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RESEARCH OPINIONS IN ANIMAL & VETERINARY SCIENCES Comparison of an Alzheimer disease drug ability to bind acetylcholinesterase
using both electrochemical and spectrophotometric assays
Miroslav Pohanka1,2*, Vojtech Adam3,4 and Rene Kizek3,4
1Faculty of Military Health Sciences, University of Defence, Trebesska 1575, CZ-500 01 Hradec Kralove, Czech Republic, European Union; 2Karel English College in Brno, Sujanovo namesti 356/1, 60200 Brno, Czech Republic 3Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic, European Union; 4Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic, European Union Abstract
Acetylcholinesterase (AChE) is an enzyme playing important role in termination of neurotransmission within cholinergic system. The enzyme is targeted by many compounds including nerve agents used for chemical warfare,
natural toxins and drugs for Alzheimer disease. Thousands of new inhibitors are prepared for pharmaceutical
purposes over the year and necessity to characterize them is given for this reason. In this work, standard
spectrophotometric test is compared to electrochemical test in order to assay AChE activity and measure its
inhibition. The both test were performed for characterization of an Alzheimer disease drug galantamine. The
spectrophotometric test was based on standard Ellman´s reaction. Electrochemical test used enzymatic hydrolysis to
acetylthiocholine with consequent electrochemical oxidation. In the experiments, we proved that electrochemical
test was able to reach better limit of detection for galantamine than the standard spectrophotometric test. Calculated
median inhibitory concentration was same for electrochemical and spectrophotometric tests. Interference in the
assay was tested as well. We proved that electrochemical test is reliable and more sensitive than the standard
spectrophotometric test. The electrochemical test would be preferred when median inhibitory concentration is the
assayed output.

acetylcholinesterase; Alzheimer disease; myasthenia gravis; huperzine; galantamine; donepezil; biosensor

To cite this article: Pohanka M, V Adam and R Kizek, 2014. Comparison of an Alzheimer disease drug ability to
bind acetylcholinesterase using both electrochemical and spectrophotometric assays. Res. Opin. Anim. Vet. Sci.,
4(4): 203-207.


galantamine, huperzine), and drugs for myasthenia gravis (neostigmine, pyridostigmine) can be exampled Enzyme acetylcholinesterase (AChE; E.C. as inhibitors of AChE (Holzgrabe et al., 2007; Pohanka, is involved in the regulation of cholinergic nerves 2011, 2012b; da Silva et al., 2011; de los Rios, 2012). where it terminates neurotransmission by hydrolysis of Necessity to analyze newly prepared drugs is given neurotransmitter acetylcholine (Wessler and in pharmacological research. In the drug selection, Kirkpatrick, 2008; Pohanka, 2012a). The enzyme has a AChE activity is analyzed in the presence of the tested wide pharmacological and toxicological significance. compound and the most potent inhibitors are given to Many compounds including nerve agents used as the next stages of preclinical tests. Currently, use of chemical warfare (sarin, soman, tabun, VX), secondary acetylthiocholine as a substrate and 5,5´-dithiobis-(2- metabolites such as caffeine or aflatoxins, some drugs nitrobenzoic acid) (further abbreviated as DTNB) as a for Alzheimer disease (donepezil, rivastigmine, chromogen are used for spectrophotometric assay of
*Corresponding author: Miroslav Pohanka, Faculty of Military Health Sciences, University of Defence, Trebesska
1575, CZ-500 01 Hradec Kralove, Czech Republic, European Union. E-Mail:; Tel: +420-973-251-519; Fax: +420-495-518-094.
Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207. AChE activity (Pohanka 2013c). The reaction is called as Ellman´s reaction in honour to team of doctor Ellman who firstly suggested the application (Ellman et al., 1961; Gorun et al., 1978; George and Abernethy, 1983; Runion et al., 1984). Though the assay is simple, sensitive and readily to use in the both laboratory and field conditions, there are some drawbacks. Beside instability of DTNB in water conditions, interference of nucleophilic substances including thiol, oxime and the other chemical groups can be expected (Pohanka, 2012b, 2013b; Pohanka et al., Fig. 1: Principle of assay based on Ellman´s reaction. The
first step is catalyzed by acetylcholinesterase
electrochemical evaluation of AChE activity assay is proposed as an alternative to the standard spectrophot- ometric assay. It is believed that the new test can avoid the drawbacks typical for Ellman´s reaction. For the experiment purposes, standard drug galantamine, compound with proper chemical name (4aS,6R,8aS)- 5,6,9,10,11,12- hexahydro- 3-methoxy- 11-methyl- 4aH- [1] benzofuro[3a,3,2-ef] [2] benzazepin- 6-ol, for Alzheimer disease was chosen as compound inhibiting AChE and being measured by the standard test. Materials and Methods
Fig. 2: Principle of electrochemical assay of acetylcholineste-
Experimental Section
rase (AChE) activity.
Spectrophotometric assay
The spectrophotometric assay was based on the from BVT Technologies (Brno, Czech Republic). The aforementioned application of acetylthiocholine chloride sensors were sized 25.4×7.3×0.6 mm and they consisted and DTNB as a chromogenic substance. Principle of the from platinum working (dot shaped with diameter 1 mm), assay is depicted in Figure 3. For the assay purposes, silver covered with silver chloride reference, and platinum lyophilized human recombinant AChE expressed in auxiliary electrodes. Reagents used for the electro- HEK293 cells with activity above 1,500 U per milligram chemical assay were the same like the used in of protein was purchased from Sigma Aldrich (Saint Louis, Missouri, USA). AChE was solved in phosphate Prior to the assay, sensor was linked with PalmSens buffered saline (PBS) and activity was adjusted up 0.5 device (Palm Sens BV, Houten, Netherlands), inserted U/µl for 1 mmol/l acetylthiocholine chloride. Reaction into a disposable plastic cuvette with maximal volume 2 was performed using standard disposable PS cuvettes. ml and operated by a computer using PsLite 1.8 The following reagents were subsequently given into the (PalmSens BV) software. Into the cuvette, the following cuvette: 100 µl of the tested solution in PBS, 200 µl of 0.4 reagents were added: 100 µl of the tested solution in PBS, mg/ml DTNB in PBS, 590 µl of PBS and the solution was 790 µl of PBS and the solution was shaken and let to shaken and let to incubate for 10 minutes. After that, 100 incubate for 10 minutes. After that, 100 µl of 10 mmol/l µl of 10 mmol/l acetylthiocholine chloride was given into acetylthiocholine chloride was injected. The mixture was the cuvette, shaken and absorbance was measured at 412 shaken and current was measured chronoampero- nm immediately after splitting and then after two minutes. metrically after two minutes. Applied potential 640 mV Mixture with acetylthiocholine replaced by pure PBS was was given in order to receive oxidation of the released used as a blank. Control measurement with sample thiocholine. The potential was chosen using experiences replaced by PBS was done as well. Galantamine was used from the former experiment (Pohanka et al., 2013). In as a standard compound inhibiting AChE. The all control measurement, sample was replaced by PBS. aforementioned reagents and standards used for the assay purpose were achieved from Sigma-Aldrich. Data processing and statistics
All samples were assayed in pentaplicate and the Electrochemical assay
both mean and standard deviation were calculated for Electrochemical assay was done in similar conditions each group of measurements. From the experimental data, like the spectrophotometric one. Scheme of the assay is percent of inhibition I was calculated using following depicted as figure 4. Screen printed sensors were purchased Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207. Where SC is a signal received by either spectro- photometric (absorbance) or electrochemical (current) assay for the control measurement. SI is a signal found for assay of the tested compound in given concentration. Software Origin 8 (OriginLab Corporation, Northampton, MA, USA) was used for data processing
and statistics evaluation. Limit of detection was calculated
from confidence interval (95 %) in calibration plot.
