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Journal of Chromatography A, 1144 (2007) 102–110

Characterization of reversible and pseudo-irreversible acetylcholinesteraseinhibitors by means of an immobilized enzyme reactor

Manuela Bartolini, Vanni Cavrini, Vincenza Andrisano ∗Department of Pharmaceutical Sciences, Via Belmeloro 6, University of Bologna, 40126 Bologna, Italy

Available online 28 November 2006

bstract

The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized onmonolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For

his application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction MediaCIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, lowonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms ofction of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver–Burk plot analysis.nalyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak

rea. AChE-CIM-IMER kinetic parameters (Kappm and v

appmax) were derived as well as inhibitory constants (Kapp

i ) of selected compounds. Moreover,oteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylationteps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool toetermine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained withhe classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-MER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the

rst phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracynd reproducibility and with saving of time and materials.

2006 Elsevier B.V. All rights reserved.

eywords: Acetylcholinesterase; Immobilization; CIM monolithic disk; Liquid chromatography; Mechanism of action; Reversible and pseudo-irreversible inhibitors;

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inetic constants

. Introduction

Fast, accurate and reproducible analyses, alternative to thelassic methods are required in new lead selection and drugiscovery. Lately, immobilization of target proteins and theirnsertion in flow-through systems have been quite spread out, asigand high-throughput screening methodologies [1–7]. Recentpplications concern the frontal affinity chromatography (FAC)ethodologies [8,9], continuous flow microfluidic systems [10],

isplacement [7] and zonal elution chromatography [11,12].In this context, the preparation of an AChE-CIM-IMER

immobilized human recombinant acetylcholinesterase-based

∗ Corresponding author. Tel.: +39 051 2099742; fax: +39 051 2099734.E-mail address: vincenza.andrisano@unibo.it (V. Andrisano).

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021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2006.11.029

icro enzyme reactor) for inhibitor screening was previouslyptimized by us [5,6]. This bioreactor (3 mm × 12 mm I.D.) washaracterized in terms of rate of immobilization, stability, con-itioning time for HPLC analyses, optimum mobile phase andeaks shape as well as nonspecific interactions and costs. Cova-ent immobilization through Schiff bases linkage gave a stableeactor without any significant change in the enzyme behav-or. The chosen Convective Interaction Media (CIM) monolithic

atrix guaranteed very short conditioning time (5 min), suit-ble time of analysis (complete elution of the product in 2 min)nd fast recovery of the enzymatic activity that represent verymportant features in high-throughput analysis. The usability

f the AChE-CIM-IMER in inhibition studies was assessed bytudying selected well-known AChE inhibitors. In particular,C50 values were assessed by injecting increasing concentrationsf the tested inhibitor and saturating concentration of substrate

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conversion of substrate into product lower than 2%). The reduc-ion of the product area was correlated to the inhibition of thenzymatic activity.

However, in a second stage following the screening step,elected hits need to be further characterized in terms of mech-nism of action (reversible inhibition) and kinetic parameterspseudo-irreversible inhibition).

The understanding of selected potent AChE inhibitors mech-nism of action is a key information to rationally design newompounds, as candidates for the treatment of Alzheimer’s dis-ase. In particular, inhibition mode is of key importance in thease of reversible AChE inhibitors because of the potential rolef the AChE peripheral binding site in inducing beta-amyloidggregation and senile plaques formation [13–17]. To this pur-ose, we here report the application of the AChE-CIM-IMER tohe determination of both the mechanism of action and inhibitoryonstants of selected AChE reversible in a highly reliable andutomated mode.

On the other hand, inhibition by pseudo-irreversiblenhibitors is time dependent, therefore, the determination of theinetic parameters is of utmost importance to assess time ofction [18–20]. Although this need, at our knowledge no spe-ific online kinetic study on pseudo-irreversible inhibitors isresent in literature, which might be related to the difficultiesn regenerating inhibited enzyme.

The here presented automated, accurate, precise method, ishe first reported HPLC-based method for the on-line kineticetermination of pseudo-irreversible inhibitors mode of action.he fact that the amount of enzyme is stable for months guar-nteed the fast and reliable characterization of new inhibitors.

