left jugularvein for control (Day 1) or probenecid infusion (Day 2),and the left femoral artery for blood sampling. A CMA/20probe with 10 mm polyarylethersulphone (PAES) mem-brane was inserted into the right jugular vein. The rat wasthen fixed on a stereotaxic frame equipped with ananesthesia mask. Two guide cannulas were implanted intothe brain striatum (ST coordinates, +0.2 mm anteroposter-ior, −4.7 mm lateral, −3.8 mm dorsoventral with anangle of 15° at the coronal plane towards midline) andlateral ventricle (LV coordinates, −0.9 mm anteropos-terior, +1.6 mm lateral, −2.9 mm dorsoventral), andfixed to the skull by a screw and dental cement. ACMA 12 probe with 3 mm PAES membrane wasinserted into the striatal guide cannula for monitoringbrain ECF and a CMA 12 probe with 1 mm PAESmembrane was inserted into the ventricular guide cannulafor CSF sampling. At the end of the surgery, the rat wasplaced in a CMA 120 system for freely moving animals inwhich it had free access to food and water, and allowed torecover for 24 hours before experimentation.On Day 1, a 90-min stabilization period was per-

formed in which Ringer’s solution, containing cefadroxil-D4, was perfused through the microdialysis probes bypump (CMA 400, Solna, Sweden) at a flow rate of0.5 μL/min. During this period, and throughout theentire experiment (another 420 min), microdialysis sam-ples (10 μL each) were collected every 20 min using afraction collector (CMA 142, Solna, Sweden) and storedat 4°C until analysis. To quantify unbound drug concen-trations in brain and blood, cefadroxil-D4 was used tocalibrate the probes using retrodialysis [32]. Becausecefadroxil levels in brain and blood were quite different,1 μg/mL cefadroxil-D4 was used to perfuse the blood

probe and 0.1 μg/mL for the brain probe. At 90 min,cefadroxil solution (6 mg/mL in normal saline) was ad-ministered intravenously (iv) as a bolus infusion of0.3 mg/kg/min for 20 min followed by a constant-rateinfusion of 0.15 mg/kg/min for 160 min (for a total of180 min). In addition to the microdialysis samples, arter-ial blood samples (100 μL) were drawn predose and at 5,18, 90, 150, 185, 190, 210, 240, 300, and 420 min afterinitiating the cefadroxil bolus infusion. Plasma was har-vested from blood after centrifuging at 7200 g for 5 minand then frozen at −20°C until analysis. On Day 2, thecefadroxil experiment was repeated, however, 15 mg/mLprobenecid in 5% NaHCO3 in saline (as opposed to 5%NaHCO3 in saline only on Day 1) was added as a20 mg/kg bolus followed by 20 mg/kg/hr infusion for420 min (i.e., cefadroxil in the presence of probenecid).

Microdialysis study of cefadroxil in the absence andpresence of Ala-AlaThe surgery and microdialysis method for this study wassimilar to that described before for probenecid except, inthis case, the dipeptide Ala-Ala was administered insteadand by intracerebroventricular (icv) infusion. In order toperform the microdialysis sampling and icv infusion sim-ultaneously, a microdialysis probe with an additionalinfusion cannula passing through the lumen of probe (IBRcombination probe with 1 mm polyacrylanitrile mem-brane, BASi, West Lafayette, IN, USA) was implanted intothe lateral ventricle (coordinates, −0.9 mm anteroposter-ior, −1.6 mm lateral, −2.9 mm dorsoventral). For thesestudies (i.e., cefadroxil in the absence and presence of Ala-Ala), the experiment was performed in one day. In brief,following the 90-min stabilization period, cefadroxil salinesolution was infused iv at 0.3 mg/kg/min for 20 minfollowed by 0.15 mg/kg/min for 400 min (for a total of420 min). An icv infusion of Ringer’s solution, 0.3 μL/min,was started 30 min prior to cefadroxil administration andmaintained for another 240 min (control phase). At thistime, an icv infusion of 0.32 mg/mL Ala-Ala in Ringer’ssolution was started and then maintained for another180 min.

In vitro brain slice studyThe brain slice protocol was based on a previously pub-lished method with minor modifications [33]. Briefly,fresh brains were collected in which six 300-μm coronalslices were prepared from each animal using a micro-tome (Leica VT1200, Leica Microsystems AB, Sweden).Resultant slices were transferred to an 80-mm diameterbeaker with 15 mL aECF containing 0.8 μM cefadroxilwith or without 5 mM GlySar, 5 mM Ala-Ala, or 1 mMprobenecid. Covered by a lid comprised of a Teflon fluo-rinated ethylene-propylene film (DuPont, Katco Ltd,UK), the beaker was incubated in a shaker (MaxQ4450,


Thermo Fisher Scientific, Nino Lab, Sweden) at 45 rpm,37°C, for 2 hr. Throughout the incubation, there wasa constant flow of oxygen into the shaking chamberto maintain slice viability. After incubation, 200 μLof blank rat brain homogenate without cefadroxilwas added to 200 μL of buffer sample to keep thematrix consistent among all the samples for the fol-lowing analysis. The brain slices were then weighed,after drying on filter paper, and homogenized indi-vidually in aECF (9:1 ratio, w/v) using an ultrasonicprocessor (VCX-130, Sonics, Chemical InstrumentsAB, Sweden). All samples were stored at −20°C untilanalysis.In all experiments, coronal slices were prepared

from the same anatomical plane corresponding to thestriatal region (no midbrain structures) in order toavoid potential discrepancies in the assessment of theunbound volume of distribution of cefadroxil in brain(Vu,brain). In our studies, the Vu,brain values of cefa-droxil were similar in each rat with little variability(mean coefficient of variation ≤5.4%). Potential re-gional differences in the Vu,brain of cefadroxil werenot studied.

