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Analytica Chimica Acta 681 (2010) 1–7

Contents lists available at ScienceDirect

Analytica Chimica Acta

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

olyion-sensitive membrane-based electrodes for heparin-bindingoldamer analysis

aitlin Kellya, Sobia Khajaa, April Venaa, Andrew Yub, Joan M. Essonb,∗

Department of Chemistry, Kalamazoo College, Kalamazoo, MI 49008, USADepartment of Chemistry and Biochemistry, Otterbein University, Westerville, OH 43081, USA

r t i c l e i n f o

rticle history:eceived 8 July 2010eceived in revised form 2 September 2010

a b s t r a c t

Polymer membrane-based electrodes sensitive to low molecular weight heparin (LMWH) have beenused to examine the binding between several preparations of LMWH and heparin-binding foldamers,which have recently been developed as potential inhibitors of the anticoagulant activity of LMWHs. It

ccepted 7 September 2010vailable online 16 September 2010

eywords:olyion-sensitive electrodeotentiometry

was found that the structure of the heparin-binding foldamer affects the equilibrium binding constant,Keq, determined by analysis of the titration curves of the foldamers with LMWHs monitored with theseelectrodes, and further, the strength of binding depends on the specific LMWH preparation. Additionally,polymer membrane-based electrodes utilizing dinonylnaphthalene sulfonate as the ion-exchanger weredeveloped to measure the heparin-binding foldamers directly in whole blood, and the response was

poph

ow molecular weight heparineparin-binding foldamer

found to depend on the li

. Introduction

Heparin, a highly sulfated polyanionic polysaccharide, is an anti-oagulant used in a wide variety of clinical procedures [1]. Itsctivity arises from its ability to bind to and increase the activ-ty of endogenous antithrombin, which inhibits several enzymesn the coagulation cascade. Treatment with heparin must provideevels that control thrombus formation without causing severeleeding. To reverse the activity of heparin, the antidote pro-amine, a polycationic peptide, is administered, which forms annactive heparin–protamine complex. However, due to bleedingomplications associated with unfractionated heparin (UFH), fre-uent monitoring is necessary to minimize the risk of hemorrhage2]. Further, for some treatments, such as deep vein thrombosisDVT), the heparin antagonizing effects wear off within a shortime period, thus necessitating continuous monitoring of the anti-oagulant ability. More recently, low molecular weight heparinsLMWHs) have been introduced to treat DVT because of their higherioavailability and longer half-life [3]. Because of their difference

n size, their biological activity is modified, inhibiting coagulationnly early in the cascade, which presumably reduces the bleeding

ide effects seen with UFH. Such characteristics allow them to bedministered subcutaneously and obviate the need for continualatient monitoring since a single dose treatment is possible [4].owever, protamine is not prescribed for use with LMWH ther-

∗ 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.2010.09.012

ilicity and charge density of the foldamer.© 2010 Elsevier B.V. All rights reserved.

apy, and further, protamine toxicity can occur and ranges frommild hypotension to severe, or even fatal, cardiac arrest [5]. Thus,there is a need for safer and more effective agents to neutralize theanticoagulant effects of LMWHs. Protamine analogues, antithrom-bin peptides, and polyarginine peptides have been proposed aspotential heparin antidotes [6–8]. However, it has been suggestedthat antidotes prepared by altering the protamine structure alonemay not provide complete reversibility of LMWH activities. Conse-quently, smaller molecule antidotes may possess advantages, suchas increased proteolytic stability, distribution, improved rever-sal of LMWH activity, and ability to scale-up synthesis. Recently,polycationic calixarenes and heparin-binding foldamers have beendeveloped as small molecule inhibitors of heparin’s anticoagulantactivity [9,10].