. Materials and methods

.1. Materials

EDA-CIM disks (3 mm × 12 mm I.D., 0.34 mL internalolume) were kindly donated by BIA Separations (Ljubl-ana, Slovenia). 5,5′-Dithio-bis(2-nitrobenzoic acid) (DTNB;llman’s reagent), glutaraldehyde 70% aqueous solution,hysostigmine and human recombinant acetylcholinesteraseAChE, EC 3.1.1.7) lyophilized powder and its substrate (S)-cetylthiocholine iodide (ATCh) were purchased from SigmaMilan, Italy). Tacrine (9-amino-1,2,3,4-tetrahydroacridineydrochloride), edrophonium chloride and monoethanolamineere obtained from Aldrich (Milan, Italy). Donepezil was aind gift from Pfizer. Ambenonium chloride pentahydrate wasurchased from Tocris Cookson (UK). Potassium chlorate andodium cyanoborohydride were obtained from Fluka (Milan,taly) and magnesium sulfate from Merck (Darmstadt, Ger-any). Rivastigmine was extracted from Exelon 3 mg, tablets

Novartis, Basel, Switzerland), and its purity confirmed byPLC (98%).HPLC-grade methanol (Romil, UK) or bidistilled water was

sed to prepare inhibitors’ solutions. Purified water from Milli-X system (Millipore, Milford, MA, USA) was used to prepareuffers and standard solutions. To prepare buffer solutionsotassium dihydrogenphosphate, dipotassium hydrogenphos-

2

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r. A 1144 (2007) 102–110 103

hate trihydrate, Tris–HCl (Carlo Erba, Milan, Italy) of analysisuality were used.

The buffer solutions were filtered through a 0.45 �m mem-rane filter and degassed before their use for HPLC.

Stock solutions of reference inhibitors were prepared in waterr methanol (1–10 mM) and further diluted in water.

.2. Apparatus

Spectrophotometric determinations with AChE in solutionere performed using a Jasco double beam V-530 UV–vis spec-

rophotometer, with a slit width of 2 nm and 0.5 s data pitch.AChE-CIM-IMER was inserted in a HPLC system consisting

f a Jasco BIP-I HPLC pump equipped with a Rheodyne Model125 injector with a 10 �L sample loop. The eluates were moni-ored by a Jasco 875-UV Intelligent UV–vis detector connectedo a computer station (JCL 6000 program for chromatographicata acquisition). For routine analyses the detector wavelengthas set at 412 or 450 nm in order to monitor the yellow productf the enzymatic reaction.

.3. Chromatographic conditions

The chromatographic analyses on AChE-CIM-IMER wereerformed at 25 ◦C unless otherwise stated.

Optimal chromatographic conditions were obtained with aobile phase consisting of 0.1 M Tris–HCl pH 8.0 containing

00 mM KClO3 as selective competitive anion for the cationicites on the matrix, 10 mM MgSO4, 1.26 × 10−4 M Ellman’seagent (Buffer A) [5,6].

.4. AChE immobilization

AChE-CIM-IMER was prepared by linking human recom-inant AChE to a previously activated EDA-CIM disk [6]. Inrief, the EDA-CIM disk was first activated by a 10% glu-araldehyde solution in phosphate buffer (50 mM, pH 6.0) (6 h,n the dark). The reacted matrix was then washed with phosphateuffer (50 mM, pH 6.0). 12 U of AChE in 800 �L of phosphateuffer (50 mM, pH 6.0) were added to the matrix and left to reactvernight.

The Schiff bases were reduced by a 0.1 M cyanoborohydrideolution in phosphate buffer (50 mM, pH 6.0) (2 h at room tem-erature). The matrix was then washed and unreacted aldehydicroups were quenched by a 0.2 M monoethanolamine solutionn phosphate buffer (50 mM, pH 6.0) (3 h at room temperature)5].

The AChE-CIM-IMER was then washed with phosphateuffer 0.1 M pH 8, inserted in the appropriate holder and con-ected to the HPLC system.

The amount of active units retained after immobilizationesulted to be 0.22 ± 0.01.