Chemical analysisThe analysis of cefadroxil (and cefadroxil D-4) was car-ried out using liquid chromatography–tandem massspectrometry (LC-MS/MS). Specifically, 5 μL microdi-alysis samples were injected into the LC-MS/MS afteradding amoxicillin solution as an internal standard.For plasma and homogenate samples, the proteinswere precipitated by adding acetonitrile at a ratio of1:3. After centrifuging at 7200 g for 3 min, the super-natant was diluted with 0.1% formic acid before inject-ing into the LC-MS/MS. Standard curves and qualitycontrol samples were used to quantify and validate theconcentrations of cefadroxil in all biological matricesfrom the study.Chromatographic separation was achieved on a HyPur-

ity C18 column (50 × 4.6 mm, particle size 3 μm) pro-tected by a HyPurity C18 guard-column (10 × 4.0 mm,particle size 3 μm; Thermo Hypersil-Keystone, PA, USA).A gradient elution involving mobile phase A (0.1% formicacid) and mobile phase B (0.1% formic acid in 1:1 aceto-nitrile:water) was delivered by two Shimadzu LC-10ADvppumps (Shimadzu, Kyoto, Japan) at 0.8 mL/min, whichwas split to 0.3 mL/min before entering the MS detector.A Quattro Ultima Pt mass spectrometer (Waters, Milford,MA, USA) was used for detection on positive electrosprayionization (ESI+) mode. The transition mode was m/z363.9→ 207.9 for cefadroxil, m/z 368.0→ 212.0 forcefadroxil-D4, and m/z 366.0→ 348.9 for amoxicillin. Alldata were acquired and processed using Masslynx 4.1(Waters, Milford, MA, USA).

Data analysisThe relative recovery of cefadroxil in each microdialysisprobe was estimated from retrodialysis of the calibrator,cefadroxil-D4, and calculated as:

Re covery ¼ Cin;CEF−D4 −Cout;CEF−D4

Cin;CEF−D4ð1Þ

where Cin,CEF-D4 is the concentration of cefadroxil-D4 inperfusate and Cout,CEF-D4 is the concentration ofcefadroxil-D4 in dialysate. The unbound concentrationsof cefadroxil in blood (Cu,blood), brain ECF (Cu,ECF), andCSF (Cu,CSF) were calculated from their respective con-centrations in dialysate (Cdialysate) as:

Cu ¼ Cdialysate

Recoveryð2Þ

For the microdialysis study of cefadroxil (with andwithout probenecid), the trapezoidal method was usedto calculate area under the curve for unbound cefadroxil(AUCu) in blood, ECF, and CSF from 0–420 min. AUCu

values from 420 min to infinity were determined by ex-trapolation from the time of the last measured concen-tration Clast according to AUCextrapolated ¼ Clast

λz, in which

λz is the terminal rate constant obtained from the slopeof the last 7 observations. The blood concentration ofcefadroxil at steady-state (Cu,ss,blood) was calculated fromthe average of concentrations during the 120–180 mintime period. The unbound partition coefficient of cefa-droxil in brain ECF (Kp,uu,ECF) and CSF (Kp,uu,CSF) wasobtained as follows:

Kp;uu;ECF ¼ AUCu;ECF

AUCu;bloodð3Þ

Kp;uu;CSF ¼ AUCu;CSF

AUCu;bloodð4Þ

Non-compartmental analyses were performed usingthe microdialysis samples from blood to obtain thepharmacokinetic parameters of unbound cefadroxil,in which area under the moment curve (AUMCu)was also obtained by trapezoidal method. The meaninput time (MIT) was 66 min calculated from MIT ¼R01�Tin21þR02�Tin22

2� R01�Tin1þR02�Tin2ð Þ , where R0 and Tin denote the infu-

sion rate and infusion time of the two consecutivecefadroxil infusions. With the correction of MIT, themean residence time with an iv bolus (MRTiv) wasobtained:

MRTiv ¼ AUMCu;0− inf

AUCu;0− inf−MIT ð5Þ

The total clearance (CL), volume of distributionsteady-state (Vss), and half-life (t1/2) were calculated


based on the total cefadroxil dose (30 mg/kg, whichincludes both the bolus and constant-rate infusions),along with AUCu and AUMCu from times zero to infin-ity (inf ):

CL ¼ Dose

AUCu;0− infð6Þ

Vss ¼ CL �MRTiv ð7Þt1=2 ¼ 0:693 �MRTiv ð8Þ

For the microdialysis study of cefadroxil with andwithout Ala-Ala, Kp,uu was calculated from the unboundconcentration of drug at steady-state (Cu,ss,ECF or Cu,ss,CSF)by:

Kp;uu ¼ Cu;ss:ECF or Cu;ss;CSF� �

Cu;ss;Bloodð9Þ

where Cu,ss was calculated during the 120–200 min timeperiod for the control phase (i.e., without Ala-Ala) andduring the 320–420 min time period for the dipeptidephase (i.e., with Ala-Ala).In analyzing brain slice data, the unbound volume of

distribution in brain (Vu,brain, in mL/g brain) was calcu-lated for cefadroxil as:

Vu;brain ¼ Abrain − V i � Cbuffer

Cbuffer 1−V ið Þ ð10Þ

where Abrain is the total amount of cefadroxil in brainslice, Cbuffer is the concentration of cefadroxil in bufferat the end of incubation, and Vi is the volume of bufferfilm surrounding the brain slice because of incompleteadsorption by the filter paper; Vi was reported as0.094 mL/g brain [34].

Statistical analysisData are expressed as mean ± SEM. A two-tailed pairedt-test was used to compare cefadroxil parameters be-tween the control and inhibition phases. A value ofp <0.05 was considered statistically significant. For thebrain slice study, a one-way ANOVA with Dunnett’s testwas performed to compare each treatment group to thecontrol. GraphPad Prism v5.04 (GraphPad Software Inc.,San Diego, CA) was used for all statistical analyses.