Foldamers are nonbiological oligomers with well-defined sec-ondary or tertiary structures that mimic proteins. Specifically, theseheparin-binding foldamers consist of a 1,3-substituted aryl amideoligomer possessing additional hydrogen-bonding substituentsthat increase solubility, conformational rigidity, and have chargecomplementarity with the pentasaccharide sequence of heparinthat is responsible for binding to antithrombin. The structures ofthe specific heparin-binding foldamers and other compounds orig-inally synthesized as potential antimicrobial foldamers but alsoinvestigated in this study for their ability to bind to heparin are

shown in Fig. 1.

Measurements of the activity of LMWH, and even more so ofthe effect of and binding to possible antidotes, is difficult becausetypical clotting time-based assays are not always sensitive towardLMWH [11]. A previous study of three of these heparin-binding

2 C. Kelly et al. / Analytica Chimica Acta 681 (2010) 1–7

Fig. 1. Structure of heparin-binding foldamers.

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oldamers examined their ability to reverse the anticoagulantffects of heparin using an aPPT assay [10]. However, due to thensensitivity of this assay toward LMWH, this experiment couldnly be completed using unfractionated heparin. The study did gon to further examine the ability of the heparin-binding foldamerso reverse the effects of the LMWH Lovenox on Factor Xa, which was

easured using a chromogenic anti-Factor Xa assay. Although thistudy demonstrated the ability to reverse LMWH activity in vitro,hey were not able to directly quantify the interaction betweenovenox and the heparin-binding foldamers, were limited to use inlasma, and further, only examined the foldamer interaction withne type of LMWH. Additionally, no sensors to directly monitor theeparin-binding foldamers currently exist. This is especially impor-ant as an arylamide foldamer related to those examined in thistudy is entering Phase II clinical trials [12].

Polymer membrane-based potentiometric polyion-sensitivelectrodes have been previously developed for measuring bothnfractionated and low molecular weight heparins in wholelood [13,14]. These electrodes are capable of generating large,on-equilibrium steady-state potentiometric responses to clin-

cally important heparin concentrations [15]. The response isriven by cooperative ion-pairing of the polyion with the ion-xchanger in the polymeric membrane phase [16]. Since theselectrodes respond only to free polyion, previous studies have usedeparin–protamine titrations monitored with these electrodes toharacterize the binding of protamine with both UFH and LMWH14]. More recently, polyion sensing has been achieved using otherlectrochemical methods, such as pulsed chronopotentiometryeveloped by Bakker [17–20], and voltammetric and amperometricechniques developed by the Amemiya and Samec groups, respec-ively [21–27]; however, these methods have not been employedo characterize the binding between polyions and selected targets.he advantages of using potentiometric polyion-sensitive elec-rodes over the Factor Xa assay are that not only can studies beompleted in whole blood, but the direct interaction between therug and antidote can also be monitored. However, no studies havexamined the ability of using heparin-sensitive electrodes to char-cterize the interaction of LMWH with heparin-binding foldamers,or for the direct analysis of heparin-binding foldamers.

The purpose of this study is to utilize potentiometriceparin-sensitive electrodes to examine the binding betweeneparin-binding foldamers and various preparations of LMWHs byitrating the foldamers with LMWH and performing subsequentcatchard plot analysis. This information is used to determinehe structural features of the heparin-binding foldamers that areecessary for strong interactions with LMWHs. Additionally, theevelopment of polymer-membrane based potentiometric elec-rodes for direct foldamer detection is also demonstrated for therst time.

. Materials and methods

.1. Materials

Tridodecylmethylammonium chloride (TDMAC), nitrophenylctyl ether (NPOE), and high molecular weight poly(vinyl chlo-ide) (PVC), bis(2-ethylhexyl) sebacate (DOS), low moleculareight heparin, and protamine (from salmon) were purchased

rom Sigma–Aldrich (St. Louis, MO). DinonylnaphthalenesulfonateDNNS) was supplied from King Industries, while Lovenox and

ragmin were gifts. Pellethane (2363-80AE) was donated byreelin-Wade (McMinnville, OR), while polyurethane (M48) wasonated by Medtronic, Inc. (Minneapolis, MN). Heparin-bindingoldamers were provided by the DeGrado laboratory at the Uni-ersity of Pennsylvania [10]. Foldamers 14 and 15 were prepared

ca Acta 681 (2010) 1–7 3

by polymerization and the value of n subunits is the averagedetermined by gel permeation chromatography and/or mass spec-trometry. All other reagents were of analytical grade.

