pb,

k, In, Inn

Keywords:ThiomersPEGPEI-thiobutyrolactoneEfux pumpsP-glycoprotein

wasy cnally thiolation with -thiobutyrolatone. Furthermore, the potential of this novel

Journal of Controlled Release 147 (2010) 6269

Contents lists available at ScienceDirect

Journal of Contro

j ourna l homepage: www.e lsevsuch as P-gp. In order to overcome this problem various types of efuxpump inhibitors have been developed. So far, however, none of themhas been approved in an oral delivery system mainly because ofsystemic side effects. An alternative might be polymeric efux pumpinhibitors showing on the one hand efciently high inhibitory activityand on the other hand being not absorbed from the GI-tract because oftheir high molecular mass.

Firstly, it has been reported previously by several research groups

Secondly, thiolated polymers or thiomers are supposed to be apromising tool in the development of drug delivery systems, as theimmobilization of thiol groups on well-established polymers results intremendous changes in terms of improved mucoadhesive, cohesive,enzyme inhibitory and permeation enhancing properties [35]. Recent-ly,Werle and Hoffer [6] reported a signicantly improved transmucosaltransport of P-gpsubstrate rhodamine-123(Rho-123) in thepresenceofthiolated chitosan as a result of P-gp efux pump inhibition. The efuxthat PEGs can inhibit efux pumps. For inimproved the permeation of efux pump subdoxorubicin by the inhibition of P-gp in

Corresponding author. Institute of Pharmacy,Innsbruck, Josef-Mller-Haus, Innrain 52c, A-6020 Inn507 5383; fax: +43 512 507 2933.

E-mail address: andreas.bernkop@uibk.ac.at (A. Bern

0168-3659/$ see front matter 2010 Elsevier B.V. Adoi:10.1016/j.jconrel.2010.06.023ally because of poor and/ation due to efuxpumps

of PEG 400 signicantly decrease the basolateral to apical transport ofdigoxin through stripped rat jejunal mucosa, indicating efux pumpinhibition.Numerous drugs cannot be administered oror highly variable uptake in the systemic circul1. Introductionwere calculated and compared with values gained from experiments with the well-established P-gp inhibitorsverapamil, reducedGSH, 6-mercatopurine and vitamin E-TPGS and the structurally similar compounds,myrj 52and brij 35. The thiolated co-polymer displayed 145.071.64 mol/g of remaining primary amino groups,84.305.43 mol/g of immobilized thiol groups and 12.741.57 mol/g of disulde bonds. The approximatemolecularmass of the thiolated co-polymerwas 16,000 Da. The 1H-NMR spectrum of PEG-g-PEI co-polymerwascharacterized by the presence of signal groups of PEG, hexamethylene diisocyanate (HMDI) and PEIsubstructures. Studies with Caco-2 cells revealed that the thiolated co-polymer shows 6.690.27% ofcytotoxicity by LDH assay and 93.330.07% of cell viability by MTT assay. The thiolated co-polymer in aconcentration of 0.5% (w/v) displayed a more pronounced effect on the absorptive transport of Rho-123(Papp=15.21.0106 cm/s) in comparison to reduced GSH, 6-mercatopurine, vitamin E-TPGS, myrj 52 andbrij 35. The thiolated co-polymer increased the transport of Rho-123 up to 3.3-fold in comparison to Rho-123without any inhibitor used as control (Papp=4.70.110

6 cm/s). The thiolated co-polymer applied in aconcentration of 0.1%, 0.25% and 0. 5% (w/v) did not only enhance the absorption but also decreased the secretorytransport of Rho-123 resulting in efux ratios (secretory Papp/absorptive Papp) of 1.0, 1.4 and 2.0, respectively.Because of these features the novel thiolated PEG-g-PEI co-polymer seems to exhibit promising properties asnovel P-gp inhibitor.

2010 Elsevier B.V. All rights reserved.

Furthermore, Johnson et al. [2] showed that concentrations of 120%stance, PEG signicantlystrates such as taxol andCaco-2 monolayers [1].

pump inhibitorythiol groups. Becabsorbed from that the site of drugby the use of the

Combining bsystem might lepotential than ea

Leopold-Franzens-Universitysbruck, Austria. Tel.: +43 512

kop-Schnrch).

ll rights reserved.ng-type chambers. Apparent permeability coefcients (Papp)Available online 31 July 2010 thiolated PEG-g-PEI co-polymer on the transport of rhodamine-123 (Rho-123) as P-gp substrate across freshlyexcised rat intestinal mucosa was evaluated in UssiAccepted 27 June 2010 polyethylenimine (PEI) andDesign and in vitro evaluation of a novel

Javed Iqbal a, Juliane Hombach a, Barbara Matuszczaka Department of Pharmaceutical Technology, Institute of Pharmacy, University of Innsbrucb Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck

a b s t r a c ta r t i c l e i n f o

Article history:Received 10 March 2010

The aimof the present studyin drug delivery systems bolymeric P-glycoprotein (P-gp) inhibitor

Andreas Bernkop-Schnrch a,nrain 52c, Josef Mller Haus, 6020 Innsbruck, Austriarain 52a, 6020 Innsbruck, Austria

to improve the inhibitory properties of poly(ethylene glycol) (PEG) as excipientovalent attachment of thiol moieties. This was achieved by grafting PEG to

lled Release

i e r.com/ locate / jconre lactivity of thiomers was shown to be mediated by theause of their high molecular mass, thiomers are note GI-tract and remain therefore located in the GI-tractabsorption. Thus systemic side effects can be excludedse thiolated polymeric excipients as drug carrier.oth systemsPEG and thiolated polymersto onead to novel efux pump inhibitors of even higherch single system.