.5. Apparent kinetic constants variation on flow rate

Apparent Kappm and v

appmax values for AChE-CIM-IMER

ere determined at increasing flow rates (0.2–1.2 mL/min) by

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njecting in duplicate 10 �L aliquots of ATCh aqueous solutionconcentrations comprised in the range 3.1–200 mM) with UVetection at 450 nm at constant temperature (25.0 ± 0.8 ◦C). Bylotting the micromoles of product formed per minute (v) versushe normalized substrate concentrations (range 0.09–12 mM),

ichaelis–Menten plots [21,22] were obtained and Kappm

nd vappmax derived at each flow rate. Normalized substrate

oncentration was calculated by the following formula:

ATCh]normalized = Cinj × Vinj

BV

here Cinj is the injected substrate concentration, Vinj ishe injected volume and BV is the bed volume of theChE-CIM-IMER.

.6. Inhibition studies

.6.1. Determination of steady-state inhibition constantK

appi ) and mechanism of actionTo obtain estimates of the competitive inhibition constant

appi , reciprocal plots of 1/v versus 1/[S] were constructed at

elatively low concentration of substrate by injecting in duplicateubstrate solutions (ATCh, 12–100 mM), containing increasingeversible inhibitor concentrations, and using the chromato-raphic conditions reported in Section 2.3. The flow rate was.0 mL/min and UV detection was at 412 nm. Product forma-ion rates (v) were estimated by integrating the resulting producteak areas [5,6]. The plots were assessed by a weighted least-quares analysis that assumed the variance of v to be a constantercentage of v for the entire data set. Slopes of these reciprocallots were then plotted against normalized inhibitor concentra-ion (considering the injected amount in 0.34 mL CIM disk voidolume) in a similar weighted analysis, andK

appi was determined

s the ratio of the replot intercept to the replot slope.Mechanism of action was evaluated by qualitatively compar-

ng Lineweaver–Burk plot trends to the theoretical ones [21].

.6.2. Determination of carbamoylation rate constants forseudo-irreversible inhibitors (physostigmine, rivastigmine)

AChE-CIM–IMER was first equilibrated with Buffer A at.0 mL/min with UV detection at 412 nm. Reference immo-ilized enzyme activity was assessed by injecting saturatingubstrate concentration (ACTh, 200 mM) in duplicate andetermining the average product peak area (A0). Then theseudo-irreversible inhibitor, previously dissolved in methanol,as added at the selected concentration to the mobile phase.he new mobile phase was run through the AChE-CIM-IMER

t = 0, carbamoylation phase). Every 6 min aliquots of 10 �Lf saturating substrate were injected and the time dependentecreasing product peak area (Ai) was monitored down to aonstant plateau value, indicative of a concentration depen-ent enzyme inhibition. This experiment was carried out forour different concentrations of physostigmine comprised in the

ange 0.02–2.0 �M and for two concentrations of rivastigmine (2nd 50 �M). Residual percent enzyme activity [(Ai/A0) × 100]as plotted versus time. The data were fitted to Eq. (1), andseudo first order rate constant kobs values were calculated

fnS

r. A 1144 (2007) 102–110

ccordingly [28].

= R0 e−kobs t + R∝ (1)

here R, R0 and R∝ are ratios of the inhibited enzyme activityAi) to the control activity (A0) at times t, 0 and ∝, respectively.

In this way, the values of the observed pseudo-first ordernhibition rate constant (kobs) for four concentrations of testednhibitor were obtained. Double reciprocal plots of kobs versusnhibitor concentration ([I]) were then used to compute k2 fromhe intercept, and from the ratio of the slope to the intercept,espectively, according to the equation:

1

kobs= Kc

k2

1

[I]+ 1

k2(2)

.6.3. Determination of decarbamoylation rate constantsor pseudo-irreversible inhibitors (physostigmine,ivastigmine)

Once a stable inhibition plateau was reached, buffer A wasgain flushed through AChE-CIM-IMER and the recovery ofChE activity over time was followed by injecting aliquots of

aturating substrate at fixed time intervals.Decarbamoylation rate was determined according to Perola

t al. [31] by using an inhibitor concentration able to give anlmost complete inhibition of the enzyme activity (0.6 �M and0 �M for physostigmine and rivastigmine, respectively) andlotting ln(At/Ap) versus time, where Ap is the percent inhibitiont plateau product peak area, that is t = 0 in the decarbamoylationhase (Ap = {100 − [100(Ai/A0)]}t=zero) and At is the percentnhibition determined over time, during the activity recoveryhase (At = {100 − [100(Ai/A0)]}t=t).