2.2. Preparation of polyanion-sensitive membrane electrodes

Disposable heparin-sensitive cylindrical membrane electrodeswere cast from a membrane cocktail consisting of 300 mg of com-ponents made of 1.5 wt% TDMAC, 32.5 wt% DOS, and 66.0 wt% PVC,dissolved in 3 mL THF. Electrodes were prepared by dipcoating thismixture onto the end of rounded glass capillary tubes protrudingout of narrow-bore Tygon tubing (i.d. 1/16′′, o.d. 1/8′′) 12 timesevery 10 min, and dried overnight. The glass capillaries were thenremoved and the tubes were filled with 10 mM NaCl. A Ag/AgCl wirewas then inserted into the inner bore of the tube to complete thefabrication of the electrode. Foldamer-sensitive membrane elec-trodes were made following the same procedure, except that themembrane cocktail contained 1 wt% DNNS, 49 wt% NPOE, 30 wt%pellethane, and 20 wt% M48. Prior to use all heparin-sensitiveelectrodes were conditioned in 10 mM NaCl, while 120 mM NaCl,0.050 M Tris, pH 7.2, was used for foldamer-sensitive electrodes.

2.3. EMF measurements with polyion-sensitive electrodes

Potentiometric responses were measured against a minia-ture Ag/AgCl reference electrode (Bioanalytical Systems Inc., WestLayfayette, IN) using a Gateway E-4200 computer with a PCI-6052E input-output board (National Instruments, Austin, TX) and acustom-built electrode interface module controlled by LabView 6.1software. Responses of a minimum of three TDMAC-based mem-brane electrodes were taken in 3 mL of 120 mM NaCl, 50 mM Tris,pH 7.2, with and without 0.1 mg of heparin-binding foldamer or0.6 mg protamine, unless otherwise specified, to which aliquots ofeach low molecular weight heparin were added to give concen-trations of heparin ranging from 1 × 10−9 to 1 × 10−4 M. Readingswere taken 5 min after each addition. Graphs were then made ofthe change in potential (�EMF) versus the concentration of lowmolecular weight heparin. A similar procedure was utilized forDNNS-based membrane electrodes. The potentiometric responsewas recorded 5 min after aliquots of various heparin-bindingfoldamers were added to 3 mL of undiluted whole blood.

2.4. Binding analysis between heparin-binding foldamers andLMWHs

The equilibrium binding constant and binding stoichiometrybetween the low molecular weight heparins and heparin-bindingfoldamers was determined using Scatchard plot analysis. The freelow molecular weight heparin concentration (F) for each point onthe titration curve was graphically determined by extrapolationonto the calibration curve. The bound LMWH concentration wasdetermined by subtracting the free LMWH concentration from thetotal amount of heparin added. The moles of bound LMWH per moleof heparin-binding foldamer (B′) was plotted versus B′/F, whichallowed determination of the binding constant, Keq, and the numberof LMWH binding sites per mole of heparin-binding foldamer (n)from the negative slope and x-intercept, respectively, of the linearleast squares fit.

3. Results and discussion

3.1. Binding of low molecular weight heparins with protamine

Initially, heparin-sensitive polymer membrane-based elec-trodes using TDMAC as the ion-exchanger were used to monitor the

4 C. Kelly et al. / Analytica Chimica Acta 681 (2010) 1–7

Table 1Summary of results of binding studies between polycations and various LMWHpreparations.