Itwas therefore the aim of this study to generate such a polymer andto investigate its efux pump inhibitory properties. Synthesis of anaccording thiolated co-polymer was achieved in three steps. In the rststep, the terminal hydroxyl groups present in homo functional poly(ethylene glycol) (HO-PEG-OH) were activated using hexamethylenediisocyanate (HMDI), followed by grafting of activated PEG withpolyethylenimine (PEI) and nally thiolation using -thiobutyrolatoneas outlined in Schemes 1 and 2. Cytotoxity of the novel polymer wasevaluated on Caco-2 monolayer. Moreover, the potential of the novelpolymer on the transport of the P-gp substrate Rho-123 across freshlyexcised rat intestinalmucosawas evaluated inUssing-type chambers onone hand in comparisonwith the classical P-gp inhibitor verapamil andsulfhydryl compounds including reduced GSH and 6-mercatopurinewhile on the other hand against several poly(ethylene glycol)derivatives such as vitamin E-TPGS, myrj 52 and brij 35.

(4x CH2 of the HMDI moiety).

2.2.3. Synthesis of Thiolated PEG-g-PEI Co-Polymer2.6 g of PEG-g-PEI co-polymer were dissolved in 50 ml of

distilled water. pH value was adjusted to 5.75 with 1 MHCl followedby the addition of 1000 l of -thiobutyrolactone. The system waskept isolated and stirred for 2 h. The pH value after thiolation was5.30.

2.2.4. PuricationDialysis was performed by using Spectra/Por 3 membrane

(MWCO: 1200) at (low acidic) pH~3 according to method asdescribed previously [7]. Briey the resulting thiolated co-polymerwas dialyzed against different aqueous media: 8 h against 5 l of5 mmol/l HCl, 8 h against 5 l of 5 mmol/l HCl containing 1% NaCl twotimes, 8 h against 5 l of 5 mmol/l HCl and nally, 8 h against 5 l of1 mmol/l HCl (40 h in total). The pH of the thiolated co-polymersolution after dialysis was about 4.3. The dialyzed product was freeze-dried for 3 days at80 C under reduced pressure and stored at 4 Cuntil use.

2.3. TNBS Assay

Primary amino content after each stage of modication wasdetermined by using a colorimetric assay with 2,4,6-trinitrobenzene-

63J. Iqbal et al. / Journal of Controlled Release 147 (2010) 62692. Materials and Methods

2.1. Materials

Polyethylenimine 600 Da (99% pure; specic gravity 1.0291.038)was purchased fromPolysciences. Homo-functional polyethylene glycol(OH-PEG-OH) 6000 Da was from Rapp Polymere GmbH, Germany.Hexamethylene diisocyanate [OCN(CH2)6NCO] (1,6-diisocyanate)99.0% pure was purchased from Fluka. D--tocopherol polyethyleneglycol 1000 succinate (vitamin E-TPGS), polyoxyethylene 40 stearate(myrj 52), brij 35 (polyethylene glycol dodecyl ether), L-glutathionereduced (reduced GSH), ()-verapamil hydrochloride, -thiobutyro-lactone and thioglycolic acid (TGA) was purchased from Sigma.Chloroformwas obtained fromRiedel-de-Han. Petroleum ether havingboiling point 4060 C was from Acros Organics.

2.2. Methods

2.2.1. Activation of OHPEG-OH (6000 Da)Four grams (0.67 mmol) of PEG were dissolved in 40 ml of

chloroform followed by the addition of 36 ml (224.1 mmol) ofhexamethylene diisocyanate (HMDI) in a 250 ml ask tted withthe condenser and the mixture was reuxed for 48 h. The activatedpolymer formed was precipitated by addition of 600 ml of petroleumether and the resulting solid product was re-dissolved in 15 ml ofchloroform. Precipitation was repeated four times more. The acti-vated polymer was vacuum dried and weighed. Finally 3.8 g of theactivated product was obtained corresponding to 95% yield. 1H NMRspectra were recorded on a Varian Gemini 200 spectrometer (1H:199.98 MHz). The TMS signal at 0.00 ppm was used as internalstandard (chemical shifts in ppm).

1H spectra (CDCl3): 4.404.28, 4.244.18 (C( O)OCH2 of the PEGmoiety), 4.023.97 (OCH2 of the PEG moiety), 3.843.45 (PEG part),Scheme 1. Reaction scheme for the synthesis of (a) activatedPEG; (b)PEG-g-PEI co-polymer.3.323.26 (PEG part), 3.23.1 (2x NCH2 of the HMDI moiety), 1.61.2(4x CH2 of the HMDI moiety).

2.2.2. Synthesis of PEG-g-PEI Co-PolymerA solution of 4.5 g of PEI 600 Da in 50 ml CHCl3 was added drop-

wise (with continuous stirring) to a solution of 3.8 g of activated PEGin 50 ml CHCl3. The mixture was reuxed for 48 h. Solvent volumewas reduced to 30 ml by evaporation at the same temperature and theco-polymer was precipitated by the addition of 500 ml of petroleumether. Finally 6.67 g of the co-polymer was obtained corresponding to80% yield.

1H spectra (CDCl3): 4.404.28, 4.244.18 (C(=O)OCH2 of the PEGmoiety), 4.023.97 (OCH2 of the PEG moiety), 3.843.45 (PEG part),3.323.26 (PEG part), 3.23.1 (2x NCH2 of the HMDI moiety), 1.61.2

Scheme 2. Reaction scheme for the synthesis of thiolated PEG-g-PEI co-polymer.sulphonic acid (TNBS) [8]. Briey, 0.5 mg of the polymerwas hydrated

64 J. Iqbal et al. / Journal of Controlled Release 147 (2010) 6269in 500 l 0.5% NaCl solution. After 30 min of incubation at roomtemperature, 500 l of 0.1% TNBS (picrylsulfonic acid solution)containing 4% NaHCO3 were added to each diluted aliquot. Absor-bance was measured at 450 nm after 2 h of incubation at 37 C andcentrifugation at 13,400 rpm for 5 min using microtitration platereader (Tecan innite M200 spectrophotometer, Grdig, Austria). L-Cysteine HCl (Sigma, St. Louis, MO) was used to make calibrationcurve for the calculations [9].

