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Analytica Chimica Acta 686 (2011) 76–80

Contents lists available at ScienceDirect

Analytica Chimica Acta

journa l homepage: www.e lsev ier .com/ locate /aca

evelopment of polymer-membrane based electrodes for suramin

ndrew Yu, Brandon Shepherd, Meghan Wagner, Jamie Clapper, Joan M. Esson ∗

epartment of Chemistry and Biochemistry, Otterbein University, Westerville, OH 43081, United States

r t i c l e i n f o

rticle history:eceived 27 August 2010eceived in revised form5 November 2010

a b s t r a c t

The development of a polymer membrane-based electrode to measure the anionic drug suramin inbuffered saline and biological samples is described. A large non-equilibrium, steady state EMF response isobserved toward suramin, and judicious choice of the polymer membrane components allows for adjust-ment of the dynamic range of the electrode. The optimized membrane for use in the toxic suramin range

ccepted 16 November 2010vailable online 23 November 2010

eywords:uraminolymer membrane-based electrodeolyanion-sensitive electrode

consists of 25 wt% tridodecylmethyl ammonium chloride, 55 wt% bis-2-ethylhexyl sebacate, and 20 wt%Pellethane. Although this electrode can be used to directly quantify suramin in human plasma, determi-nation of suramin that is not affected by the background concentration of small anions is best achievedby simple potentiometric titrations with polycationic protamine monitored with a protamine-sensitiveelectrode.

© 2010 Elsevier B.V. All rights reserved.

otentiometry

. Introduction

Suramin, a symmetrical polysulfonated naphthylurea shown inig. 1, has been historically used to treat human African trypanoso-iasis (HAT), also known as sleeping sickness [1]. Although HATas almost eradicated in the 1960s, the relaxation of surveillanceas led to a resurgence of this disease with an estimated 70,000eople affected each year [2]. Suramin is also used as the main treat-ent for onchocerciasis, more commonly called river blindness, a

arasitic disease prevalent in Africa and Latin America estimatedo affect half a million people annually [3,4], and has been moreecently explored as an inhibitor of snake venom mytotoxicity and aossible treatment against Trypanosoma cruzi, a parasitic protozoanhat is the etiological agent of Chagas disease, which is highly preva-ent in Latin America [5–8]. Additionally, since naturally occurringolyanions have been implicated in several pathological processesuch as angiogenesis and tumor growth, the polyanionic charac-eristics of suramin allow it to function as an antagonistic anti-viralnd chemotherapeutic drug [9,10]. More specifically, it has beensed in combination with other drugs in phase II clinical trials to

ncrease the effectiveness of chemotherapy treatment for renal cellarcinoma, to reverse chemotherapy resistance in chemoresistant

on-small-cell lung cancer, and to inhibit HIV infection [11–14].

Despite its use for treating a wide range of diseases, there isdose-related toxicity associated with suramin that is observed

t high suramin concentrations ranging from 200 to 350 �g mL−1

∗ Corresponding author. Tel.: +1 614 823 1716; fax: +1 614 823 1968.E-mail address: jesson@otterbein.edu (J.M. Esson).

003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2010.11.032

[15]. Although suramin has been used in clinical trials for sev-eral metastatic cancers, such as renal and prostate carcinomas andlymphomas, it has not been considerably successful because of itsbroad spectrum of toxicity, including adrenal and renal insuffi-ciency, coagulation factor abnormalities, immunosuppression, andpoly-neuropathy [16–19]. Indeed, the adverse effects reported withsuramin were even sufficient for the US Food and Drug Adminis-tration to block approval for use in prostate cancer, although HATregimens are short enough to make safety tolerable [20]. Becauseof its toxicity at high concentrations, the use of suramin treatmentsis being examined at concentrations lower than 65 �g mL−1 whereit can still inhibit basic fibroblast growth factor, which has beenimplicated in neovascularization and tumor growth, and it has alsobeen explored in combination chemotherapeutic treatment withother drugs at noncytotoxic doses [21,22,11,12,23–25].