LMWH Polycation Keq, M−1 (×105) n

Lovenoxa Protamine (herring) 54 ± 11 0.47 ± 0.03Lovenox Protamine (salmon) 3.1 ± 0.2 0.17 ± 0.02Lovenox Foldamer 1 0.72 ± 0.21 2.9 ± 1.0Lovenox Foldamer 2 3.6 ± 1.0 2.2 ± 0.8Lovenox Foldamer 3 1.1 ± 0.2 2.9 ± 0.5Lovenox Foldamer 4 1.6 ± 1.3 3.0 ± 3.0Lovenox Foldamer 5 NDb NDb

Lovenox Foldamer 6 2.2 ± 0.9 1.9 ± 1.0Lovenox Foldamer 7 2.1 ± 0.3 3.3 ± 0.6Lovenox Foldamer 8 0.76 ± 0.51 2.8 ± 2.0Lovenox Foldamer 10 1.4 ± 1.1 3.2 ± 3.0Lovenox Foldamer 13 4.0 ± 1.0 3.2 ± 1.0Lovenox Foldamer 14 7.2 ± 0.8 4.8 ± 0.9Lovenox Foldamer 15 15.7 ± 0.2 3.6 ± 0.6Lovenox Foldamer 16 9.0 ± 1.6 2.1 ± 0.5Lovenox Foldamer 18 0.29 ± 0.30 6.8 ± 0.7Lovenox Foldamer 20 1.5 ± 0.8 2.4 ± 1.5Lovenox Foldamer 21 7.4 ± 1.6 1.1 ± 0.3Lovenox Foldamer 22 7.3 ± 0.5 0.1 ± 0.0Fragmina Protamine (herring) 349 ± 55 0.64 ± 0.02Fragmin Protamine (salmon) 54.8 ± 1.4 0.19 ± 0.05Fragmin Foldamer 6 58.2 ± 1.3 0.18 ± 0.05Sigma Protamine (salmon) 0.72 ± 0.6 0.63 ± 0.13Sigma Foldamer 2 4.25 ± 0.42 2.06 ± 0.01Sigma Foldamer 3 3.50 ± 0.42 0.54 ± 0.45

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ΔEM

F, m

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Log [Lovenox], mg/mL

Lovenox

Foldamer 1

Foldamer 2

Foldamer 3

Protamine

a Data taken from Ref. [13].b The potentiometric titration curve with Foldamer 5 was the same as that of

ovenox alone, and, thus, the binding results could not be determined.

itrations of protamine with a variety of low molecular weight hep-rin preparations, including Lovenox, Fragmin, and Sigma LMWHdata not shown, Scatchard analysis results shown in Table 1).he equilibrium binding constant, Keq, of protamine with Lovenoxnd Fragmin showed similar trends as those reported in previoustudies with larger Keq values observed with Fragmin comparedo Lovenox [14]. This is attributed to the higher average molecu-ar weight and increased percentage of higher molecular weightomponents within the Fragmin preparation compared to Lovenox28–31]. However, the values of the equilibrium binding constantere different, which may be due to the fact the current study usedrotamine from salmon while the earlier studies used herring ashe source. Regardless, protamine was found to bind more stronglyo Fragmin than Lovenox in both cases, which is likely due to theifferences in structure and molecular weight of the LMWHs. Thequilibrium binding constant of protamine with Sigma LMWH wasess than that to either Lovenox or Fragmin (Table 1). Less informa-ion is available on the composition of the Sigma LMWH comparedo the more widely used, clinically important Lovenox and Fragmin;owever, its average molecular weight is similar to that of Lovenox4500 Da). It is likely that the lower binding strength may be dueo the differences in structure that arise during different depoly-

erization methods of UFH to create LMWHs [32], or it is possiblehat the Sigma LMWH is not as polydisperse and has fewer higher

olecular weight components.