By linear regression analysis of the obtained linear plots,ecarbamoylation rate constants (k3) were derived as the slopealues.

. Results and discussion

.1. Reversible inhibition studies

Other that its role in the synaptic breakdown of the neu-otransmitter acetylcholine, AChE may play a key role in theevelopment of the senile plaques of Alzheimer’s disease, asevealed by the finding that AChE accelerates �-amyloid pep-ide (�A) deposition [14–16]. This non classical role of AChEn the central nervous system was shown to be affected in vitroy peripheral anionic binding site ligands such as propidiumr some new dual binding site inhibitors [14–17], while pureompetitive inhibitors resulted inefficacious in blocking this pro-oting effect. This finding suggests clearly that is extremely

mportant to verify mechanism of action of selected ligands ableo interact simultaneously with AChE catalytic and peripheralites since these compounds may possess advantages over thenown AChE inhibitors, as more promising candidate drugs.

Therefore, the first part of this work was focused on theunction-based use of the AChE-CIM-IMER for the determi-ation of the mechanism of action and inhibition constants.everal approaches are reported for full reversible inhibitor

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M. Bartolini et al. / J. Chro

haracterization. Frontal affinity chromatography with masspectrometric detection (FAC/MS) is a typical application forigand binding studies [8,9]. In this method a compound mix-ure is continuously infused through a column containing aound protein target. The dissociation constant for ligand can beetermined from the breakthrough curve without knowing theoncentration of the ligand. By using tandem MS methods, it isossible to identify the compounds that are retained on column8].

However, by using FAC, the retention data do not providenformation on where the compound binds to the enzyme, asinding could occur at non-functional site. This issue could beddressed by employing a suitable competitor which however isot always available.

These problems can be solved by using immobilized enzymeeactors for function-based screening. Concerning the exam-nation of IMER inhibition, there are several reports in theiterature [1–12], but only Wainer and Brennan investigated the

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Fig. 1. Structure of selected reversible and pseudo-irreversible inhibitors of acet

r. A 1144 (2007) 102–110 105

nhibitor mechanism of action. For instance, Wainer initiallyeported displacement chromatography on a �-chymotrypsin-MER. Studies were carried out by adding the inhibitor to theobile phase, equilibrating the IMER with it and injecting the

ubstrate. The results of the displacement chromatographic stud-es were analysed using Lineweaver and Burk plots [7].

More recently, Brennan reported the simultaneous infusionf substrate and inhibitor through a capillary monolithic IMERnd the determination of Ki for competitive inhibitor by MS [4].owever, both the methods suffer from some drawbacks due to

he simultaneous presence in the mobile phase of both substratend inhibitor which might show a too high absorbance at theetector in the case of UV–vis detection (the former case) andon suppression in the case of MS detector (Brennan’s method).

oreover, both methods do not allow automation.We choose the injection of substrate and inhibitor together

ecause our system does operate in equilibrium conditions sinceubstrate, product of the enzymatic reaction and inhibitor have

ylcholinesterase. For the traded compounds the trade name is also shown.

106 M. Bartolini et al. / J. Chromatogr. A 1144 (2007) 102–110

Table 1Kinetic parameters for AChE-CIM-IMER. Km and vmax were determined at constant temperature t = (25.0 ± 0.8) ◦C using buffer A as mobile phase

Flow rate (mL/min) Contact time (min) Kappm (normalized) (mM) vmax (�mol/min)

0.4 0.85 0.338 0.0460.6 0.57 0.398 0.044011

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Concerning vmax, its trend was reversed: increasing the flowrate the amount of substrate hydrolyzed per minute slightlydiminished (Table 1), in accordance with Sutherlin work [23].Nevertheless, working at 1.0 mL/min the rate of hydrolysis

.8 0.42

.0 0.34

.2 0.28

he same retention time, due to the small dimensions of the CIMatrix, mobile phase composition and the rapid mass transfer

n monolithic materials. Therefore, as Wainer stated [7], sincehe contact between all the reactants is maintained through theength of the AChE-CIM-IMER, injecting a mixture of a sub-trate and a reversible inhibitor onto a column with immobilizednzyme will give a real picture of their interaction.