Because of its cytotoxicity and its use over a wide rangeof dosage concentrations, there is a need for a fast, quantita-tive method to measure suramin in biological samples. Indeed,studies have recommended serial measurements of suramin dueto poly-neuropathy encountered during suramin therapy [26].Suramin levels have been determined in plasma colorimetricallyafter hydrolysis, and in plasma and serum via reversed phaseHPLC with detection limits of 7 ng mL−1 and 0.5 �g mL−1, respec-tively, well below the toxic level [27–31]. Suramin has also beenquantified in serum over the range of 48–524 �g mL−1 using

capillary electrophoresis [32–34]. However, there exists no fast,reliable method to determine suramin directly in complex mediawithout extensive sample preparation. Further, electrochemicalmethods for suramin measurement have not previously beenreported.

A. Yu et al. / Analytica Chimica Acta 686 (2011) 76–80 77

cture

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was repeatedly dipped into the membrane cocktail approximately12 times every 10 min to form the polymer membrane. The cap-illary tube was removed and the resulting tube filled with andconditioned in 10 mM NaCl for 24 h, unless otherwise stated. To

Table 1Composition of polymer membranes.

Membrane Ion-exchanger Plasticizer Polymer

A 1.5 wt% TDMA 32.5 wt% DOS 66.0 wt% PVCB 1.5 wt% TDA 32.5 wt% DOS 66.0 wt% PVCC 1.5 wt% DDDA 32.5 wt% DOS 66.0 wt% PVCD 1.5 wt% MTOA 32.5 wt% DOS 66.0 wt% PVCE 1.5 wt% BDDA 32.5 wt% DOS 66.0 wt% PVC

Fig. 1. Stru

Ion-selective electrodes have been routinely used for direct clin-cal measurements of small ions, such as Na+ and Cl−, in wholelood since the 1960s because of their low cost, simplicity, andpeed [35]. The potential (EMF) of these electrodes when at equilib-ium with the ion of interest is described by the Nikolsky–Eisenmanquation, Eq. (1)

= Eo + RT

ziFln[ai + kpot

i,jazi/zj

j ] (1)

here R and F are constants; T is temperature; z is the charge ofhe analyte ion i or interfering ion j; and kpot

i,jis the selectivity

oefficient. More recently, polymer membrane-based potentiomet-ic electrodes when operated under non-equilibrium, steady-stateonditions have been used for the measurement of macromolec-lar polyions, such as heparin and protamine, in whole blood,nd polyionic drugs, such as pentosan polysulfate, in undilutedlasma [36–38]. The large signals observed toward these polyionsas been attributed to the favorable extraction of the hydrophilicolyion into the organic membrane due to cooperative ion-pairingetween the polyion and the lipophilic ion-exchanger in the poly-er membrane and formation of a reverse micelle structure [39].

he non-equilibrium steady-state potential that develops at theample-membrane interface during short measurement times andow polyion concentrations is driven by kinetic factors and can beescribed by Eq. (2) [40]

EMF = RT

Fln

(1 − zDaım

RT+Dmıa

cp

)(2)

here R and F are the gas and Faraday constants, respectively; RT+

s the concentration of ion-exchanger in the membrane; D is theiffusion constant and ı the diffusion layer thickness in the aqueousa) and membrane phases (m); and cp is the concentration of theolyion in the sample.

In this study, the development of a polymer membrane-basedlectrode that is capable of measuring suramin directly in phys-ological samples in the clinically relevant concentration ranges described. In addition to examining the response characteris-ics of this electrode, its response mechanism is also explored toetermine if it is capable of generating large, analytically usefulignals when operated under non-equilibrium conditions. This isspecially interesting since suramin is a multiply charged ionicrug; however, it is much smaller than polyions for which otherolyion-sensitive electrodes have been developed. Suramin has a

olecular weight of roughly 1300 Da while polyanions for which

lectrodes have been previously developed have molecular weightsreater than 4000 Da. Finally, the quantification of suramin atg mL−1 levels in physiological samples by direct potentiometryith the optimized suramin polymer membrane-based electrode

of suramin.

or via titration with protamine as monitored with a protamine-sensitive electrode is described.