ResultsMicrodialysis study of cefadroxil in the absence andpresence of probenecidThere were no significant differences in probe relativerecoveries between the two days. The recoveries were14 ± 1% for the 3-mm probe in brain ECF, 6.7 ± 1.1% forthe 1-mm probe in lateral ventricle, and 71 ± 2% for the10-mm probe in blood. As shown in Figure 1A, steady-

state concentrations of cefadroxil in blood were quicklyachieved after the bolus infusion of 0.3 mg/kg/min for20 min followed by the constant-rate infusion of0.15 mg/kg/min for 160 min. Compared to Day 1 (con-trol phase), probenecid infusion increased Cu,ss,blood

and AUCu of cefadroxil by ~60%. The elevated sys-temic exposure probably resulted from a decrease incefadroxil clearance from 16.9 ± 1.0 to 10.7 ± 0.7 mL/min/kg (Table 1). However, the MRT and t1/2 did notdiffer significantly between days, reflecting a reducedvolume of distribution (Vss) with probenecid, indicat-ing probenecid may decrease the accumulation ofcefadroxil in certain tissues. Plasma cefadroxil concen-trations (data not shown) were comparable to theunbound blood concentrations from microdialysis, con-sistent with previous studies showing that the unboundfraction of cefadroxil in plasma (fu) is nearly 1.0 [35].In addition to increasing unbound cefadroxil blood

concentrations, probenecid increased the AUCu of drugin brain ECF 4-fold (p <0.05) and the AUCu of drug inCSF 2-fold (p >0.05) (Figures 1B and 1C, and Table 1).To determine if cefadroxil penetration into brain wasaffected by probenecid, brain drug concentrations werecorrected by the corresponding values in blood (Figure 2).During probenecid infusion (Day 2), the Cu,ECF values ofcefadroxil, relative to blood, were higher than control(Day 1) at all time points. In contrast, the Cu,CSF values ofcefadroxil, relative to blood, were comparable. To evaluatethe effect of probenecid on cefadroxil penetration intobrain, the unbound partition coefficient Kp,uu was calcu-lated for both brain ECF and CSF using AUCu values from0–420 min and from 0-infinity (Figure 3). Kp,uu wasaround 0.02 in both brain ECF and CSF in the controlsituation, indicating limited penetration of cefadroxil intobrain and extensive efflux at the BBB (Table 1). Kp,uu,ECF

values were about 2.5-fold greater with probenecid treat-ment as compared to control. In contrast, there were nosignificant differences in Kp,uu,CSF between control andprobenecid treatments. This may reflect, in part, greaterexperimental variability in the direction of change for thisparameter (Figures 3C and 3D).

Microdialysis study of cefadroxil in the absence andpresence of Ala-AlaRecoveries were 16 ± 2%, 12 ± 1%, and 72 ± 1% forprobes in the striatum, lateral ventricle and blood, re-spectively. Ala-Ala is a natural dipeptide that can be de-graded in the body; thus, Ala-Ala was infused by the icvroute in order to achieve high concentrations in CSF.The goal of the study was to determine if Ala-Ala affectsthe distribution of cefadroxil by comparing levels in ECFand CSF between vehicle control phase and duringAla-Ala infusions. As shown in Figure 4, the unboundconcentrations of cefadroxil did not change substantially

Figure 1 The concentration-time profiles of unbound cefadroxilin rat blood (A), brain ECF (B), and CSF (C) in the absence andpresence of probenecid. Open circles represent the results fromDay 1 (no probenecid) and solid circles the results from Day 2 (withprobenecid). Data are expressed as mean ± SEM (n = 6).


in brain ECF or CSF during Ala-Ala infusions. Further-more, there was no significant difference betweencontrol and Ala-Ala infusions in Kp,uu,ECF (0.033 ± 0.004to 0.041 ± 0.008, p = 0.15) or Kp,uu,CSF (0.038 ± 0.017 to0.043 ± 0.016, p = 0.43).

In vitro brain slice studyVu,brain describes the relationship between the totalamount of drug in brain and the unbound concentrationof drug in ECF, and is useful as a measure of intra-parenchymal distribution [36]. A higher value Vu,brain

suggests that more drug accumulates inside the braincells. For control brain slices, the Vu,brain of cefadroxilwas 3.67 ± 0.23 mL/g brain (Figure 5). Two PEPT2substrates, Ala-Ala and GlySar, reduced the Vu,brain of

Table 1 Pharmacokinetic parameters of unboundcefadroxil in rat blood and brain on Day 1 (Control, Ctrl)and Day 2 (with probenecid, Pro)