In order to validate our method, reversible referencenhibitors with known potency and mechanism of actionere chosen (Fig. 1). In particular two compounds approved

or AD treatment (donepezil and tacrine) and two knowneversible AChE inhibitors (edrophonium and ambenonium)ere selected.For the determination of the inhibitory constant (Kapp

i ) ofeversible inhibitors by AChE-CIM-IMER, apparent kineticonstants (Kapp

m and vappmax) were first studied as a function of

ow rate. Ten microliter aliquots of ATCh aqueous solutionst increasing concentration (3.1–200 mM) were injected in theMER under the optimized chromatographic conditions reportedn Section 2.3. Substrate is hydrolyzed by immobilized AChE,iving raise to thiocholine which reacts with Ellman’s reagentissolved in the mobile phase, stoichiometrically forming 5-hio-2-nitro-benzoic acid (yellow anion), detected as elutingeak by UV detection at 412–450 nm [5,6]. Product peaklution is complete in 2 min. Michaelis and Menten curvesere obtained by plotting velocity (micromoles of product

ormed per minute) versus normalized substrate concentration0.09–12 mM), that is the concentration of substrate in theChE-IMER (0.34 mL). As reported in Table 1 and shown inig. 2, K

appm values were found slightly increasing with flow

ate. However, by extrapolating at zero flow rate, Kappm 0 value was

ound to be 254 ± 30 �M, slightly higher but in the same rangef the value obtained with the enzyme in solution (170 ± 15 �M)8–11]. It was already previously noticed that enzyme immobi-

Fig. 2. Trend of kappm as a function of flow rate.

FCatd

0.435 0.0430.443 0.0360.536 0.026

ization could increase Kappm [2,10–12], as the lower substrate

ffinity could simply be due to reduced accessibility of the sub-trate to the bound enzyme. This phenomenon can be morevident in a flow-through system when increasing the flow rate.owever, when working at 1.0 mL/min, Kapp

m value was just lesshan two times higher than the extrapolated K

appm 0 value.

ig. 3. Inhibition of AChE hydrolysis of acetylthiocholine (ATCh) by AChE-IM-IMER flow through system. Lineweaver–Burk reciprocal plots of velocitynd substrate concentrations are presented for (A) edrophonium (EDRO), (B)acrine (TAC). Lines were derived from a weighted least-squares analysis of theata points.

M. Bartolini et al. / J. Chromatogr. A 1144 (2007) 102–110 107

Table 2Ki values obtained with AChE-CIM-IMER and free human recombinant AChE in solution by Ellman spectrophotometric method [22]

Compound Ki(normalized) (AChE-CIM-IMER) (M) ± SEM Ki (Free) (M) ± SEM Mechanism of action

Edrophonium (5.38 ± 0.52)10−6 (1.63 ± 0.23)10−6 CompetitiveTacrine (2.40 ± 0.36)10−6 (1.51 ± 0.16)10−7 MixedDonepezil (2.45 ± 0.75)10−7 (2.05 ± 0.33)10−8 MixedAL

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pagm[mtherefore necessary for SAR studies and to predict their in vivobehavior.

The inhibition of AChE (EH) by carbamates (AB) involvesa reversible complex (EH–AB) formation, followed by car-

mbenonium (1.30 ± 0.98)10−8

AM 4 (7.65 ± 0.16)10−8

a Ki value for ambenonium in solution was determined on human purified ery

as 75% of the velocity extrapolated at zero flow rate, andhe complete elution of the yellow anion was achieved in justmin. Therefore, considering these results, we can assert that

mmobilization did not markedly affect AChE catalytic activity.he overall chromatographic conditions were found appropri-te to carry out the determination of inhibition constants for theelected inhibitors, because a satisfactory response in term ofeak area (i.e. hydrolysis rate) and reasonable time for anionlution were achieved.