2. Experimental

2.1. Reagents

Suramin hexasodium salt was purchased from A.G. Scien-tific, Inc. (San Diego, CA). Didodecyldimethylammonium bromide(DDDA), tridodecylmethylammonium chloride (TDMA), tetrado-decylammonium chloride (TDA), methyltrioctadecylammoniumbromide (MTOA), benzyldimethyldodecylammonium bromideion-exchangers (BDDA), bis(2-ethylhexyl) sebacate (DOS), 2-nitrophenyl octyl ether (NPOE), and poly(vinyl chloride) highmolecular weight (PVC), were purchased from Fluka (St. Louis, MO).Tris Buffer, 2 M Solution was purchased from Fisher BioReagents(Pittsburgh, PA). M48 polyurethane was provided by Medtronic(Minneapolis, MN, USA), while Pellethane 2363-80AE was donatedby Freelin-Wade (McMinnville, OR). Calcium dinonylnaphthalenesulfonate (DNNS) was a gift from King Industries (Norwalk, CT,USA). All other reagents were analytical grade or better. All solu-tions were prepared using deionized water (>18 M�) purified usinga Milli-Q system (Millipore, Billerica, MA, USA).

2.2. Suramin electrode fabrication and EMF measurements

The polymer membranes for suramin were prepared by creatinga 0.07 g mL−1 membrane cocktail composed of polymer, plasti-cizer, and ion-exchanger (about 400 mg total) dissolved in THF(see Table 1 for a detailed list of membrane compositions). A smallcapillary tube inserted into Tygon tubing (i.d. 1/16 in., o.d. 1/8 in.)

F 3.0 wt% TDMA 31.0 wt% DOS 66.0 wt% PVCG 5.0 wt% TDMA 60.0 wt% DOS 35.0 wt% PVCH 10.0 wt% TDMA 70.0 wt% DOS 20.0 wt% PVCI 10.0 wt% TDMA 70.0 wt% DOS 20.0 wt% PellethaneJ 25.0 wt% TDMA 55.0 wt% DOS 20.0 wt% Pellethane

7 imica Acta 686 (2011) 76–80

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A series of lipophilic quaternary ammonium salts were investi-gated as possible ion-exchangers for a suramin electrode, and theresponse of electrodes incorporating these salts is shown in Fig. 2.The electrodes incorporating TDMA as the ion-exchanger (Mem-brane A) elicited the largest responses toward suramin, followed

8 A. Yu et al. / Analytica Ch

omplete the electrode fabrication, a Ag/AgCl wire was inserted intohe tube. The potentials of the electrodes were measured against

miniature Ag/AgCl reference electrode (Bioanalytical Systemsnc., West Lafayette, IN) using a Gateway E-4200 computer withPCI-6052E input-output board (National Instruments, Austin, TX)nd a custom-built electrode interface module controlled by Lab-iew 6.1 software. When the polymer membrane electrodes weresed during membrane optimization experiments, the response ofminimum of three electrodes to aliquots of suramin was mea-

ured every 6 min in 10 mL of 120 mM NaCl, 50 mM Tris, pH 7.2. Toimulate real biological samples, given amounts of suramin werepiked into 0.5 mL of human plasma. Additional aliquots of suraminere then added directly to the solution and monitored with the

ptimized suramin-sensitive electrode. This procedure was donen triplicate.

.3. Protamine electrode fabrication and EMF measurements

The protamine-sensitive electrode was fabricated in a similarashion to what was done for suramin, except that the membraneonsisted of 1 wt% DNNS, 49 wt% NPOE, 30 wt% Pellethane, and0 wt% M48 [41]. Using the same measurement system as the onehat was used for the suramin electrode, the EMF change of a min-mum of two electrodes was monitored every two min as aliquotsf 0.513 mg mL−1 protamine were added to a simulated biologicalample, which was made by spiking suramin into 4 mL of humanlasma.

. Results and discussion

.1. Characterization of response mechanism

The EMF response of polymer membrane-based electrodesmploying Membrane A, whose composition is listed in Table 1,ere evaluated under both equilibrium and non-equilibrium con-itions. As the Nernst equation (Eq. (1)) dictates, the response of thelectrode toward suramin under equilibrium conditions, in whichhe electrode is conditioned in suramin prior to its use, would bexpected to be roughly −10 mV decade−1 change in concentrationince the polyionic drug is likely deprotonated at a pH of 7.2 to giveuramin a charge of −6 given that the pKa of 1-naphthalenesulfoniccid is 0.2 [41]. Indeed, when the electrode was conditioned inuramin before use, the signal changed −11.5 ± 1.8 mV decade−1

hange in concentration (data not shown). This small signal changeimits the analytical use of this electrode when under equilibriumonditions. However, when the electrode is operated under non-quilibrium conditions in which the electrode is exposed to only0 mM NaCl before use, the potential elicited a super-Nernstianesponse. Over the narrow concentration range of 1.8 × 10−7 to.1 × 10−7 M, the potential changed −134.5 ± 3.2 mV per decadehange in concentration (Fig. 2). Thus, polymer membrane-basedlectrodes can be used under non-equilibrium, steady-state con-itions to generate large, analytically useful responses towarduramin. Further, because of this, the dynamic range of the elec-rode is able to be modified by judicious choice of membraneomponents (see Section 3.3).