.2. Binding of Lovenox with heparin-binding foldamers

The response of heparin-sensitive electrodes to Lovenox inhe absence and presence of various heparin-binding foldamersevealed that Lovenox binds to the foldamers (see Fig. 2, selectata shown). Heparin is known to be the biological macromolecule

ith the highest negative charge density. Therefore, the cationic

oldamers and Lovenox are expected to interact primarily throughlectrostatic interactions between the negatively charged groups ofeparin and the positively charged substituents of the foldamers.nly unbound Lovenox is able to enter the polymer membrane and

Fig. 2. Typical titration curves obtained with heparin-sensitive electrodes. The aver-ages of a minimum of three titrations of 0.1 mg of various heparin-binding foldamersand 0.6 mg of protamine with Lovenox in 120 mM NaCl, 50 mM Tris, pH 7.2, areshown. The error bars indicate the standard deviation.

elicit a signal. When the charge on Lovenox is neutralized by thefoldamers, its extraction into the membrane is prevented. Assum-ing that the dissociation rate of the complex is slow, it generatesno signal. Thus, a large signal change is observed only once enoughLovenox is added such that there is free Lovenox in solution. The factthat the interaction between LMWH and the foldamers is electro-static in nature is supported by the fact that the binding of Lovenoxto heparin-binding Foldamer 2 was affected by the backgroundsalt concentration. As the salt concentration increased from 50 to120 mM NaCl, Keq for Lovenox binding to 2 decreased by a factorof 10 (data not shown), which is likely due to the disruption of theelectrostatic interaction between the LMWH and the cationic siteson the antidote by the ions in the background solution.

Of the three foldamers included in Fig. 2, only one was foundto have a titration curve that overlaps significantly with that ofprotamine, heparin-binding Foldamer 2. This suggests equivalentbinding efficiencies to Lovenox by Foldamer 2 and protamine orperhaps even greater interactions with Foldamer 2 consideringthat protamine is present at 0.6 mg, while Foldamer 2 is presentat 0.1 mg. The other two, Foldamers 1 and 3, bind more weaklysince less Lovenox was needed to achieve complete neutralizationand reach the endpoint of the titration curve. These observationsare further supported by the results of the Scatchard plot anal-ysis that shows Foldamer 2 has an equilibrium binding constant(Keq) slightly larger than protamine, while Foldamers 1 and 3 havesmaller values (Table 1). These findings are consistent with a previ-ous study that used a traditional chromogenic anti-Factor Xa assayto examine the effect of these three heparin-binding foldamers onthe inhibition of Factor Xa activity by Lovenox [10]. The previousstudy found that Foldamer 3 had an increased inhibition potencycompared to Foldamer 1, albeit not as great as that of Foldamer 2compared to Foldamer 1, which was an approximately threefoldincrease [10]. These previous results correlate with the increase inthe equilibrium binding constant found in this study from Foldamer1 to Foldamer 3, and the even larger increase from Foldamer 1 toFoldamer 2, which is approximately threefold (Table 1).

Additional heparin-binding foldamers were investigated in thisstudy that were not analyzed previously. The equilibrium bindingconstant of these foldamers was found to depend on their struc-tural features. An increase in molecular weight of the foldamerswas found to increase Keq (R2 = 0.79, excluding Foldamers 21 and22 that have significant structural differences), as did an increase inthe number of positively charged substituents (R2 = 0.79, excluding

Foldamers 21 and 22, and assuming that the basic functional groupsare all protonated at pH 7.2). This is perhaps best observed byconsidering Foldamer 15, which has the highest molecular weightand number of charges out of the 17 heparin-binding foldamersexamined, and also has the largest equilibrium binding constant

C. Kelly et al. / Analytica Chimi

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Lovenox

Foldamer 4

Foldamer 5

Foldamer 18

Fig. 3. Potentiometric responses of heparin-sensitive electrodes to Lovenox in thearN

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The results from this study further support the need to consider

bsence and presence of 0.1 mg of heparin-binding Foldamers 4, 5, and 18. Theesponses are an average from a minimum of three electrodes taken in 120 mMaCl, 50 mM Tris, pH 7.2. The error bars indicate the standard deviation.