Results and Discussion
Galantamine was chosen as a representative Fig. 3: Spectrophotometric assay of galantamine. Error bars
compound used for Alzheimer disease therapy. It is well indicate standard deviation for n=5. The data were
tolerated by patients and it has good efficacy in the fitted (full line) and confidence interval for
therapy (Bond et al., 2012). It was discovered by probability level 0.05
Mashkovsky and Kruglikova-Lvova as a secondary metabolite in Caucasian snowdrops Galanthus sp. in early 1950s (Heinrich and Teoh, 2004). Currently, galantamine is sold over the world under trade names RazadyneTM, RazadyneTMER, ReminylTMER, and Reminyl®, and it remains as one of the most relevant compounds used for the therapy. Calibration for galantamine was done by the both spectrophotometry and electrochemistry in concentration range 10-9 – 10-2 mol/l (final concentration in the cuvette). PBS was used for control measurement. Calibration is depicted as figure 1 for the spectrophotometry and figure 2 for the electrochemistry. When searched limit of detection, electrochemical assay was able to detect as low as 5×10-9 mol/l of galantamine. Comparing to it, spectrophotometric assay have limit of detection 2×10-9 mol/l for galantamine. When comparing the two limits of Fig. 4: Electrochemical assay of galantamine. Description is
detection, the electrochemical method was able to detect the same as in figure 1.
3.7 times lower amount of galantamine. However, the lower limit of detection does not privilege any method if not immobilized for the assay purposes. Decision not to accuracy to estimate median inhibitory concentration is use immobilization was initiated by decision to establish method close to standard spectrophotometric test. For the above mentioned reason, the both methods Immobilization of AChE would lead to preparation of a were fitted by Boltzmann function and median inhibitory biosensor and further improvement to limits of detection. concentration was calculated. The median inhibitory However, comparison of such experimental data and data concentration for galantamine was 2.19×10-6 mol/l when from spectrophotometric test would be complicated. used spectrophotometry and 2.09×10-6 mol/l when used Limits of detection known from literature about AChE electrochemistry. The data are insignificant one to each based biosensors are slightly lower than the here other when used analysis of variance and probability level described device. In an example, AChE based 0.05. The two methods were correlated one to each other. electrochemical biosensor using Co phtalocyanine and Coefficient of determination R2 was 0.997. Slope for the carbon electrodes was used for assay of organophosphate correlation was 1.04. Considering the calculated median pesticides with limit of detection 2 ppb for malaoxon inhibitory concentration, we can claim that the (Ivanov et al., 2011). In another paper, biosensor electrochemical assay is reliable and very close in the containing AChE immobilized on carbon nanotubes was used to detect chosen pesticides and nerve agents (Liu and The reached limits of detection are quite good for Lin, 2006). Limit of detect for organophosphate paraoxon characterization of drugs. On the other hand, AChE was Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207. Table 1: Interference in the assays in percent of control
The Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged for project LH11023. Financial support from CEITEC CZ.1.05/ 1.1.00/02.0068 is highly acknowledged as well. Interference was measured using acetone oxime, References