2.4. Ellman's Test

The total amount of thiol groups attached to the thiolated co-polymer was spectrophotometrically quantied by Ellman's reagentas described by our research group previously [10]. L-Cysteine HCl(Sigma, St. Louis, MO) was employed to establish calibration curve.First accurately 500 gof the thiolated co-polymer was dissolved in500 l of 0.5 M phosphate buffer solution (pH 8.0) followed by theaddition of 500 l of Ellman's reagent (3 mg of 5,5'-dithiobis(2-nitrobenzoic acid) in 10 ml of 0.5 M phosphate buffer; pH 8.0). Theresulting solution was allowed to proceed for 90 min at roomtemperature in dark. Centrifugation was done at 13,400 rpm for5 min. The amount of thiol groups on the co-polymer was thereafterdetermined by measuring the absorbance of 100 l of the supernatantat 450 nm using Tecan innite M200 spectrophotometer, Grdig,Austria [11].

2.5. Disulde Bond Test

This test was used to determine the extent of oxidation during thethiolation step by determining the amount of disulde bonds formeddue to the oxidation by air/atmosphere.

Briey, 0.5 g of the thiolated co-polymer was taken in 15 ml falcontube for the test, dissolved in 350 l of distilled water and incubated atroom temperature for 30 min for equilibration. 150 l of 0.05 M TRISbuffer was added to each of the above tubes followed by the additionof 1 ml of 4% w/v aqueous solution of sodium borohydrate andincubation at 37 C in water bath for 60 min. Then 200 l of 5 M HClwere added to each tube to quench further reaction. Finally, theamount of remaining thiol groups was determined by Ellman'sreagent as described above [12].

2.6. Size Exclusion Chromatography

The approximate molecular mass of the thiolated PEG-g-PEI co-polymer was determined by size-exclusion chromatography (SEC).The SEC consisted of a HPLC pump L-2130 (Merck-Hitachi, Darmstadt,Germany), a Merck-Hitachi Autosampler L-2200, and a columnthermostat (25 C). The SEC column, Suprema Max 3000 used withpre-column, was from Polymer Standard Service.

Mainz, Germany. Thiolated co-polymer and eight different knownmolecular mass standards of PEG ranging from 400 Da to 12 Da weredetected by a differential refractive index (RI) detector L-2490 fromMerck-Hitachi. The retention times for each standard and thiolatedco-polymer were measured and the approximate molecular mass ofthiolated PEG-g-PEI co-polymer peak was calculated by plottingstandard curve between molecular mass and RT of known molecularmass standards. 0.1 M NaCl solution plus 0.1% triuoroacetic acid(TFA) was used as eluent. The ow rate was 1 ml/min. Thiolated PEG-g-PEI co-polymer and standard concentrations were 10 mg/ml eachand 20 l was injected for each isocratic run.

2.7. Cytotoxicity Screening

2.7.1. LDH AssayIn vitro cytotoxicity of the polymer was determined bymeasuring the release of the enzyme lactate dehydrogenase(LDH) from non-viable Caco-2 cells as a result of interaction with1% (w/v) of polymer solution in MEM without phenol red and FCS.For this approximately 100,000 cells/well were seeded to a 12-wellplate and incubated for 24 h at 37 C in 5% CO2 environment. After24 h, the cultured cells were rst washedwith 1 PBS (pre-warmedat 37 C). Test solution (1%), negative control (MEM with outphenol red) and positive control (2% (v/v) Titron X-100) wereadded in triplicate to the cell culture in 1 ml quantity. Samples attime point zero (100 l) were taken from the supernatant andstored at 4 C. The cells with mediumwere incubated again at 37 Cin 5% CO2 environment for 3 h. 100 l samples were withdrawn,centrifuged for 2 min at 5000 rpm to remove detached cells.The LDH-content of 50 l supernatant was measured at492 nm with Tecan innite M200 spectrophotometer, Grdig,Austria using a commercial test kit (Roche Diagnostics, Meylan,France) after incubation for 30 min at room temperature in dark.The apparent cytotoxicity was calculated according to the followingequation:

Cytotoxicity % = Experimental valueNegative controlPositive ControlNegative Control 100

2.7.2. MTT TestCell Vitality AssayAfter 24 h incubation of Caco-2 cell monolayers (1105 cells/well)

at 37 C in atmosphere of 5% CO2, cell monolayers were washed with1 PBS. Test solution (1%), negative control (MEM without FCS andphenol red) and positive control (2% (v/v) Titron X-100) were addedand incubated for 3 h. 1 ml of 0.5 mg/ml solution of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in MEMwithout FCS and phenol red was added to each well and the plateswere further incubated at 37 C in an incubator for 90 min.Solubilization of the formazan crystals was achieved by aspiration of1 ml of supernatant followed by addition of 1 ml 0.04 M HCl inisopropanol. The dye solution was removed and centrifuged at13,000 rpm for 2 min. The absorbance (OD) of supernatant at570 nm was measured with background subtraction at 650 nmusing microplate reader (Tecan innite M200 spectrophotometer,Grdig, Austria) [12].