Table 1). The greater number of positive charges likely increaseshe overall strength of the electrostatic interactions with the neg-tively charged Lovenox.

More subtle changes in the chemical structure, such as choicef terminal anchoring groups, substituent changes, and backbonehanges, were also found to affect the equilibrium binding con-tant. For example, changing from a guanidinylated terminal groupFoldamer 4) to an amine terminus (Foldamer 5) affected thenteraction with Lovenox to such an extent that the titrationf Foldamer 5 showed no shift from the calibration curve withovenox alone (Fig. 3). However, a change from a C3 terminalpacer in Foldamer 5 to a C4 spacer in Foldamer 18 facilitated weakinding to Lovenox (Table 1). Previous molecular dynamic stud-

es have shown that binding occurs when the terminal cationicubstituents anchor the foldamer via electrostatic interactions tohe pentasaccharide sequence in heparin responsible for its anti-oagulant activity and then align other positive charges of theoldamer with negative charges of heparin [10]. Thus, structuralhanges that affect the strength of the anchoring electrostaticnteractions affect the equilibrium binding constant. Indeed, its known that guanidinium binds more strongly to sulfonateroups than amines [33], which supports the larger Keq observedith the guanidinylated Foldamer 4 compared to the aminated

oldamers 5 and 18 (Table 1). Although guanidinylated terminalroups enhance the binding of some foldamers containing theseroups, not all foldamers possessing these anchors have high Keq,hich is likely due to other structural features of these com-ounds.

Substituent changes along the aryl amide backbone and to theackbone itself were also found to affect the equilibrium bindingonstant. For example, the addition of a t-butyl group as a sub-tituent to the aryl amide backbone to the R4 position in Foldamer

that possesses guanidinylated groups in the R2 and R3 posi-ions to give Foldamer 20 was found to increase Keq by a factorf two. However, the addition of a t-butyl group to Foldamer 3,hich possesses aminated groups in the R2 and R3 positions, to

ive Foldamer 8 results in a decrease in Keq. This is consistentith previous studies of similar amphiphilic arylamide foldamers

or use as biomimetic antimicrobial compounds that show dif-erential effects on the minimal inhibitory concentration (MIC)equired to fully inhibit the growth of bacteria [34]. Changing fromn amine to a guanidinium group elicited different changes inhe MIC of two different bacteria, which suggests that differen-ial effects can be observed with biological targets despite bothets of foldamers having t-butyl groups that is dependent on the

xact structure–activity relationships between the foldamer andhe binding target. Further changes to the foldamer structure alsoffected the observed Keq. Changing the t-butyl group in Foldamer0 to an ester derivative in Foldamer 13 increased the equilib-

ca Acta 681 (2010) 1–7 5

rium binding constant threefold. Changing the backbone itself fromFoldamers 20 to 21 was found to increase Keq by a factor of five.These structural changes likely affect the flexibility of the ary-lamide oligomers and the ability of the positive charges on thefoldamer to align with the negative charges on heparin, whichin turn changes the observed equilibrium binding constants. Thisis supported by molecular dynamic studies that show the sub-stituents on the arylamide backbone affect the flexibility of theheparin-binding foldamers [35].

Of the 17 heparin-binding foldamers examined, seven werefound to bind more strongly to Lovenox than protamine. Foldamers2 and 13 both have guanidinylated terminal groups that enhancethe anchoring interactions to Lovenox; Foldamers 14, 15, and16 have much higher molecular weights and number of positivecharges; while Foldamers 20 and 21 have significant alterations inthe backbone structure.