.2. Response time

Because the electrode is operating under a non-equilibrium,teady-state response mechanism, the potential will decrease

apidly when first exposed to suramin as the drug passes throughhe sample-membrane interface, followed by a smaller change inhe signal as suramin slowly diffuses into the membrane to reachts maximum equilibrium potential. Therefore, it is necessary to beonsistent in the time that the electrode is exposed to the sample

Fig. 2. Potentiometric responses of polymer membrane-based electrodes towardsuramin. (closed diamond) Membrane A, (�) Membrane B, (�) Membrane C, (opendiamond) Membrane D, and (�) Membrane E.

solution. In this study, 6 min was found to be optimal for suraminmeasurements using Membrane A since the signal was rapidlychanging at shorter time periods, but longer time periods did notsignificantly enhance the signal change (see Fig. 3). Indeed, 55% ofthe response at ten min is obtained within 6 min of exposure toconcentrations equal to or below 1.1 × 10−7 M and 75% to concen-trations greater than 1.8 × 10−7 M. Thus, responses were recordedevery 6 min in subsequent studies.

3.3. Optimization of the membrane composition

The identity and weight percentages of plasticizer, ion-exchanger, and polymer were varied to optimize the polymermembrane composition used in potentiometric electrodes withrespect to generating the largest signal change toward suraminin its clinically relevant concentration range. Since the electroderesponse is driven by ion-pairing between the ion-exchanger andthe analyte and is due to a non-equilibrium steady-state that devel-ops at the sample-membrane interface, the response is influencedboth by the choice of ion-exchanger and by kinetic parameters thataffect the short-term steady-state response described by Eq. (2).

Electrodes containing tridodecylmethylammonium (TDMA) asthe ion-exchanger but varying in plasticizer identity between bis(2-ethylhexyl) sebacate (DOS) and 2-nitrophenyl octyl ether werefound to elicit similar responses (data not shown). However, thereproducibility was slightly improved with DOS. Therefore, DOSwas used as the plasticizer in all later tests.

Fig. 3. Time profile of potentiometric responses of polymer membrane-based elec-trodes incorporating Membrane A toward 1.1 × 10−7, 1.8 × 10−7, and 2.6 × 10−7 Msuramin, added at times denoted by *.

A. Yu et al. / Analytica Chimica Acta 686 (2011) 76–80 79

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edly calibrated toward suramin with reconditioning in 3 M NaClfor 72 h between each use. The response of the electrode decreasesand an unwanted shift in the dynamic range occurs upon repeatedcalibration. These data suggest that the polymer membrane-basedelectrodes for suramin should be employed as single use devices

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ig. 4. Potentiometric responses of polymer membrane-based electrodes towarduramin. (closed diamond) Membrane A, (�) Membrane F, (�) Membrane G, (�)embrane H, (open diamond) Membrane I, and (�) Membrane J.

y electrodes based on tetradodecylmethylammonium chlorideMembrane B), and didodecylmethylammonium chloride (Mem-rane C). The response of polymer membrane-based electrodes forolyions has been previously shown to be affected by the stabil-

ty of the ion-pair that is formed between the ion-exchanger andhe polyion, and the cooperative interactions between neighbor-ng ion-exchangers that facilitate formation of the reverse micelletructure that stabilizes the hydrophilic polyion in the hydrophobicembrane phase [39]. Similar to these previous studies, TDMA was

ound to be the most effective ion-exchanger to measure suramin,ikely due to the more effective electrostatic shielding provided byhe three dodecyl chains compared to shielding provided by thelternative ion-exchanger structures [42].