Parameters Unit Day 1 (Ctrl) Day 2 (Pro) Pro/Ctrl

Blood

AUCu (0–420) μg*min/mL 1747 ± 90 2801 ± 175*** 1.60

AUCu (0-inf) μg*min/mL 1802 ± 97 2873 ± 177*** 1.59

Cu,ss,blood μg/mL 8.5 ± 0.4 13.8 ± 0.9*** 1.62

MRTiv min 71 ± 4 77 ± 4 1.05

t1/2 min 49 ± 2 53 ± 3 1.09

CL mL/min/kg 16.9 ± 1.0 10.7 ± 0.7*** 0.63

Vss L/kg 1.19 ± 0.12 0.82 ± 0.10*** 0.69

Brain ECF

AUCu (0–420) μg*min/mL 31 ± 5 122 ± 31* 3.93

AUCu (0-inf) μg*min/mL 40 ± 7 174 ± 35** 4.37

Kp,uu,ECF (0–420) 0.018 ± 0.003 0.042 ± 0.009* 2.35

Kp,uu,ECF (0-inf) 0.022 ± 0.003 0.058 ± 0.009* 2.63

Brain CSF

AUCu (0–420) μg*min/mL 39 ± 12 73 ± 27 1.88

AUCu (0-inf) μg*min/mL 57 ± 15 117 ± 50 2.04

Kp,uu,CSF (0–420) 0.022 ± 0.006 0.024 ± 0.008 1.13

Kp,uu,CSF (0-inf) 0.031 ± 0.007 0.039 ± 0.015 1.26

Data are expressed as mean ± SEM (n = 6). A paired t-test was performed tocompare cefadroxil parameters between the control (without probenecid) andtreatment (with probenecid) phases of the study. *p < 0.05, **p < 0.01, and***p < 0.001. Abbreviations: AUCu, Area under the unbound concentration-timecurve from time zero to 420 min (0–420) or from time zero to infinity (0-inf);Cu,ss,blood, Unbound steady-state blood concentration; MRTiv, Mean residencetime; t1/2, Half-life; CL, Total clearance; Vss, Volume of distribution steady-state;Kp,uu,ECF, Ratio of AUCu in brain ECF to AUCu in blood; and Kp,uu,CSF, Ratio ofAUCu in CSF to AUCu in blood.

Figure 2 The ratio of unbound cefadroxil in rat brain ECF (A) orCSF (B) to that in blood versus time. Open circles represent theresults from Day 1 (no probenecid) and solid circles the results fromDay 2 (with probenecid). Data are expressed as mean ± SEM (n = 6).


cefadroxil to 0.95 ± 0.45 and 1.10 ± 0.05 mL/g brain, re-spectively, indicating that they reduced the accumulationof cefadroxil inside brain cells (p < 0.001). In contrast,probenecid increased the Vu,brain of cefadroxil to 6.06 ±0.15 mL/g brain, suggesting that probenecid led to moreaccumulation of cefadroxil inside brain cells (p < 0.001).

DiscussionThe current study used microdialysis and brain slicemethods to examine the transport mechanisms affectingthe distribution of cefadroxil, a cephalosporin antibiotic, inbrain. The results demonstrated that: 1) co-administrationof probenecid increased blood cefadroxil levels; 2) probene-cid markedly increased brain ECF cefadroxil concentra-tions; 3) the probenecid effect on brain ECF levels werepartially due to increased blood concentrations but also

due to inhibition of cefadroxil efflux at the BBB (OATs,OATPs and/or MRPs); 4) in contrast, increased CSFcefadroxil concentrations with probenecid were onlydue to elevated blood concentrations of antibiotic; 5)intracerebroventricular infusion of the PEPT2 sub-strate, Ala-Ala, did not increase brain ECF or CSFcefadroxil levels; and 6) brain slice experiments dem-onstrated that PEPT2 was involved in the uptake ofcefadroxil into brain cells and that probenecid blockeda mechanism transporting cefadroxil out of cells.In the interaction study between cefadroxil and pro-

benecid, intravenous co-administration of probenecid re-duced the clearance of cefadroxil. This finding wasconsistent with previous studies [20,25] showing thatprobenecid inhibits the renal secretion of many cephalo-sporins by OATs (and perhaps MRPs and OATPs) at thekidney proximal tubule [37]. Even though steady-stateconcentrations were achieved quickly for unbound cefa-droxil in blood, steady-state concentrations in brain ECFwere not fully reached within the infusion period of3 hr. As a consequence, Cu,ECF decreased more slowlythan Cu,blood after termination of the cefadroxil infusion.The above phenomenon may be due to low permeabilityof passive diffusion of cefadroxil at the BBB, consideringits high hydrophilicity. The Kp,uu of brain ECF is deter-mined by the net influx and efflux clearances at the BBB,as Kp,uu = CLin/CLout [36]. If only passive transport oc-curs at the BBB, Kp,uu is equal to unity due to the equalvalues for CLin and CLout. However, the Kp,uu,ECF of cefa-droxil was about 0.02, indicating that cefadroxil CLout ismuch higher than CLin. Thus, it appears that there is netefflux transport for cefadroxil at the BBB. It has been re-ported that cefadroxil is a substrate of OATs and MRPs[6,21,22]. Specifically, OAT3 located at the basolateral(abluminal) side of the BBB and MRPs at the apical (lu-minal) side of the BBB mediate brain-to-blood transportas efflux transporters, thus possibly contributing the lowKp,uu,ECF of cefadroxil [13,15,38]. Inhibition of OAT3and/or MRPs at the BBB is the probable reason whyprobenecid increased the Kp,uu,ECF of cefadroxil ~2.5fold. In addition to OATs and MRPs, cefadroxil was re-ported to be a substrate of OATPs. However, OATPs arebidirectional transporters [12,17,18] and their net effecton cefadroxil transport at the BBB is unknown. A sche-matic representation of the membrane transporters in-volved in the CNS distribution of cefadroxil is shown inFigure 6.OATs and MRPs [13,14] are also responsible for the

transport of substrates from CSF to blood at the BCSFB.Therefore, it was expected that inhibition of OATs andMRPs by probenecid would increase the Kp,uu,CSF ofcefadroxil. However, no significant change was foundfor this parameter. The differential effect of transporterinhibition by probenecid on the distribution of cefadroxil

Figure 3 The unbound partition coefficient (Kp,uu) of cefadroxil in rat brain ECF (A, B) and CSF (C, D) for each of the six animals. CEFrepresents the study in which cefadroxil is given alone (Day 1) and CEF + PRO is when cefadroxil is given in the presence of probenecid (Day 2).See Table 1 for statistical analyses.