2.8. Efux Pump Inhibition Studies

Non-fasting male Sprague Dawley rats weighing 250300 g wereused for efux pump inhibition studies. After sacricing by cervicaldislocation, the distal ileum (20 cm) was removed immediately andpreserved in 200 ml of 0.9% NaCl solution (w/v). The tissue was cutinto strips of 1.5 to 2 cm, rinsed free of luminal contents and mountedin Ussing chambers (surface area=0.64 cm2) without stripping offthe underlying muscle layer. The incubation medium was preheatedat 37 C containing 250 mM NaCl, 2.6 mMMgSO4, 10 mM KCl, 40 mMglucose and 50 mM NaHCO3 buffered with 50 mM Hepes (N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid)]; pH 7.2 wasadded to apical (AP) and basolateral (BL) chambers in a quantity of1 ml in each chamber. The chambers were screwed tight and thewhole assembly was kept in a water bath tomaintain the temperatureinside the chambers at 37 C. An equilibrium period of 30 min wasallowed to the whole assembly prior to start the efux pumpinhibition studies. All the measurements for control and sampleswere performed in triplicates [13].

2.8.1. Apical to Basolateral (AB) TransportIn control studies, Rho-123 in a nal concentration of 0.001% (w/v)

was applied to apical compartment for absorptive (AP to BL)transport. The transport of Rho-123 was investigated in the absenceand presence of test compounds 0.5%, 0.25%, 0.1% (w/v) thiolated

PEG-g-PEI co-polymer, 0.5% (w/v) myrj 52 , 0.5% (w/v) reduced GSH,

commonly used sink condition based calculation [14]. The advantage

VD + VR VD + VR

65J. Iqbal et al. / Journal of Controlled Release 147 (2010) 6269where VD is the volume in the donor compartment (1.0 mL), VR is thevolume in the receiver compartment (1.0 ml), A is the area of theUssing-type chambers (0.64 cm2),M is the total amount of drug in thesystem, CR,0 is the drug concentration in the receiver compartment atthe start of the time interval, and CR(t) is the drug concentration in thereceiver compartment at time t measured from the start of theinterval. The sampling procedure necessitates the recalculation of Mand CR,0 for each interval. Transport enhancement ratios (R) werecalculated from Papp values as follows:

R=Papp (test compound)/Papp (buffer).

2.8.2. Basolateral to Apical (BA) TransportIn control studies, Rho-123 in a nal concentration of 0.001% (w/v)

was applied to basolateral compartment for secretary (BL to AP)transport. The transport of Rho-123 was investigated in the absenceand presence of test compounds 0.5%, 0.25% and 0.1% (w/v) thilolatedPEG-g-PEI co-polymer. Apparent permeability coefcients (Papp) forRho-123 were calculated as already described above [14].

2.9. Reversibility of Intestinal P-gp Inhibition Studies

For reversibility of P-gp inhibition studies, the method statedabovewas followedwith slightmodications. The thiolated PEG-g-PEIpolymer (0.5% w/v) was dissolved in Hepes buffer and added to APchamber. After 60 min of incubation time, the test compound wasremoved from the AP chamber, the chamber was rinsed three timeswith 1 ml Hepes buffer and then Rho-123, dissolved in Hepes buffer ina nal concentration of 0.001% (w/v), was added to AP side forabsorptive transport. As negative control, the chambers wereincubated for 60 min with Hepes buffer and then Rho-123 in a nalconcentration of 0.001% (w/v) was added to AP side. For positivecontrol the chambers were incubated with Hepes buffer as well, butafter 60 min of incubation time, Rho-123 containing the according testcompound in the same concentration as mentioned above, was addedto AP chamber. Samples were withdrawn every 30 min for 3 h andof the nonsink condition analysis is that it is applicable also when theexperiment does not exhibit linear drug ux, e.g., for highlypermeable compounds. Papp values for the studied substances weredetermined by nonlinear curve tting of following equation to theexperimental data.

CR t =M

+ CR;0M ePappA1=VD + 1=VRt0.5% (w/v) D--tocopherol polyethylene glycol 1000 succinate(Vitamin E-TPGS), 0.5% (w/v) brij 35, verapamil (100 M), 6-mercaptopurine (100 M). Samples were withdrawn in a quantityof 100 l at exactly 30-min intervals over a time period of 3 h from theacceptor chamber and transferred to a 96-well microtitration plate.The buffer quantity in acceptor chamber was lled up immediatelyagain to 1.0 ml by the replacement with transport medium pre heatedat 37 C. Rho-123 was measured at 485 nm (extinction) and 520 nm(emission) and the amount of permeated Rho-123 was calculated byinterpolation from an according standard curve using a uorimeter(SLT; Spectra Fluor, Tecan, Austria). Cumulative corrections weremade for previously removed samples. Apparent permeabilitycoefcients (Papp) for Rho-123 were calculated at 30, 60, 90, 120and 180 min and reported as Papp values at 180 min. Papp values werecalculated using the generally applicable nonsink condition analysisthat imposes less restriction on the experimental conditions than theevaluated as described above.3. Statistical Data Analysis

Statistical data analyses were performed using the Student's t-testwith pb0.05 as the minimal level of signicance. All values areexpressed as the meansS.D.

4. Results and Discussions

4.1. Co-Polymer Synthesis

Synthesis of PEG-g-PEI polymeric backbone was achieved bycoupling homo-functional linear PEG to branched PEI molecules. Theoverall synthetic pathway is shown in Schemes 1 and 2.

The rst reaction step involves the activation of homo-functionalPEG. Both terminal functional hydroxyl groups of PEG were activatedby using HMDI in order to perform coupling reaction in the secondstep (with primary amino groups present in PEI). HMDI was found tobe very suitable for the activation reaction, since it has two terminalfunctional isocyanate groups which were found equally reactivetowards both the terminal hydroxyl groups of PEG as well as primaryamino groups present in PEI. One isocyanate group of HMDI was usedto transform the terminal hydroxyl functional group of PEG intofunctionality and the other one was used to connect activated PEG toPEI via one of the primary amino groups present in PEI. The use ofHMDI makes the addition reaction feasible without any side productswhich results in facile isolation of PEG-g-PEI polymeric backbone,needed for thiolation. On the other hand, the great stability of ureaand urethane groups against hydrolytic cleavage makes the polymericbackbone good soluble in water. The possible side reaction duringactivation step which might result in the formation of a dimericproduct was avoided by the use of an excess of HMDI. After activation,the excess of HMDI has to be removed carefully before performing thecoupling reactionwith PEI, as it might result otherwise in cross linkingof PEI molecules leading to decreased solubility. This was achieved byexcessive washing with petroleum ether. Due to low stability, theactivated PEG was immediately coupled to PEI. Finally, thiolation wasdone by using -thiobutyrolactone in isolated conditions to form theco-polymer. The nal thiolated product was dialyzed to remove theexcess of un-reacted reactants and stored at 4 C in isolated conditionsto minimize the air contact and subsequent chances of disulde bondformation by oxidation [7].