Hence, evaluation of mechanism of action was performed forll the selected reversible inhibitors as described in Section 2.6,y co-injecting substrate and inhibitors and evaluating the result-ng peak area. In Fig. 3, Lineweaver–Burk plots obtained foracrine and edrophonium are reported as examples of different

echanisms of action. Reciprocal plots for edrophonium inhibi-ion (Fig. 3A) showed unvaried v

appmax and increasing x-intercepts

higher Kappm ) at increasing inhibitor concentrations as a pure

ompetitive AChE inhibitor. Reciprocal plots for tacrine inhibi-ion showed instead both increasing slopes (decreased v

appmax at

ncreasing inhibitor concentrations) and increasing interceptshigher K

appm ) with higher inhibitor concentration (Fig. 3B).

his pattern indicates a mixed-type inhibition. These results aren agreement with patterns obtained with the same enzyme inolution [24–26].

Replots of the slope versus the concentration of inhibitor gavestimate of the competitive inhibition constant (Kapp

i ) reportedn Table 2 along with K

appi values obtained for ambenonium and

onepezil. Ki values obtained by the classical spectrophotomet-ic method with the human recombinant AChE in solution arelso presented for comparison.

From Lineweaver–Burk plots it resulted that ambenoniumnd donepezil caused a mixed-type inhibition, in agreement withata reported in literature for the enzyme in solution [26,27].

In addition, an analysis of Kappi values revealed a good linear

orrelation (y = 0.677x + 1.292; r2 = 0.9621) between the valuesbtained with the immobilized enzyme and the enzyme in solu-ion. The K

appi values obtained with AChE-CIM-IMER were

herefore found to well correlate with the values reported in theiterature.

The proposed new on-line method was further validated byetermining the K

appi value of the non-classical inhibitor LAM

. LAM 4 (6-[ethyl-(2-methoxybenzyl)amino]-1-(1′-{6-[ethyl-2-methoxybenzyl)amino]hexanoyl}[4,4′]bipiperidinyl-1-yl)-

exan-1-one) is a recently synthesized potent diamine-diamideChE inhibitor (see Fig. 1 for structure) endowed with aanomolar inhibitory potency and a mixed-type inhibition15]. Mechanism of action determined by AChE-CIM-IMER

Fmeta

(1.20 ± 0.30)10−10a Mixed(5.67 ± 0.15)10−9 Mixed

yte AChE. From ref. [26].

esulted in agreement with that obtained with the free enzymend its K

appi value (see Table 2) fitted well in the previously

btained correlation curve. Therefore, AChE-CIM-IMER cane applied to the determination of mechanism of action of neweversible inhibitors with advantages in terms of automation,ccuracy and precision, time and costs saving.

.2. Pseudo-irreversible inhibition studies

A further class of AChE inhibitors is represented by com-ounds endowed with a carbammic moiety [28–33]. After thepproval in 2000 of Rivastigmine (Exelon, Fig. 1), a neweneration-AChE inhibitor with a carbamate function, carba-ates regained interest for the treatment of Alzheimer’s disease

33]. Accurate and automated methodologies to define theirechanism of action and to assess their kinetic constants are

ig. 4. AChE-CIM-IMER time dependent carbamoylation (0.6 �M physostig-ine in the mobile phase) and decarbamoylation (buffer A as mobile phase)

xperiments and mechanism of action of pseudo-irreversible inhibitors. Inhibi-ion and regeneration of AChE-CIM-IMER is expressed as percent product peakrea.

108 M. Bartolini et al. / J. Chromatogr. A 1144 (2007) 102–110

Fig. 5. (A) Time course of AChE-CIM-IMER carbamoylation by increasingpom

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CsbrftimpotbbsAi

Table 3IC50 values for the selected pseudo-irreversible inhibitors obtained withAChE-CIM-IMER and human recombinant AChE in solution by Ellman’s spec-trophotometric method [22]

Compound IC50 (Free) (M) ± SEM IC50 (AChE-CIM-IMER)(M) ± SEM

Physostigmine (1.34 ± 0.54)10−8 (2.01 ± 0.41)10−8

Rivastigmine (1.53 ± 0.64)10−6a (2.86 ± 0.26)10−6

Pc

ofwc[k0v(pptcrptultemtlrstip

bcemit

plap

hysostigmine concentration in the mobile phase. (B) Trend of kobs dependencen physostigmine concentration. Double reciprocal plots of kobs vs. physostig-ine concentration ([I]) were then used to compute k2 (Eq. (2)).