Figure 4 The concentration-time profiles of unbound cefadroxilin rat blood, brain ECF, and CSF in the absence and presenceof Ala-Ala. Solid squares represent the results in blood, open circlesthe results in brain ECF, and solid triangles the results in CSF. Thevertical dashed line separates the two treatment phases (CEF ± Ala-Ala).Data are expressed as mean ± SEM (n = 7).


in brain ECF and CSF may be related to the physio-logical and structural differences between BBB andBCSFB. The complement of efflux transporters, their ex-pression levels, and cellular location may affect the rela-tive importance of individual transporters in each of thetwo systems. In addition, the endothelial BBB is tighterthan the epithelial BCSFB (choroid plexus), affectingparacellular diffusion [42]. A recent study on the effectsof probenecid on methotrexate transport found a differ-ent modulation of methotrexate distribution in brainECF and CSF [43]. There was a dose-dependent effect,in which probenecid increased the brain ECF-to-plasmaratio for two dose regimens of methotrexate (40 mg/kgand 80 mg/kg), whereas probenecid only significantly in-creased the CSF-to-plasma ratio at the higher dose [43].The differential effects of probenecid on cefadroxil atthe BBB and BCSFB in our study are unlikely to be dueto differences in inhibitor concentration at the two sitesas Deguchi et al. [44] found higher probenecid concen-trations in CSF than ECF after systemic dosing.In a previous study, PEPT2 ablation resulted in a

marked increase in the CSF-to-blood concentration ratioof cefadroxil, indicating the importance of PEPT2 ineliminating cefadroxil from CSF at the BCSFB [20].However, in the present study, an icv infusion of thePEPT2 substrate Ala-Ala did not significantly changeCSF cefadroxil concentrations. This lack of effect mayreflect insufficient concentrations of Ala-Ala reaching

the BCSFB. Ala-Ala was chosen to inhibit PEPT2 be-cause it has a relatively high affinity for that transporter(Ki = 6.3 μM, similar to that of cefadroxil with a Ki =3.0 μM) [2]. However, Ala-Ala has the disadvantage ofbeing degraded by peptidases, many of which are foundin the choroid plexus and brain [45].

Figure 5 The unbound volume of distribution of cefadroxil(Vu,brain) in rat brain slices. Studies were performed with 0.8 μMcefadroxil alone (Control) and in the presence of inhibitors (Ala-Ala,GlySar and Probenecid treatments). Data are expressed as mean ±SEM (n = 3-4). One-way ANOVA followed by the Dunnett’s test wasperformed to compare the inhibitor and control phases. ***p < 0.001compared to control.


Vu,brain is a measure of drug distribution within brainparenchyma. The water volume in brain parenchyma is0.8 mL/g brain and a Vu,brain of around 0.8 mL/g brainindicates a drug is distributed evenly through the wholebrain tissue [36]. From a previous study using equilibrium

Figure 6 Membrane transporters (potentially) involved in the CNS disschematic representation [16,39-41]. There is much debate regarding the isthough, considerable evidence for some MRPs having an apical distribution

evidence for the probenecid-inhibitable efflux of cefadroxil from brain cells

MRP and/or OATP transporters. BL represents the basolateral membrane, A

dialysis in brain homogenate (data not published), cefa-droxil had a fraction unbound (fu) of nearly 1, indicatinglittle, if any, drug binding to brain tissues. This, togetherwith the cefadroxil Vu,brain of 3.67 mL/g brain in the presentstudy indicates the presence of uptake transporter(s) at themembrane of brain cells. The PEPT2 substrates, 5 mMAla-Ala and GlySar, reduced the Vu,brain of cefadroxil, indi-cating that competitive inhibition of PEPT2 decreased theuptake of cefadroxil into brain cells. This is consistent withprevious findings that PEPT2 is expressed on neurons andresponsible for cellular uptake [46]. In contrast, probenecidincreased the Vu,brain of cefadroxil, indicating there may alsobe efflux transporters (e.g., OATs, MRPs or OATPs) remov-ing cefadroxil from brain cells. Interestingly, a previousstudy demonstrated that probenecid increased the intracel-lular levels of valproic acid by 1.5-fold in rabbit brain dur-ing in vivomicrodialysis [47].By using intracerebral microdialysis in vivo and brain

slices in vitro, a better understanding was obtained aboutthe effect of transporters on cefadroxil distribution inbrain and, specifically, in brain extracellular and intracellu-lar fluids, and CSF. From our study, it appears that trans-porters which are probenecid inhibitable (i.e., OATs,MRPs and/or OATPs) move cefadroxil in a vectorial direc-tion from brain ECF to blood, and that PEPT2 transportscefadroxil into brain cells. In addition, as probenecid in-creased cefadroxil uptake into brain slices, there is an asyet unidentified cefadroxil transporter effluxing this ceph-alosporin from brain cells. It is concluded that multipletransporters play a role in the distribution of cefadroxil

tribution of cefadroxil. Several references were used to inform thisoforms and membrane localization of MRPs at the BBB. There is,clearing substrates to blood as depicted. There is also functional

, the nature of which is uncertain but may include OAT,

P the apical membrane, and EP the ependyma.