The synthesis of the co-polymer was monitored by 1H NMRspectroscopy. Activation of PEG with HMDI is clearly indicated sincethe signals for both, the protons of the PEG part and those of the HMDIsubunit appeared at different chemical shifts. The new multiplets atchemical shifts of ~1.4 ppm and ~1.5 ppmwhich represent the signalsof the four methylene groups of the HMDI part, the signal at ~3.1 ppm(methylene protons ofCH2N C O and CH2-NHCOO) as well as thesignal at ~4.2 ppm verify successful activation. The latter signalrepresents those of the two methylene protons of the PEG partneighboring the hydroxy group which has been substituted. Thesechemical shifts, however, are in accordance with the data described inthe literature [15]. The 1H NMR spectrum of the product formed byreaction of activated PEGwith PEI was characterized by the additionalsignals; especially the broad signal in the range of ~2.4 to 2.9 ppmindicates the presence of the PEI part. Whereas the 1H NMR spectraconrmed the polymer structure, this technique could not be used todetect the cysteine part of the nal products due to the highmolecularmass of the polymeric part.

4.2. TNBS Assay

Since primary amino functional groups present in PEI were themost reactive, TNBS assay was adapted to quantify them after eachstep of synthesis. Results are shown in Fig. 1. The results from TNBS

assay revealed that unmodied PEI contained 856.1421.68 mol/g

Fig. 2. LDH assay on Caco-2 cells as a percentage of total cell LDH activity. The cells wereincubated with indicated test compound and the percentage of LDH release wasdetermined after t=3 h (white bars) of incubation period. Each bar represents themeanS.D. of three experiments.

66 J. Iqbal et al. / Journal of Controlled Release 147 (2010) 6269of primary amino groups. The amount of these primary amino groupswas found to be decreased after completion of each step of synthesis.Thus, proving successful completion of each reaction step by thecoupling of reagents utilizing primary amino groups. PEG-g-PEIpolymeric backbone displayed 336.4613.07 mol/g of remainingprimary amino groups and the nal thiolated PEG-g-PEI co-polymerhad only 145.071.64 mol/g of primary amino groups left.

4.3. Ellman's Test and Disulde Bond Test

Thiolation was achieved by using -thiobutyrolactone which iswidely used as thiolating agent for PEI [16]. The resulting thiolatedpolymer was found to display 84.305.43 mol/g of thiol goups.

The inter- and/or intramolecular disulde bonds were 12.741.57 mol/g. The low percentage of disulde bonds indicated lowextent of oxidation during the coupling reaction thus demonstratingefciency of the thiolation step.

4.4. Determination of Relative Molecular Mass

The approximate molecular mass (number average molecularweight) of the thiolated PEG-g-PEI co-polymer was determined bysize exclusion chromatography. SEC elugrams showed that thepolymer with high molecular mass was eluted earlier compared tothose with low molecular mass. Polymer peak appeared at 12.5 minon RI detector representing a molecular mass of approximately16,000 Da. The isocratic acidic eluent caused amaximum expansion ofthe polycation and allowed sufcient separation on the column.

Fig. 1. Determination of free amino groups in the indicated test compounds (TNBSassay). The quantitative determination of amino groups was done with 2,4,6-trinitrobenzenesulfonic acid (TNBS). The absorbance was measured after 2 hincubation at 37 C. Each bar represents the meanS.D. of three experiments.4.5. Cytotoxicity Screening

In vitro cytotoxicity of the polymer was determined by measuringthe release of the enzyme lactate dehydrogenase (LDH) from damagedCaco-2 cells. In vitro cytotoxicity results are presented in Fig. 2. Theseresults showed that negative control (MEM without FCS and phenolred) and positive control (2% v/v Titron-X) indicate 0.200.06% and1000.63% cytotoxity, respectively. The resulting thiolated PEG-g-PEIco-polymer (1% w/v) exhibited only 6.690.27% of toxicity. PEG-g-PEI co-polymer was found 5.740.25% toxic. Activated PEG showed31.660.78% cytotoxicity which might be due to the presence ofisocyanate groups present on the activated adduct.

Tetrazolium-based MTT colorimetric assay was performed onCaco-2 cells to verify the results obtained from LDH assay in terms ofcell viability. Results obtained fromMTT test as shown in Fig. 3were ingood correlation with those from the LDH assay. With thiolated co-polymer, cell survival decreased by 93.330.07% while PEI alonedisplayed 86.590.59% cell viability. Activated PEG was found to bemost toxic by demonstrating cell viability of 68.390.15%. The reasonmight be the same as described above.

In general, small molecule efux pump inhibitors (SMIs) arepharmacologically active compounds and commonly bear the risk ofSMI mediated toxicity, accumulation or anti-targeting. The use ofnovel thiolated PEG-g-PEI co-polymer would avoid these undesiredand toxic side effects. Because of the high molecular mass, it shouldnot be absorbed from the GI-tract and should remain concentrated inthe GI-tract at the site of drug absorption.