amoylation of the enzyme, and production of a covalent adductEA) [28–30]. The carbamoylated enzyme is then hydrolyzed toegenerate the free enzyme. The whole mechanism is presentedn Fig. 4. After the reversible complex formation (EH–AB; equi-ibrium constant: KC ≡ k1/k−1), the carbamoylation phase ofhe reaction is considerably rapider than the decarbamoylationhase (i.e. k2 > k3), and the two phases can be characterizedeparately. When studies are carried out in solution, k2 and k3an be evaluated by using several techniques, among whichtop-time assays and dialysis are respectively the most used30,31]. Both techniques are time and material consuming andhe two constants need to be determined in two different sets ofxperiments.

On the basis of these considerations, we thought AChE-IM-IMER could represent a powerful tool to evaluate in one

ingle experiment both the carbamoylation (k2) and the decar-amoylation (k3) constants. To this purpose, physostigmine andivastigmine (Fig. 1) were selected as reference compounds. Byollowing the procedure reported in Section 2.6, after assessinghe initial enzyme activity, the reference inhibitor was dissolvedn the mobile phase and the progressive enzyme inhibition was

onitored by following the time dependent decrease of theroduct peak area. In Fig. 4, the time dependent inactivationf immobilized AChE by physostigmine at a 0.6 �M concen-ration is reported. Once the inhibition plateau was reached,y switching the mobile phase back to buffer A, it was possi-

le to regenerate the inactivated enzyme (see decabamoylationection). In Fig. 5, the graphical representation of immobilizedChE inactivation by increasing physostigmine concentrations

s reported.

epb

ercent inhibition for each inhibitor concentration was estimated at a timeorresponding to the plateau phase in the time dependent profile of inhibition.a Data from ref. [34].

By using Eq. (1) (see Section 2.6), the values of thebserved pseudo-first order inhibition rate constants (kobs) forour concentrations of physostigmine in the range 0.02–2.0 �Mere obtained. Double reciprocal plots of kobs versus inhibitor

oncentration ([I]), according to Feaster and Quinn method28], were obtained. The value of carbamoylation constant2 for physostigmine on AChE-CIM-IMER was found to be.40 ± 0.06 (min−1) ± SEM, in perfect correlation with thealue obtained by the classic stopped time method [0.41 ± 0.05min−1) ± SEM] [19]. By comparing the inhibition profiles ofhysostigmine and rivastigmine at a concentration giving a com-lete AChE inhibition (0.6 �M and 50 �M, respectively, onhe basis of their different inhibitory potency), physostigminearbamoylation half-time was found to be 3.9 min, whereasivastigmine’s was 11.4 min. This is in agreement with ourrevious studies on free enzyme where we found that theime course of the inhibition is characterized by an increasentil a steady state, which is reached after a time that corre-ated with the bulkiness of the substituent on the nitrogen ofhe carbamate function, the rate constants of the carbamoyl-nzyme formation (k2) being higher for mono-substitutedethyl-derivatives [18–20]. Inhibitory potencies obtained for

he two selected pseudo-irreversible inhibitors were calcu-ated and compared with data obtained by using the sameecombinant enzyme in solution. The IC50 values are pre-ented in Table 3 and resulted in agreement, confirminghat the IMER could discriminate between pseudo-irreversiblenhibitors differing of two orders of magnitude in their inhibitoryotencies.

As previously stated, once the inhibition plateau was reached,y changing mobile phase, it was possible to hydrolyzearbamoyl-AChE adducts, completely regenerate the activenzyme and evaluate decarbamoylation rates. Typical chro-atograms obtained in the decarbamoylation phase are overlaid

n Fig. 6. It is easy to notice the increase of product area overime while the carbamoylated complex is hydrolyzed.

In Fig. 4, the profile of activity recovery after inhibition byhysostigmine 0.6 �M is reported together with the carbamoy-ation profile as example of the global pattern obtained duringcomplete experiment (carbamoylation plus decarbamoylationhases).

A 2 h flushing was required to achieve a complete recov-ry of AChE-CIM-IMER activity after complete inhibition byhysostigmine, whereas a 34 h flushing was required after inhi-ition by rivastigmine.