into and within the brain. The impact of these trans-porters on specific cephalosporins will depend on trans-porter affinities and drug levels in brain. Microdialysis is auseful tool to study the kinetics of unbound drug concen-trations in ECF and CSF [48]. The brain slice method,together with other tools like equilibrium dialysis, providesan approach to study the distribution of drugs withinbrain after passing the BBB and BCSFB [34,49].A deeper understanding of the brain distribution of

cephalosporins may aid in the better use of these anti-bacterial agents for the prophylaxis and treatment ofCNS infections. Bacterial meningitis is an inflammatoryprocess of the leptomeninges caused by bacterial infec-tions. Bacterial meningitis is the most frequent CNS in-fection with a mortality rate approaching 20% [50]. It isbelieved that bacteria enter the CNS across BBB orBCSFB via transcytosis and finally enter the CSF [50].Even though BBB permeability increases during menin-gitis [51], the barriers and their efflux transporters stillplay a role in limiting cephalosporin entry to brain. Clin-ically, the cephalosporins used for meningitis are limitedto ceftriaxone, cefotaxime, ceftazidime, and cefepime,which have high penetration into CSF [52]. AnotherCNS infection is cerebritis, a focal brain parenchyma in-fection, which is often followed by brain abscesses andpermanent damage [53]. Treatment for cerebritis andbrain abscesses also involves antibiotics. The strategy ofblocking the related efflux transporters at the BBB andBCSFB is a promising way to enhance the penetration ofrelevant cephalosporins into brain ECF and CSF.Probenecid was firstly widely used to decrease renal

clearance of penicillin during World War II, when anti-biotic supplies were low. Probenecid decreases the elim-ination rate and volume of distribution for a variety ofmedications including most cephalosporins [54]. How-ever, with easier and cheaper production of antibiotics,probenecid is now seldom used with antibiotics. Thepresent study showed that probenecid was able to in-crease the distribution of cefadroxil in brain ECF notonly by reducing the renal clearance (and increasing sys-temic exposure) but also by specifically increasing thepenetration into brain (i.e., increased Kp,uu) and furtherinto brain cells. It should be appreciated that, althoughthis study was not designed to study cefadroxil underclinical dosing conditions, the co-administration of pro-benecid allowed cefadroxil to reach the lower limit of itsminimal inhibitory concentration in brain ECF for somebacteria (i.e., about 0.4 μg/mL). Thus, the combinedtherapy of cefadroxil (or perhaps other cephalosporins)and probenecid might be useful for some cases of men-ingitis and brain abscesses. Whether or not this ap-proach is feasible would depend upon the extent of thisdrug-drug interaction in patients during different dosingcombinations of both antibiotic and the inhibitor.

Moreover, there is a delicate balance between the dose–response relationships of bacterial kill and CNS toxicity,which of course would have to be taken into account.

ConclusionsUsing in vivo microdialysis and in vitro brain slicemethods in rat, the present study demonstrated thatprobenecid increased cefadroxil distribution into brainextracellular and intracellular fluids by blocking relatedefflux transporters at the BBB and brain cells. Our find-ings suggest that the combination of probenecid andsome cephalosporins may provide a strategy to increasetherapeutic drug levels in brain for better treatment ofCNS infections like bacterial meningitis and brain ab-scesses. On the other hand, since multiple transportersare involved in transporting cephalosporins in brain,there is also the potential for drug-drug interactions toenhance cephalosporin-induced neurotoxicity.

AbbreviationsaECF: Artificial extracellular fluid; CSF: Cerebrospinal fluid; BBB: Blood–brainbarrier; BCSFB: Blood-cerebrospinal fluid barrier; CNS: Central nervous system;POT: Proton-coupled oligopeptide transporter; OAT: Organic aniontransporter; OATP: Organic anion transporting polypeptide; MRP: Multidrugresistance-associated protein; PEPT2: Peptide transporter 2; CEF: Cefadroxil;PRO: Probenecid; GlySar: Glycylsarcosine; Cu,blood: Unbound concentration ofcefadroxil in blood; Cu,ECF: Unbound concentration of cefadroxil in brainextracellular fluid; Cu,CSF: Unbound concentration of cefadroxil incerebrospinal fluid; AUC: Area under the curve; t1/2: Half-life; inf: Infinity;CL: Clearance; MRT: Mean residence time; AUMC: Area under the momentcurve; Vss: Volume of distribution steady-state; Kp,uu: Unbound partitioncoefficient; Vu,brain: Unbound volume of distribution in brain.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsXC contributed to the design of the study, carried out all experiments,performed data collection and analysis, and drafted the manuscript. ILparticipated in the brain slice study. MP participated in all experiments anddata collection. RFK, DES, MHU contributed to the design of the study, datainterpretation, and drafting of the manuscript. All authors have read andapproved the final version of the manuscript.

AcknowledgementsThis work was supported by the National Institutes of Health NationalInstitute of General Medical Sciences grant R01-GM035498 (to DES) and bythe Dr. Joan B. Kessler Award (a Rackham International Research Award) fromthe University of Michigan. The authors greatly appreciated the excellenttechnical support provided by Britt Jansson in the chemical analyses and byJessica Dunhall in the animal experiments.

Author details1Department of Pharmaceutical Sciences, College of Pharmacy, University ofMichigan, Ann Arbor, Mi 48109, USA. 2Department of PharmaceuticalBiosciences, Translational PKPD Research Group, Uppsala University, Box 591,SE-75124 Uppsala, Sweden. 3Biopharmaceutics and Pharmacokinetic Division,Department of Pharmaceutics, Faculty of Pharmacy, Tehran University ofMedical Sciences, Tehran, Iran. 4Department of Neurosurgery, University ofMichigan Health System, Ann Arbor, MI 48109, USA.

Received: 18 August 2014 Accepted: 18 October 2014Published: 14 November 2014


References1. Brunton LL, Chabner BA, Knollmann BC: Goodman & Gilman’s The

Pharmacological Basis of Therapeutics. 12th edition. New York:McGraw-Hill; 2011.

2. Brandsch M, Knutter I, Bosse-Doenecke E: Pharmaceutical andpharmacological importance of peptide transporters. J PharmPharmacol 2008, 60:543–585.

3. Burckhardt G: Drug transport by Organic Anion Transporters (OATs).Pharmacol Ther 2012, 136:106–130.

4. Nakakariya M, Shimada T, Irokawa M, Koibuchi H, Iwanaga T, Yabuuchi H,Maeda T, Tamai I: Predominant contribution of rat organic aniontransporting polypeptide-2 (Oatp2) to hepatic uptake of beta-lactamantibiotics. Pharm Res 2008, 25:578–585.