4.6. Efux Pump Inhibition Studies

The inuence of 0.5% (w/v) thiolated PEG-g-PEI and several wellknown P-gp inhibitors was evaluated with freshly excised ratintestinal mucosa mounted in Ussing-type chambers. Freshly excisedrat intestinal mucosa has been used for transport studies due to theadvantage of the presence of the mucus layer. This mucus layer isabsent in Caco-2 cells due to lack of mucin secreting cells (globletcells). The physicochemical properties of this mucus gel can inuencethe rate of diffusion from the bulk to the site of absorption. Apart fromthis diffusion process, cationic polymers can also be ionicallyimmobilized in the mucus layer due to anionic sub-structures ofmucins such as sialic acid and sulfonic acid moities. Accordingly,freshly excised intestinal mucosa is much closer to the in vivosituation than the well-established Caco-2 monolayer. Rho-123 waschosen as representative P-gp substrate to perform efux pumpinhibition studies, as its efciency to act as a suitable test compoundfor P-gp mediated transport studies had been demonstrated inFig. 3. MTT assay on Caco-2 cells. The cells were incubated with indicated testcompound for 3 h and the percentage of metabolic formation of water insoluble 1,3,5-triphenylformazan was determined after t=90 min (white bars) of incubation period.Each bar represents the meanS.D. of three experiments.

previous studies [17,18]. Results of the in vitro efux pump inhibitionstudies are shown in Fig. 4 and Table 1. GSH was used as controlbecause several studies reported that GSH can inhibit P-gp. Werle andHoffer for instance, reported an increased transport of P-gp substrateRho-123 in the presence of GSH across the rat intestinal mucosa [6].

Out of well-known P-gp inhibitors, the gold standard P-gp inhibitor,verapamil and poly(ethylene glycol) derivatives vitamin E-TPGS (0.5%w/v), myrj 52 (0.5%w/v) and brij 35 (0.5% w/v) led to an increase inthe absorption of Rho-123. A more pronounced absorption enhancingeffect of Rho-123 could be achieved in the presence of sulfhydrylcompounds, 6-mercaptopurine (100 M) and reduced GSH (0.5%, w/v).Within this study it could be demonstrated that the thiolated PEG-g-PEIco-polymer (0.5% w/v) displays the strongest effect on the transport ofRho-123 across rat intestinal mucosa in vitro in comparison with myrj

52, vitamin E-TPGS, brij 35, reduced GSH and 6-mercaptopurine. Theapparent permeability coefcient (Papp) of Rho-123 in the presence of0.5% (w/v) of thiolated PEG-g-PEI polymerwas 15.180.95106 cm/s. Thiolated PEG-g-PEI increased the Papp of Rho-123 up to 3.26-fold incomparison to Rho-123 without any inhibitor used as control(Papp=4.660.04106 cm/s).

The absorptive (AP to BL) and secretory (BL to AP) transport of0.001% (w/v) of Rho-123 across male Sprague Dawley rat ileum wasinvestigated in the presence of 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v)of thiolated PEG-g-PEI co-polymer at 37 C. As shown in Table 2 andFig. 5, due to the addition of 0.5% (w/v), 0.25% (w/v) and 0.1% (w/v) ofthiolated PEG-g-PEI co-polymer to the buffer, the absorptive Papp was

another in vivo study in which poly(acrylic acid)-cysteine solution

Table 1Comparison of the in vitro apparent permeability coefcients (Papp) of Rho-123 andimprovement ratios in presence of indicated test compounds for absorptive transport(AB) (meansSD, n=3).

Test compound (concentration) Papp(cm/s)106

Improvement ratio R=Papp(test compound)/Papp (buffer)

Buffer 4.70.1 myrj 52 (0.5% w/v) 8.51.0 1.8Verapamil (100 M) 9.22.0 2.0Vitamin E-TPGS (0.5% w/v) 9.52.0 2.0brij 35 (0.5% w/v) 10.60.2 2.3Reduced GSH (0.5% w/v) 11.00.1 2.46-mercaptopurine (100 M) 11.10.4 2.4*Thiolated PEG-g-PEI (0.5% w/v) 15.21.0 3.3

*(difference from buffer control pb0.0001).

Table 2Comparison of the absorptive and secretory apparent permeability coefcients (Papp) ofRho-123 and resulting efux ratios in the presence of different concentrations ofthiolated PEG-g-PEI co-polymer (meansSD, n=3).

Test compound Papp (cm/s)106 Efux ratio(secretoryPapp/absorptivePapp)

Transport direction

Absorptive Secretory

(AP to BL) (BL to AP)

Buffer 5.80.3 18.80.9 3.3Thiolated PEG-g-PEI (0.10% w/v) 8.30.4 16.70.8 2.0Thiolated PEG-g-PEI (0.25% w/v) 12.00.6 16.40.6 1.4Thiolated PEG-g-PEI (0.50% w/v) 15.21.0 15.40.7 1.0

67J. Iqbal et al. / Journal of Controlled Release 147 (2010) 6269Fig. 4. In vitro transport data of Rho-123 (0.001% w/v) across rat intestinal mucosa inthe absorptive direction in absence of polymer () and with thiolated PEG-g-PEI co-polymer (0.5% w/v) () in comparison to A: verapamil (100 M) (); B: 6-mercaptopurine (100 M) (); C: reduced GSH (0.5% w/v) (); D: vitamin E-TPGS(0.5% w/v) (); E: brij 35 (0.5% w/v) (); F myrj 52 (0.5% w/v) (). Transport dataare means ( S.D.) of at least 3 experiments. differs from pb0.0001. differs from

pb0.0001.increased by +162.18%, +106.91%, and +43.87%, respectively whilethe secretory Papp was decreased by18.08%,13.03%, and11.38%,respectively in comparison to control buffer.