cysteine, and ethanol. The interference is resumed in table 1. The compounds were given into the cuvette up to final Bond, M., Rogers, G., Peters, J., Anderson, R., Hoyle, M., concentration 0.1 mmol/l for acetone oxime and cysteine. Miners, A., Moxham, T., Davis, S., Thokala, P., Ethanol was added up to concentration 10% (v/v). For Wailoo, A., Jeffreys, M. and Hyde, C. 2012. The purpose of this part of experiment, volume of applied PBS effectiveness and cost-effectiveness of donepezil, described above was decreased for 100 µl and the tested galantamine, rivastigmine and memantine for the compounds were applied just in volume 100 µl. treatment of Alzheimer's disease (review of As seen in the table, cysteine was oxidized by applied Technology Appraisal No. 111): a systematic review voltage and caused interference more than 100 % when and economic model. Health Technology Assessment, applied final concentration 0.1 mmol/l in the electro- chemical assay. The same interference was found in da Silva, V.B., de Andrade, P., Kawano, D.F., Morais, spectrophotometric assay. The fact that free thiol can P.A.B., de Almeida, J.R., Carvalho, I., Taft, C.A. and interfere is not surprising, because it is well known for da Silva, C. 2011. In silico design and search for acetylcholinesterase inhibitors in Alzheimer's disease Ellman´s reaction (Prokofieva et al., 2012). It is with a suitable pharmacokinetic profile and low frequently used for the both electrochemical and toxicity. Future Medicinal Chemistry, 3: 947-960. spectrophotometric assay of thiol containing antioxidants de los Rios, C. 2012. Cholinesterase inhibitors: a patent (Pohanka, 2012c; Tipple and Rogers, 2012; Squellerio et review (2007-2011). Expert Opinion on Therapeutic al., 2012). Interference of an oxime group containing compound is not surprising as well. This kind of Ellman, G.L., Courtney, K.D., Andres, V. and compounds causes oximolysis. The oximolysis leads to Featherstone, R.M. 1961. A new and rapid spontaneous reaction with DTNB (Worek et al., 2012; colorimetric determination of acetylcholinesterase Pohanka, 2012a, 2013a). Electrochemical assay is not activity. Biochemical Pharmacology, 7: 88-95. sensitive to the interference because oxime is not redox Fekonja, O., Zorec-Karlovsek, M., El Kharbili, M., active during the measurement. Ethanol influences the Fournier, D. and Stojan, J. 2007. Inhibition and assay in course of AChE inhibition. The inhibitory effect protection of cholinesterases by methanol and is not specific and it can be anticipated for other organic ethanol. Journal of Enzyme Inhibition and Medicinal solvents as well (Turdean and Turdean, 2008; Pietsch et al., 2009). Ethanol is a typical example of such solvents George, P.M. and Abernethy, M.H. 1983. Improved (Fekonja et al., 2007). When considered the mentioned Ellman procedure for erythrocyte cholinesterase. results. We can claim that the main advantage of the electrochemical assay lays in ability to avoid interference Gorun, V., Proinov, I., Baltescu, V., Balaban, G. and by some nucleophilic substances. On the other hand, Barzu, O. 1978. Modified Ellman procedure for assay compounds inhibiting AChE and free thiol containing of cholinesterases in crude enzymatic preparations. compounds will cause the same interference in the Analytical Biochemistry, 86: 324-326. spectrophotometrical like in the electrochemical assay. Heinrich, M. and Teoh, H.L. 2004. Galanthamine from snowdrop-the development of a modern drug against Conclusions
Alzheimer's disease from local Caucasian knowledge. Electrochemical assay was introduced as a tool for a Journal of Ethnopharmacology, 92: 147-162. fast and reliable testing of drugs inhibiting enzyme AChE. Holzgrabe, U., Kapkova, P., Alptuzun, V., Scheiber, J. The new assay was found to be more sensitive than the and Kugelmann, E. 2007. Targeting acetylcholine- traditional spectrophotometric test. Moreover, the sterase to treat neurodegeneration. Expert Opinion on electrochemical assay is less sensitive to interference Therapeutic Targets, 11: 161-179. caused by nucleophilic substances. On the other hand, Ivanov, A.N., Younusov, R.R., Evtugyn, G.A., Arduini, electrochemical assay is