3.3. Binding of different low molecular weight heparinpreparations with heparin-binding foldamers

Analogous studies to those with Lovenox were completed withother LMWH preparations and select heparin-binding foldamers.The interactions of LMWH from Sigma were examined withheparin-binding Foldamers 2 and 3. Both were found to bind toSigma LMWH more strongly than protamine with Foldamer 2having a sixfold higher equilibrium binding constant (Keq) thanprotamine, and Foldamer 3 having a fivefold increase (Table 1). Incomparison, Foldamer 2 exhibited a smaller increase in Keq withLovenox compared to protamine (a factor of 1.2), while Foldamer3 showed a decrease in Keq compared to protamine (a factor of0.36).

The binding of the low molecular weight heparin Fragmin withFoldamer 6 was also examined (Table 1). The equilibrium bindingconstant Keq was found to increase slightly compared to that withprotamine (a factor of 1.1), while the Keq of Lovenox with the samefoldamer compared to that with protamine was found to decrease(a factor of 0.7). Thus, it is evident that the binding of the heparin-binding foldamer to low molecular weight heparins is dependenton the preparation of the LMWH, as is similar to the binding ofprotamine with LMWHs as was seen in Section 3.1 and in previouswork [14].

The LWMHs used in this study are prepared by different par-tial chemical depolymerization methods of UFH, thus, giving eachpreparation slightly different structural features [32,36]. Such vari-ation is due to differences in the cleavage point along the heparinpolysaccharide chain, as well as modifications of the monosac-charidic units at the site of cleavage [37]. All the preparationsare polydisperse with molecular weight ranges from 2000 to10,000. However, Lovenox and LMWH from Sigma have aver-age molecular weights of 4500, while that of Fragmin is closerto 6000 [28,29]. Additionally, the polydispersity of Fragmin islower than Lovenox; thus, Lovenox contains a higher proportionof lower molecular weight species [30,31]. The pharmacologicalcharacteristics of different preparations of LMWHs also vary. Forexample, the anti-Xa:IIa activity ratio of Lovenox is higher thanthat of Fragmin [3]. Additionally, although protamine is known toneutralize the anti-IIa activities of LMWHs similarly, protamineneutralizes the anti-Xa activity of LMWHs differently [38]. Forthese reasons, the US FDA classifies each preparation of LMWHas a distinct drug that cannot be interchanged with one another.

each LMWH to have a unique chemical identity, and, more impor-tantly, suggest that different anticoagulation inhibitors may bebest suited for the neutralization of different LMWH prepara-tions.

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1.3 mg foldamer

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ig. 4. Potentiometric titrations of Foldamer 6 at two different concentrations withovenox in 120 mM NaCl, 50 mM Tris, pH 7.2. The response is the average of threelectrodes and the error bars indicate the standard deviation in the measurements.

.4. Development of polymer-membrane electrodes foreparin-binding foldamers

As studies on the heparin-binding foldamers continue and theirse in clinical applications expands, there is a need to directlynd quickly measure the foldamers in complex media. This can bechieved either through titration of the foldamers with LMWH, ass seen in Fig. 4 where two different concentrations of Foldamer 6an be distinguished through a titration with Lovenox that is mon-tored with the heparin-sensitive electrode, or by using a polymer

embrane-based electrode sensitive toward the heparin-bindingoldamer itself.

The responses of polymer membrane-based electrodes thattilize dinonylnaphthalene sulfonate (DNNS) as the ion-exchangerere evaluated toward several different heparin-binding

oldamers and protamine in whole blood (Fig. 5). This elec-rode was not only found capable of measuring protamine, whichas expected since similar electrodes have been used previously

or protamine detection [14], but was also responsive to theeparin-binding foldamers. However, the responses toward eacholycationic analyte vary and are dependent on the charge densitynd lipophilicity of each.