The results of studies performed within our research groupshowed that chitosan-4-thiobuthylamidine can act as an inhibitor ofP-gp, thus increasing absorption of P-gp substrate Rho-123 across ratsmall intestine in vitro [17] and in vivo [6]. The mechanismresponsible for enhancing the absorption of P-gp substrate by non-absorbable sulfhydryl compounds like thiolated polymers seems notto be based on substrate competition or ATP-depletion, as reported forother multidrug resistance reversing agents [19], since thiomers arenot uptaken from cells due to their high molecular mass. It seemsmore likely, that thiomers act via: (1) changes of the membranestructure, which affect the conformations of the efux transportproteins, and/or (2) the incorporation of the bulky polymer may leadto a steric hindrance which limits active efux. In fact, P-gp activitywas reported to highly depend on themembranemicroviscosity and itwas furthermore suggested that the ability of, for example, Pluronicsto affect P-gp activity is mediated by effects on the membranestructure [20]. In addition, thiomers like chitosan-TGA might alsoaffect the P-gp activity through a covalent interaction between thiolgroups on the polymer and cysteine-residues of P-gp. Al-Shawi et al.[21] could demonstrate a covalent interaction between thiolatedcompounds and cysteine-residues of P-gp. This hypothesis is furtherconrmed by ndings within this study, showing that the novelthiolated PEG-g-PEI co-polymer increases the permeation of P-gpsubstrate Rho-123 across rat intestinal mucosa.

Apart from P-gp inhibition, thiolated polymers were previouslyreported tomodulate drug absorption by inhibition of intestinal efuxpumps such as multidrug resistance proteins (MRPs) as well. It hasbeen previously reported that a delivery system based on (0.5% w/v)chitosan-TBA) and (0.5% w/v) GSH improved the in vitro transport ofMRP substrate saquinavir up to 1.6-fold in Caco-2 monolayer and 2.1-fold in rat intestinal mucosa. Moreover, the in vivo Cmax was increasedup to 1.6-fold in rats [22]. These ndings were further conrmed by

68 J. Iqbal et al. / Journal of Controlled Release 147 (2010) 6269increased the area under the plasma concentration time curve(AUC012) of sulforhodamine 101 up to 3.8-fold in comparison tobuffer control [23]. These ndings seem to indicate that thiomersmight be useful tools to deliver various drugs which are affected byefux transporters such as P-gp and MRP. Taking all these aspects inmind, the novel thiolated PEG-g-PEI co-polymer may be considered aspotentially interesting candidate for P-gp inhibition.

4.7. Reversibility of Intestinal P-gp Inhibition Studies

Fig. 6 shows the results from experiments for testing the thiolatedPEG-g-PEI polymer for evaluation of reversibility of P-gp inhibition onrat intestine. It has been shown that the thiolated PEG-g-PEI polymeracts as reversible inhibitor. After 60 min of incubationwith 0.5% (w/v)thiolated PEG-g-PEI polymer, subsequent removal and three-fold

Fig. 5. Tansepithelial transport of Rho-123 (0.001%w/v) in the absorptive (lled symbols)and secretory direction (open symbols) in the absence (,) or presence (,) of A: 0.5%(w/v) of thiolated PEG-g-PEI co-polymer; B: 0.25% (w/v) of thiolated PEG-g-PEI co-polymer; C: 0.1% (w/v) of thiolated PEG-g-PEI co-polymer. Transport data is expressed aspercentageof the total dose of Rho-123 (0.001%w/v) applied toapical or basolateral sideofmucosa. Each value represents the meanS.D. of at least 3 trials. differs from pb0.0001. differs from pb0.049.washing, the inhibitory activity of thiolated polymerwas reversed anda total inversion of the transport of Rho-123 was observed.

5. Conclusion

Based on experimental results, a novel thiolated co-polymer, namelya thiolated PEG-g-PEI co-polymer has been synthesized and character-ized successfully. This novel thiomer displayed a more pronouncedeffect on the absorptive transport of Rho-123 in comparison withseveral well-known P-gp inhibitors including, reduced GSH, 6-merca-topurine and vitamin E-TPGS as well as the structurally similarcompounds myrj 52 and brij 35. Moreover, the novel thiolated PEG-g-PEI co-polymer increased the transport of Rho-123 up to 3.26-fold incomparison to Rho-123 without any inhibitor used as control. Thethiolated PEG-g-PEI co-polymer did not only improve the transport ofRho-123 in the absorptive direction but also reduced the basolateral toapical transport of Rho-123 across rat intestinalmucosa. Thus, this studysuggests that the novel polymer might turn out to be a valuable tool inorder to improve the intestinal uptake of P-gp substrates.

Fig. 6. Permeation studies on freshly excised rat intestinal mucosa mounted on Ussing-type chambers. Effect of 0.5% (w/v) of thiolated PEG-g-PEI polymer () on Rho-123uptake (A to B) in comparison to Rho-123 control (0.001% w/v) () and 60 min ofprevious incubation with 0.5% (w/v) of thiolated PEG-g-PEI polymer (). Each valuerepresents the meanS.D. of at least 3 trials. differs from and pb0.0001.Acknowledgements

The authors wish to thank Higher Education Commission Pakistanand Austrian Agency for International Cooperation in Education andResearch (AD) for their support and funding.

Appendix A. Supplementary Data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.jconrel.2010.06.023.

References

[1] E.D. Hugger, K.L. Audus, R.T. Borchardt, Effects of poly (ethylene glycol) on efuxtransporter activity in Caco-2 cell monolayers, J. Pharm. Sci. 91 (2002) 19801990.

[2] B.M. Johnson, W.N. Charman, C.J.H. Porter, An in vitro examination of the impactof polyehtylene glycol 400, pluronic P 85 and vitamin E D-a-tocopherylpolyethylene glycol 1000 succinate on p-glycoprotein efux and enterocyte-based metabolism in excised rat intestine, AAPS PharmSci 4 (2002) 113.

[3] A. Bernkop-Schnrch, V. Schwarz, S. Steininger, Polymers with thiol groups: anew generation of mucoadhesive polymers? Pharm. Res. 16 (1999) 876881.