5. Nakakariya M, Shimada T, Irokawa M, Maeda T, Tamai I: Identification andspecies similarity of OATP transporters responsible for hepatic uptake ofbeta-lactam antibiotics. Drug Metab Pharmacokinet 2008, 23:347–355.

6. Akanuma S, Uchida Y, Ohtsuki S, Kamiie J, Tachikawa M, Terasaki T, HosoyaK: Molecular-weight-dependent, anionic-substrate-preferential transportof beta-lactam antibiotics via multidrug resistance-associated protein 4.Drug Metab Pharmacokinet 2011, 26:602–611.

7. Kato Y, Takahara S, Kato S, Kubo Y, Sai Y, Tamai I, Yabuuchi H, Tsuji A:Involvement of multidrug resistance-associated protein 2 (Abcc2) inmolecular weight-dependent biliary excretion of beta-lactam antibiotics.Drug Metab Dispos 2008, 36:1088–1096.

8. Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, DahlinA, Evers R, Fischer V, Hillgren KM, Hoffmaster KA, Ishikawa T, Keppler D, KimRB, Lee CA, Niemi M, Polli JW, Sugiyama Y, Swaan PW, Ware JA, Wright SH,Yee SW, Zamek-Gliszczynski MJ, Zhang L: Membrane transporters in drugdevelopment. Nat Rev Drug Discov 2010, 9:215–236.

9. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ: Structure andfunction of the blood–brain barrier. Neurobiol Dis 2010, 37:13–25.

10. Abbott NJ: Blood–brain barrier structure and function and the challengesfor CNS drug delivery. J Inherit Metab Dis 2013, 36:437–449.

11. Saunders NR, Habgood MD, Dziegielewska KM: Barrier mechanisms in thebrain, I. adult brain. Clin Exp Pharmacol Physiol 1999, 26:11–19.

12. Kusuhara H, Sugiyama Y: Efflux transport systems at the blood–brainbarrier and blood CSF barrier. In Drug Transport(ers) and the Diseased Brain,International Congress Series. 1277th edition. Edited by DeBoer AG.2005:111–122.

13. Hartz AMS, Bauer B: ABC Transporters in the CNS - an inventory. Curr PharmBiotechno 2011, 12:656–673.

14. Farthing CA, Sweet DH: Expression and function of organic cation andanion transporters (SLC22 family) in the CNS. Curr Pharm Design 2014,20:1472–1486.

15. Loscher W, Potschka H: Role of drug efflux transporters in the brain fordrug disposition and treatment of brain diseases. Prog Neurobiol 2005,76:22–76.

16. Smith DE, Johanson CE, Keep RF: Peptide and peptide analog transportsystems at the blood-CSF barrier. Adv Drug Deliv Rev 2004, 56:1765–1791.

17. Westholm DE, Rumbley JN, Salo DR, Rich TP, Anderson GW: Organic anion-transporting polypeptides at the blood–brain and blood-cerebrospinalfluid barriers. Curr Top Dev Biol 2008, 80:135–170.

18. Ronaldson PT, Davis TP: Targeted drug delivery to treat pain and cerebralhypoxia. Pharmacol Rev 2013, 65:291–314.

19. Xiang J, Chiang PP, Hu Y, Smith DE, Keep RF: Role of PEPT2 inglycylsarcosine transport in astrocyte and glioma cultures. Neurosci Lett2006, 396:225–229.

20. Shen H, Ocheltree SM, Hu Y, Keep RF, Smith DE: Impact of geneticknockout of PEPT2 on cefadroxil pharmacokinetics, renal tubularreabsorption, and brain penetration in mice. Drug Metab Dispos 2007,35:1209–1216.

21. Khamdang S, Takeda M, Babu E, Noshiro R, Onozato ML, Tojo A, Enomoto A,Huang XL, Narikawa S, Anzai N, Piyachaturawat P, Endou H: Interaction ofhuman and rat organic anion transporter 2 with various cephalosporinantibiotics. Eur J Pharmacol 2003, 465:1–7.

22. de Waart DR, van de Wetering K, Kunne C, Duijst S, Paulusma CC, OudeElferink RP: Oral availability of cefadroxil depends on ABCC3 and ABCC4.Drug Metab Dispos 2012, 40:515–521.

23. Garcia-Carbonell MC, Granero L, Torres-Molina F, Aristorena JC, Chesa-JimenezJ, Pla-Delfina JM, Peris-Ribera JE: Nonlinear pharmacokinetics of cefadroxil inthe rat. Drug Metab Dispos 1993, 21:215–217.

24. Posada MM, Smith DE: Relevance of PepT1 in the intestinal permeabilityand oral absorption of Cefadroxil. Pharm Res 2013, 30:1017–1025.

25. Marino EL, Dominguezgil A: The pharmacokinetics of Cefadroxilassociated with probenecid. Int J Clin Pharm Th 1981, 19:506–508.

26. Sugiyama D, Kusuhara H, Shitara Y, Abe T, Meier PJ, Sekine T, Endou H,Suzuki H, Sugiyama Y: Characterization of the efflux transport of17beta-estradiol-D-17beta-glucuronide from the brain across theblood–brain barrier. J Pharmacol Exp Ther 2001, 298:316–322.

27. Luna-Tortos C, Fedrowitz M, Loscher W: Evaluation of transport ofcommon antiepileptic drugs by human multidrug resistance-associatedproteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistantepilepsy. Neuropharmacology 2010, 58:1019–1032.