Protamine is a small, highly basic protein with a moleculareight of roughly 5000 Da and approximately 2/3 of its com-osition is in the form of arginine residues. In contrast, theeparin-binding foldamers are smaller, having molecular weightsarying from approximately 500 to 4200 Da depending on the spe-

ific foldamer, and possess a backbone made of benzene ringsonnected by peptide bonds, enhancing the lipophilicity of theseompounds relative to protamine. As seen in Fig. 5, the non-quilibrium, steady-state response of the DNNS-based electrode

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

2.001.501.000.500.00-0.50-1.00-1.50

ΔEM

F, m

V

μg/mL

Foldamer 15

Foldamer 16

Foldamer 20

Protamine

ig. 5. Potentiometric response of DNNS-based polymer membrane-based elec-rodes toward various polycations in undiluted, whole blood. The response is theverage of three electrodes and the error bars indicate the standard deviation in theeasurements.

ca Acta 681 (2010) 1–7

to Foldamer 20 is slightly larger and significantly more sensi-tive than the other foldamers examined, which is likely due tothe fact that it has the highest charge density. Previous stud-ies have shown that polyion-sensitive polymer membrane-basedelectrodes give large, analytically useful signals when operatedunder non-equilibrium conditions that are driven by ion-pairingbetween the ion-exchanger and the analyte [16]. The extraction ofrather hydrophilic polyions into the hydrophobic polymer mem-brane phase is facilitated by this ion-pairing interaction followedby the formation of a reversed micelle in the membrane createdby the cooperative interactions of hydrophobic portions of theion-exchanger. This cooperativity increases with increasing chargedensity of the polyion [16]. Thus, foldamers with higher charge den-sity are expected to form more stable ion-pairs in the membranephase. The difference in sensitivities observed for the various poly-cationic analytes may also be attributed to the difference in thediffusion coefficients of each of the DNNS–polycation complexesin the polymer membrane phase. Further, Foldamer 20 is moreamphiphilic compared to the other hydrophilic polycations, whichmay alter its interactions with the ion-exchanger and enhance itsextraction into the polymer membrane.

Of Foldamers 15 and 16, which have similar charge densities,heparin-binding Foldamer 16 elicits a larger and more sensitiveresponse. Foldamer 16 contains an additional benzyl group ateach terminus of the foldamer that increases the lipophilicity ofthe compound relative to Foldamer 15, and likely enhances itsextraction into the organic membrane phase. Although protamineis less lipophilic than the latter two foldamers, it still elicits amore sensitive response, which is likely due to its higher chargedensity compared to these foldamers that enhances ion-pairingwithin the membrane phase. Regardless, the DNNS-based poly-mer membrane-based electrodes are capable of directly measuringheparin-binding foldamers in whole blood. This is a significant andimportant finding as it is the first time direct sensing of heparin-binding foldamers has been achieved, and a polycationic arylamidecompound similar to the ones examined in this study is currentlyentering Phase II clinical trials [12].

3.5. Conclusions

These results demonstrate that heparin-sensitive polymermembrane-based electrodes can be effectively used as a tool fordetermining the potency of arylamide oligomers as potential anti-dotes to low molecular weight heparins. Indeed, this method is thefirst that is capable of directly monitoring the binding betweenLMWHs and heparin-binding foldamers, and, further, has identi-fied several candidates that may prove to be more effective thanprotamine at reversing the anticoagulant effects of low molec-ular weight heparin due to their increased binding to heparin.Results from this work further support the classification of differentpreparations of LMWHs as unique drugs, and suggest that differ-ent antidotes may be more effective for different LMWHs. Finally,direct sensing of heparin-binding foldamers using potentiomet-ric polymer-membrane electrodes has been demonstrated for thefirst time. Future studies may involve expanding the use of thesepotentiometric electrodes, or ones based on other electrochemi-cal methods, to ongoing studies with polycationic arylamide drugscurrently under development.

Acknowledgements

This work was funded by Otterbein University and KalamazooCollege. The authors would also like to thank William F. DeGradoand co-workers at the University of Pennsylvania for helpful dis-cussions throughout this work.

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