[4] A.E. Clausen, A. Bernkop-Schnrch, In vitro evaluation of the permeation-enhancing effect of thiolated polycarbophil, J. Pharm. Sci. 89 (2000) 12531261.

[5] A. Bernkop-Schnrch, Thiomers: a new generation of mucoadhesive polymers,Adv. Drug Deliv. Rev. 57 (2005) 15691582.

[6] M. Werle, M. Hoffer, Glutathione and thiolated chitosan inhibit multidrugresistance P-glycoprotein activity in excised small intestine, J. Control. Rel. 111(2006) 4146.

[7] I. Bravo-Osuna, T. Schmitz, A. Bernkop-Schnrch, Elaboration and characterizationof thiolated chitosan-coated acrylic nanoparticles, Int. J. Pharm. 316 (2006)170175.

[8] S.L. Snyder, P.Z. Sobocinski, An improved 2, 4, 6-trinitrobenzenesufonic acidmethod for the determination of amines, Anal. Biochem. 64 (1975) 284288.

[9] M.K. Marschtz, F.M. Veronese, A. Bernkop-Schnrch, Inuence of the spacer onthe inhibitory effect of different polycarbophil-protease inhibitor conjugates, Eur.J. Pharm. Biopharm. 52 (2001) 137144.

[10] A.H. Krauland, V.M. Leitner, A. Bernkop-Schnrch, Improvement in the in situgelling properties of deacetylated gellan gum by the immobilization of thiolgroups, J. Pharm. Sci. 92 (2002) 12341241.

[11] A. Bernkop-Schnrch, M. Hornof, T. Zoidl, Thiolated polymers-thiomers: synthesisand in vitro evaluation of chitosan-2-iminothiolane conjugates, Int. J. Pharm. 260(2003) 229237.

[12] T. Mosmann, Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays, J. Immunol. Meth. 65(1983) 5563.

[13] F. Fger, H. Hoyer, K. Kafedjiiski, M. Thaurer, A. Bernkop-Schnrch, In vivocomparison of various polymeric and low molecular mass inhibitors of intestinalP-glycoprotein, Biomaterials 27 (2006) 58555860.

[14] S. Tavelin, J. Grsj, J. Taipalensuu, G. Ocklind, P. Artursson, Applications ofepithelial cell culture in studies of drug transport, Meth. Mol. Biol. 188 (2002)233272.

[15] H. Petersen, K. Kunath, A.L. Martin, S. Stolnik, C.J. Roberts, M.C. Davies, T. Kissel,Star-shaped poly(ethylene glycol)-block-polyethylenimine co-polymers enhanceDNA condensation of low molecular weight polyethylenimines, Biomacromole-cules 3 (2002) 926936.

[16] U. Hellberg, J.P. Ivarsson, B.L. Johansson, Characteristics of superdex prep grademedia for gel ltration chromatography of proteins and peptides, ProcessBiochem. 31 (1996) 163172.

[17] F. Fger, T. Schmitz, A. Bernkop-Schnrch, In vivo evaluation of polymeric deliverysystems for P-glycoprotein substrates, Biomaterials 27 (2006) 42504255.

[18] M. Kageyama, H. Namiki, H. Fukushima, Y. Ito, N. Shibata, K. Takada, In vivo effectsof cyclosporine A and ketoconazole on the pharmacokinetics of representativesubstrates for P-glycoprotein and cytochrome P450 (CYP) 3A in rats, Biol. Pharm.Bull. 28 (2005) 316322.

[19] Y.L. Lo, J.D. Huang, Effects of sodium deoxycholate and sodium caprate on thetransport of epirubicin in human intestinal epithelial Caco-2 cell layers andeverted gut sacs of rats, Biochem. Pharmacol. 59 (2000) 665672.

[20] T. Demina, I. Grozdova, O. Krylova, A. Zhirnov, V. Istratov, H. Frey, H. Kautz, N.Melik-Nubarov, Relationship between the structure of amphiphilic copolymersand their ability to disturb lipid bilayers, Biochemistry 44 (2005) 40424054.

[21] M.K. Al-Shawi, I.L. Urbatsch, A.E. Senior, Covalent inhibitors of P-glycoproteinATPase activity, J. Biol. Chem. 269 (1994) 89868992.

[22] F. Fger, K. Kafedjiiski, H. Hoyer, B. Loretz, A. Bernkop-Schnrch, Enhancedtransport of P-glycoprotein substrate saquinavir in presence of thiolated chitosan,J. Drug Target. 15 (2007) 132139.

[23] M. Greindl, F. Fger, J. Hombach, A. Bernkop-Schnrch, In vivo evaluation ofthiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efuxpumps substrates, Eur. J. Pharm. Biopharm. 72 (2009) 561566.

69J. Iqbal et al. / Journal of Controlled Release 147 (2010) 6269

Design and in vitro evaluation of a novel polymeric P-glycoprotein (P-gp) inhibitorIntroductionMaterials and MethodsMaterialsMethodsActivation of OHPEG-OH (6000Da)Synthesis of PEG-g-PEI Co-PolymerSynthesis of Thiolated PEG-g-PEI Co-PolymerPurification

TNBS AssayEllman's TestDisulfide Bond TestSize Exclusion ChromatographyCytotoxicity ScreeningLDH AssayMTT TestCell Vitality Assay

Efflux Pump Inhibition StudiesApical to Basolateral (AB) TransportBasolateral to Apical (BA) Transport

Reversibility of Intestinal P-gp Inhibition Studies

Statistical Data AnalysisResults and DiscussionsCo-Polymer SynthesisTNBS AssayEllman's Test and Disulfide Bond TestDetermination of Relative Molecular MassCytotoxicity ScreeningEfflux Pump Inhibition StudiesReversibility of Intestinal P-gp Inhibition Studies

ConclusionAcknowledgementsSupplementary DataReferences