28. Ocheltree SM, Shen H, Hu Y, Xiang J, Keep RF, Smith DE: Mechanisms ofcefadroxil uptake in the choroid plexus: studies in wild-type and PEPT2knockout mice. J Pharmacol Exp Ther 2004, 308:462–467.

29. Jiang H, Hu Y, Keep RF, Smith DE: Enhanced antinociceptive response tointracerebroventricular kyotorphin in Pept2 null mice. J Neurochem 2009,109:1536–1543.

30. Xiang J, Jiang H, Hu Y, Smith DE, Keep RF: Kyotorphin transport andmetabolism in rat and mouse neonatal astrocytes. Brain Res 2010,1347:11–18.

31. Xie RJ, Bouw MR, Hammarlund-Udenaes M: Modelling of the blood–brainbarrier transport of morphine-3-glucuronide studied using microdialysisin the rat: involvement of probenecid-sensitive transport. Br J Pharmacol2000, 131:1784–1792.

32. Bengtsson J, Bostrom E, Hammarlund-Udenaes M: The use of a deuteratedcalibrator for in vivo recovery estimations in microdialysis studies.J Pharm Sci 2008, 97:3433–3441.

33. Loryan I, Friden M, Hammarlund-Udenaes M: The brain slice method forstudying drug distribution in the CNS. Fluids Barriers CNS 2013, 10:6.

34. Friden M, Ducrozet F, Middleton B, Antonsson M, Bredberg U, Hammarlund-Udenaes M: Development of a high-throughput brain slice method forstudying drug distribution in the central nervous system. Drug MetabDispos 2009, 37:1226–1233.

35. Huh Y, Keep RF, Smith DE: Impact of lipopolysaccharide-induced inflammationon the disposition of the aminocephalosporin cefadroxil. Antimicrob AgentsChemother 2013, 57:6171–6178.

36. Hammarlund-Udenaes M, Friden M, Syvanen S, Gupta A: On the rate andextent of drug delivery to the brain. Pharm Res 2008, 25:1737–1750.

37. Giacomini KM, Huang SM: Transporters in drug development and clinicalpharmacology. Clin Pharmacol Ther 2013, 94:3–9.

38. Mahringer A, Ott M, Reimold I, Reichel V, Fricker G: The ABC of theblood–brain barrier - regulation of drug efflux pumps. Curr PharmDesign 2011, 17:2762–2770.

39. Keep RF, Smith DE: Choroid plexus transport: gene deletion studies.Fluids Barriers CNS 2011, 8:26.

40. Redzic Z:Molecular biology of the blood–brain and the blood-cerebrospinalfluid barriers: similarities and differences. Fluids Barriers CNS 2011, 8:3.

41. Miller DS: ABC transporters at the blood–brain barrier. Top Med Chem2013. doi:10.1007/7355_2013_31.

42. Abbott NJ, Ronnback L, Hansson E: Astrocyte-endothelial interactions atthe blood–brain barrier. Nat Rev Neurosci 2006, 7:41–53.

43. Westerhout J, van den Berg DJ, Hartman R, Danhof M, de Lange EC:Prediction of methotrexate CNS distribution in different species - influenceof disease conditions. Eur J Pharm Sci 2014, 57:11–24.

44. Deguchi Y, Nozawa K, Yamada S, Yokoyama Y, Kimura R: Quantitative evaluationof brain distribution and blood–brain barrier efflux transport of probenecid inrats by microdialysis: possible involvement of the monocarboxylic acidtransport system. J Pharmacol Exp Ther 1997, 280:551–560.

45. Bourne A, Barnes K, Taylor BA, Turner AJ, Kenny AJ: Membrane peptidasesin the pig choroid plexus and on other cell surfaces in contact with thecerebrospinal fluid. Biochem J 1989, 259:69–80.

46. Shen H, Smith DE, Keep RF, Brosius FC 3rd: Immunolocalization of theproton-coupled oligopeptide transporter PEPT2 in developing rat brain.Mol Pharm 2004, 1:248–256.

47. Scism JL, Powers KM, Artru AA, Lewis L, Shen DD: Probenecid-inhibitableefflux transport of valproic acid in the brain parenchymal cells of rabbits:a microdialysis study. Brain Res 2000, 884:77–86.

48. Hammarlund-Udenaes M: The use of microdialysis in CNS drug deliverystudies - pharmacokinetic perspectives and results with analgesics andantiepileptics. Adv Drug Deliv Rev 2000, 45:283–294.


49. Friden M, Gupta A, Antonsson M, Bredberg U, Hammarlund-Udenaes M: In vitromethods for estimating unbound drug concentrations in the brain interstitialand intracellular fluids. Drug Metab Dispos 2007, 35:1711–1719.

50. Meli DN, Christen S, Leib SL, Tauber MG: Current concepts in thepathogenesis of meningitis caused by Streptococcus pneumoniae.Curr Opin Infect Dis 2002, 15:253–257.

51. Tunkel AR, Scheld WM: Pathogenesis and pathophysiology of bacterial-meningitis. Clin Microbiol Rev 1993, 6:118–136.

52. Tunkel AR, Hartman BJ, Kaplan SL, Kaufman BA, Roos KL, Scheld WM,Whitley RJ: Practice guidelines for the management of bacterialmeningitis. Clin Infect Dis 2004, 39:1267–1284.

53. Kumar V, Abbas AK, Fausto N, Robbins SL, Cotran RS: Robbins and CotranPathologic Basis of Disease. Philadelphia: Elsevier Saunders; 2005.

54. Robbins N, Koch SE, Tranter M, Rubinstein J: The history and future ofprobenecid. Cardiovasc Toxicol 2012, 12:1–9.

doi:10.1186/2045-8118-11-25Cite this article as: Chen et al.: Effect of transporter inhibition on thedistribution of cefadroxil in rat brain. Fluids and Barriers of the CNS2014 11:25.

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