As is seen in Fig. 2, a large signal change toward suramin isbserved with the electrode incorporating Membrane A until a con-entration of 6.3 × 10−7 M (0.82 �g mL−1) is reached, after whichnly small changes in the signal occur as the surface of the mem-rane becomes fully equilibrated with suramin in the sample phasend the response becomes governed by the Nernst equation. Unfor-unately, the response from electrodes containing Membrane Aoes not include the concentration at which suramin shows toxicffects (200–350 �g mL−1 or 1.54 × 10−4 to 2.70 × 10−4 M) nor doest encompass the concentrations being explored to treatments inhe noncytotoxic range (<65 �g mL−1) [15,21]. Because this poly-

er membrane-based electrode for suramin responds under aon-equilibrium, steady-state response mechanism, kinetic fac-ors affect the signal and it is possible to shift the dynamic rangef the electrode into the clinically important concentration rangey altering the composition of the polymer membrane. As seen

n Eq. (2), changing the concentration of the ion-exchanger andhe diffusion layer thickness or diffusion coefficient in the mem-rane phase, which is done by changing the ratio or identity ofhe plasticizer and polymer, will change the EMF response. As theoncentration of the ion-exchanger TDMA is increased from 1.5o 3.0 wt% in the polymer membrane (Membrane A compared to

embrane F, respectively), the dynamic range shifts to a higheroncentration range (Fig. 4). However, the dynamic range still doesot encompass the clinically important range. Changing to highereight percentages of plasticizer and further increasing the weightercent of TDMA moves the dynamic range to even higher concen-rations as suramin is able to diffuse further into this more flexible

embrane necessitating more suramin to enter into the polymero elicit the same response (Fig. 4, Membranes G and H). Indeed,he dynamic range of the electrode incorporating Membrane H

verlaps the noncytotoxic concentrations used in suramin ther-py. However, in order for the dynamic range to encompass theange of concentrations where cytotoxicity occurs, a change in thedentity of the polymer from PVC to Pellethane is necessary (Fig. 4,

Fig. 5. Reversibility of polymer membrane-based electrodes incorporating Mem-brane J when repeatedly immersed in buffer or 375 �g mL−1 suramin.

Membranes I and J). Since the diffusion of many types of drugs intoPellethane catheters is typically greater than PVC catheters, it isexpected that the diffusion of suramin into Pellethane will also begreater than that into PVC, which changes Dm in Eq. (2) and shiftsthe dynamic range to higher concentrations [43]. It is also possiblethat the change in hydrophobicity of the polymer membrane whenPVC is replaced by Pellethane affects the solvation of suramin inthe membrane phase, altering its extraction into the membrane.Thus, different polymer membranes can be utilized for suraminmeasurements depending on the clinical needs with Membrane Hemployed at lower doses of suramin in the noncytotoxic range andMembrane J at higher suramin concentrations where cytotoxicityoccurs.

3.4. Reversibility

In order to characterize the reversibility, electrodes incorporat-ing Membrane J were repeatedly moved between background andconcentrated suramin solutions (375 �g mL−1) over eight min timeperiods. As is seen in Fig. 5, the potential returns to the baselinevalue when the electrode is re-immersed in the buffered solution. Inspite of this, the EMF response to the same concentration of suraminincreased with subsequent exposures, which suggests that suraminis not completely removed from the membrane and is in fact accu-mulating in the polymer. More evidence for this fact comes fromFig. 6, which shows the response of electrodes that were repeat-

Log [Suramin], M

Fig. 6. Repeated calibration of polymer membrane-based electrodes incorporatingMembrane J toward suramin after storage in 3 M NaCl for 72 h between use. Theaverage and standard deviation of nine electrodes are shown.

80 A. Yu et al. / Analytica Chimica

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ig. 7. Potentiometric titrations of suramin with protamine monitored withrotamine-sensitive electrodes in undiluted human plasma. (�) no suramin, (openiamond) 43.8 �g mL−1 suramin, (�) 62.8 �g mL−1 suramin.

n order to achieve improved reproducibility due to the limitedeversibility of the electrode.

.5. Application to real-world samples

The optimized potentiometric electrode incorporating Mem-rane J was evaluated for use in biological samples by placing thelectrode in plasma spiked with suramin, and monitoring the EMFhange as additional aliquots of the drug were added. The curve thatas produced was overlaid with the calibration curve determined

n plasma lacking suramin in an effort to determine the initialmount of suramin present (data not shown). Although this methodas able to show differing plasma levels of suramin, the concentra-

ions were overestimated with a percent recovery of 159% and 151%or 38.9 and 77.8 �g mL−1 suramin, respectively. The steady-stateotentials measured with polyanion-sensitive electrodes can be

nfluenced by the background concentration of small anions, suchs chloride or salicylate, which affects the background potentialf the electrode in various plasma samples and may cause limitedccuracy with this method.