M. Bartolini et al. / J. Chromatog

Fig. 6. Overlaid chromatograms for AChE-CIM-IMER activity recovery overtime after inhibition by physostigmine 2 �M. The chromatographic peak isthe yellow anion, stechiometrically related to thiocholine production. Chro-matogram 0 → t = 0 (maximum inhibition); chromatogram 5 → t = 100 min(cfl

eaoo

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t(

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amrara

4

(ti

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complete recovery). [ATCh]inj = 200 mM; mobile phase: 0.1 M Tris–HCl pH 8.0ontaining 100 mM KClO3, 10 mM MgSO4, 1.26 × 10−4 M Ellman’s reagent;ow rate: 1.0 mL/min; λ = 412 nm.

Calculation for k3 was performed by applying Perola’s math-

matical equation [31] that assumes that, by starting from anlmost complete inhibition, k3 can be calculated from the slopef the linear plot obtained by monitoring the inhibition degreever time.

ig. 7. Linear plots obtained by applying Perola equations [31] to AChE decar-amoylation. AChE-CIM-IMER was first inhibited by 0.6 �M physostigminend 50 �M rivastigmine dissolved in the mobile phase. Slopes of the linear plotsere used to compute decarbamoylation kinetic constant (k3).

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r. A 1144 (2007) 102–110 109

In Fig. 7, linear plots obtained by applying Perola equationso physostigmine and rivastigmine AChE-CIM-IMER inhibition0.6 �M physostigmine and 50 �M rivastigmine) are reported.

According to previous studies, the velocity of this step waselated to the length of the carbamate N-alkyl chain. The k3h−1) ± SEM values were found in agreement with the bulki-ess of the substituent on the nitrogen of the carbamate functionrivastigmine 0.050 ± 0.001/h; physostigmine 0.74 ± 0.02/h).

More interestingly, rivastigmine decarbamoylation half-time11 h) was found in agreement with the pharmacodynamic half-ife in vivo (10 h) [32].

It has to be noted that this new method allowed a much moreccurate determination of the mechanism of action of rivastig-ine, in fact by classical dialysis studies, we found that AChE

egeneration occurred much more slowly (half-time 512 h), ingreement with the work of Bar-On [33]. This faster activityecovery might be related to the continuous fresh buffer supplynd parallel easier removal of the hydrolyzed carbamate moiety.

. Conclusions

Besides representing a valid tool to screen new inhibitorsIC50 or single dose inhibition), AChE-CIM-IMER can be usedo characterize active compounds, both reversible and pseudo-rreversible inhibitors, by on-line automated assays.

The proposed method has the advantage that the determina-ion of the inhibitory potency is based on changes in activityather than on simple binding, minimising the interference fromonselective binding and can be automated.

On-line evaluation of mechanism of action (competitive, nonompetitive, mixed type) and determination of the inhibitoryonstant (Ki) were here presented for selected reversiblenhibitors (donepezil, tacrine, edrophonium, ambenonium). Thebtained results were found in agreement with data determinedn the free AChE.

Noteworthy results were obtained in the application of theChE-based IMER in the characterization of the carbamoyla-

ion and decarbamoylation steps in pseudo-irreversible bindingf carbamate derivatives. AChE-CIM-IMER resulted a valid toolo determine simultaneously the kinetic constants in a reliablend fast mode. Furthermore, after inactivation by carbamates,ctivity could be fully recovered and the AChE-CIM-IMERould be reused for further studies.

Up to our knowledge, these are the first reported studiesn the on-line determination of kinetic constants for pseudo-rreversible inhibitors.

Other general advantages of this methodology are thencreased reproducibility and accuracy of AChE-CIM-IMER,nd its stability: nanomoles of the immobilized recombinantnzyme in the IMER format have been used to perform morehan two thousand and two hundred runs. Furthermore, the sameMER has been daily used for over seven months partially main-aining its initial activity.

However, it is worth to mention that this method is well suitedn cases where mixtures are not highly complex and where thedentity of the compound is known. Complex mixtures wouldequire either an additional pre-fractionation by reversed-phase

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C–MS or a previous FAC/MS to select highly bound compoundrior to IMER-LC.

cknowledgement

MIUR-COFIN and FIRB are gratefully acknowledged forheir financial support.

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