not advantageous if there is a demand to calculate enzyme activity in katals. Acetylcholinesterase biosensor based on single- Electrochemical assay should be privileged above the walled carbon nanotubes-Co phtalocyanine for spectrophotometric test when median inhibitory organophosphorus pesticides detection. Talanta, 85: Res. Opin. Anim. Vet. Sci., 2014, 4(4): 203-207. Liu, G. and Lin, Y. 2006. Biosensor based on self- J., Masarik, M., Eckschlager, T. and Kizek, R. 2012. assembling acetylcholinesterase on carbon nanotubes Voltammetry Assay for Assessment of Oxidative for flow injection/amperometric detection of organo- Stress linked Pathologies in Brain Tumor suffered phosphate pesticides and nerve agents. Analytical Childhood Patients. International Journal of Electrochemical Science, 7: 11978-11992. Pietsch, M., Christian, L., Inhester, T., Petzold, S. and Prokofieva, D.S., Jenkins, R.O. and Goncharov, N.V. Gutschow, M. 2009. Kinetics of inhibition of 2012. Microplate biochemical determination of acetylcholinesterase in the presence of acetonitrile. Russian VX: Influence of admixtures and avoidance of false negative results. Analytical Biochemistry, Pohanka, M. 2011. Cholinesterases, a target of pharmacology and toxicology. Biomedical Papers, Runion, T.H., Smith, C.L. and Stoll, R.E. 1984. Modification of Ellman technique for measuring Pohanka, M. 2012c. Acetylcholinesterase based dipsticks erythrocyte cholinesterase using a centrifichem-500 with indoxylacetate as a substrate for assay of analyzer. Clinical Chemistry, 30: 1060-1060 organophosphates and carbamates. Analytical Letters, Squellerio, I., Caruso, D., Porro, B., Veglia, F., Tremoli, E. and Cavalca, V. 2012. Direct glutathione Pohanka, M. 2012b. Acetylcholinesterase inhibitors: a quantification in human blood by LC-MS/MS: patent review (2008-present). Expert Opinion on comparison with HPLC with electrochemical Therapeutic Patents, 22: 871-886. detection. Journal of Pharmaceutical and Biomedical Pohanka, M. 2012a. Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology. Tipple, T.E. and Rogers, L.K. 2012. Methods for the International Journal of Molecular Sciences, 13: determination of plasma or tissue glutathione levels. Methods in Molecular Biology, 889: 315-324. Pohanka, M. 2013a. Cholinesterases in biorecognition and Turdean, G.L. and Turdean, M.S. 2008. Synergetic effect biosensor construction, a review. Analytical Letters, of organic solvents and paraoxon on the immobilized acetylcholinesterase. Pesticides in Biochemistry and Pohanka, M. 2013b. Spectrophotomeric assay of aflatoxin B1 using acetylcholinesterase immobilized on standard Wessler, I. and Kirkpatrick, C.J. 2008. Acetylcholine microplates. Analytical Letters, 46: 1306-1315. beyond neurons: the non-neuronal cholinergic system Pohanka, M., Fusek, J., Adam, V. and Kizek, R. 2013. in humans. British Journal of Pharmacology, 154: Carbofuran assay using gelatin based biosensor with acetylcholinesterase as a recognition element. Worek, F., Eyer, P. and Thiermann, H. 2012. International Journal of Electrochemical Science, 8: Determination of acetylcholinesterase activity by the Ellman assay: A versatile tool for in vitro research on Pohanka, M., Hynek, D., Kracmarova, A., Kruseova, J., medical countermeasures against organophosphate Ruttkay-Nedecky, B., Sochor, J., Adam, V., Hubalek, poisoning. Drug Testing and Analysis, 4: 282-291.


Microsoft word - 2010 step therapy requirements.doc

2010 Step Therapy Criteria Effective January 1, 2010 Step Therapy Group Description Step Therapy Criteria Requires member has filled at least a 60-day supply of a step 1 drug (i.e. long-acting beta agonist, Spiriva or inhaled corticosteroid) in the last 120 days before Advair will be covered Requires member must have a documented 60-day trial of either Antabuse or naltrexone before

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