Although direct potentiometry with the optimized suramin-ensitive electrode is capable of differentiating suramin levels inlasma, a titrimetric method of quantification was utilized thatoes not depend on the background salt concentration. Indeed,he most reliable determinations of other polyanionic drugs, suchs heparin, are not achieved using a heparin-sensitive electrodeut rather using a protamine-sensitive electrode as an end pointetector in a traditional titration of heparin with protamine [37].he ability to apply a similar titrimetric method for the determina-ion of suramin in biological samples was demonstrated by spikinguman plasma with varying levels of suramin, and titrating theolution with protamine while monitoring the EMF change with arotamine-sensitive electrode. As can be seen in Fig. 7, increasingmounts of suramin require greater amounts of protamine to reachhe endpoint. This indicates that suramin is binding to protaminend preventing its extraction in the polymer membrane. Only oncexcess protamine is added, the signal increases and an end points observed. Thus, protamine-sensitive electrodes can be used inclassical potentiometric titration to determine suramin levels inndiluted plasma samples.

.6. Conclusions

We have developed a polymer membrane-based electrode forhe measurement of suramin in physiological samples that offershe advantages of simplicity and speed over current suramin

easurement methods. Because this electrode operates under a

on-equilibrium, steady-state response mechanism, it is possibleo alter the dynamic range of the electrode by judicious choicef membrane components. Although this electrode can be usedo directly quantify the amount of suramin, a titrimetric methodn which the suramin is titrated with protamine and monitored

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Acta 686 (2011) 76–80

with a protamine-sensitive electrode yields results that are inde-pendent of the background concentration of small anions. We arecurrently examining the use of this electrode to characterize thebinding between suramin and various biologically important tar-gets, and to quantify suramin analogs that are being explored forclinical use.

Acknowledgement

We are grateful to Otterbein University for funding this work.

References

[1] E.L.M. Vansterkenburg, I. Coppens, J. Wilting, O.J. Bos, M.J. Fischer, L.K.H.Janssen, et al., Acta Trop. 54 (1993) 237.

[2] World Health Organization, African Trypanosomiasis (Sleeping Sickness). FactSheet 259, World Health Organization, Geneva, Switzerland, 2006.

[3] A. Abiose, Ann. Trop. Med. Parasitol. 92 (1998) S11.[4] WHO, Prevention of Blindness and Visual Impairment, 2010,

http://www.who.int/blindness/causes/priority/en/index3.html (accessed08.04.10).

[5] D.F.R. Bisaagio, C.M. Adade, T. Souto-Padron, Int. J. Antimicrobial. Agents 31(2008) 282.

[6] E.Z. Arruda, N.M. Silva, R.A. Moraes, P.A. Melo, Braz. J. Med. Biol. Res. 35 (2002)723.

[7] M.T. Murakami, E.Z. Arruda, P.A. Melo, A.B. Martinez, S. Calil-Elias, M.A. Tomaz,B. Lomonte, J.M. Gutierrez, R.K. Arni, J. Mol. Biol. 350 (2005) 416.

[8] D.N. Sifuentes, C.Z. El-Kik, H.D. Ricardo, et al., Toxicon 51 (2008) 28.[9] M. Rusnati, C. Urbinati, Curr. Pharmaceut. Des. 15 (2009) 2946.10] C. Urbinati, P. Chiodelli, M. Rusnati, Molecules 13 (2008) 2758.11] S. George, R. Dreicer, J.J.L. Au, T. Shen, B.I. Rini, S. Roman, M.M. Cooney, T.

Mekhail, P. Elson, G.M. Wientjes, R. Ganapathi, R.M. Bukowski, Clin. Genitouri-nary Cancer 6 (2008) 79.

12] M.A. Villalona-Calero, G.A. Otterson, M.G. Wientjes, F. Weber, et al., Ann. Oncol.19 (2008) 1903.

13] N. Yahi, J.M. Sabatier, P. Nickel, K. Mabrouk, F. Gonzalez-Scarano, J. Fantini, J.Biol. Chem. 269 (1994) 24349.

14] C.W. Mahoney, A. Azzi, K.P. Huang, J. Biol. Chem. 265 (1990) 5424.15] R.J. Bitton, W.D. Figg, D.J. Venzon, et al., J. Clin. Oncol. 13 (1995) 2223.16] R. Dreicer, D.C. Smith, R.D. Williams, W.A. See, Invest. New Drugs 17 (1999)

183.17] N.J. Vogelzang, T. Karrison, W.M. Stadler, J. Garcia, H. Cohn, J. Kugler, T. Troeger,

L. Giannone, R. Arrieta, M.J. Ratain, E.E. Vokes, Cancer 100 (2004) 65.18] T.E. Voogd, E.L. Vansterkenburg, J. Wilting, L.H. Janssen, Pharmacol. Rev. 45

(1993) 177.19] D.J. Cole, S.E. Ettinghausen, H.I. Pass, D.N. Danforth, M.W. Linehan, C.W. Myers,

et al., Surgery 116 (1994) 90.20] M. Kaur, E. Reed, O. Sartor, W. Dahut, W.D. Figg, Invest. New Drugs 20 (2002)

209.21] S. Song, M.G. Wientjes, Y. Gan, et al., Proc. Natl. Acad. Sci. U. S. A. 97 (2000)

8658.22] S. Song, M.G. Wientjes, C. Walsh, et al., Cancer Res. 61 (2001) 6145.23] Y. Xin, G. Lyness, D. Chen, et al., J. Urol. 174 (2005) 322.24] P. Dua, A. Ingle, R.P. Gude, Int. J. Cancer 121 (2007) 1600.25] M.P. Barrett, D.W. Boykin, R. Brun, R.R. Tidwell, Br. J. Pharmacol. 152 (2007)

1155.26] R.V. LaRocca, J. Meer, R.W. Gilliatt, C.A. Stein, et al., Neurology 40 (1990) 954.27] W.G. Dangerfield, W.E. Gaunt, A. Wormall, Biochem. J. 32 (1938) 592.28] O. Teirlynck, M.G. Bogaert, P. Demedts, H. Taelman, J. Pharm. Biomed. Anal. 7

(1989) 123.29] B. Olgemoller, T. Deufel, R. Schleicher, K.-D. Gerbitz, Z. Fresenius, Anal. Chem.

324 (1986) 356.30] M. Kassack, P. Nickel, J. Chromatogr. B: Biomed. Appl. 686 (1996) 275.31] E. Brandsteterova, V. Chovancova, I. Koza, J. Mardiak, J. Halko, Neoplasma 38

(1991) 425.32] P.C. Dabas, M.C. Vescina, C.N. Carducci, J. Capillary Electrophoresis 4 (1997) 253.33] L.L. Garcia, Z.K. Shihabi, J. Liq. Chromatogr. 16 (1993) 2049.34] L.L. Garcia, Z.K. Shihabi, J. Liq. Chromatogr. 16 (1993) 1279.35] D.M. Prantis, C. Telting-Diaz, M.E. Meyerhoff, CRC Crit. Rev. Anal. Chem. 23

(1992) 163.36] M.E. Meyerhoff, V.C. Yang, J.A. Wahr, L. Lee, J.H. Yun, B. Fu, E. Bakker, Clin. Chem.

41 (1995) 1355.37] N. Ramamurthy, N. Baliga, J.A. Wahr, U. Schaller, V.C. Yang, M.E. Meyerhoff, Clin.

Chem. 44 (1998) 606.38] N. Durust, M.E. Meyerhoff, Anal. Chim. Acta 432 (2001) 253.39] B. Fu, E. Bakker, V.C. Yang, M.E. Meyerhoff, Macromolecules 28 (1995) 5834.

40] B. Fu, E. Bakker, J.H. Yun, V.C. Yang, M.E. Meyerhoff, Anal. Chem. 66 (1994) 2250.41] N. Ramamurthy, N. Baliga, T.W. Wakefield, P.C. Andrews, V.C. Yang, M.E. Mey-

erhoff, Anal. Biochem. 266 (1999) 116.42] S.-C. Ma, V.C. Yang, B. Fu, M.E. Meyerhoff, Anal. Chem. 65 (1993) 2078.43] J.C. Smith, M.C. Davies, C.D. Melia, S.P. Denyer, M.R. Derrick, Biomaterials 17

(1996) 1469.