New Method for the Determination of the Half Inhibition Concentration (IC 50 ) of Cholinesterase Inhibitors

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1 New Method for the Determination of the Half Inhibition Concentration (IC 50 ) of Cholinesterase Inhibitors Markéta Kovářová a, Karel Komers a, *, Šárka Štěpánková b, Patrik Pařík c, and Alexander Čegan b a Department of Physical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice, Czech Republic. Fax: karel.komers@upce.cz b Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic c Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic * Author for correspondence and reprint requests Z. Naturforsch. 68 c, (2013); received February 14, 2012/February 4, 2013 A new and simple analytical method is described for the determination of the IC 50 values of the inhibitors of the hydrolysis of acetylcholine (ACh) or acetylthiocholine (ATCh) by cholinesterases. The method is based on monitoring the time course of the ph value during the uninhibited and inhibited reaction. It requires only a ph meter with a suitable ph measuring cell and a small thermostated stirred batch reactor. The method has been validated for twelve different types of cholinesterase inhibitors. The determined IC 50 values are comparable to those obtained by independent, more complicated, and expensive methods (Ellman s and ph-stat). Key words: Cholinesterase Inhibition, IC 50, Determination Introduction The IC 50 is the molar concentration of a given inhibitor (I), which reduces the respective uninhibited reaction rate to one half, and is indicative of the inhibitory potency of a compound. We investigated new inhibitors of the in vitro hydrolysis of acetylcholine (ACh) by cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Ellman s method (Ellman et al., 1961) is routinely used for the determination of the cholinesterase activity and efficiency of cholinesterase inhibitors. It is based on the measurement of the absorbance at 412 nm of the yellow complex produced by the reaction between thiocholine (TCh) and DTNB (5,5 -dithiobis-2-nitrobenzoic acid, Ellman s reagent), as described in detail in Zdražilová et al. (2004). Ellman s method has two disadvantages: 1) The composition of the yellow complex and its absorption coefficient at 412 nm depend strongly on the composition of the reaction mixture, above all on the ph value. 2) Acetylthiocholine (ATCh) instead of ACh must be used as the substrate. However, the reaction rates of the ACh and ATCh hydrolysis by AChE (or BChE) are not identical, nor are the IC 50 values (Zdražilová et al., 2006a). When using ACh, the determination of the rate of the enzymatic reaction, and hence also of IC 50, requires the quantitative determination of the concentration of one of the reaction components, i.e. ACh, choline (Ch), acetic acid (HAc), or protons from HAc, in the reaction mixture. The suitable methods, e.g. HPLC or ph-stat (Zdražilová et al., 2006b), are experimentally much more sophisticated and time-consuming. This paper describes a new simple, and fast method of IC 50 determination with the possibility of using either ACh or ATCh. The method is based on the measurement of changes in the ph value as a function of time during the enzymatic hydrolysis of ACh or ATCh, it is called the ph(t) method. Material and Methods Principle of the ph(t) method During the enzymatic hydrolysis of ACh (ATCh) by cholinesterases HAc is formed. Therefore, in a non-buffered reaction mixture the ph value decreases in time according to the kinetics of the hydrolysis and dissociation constant of the weak acid HAc in aqueous milieu. This decrease is slower in the presence of an inhibitor. From the 2013 Verlag der Zeitschrift für Naturforschung, Tübingen

2 134 M. Kovářová et al. IC 50 of Cholinesterase Inhibitors difference between the respective rates, the value of IC 50 can be calculated. As a consequence of the ph change of the reaction mixture, the activity of AChE changes continuously. We suppose that the activity changes proportionally during uninhibited and inhibited hydrolyses, respectively. So, by suitable comparison of the time courses of the two hydrolyses, under otherwise identical conditions, this change of activity can be disregarded. Realization of the measurement A ph meter (inolab, Level 2; WTW, Weinheim, Germany) with a cell glass electrode/saturated silver chloride electrode ( ; Metrohm, Herisau, Switzerland) was used for the determination of ph(t) in the reaction mixture containing either ACh or ATCh, AChE, with or without inhibitor (I). The following reaction conditions were used: 25 C; initial molarity, [ACh] 0 = [ATCh] 0 = 4 mm; catalytic activity of AChE, 0.14 U/ml reaction mixture. The initial concentrations of I were [I] 0 = μm. The initial reaction mixture was prepared in the following order: water (x ml), ACh chloride or ATCh iodide (l ml of 0.1 M aqueous solution), inhibitor ( ml of 0.01 M solution in dioxane). Dioxane increased the solubility of the inhibitors in water while it did not affect the reaction. None of the used inhibitors affected the ph value of the reaction mixture. The reaction was started by rapid addition of the aqueous AChE solution (y ml, according to its actual catalytic activity) into the well-stirred and thermostated reaction mixture. Its final volume was 25 ml. The ph value was recorded every minute up to min total reaction time. The automatic titrator 736 GP Titrino (Metrohm) was used for the ph-stat method. Calculation of IC 50 (pi 50 ) The determined ph(t) values were recalculated to give the corresponding values of [H + ]. [H + ] 0 and [H + ] are the actual molarities of protons produced during uninhibited and inhibited hydrolysis in the same reaction time t. Because HAc is a weak acid in water [K HAc (25 C) = 18 μm (Atkins, 1992)], the values [H + ] 0 and [H + ] are practically equal with the produced concentrations of HAc in the reaction mixture (e.g. in a 0.4 mm HAc aqueous solution 99% of HAc is dissociated). Provided that the variation of the AChE activity with the ph value is identical for uninhibited and inhibited ACh or ATCh hydrolysis, the ratio of the rate of uninhibited hydrolysis (v 0 ) vs. that in the presence of an inhibitor of given molarity (v) must be constant for every reaction time. IC 50 is then: IC 50 = 50 [I]/(v 0 /v), (1) where [I] is the initial molarity of a given inhibitor. A more exact determination of IC 50 is pos sible for enzyme inhibition of the fully competitive, non-competitive, uncompetitive, and mixed type: a linear relation of (v 0 /v) vs. [I] is valid (Kulhavý et al., 2002; Purich, 2010): v 0 /v = const. [I] + 1. (2) The value of const. depends on the respective type of inhibition. According to Zdražilová et al. (2006a, b) and Kovářová et al. (2010), carbamates and imidazoles inhibit AChE competitively. Therefore, for the determination of the corresponding straight line only a single experimental point {(v 0 /v) ; [I]} is theoretically required, the second one is (1 ; 0). But more experimental points for additional inhibitor concentrations, together with the point (1 ; 0), make the determination of IC 50 much more reliable. We constructed the described linear functions (v 0 /v) a vs. [I] by means of 3 to 4 experimental points in addition to the point (1 ; 0). Every used value of (v 0 /v) a is the average of a minimum of 20 experimentally determined actual ratios (v 0 /v) t calculated according to the formula (v 0 /v) t = ([H + ] 0,t+1 [H + ] 0,t )/([H + ] t+1 [H + ] t ). (3) The average deviation of the average value of (v 0 /v) t of all used inhibitors was 7.2%. Chemicals ATCh iodide, ACh chloride, 5,5 -dithiobis-2- nitrobenzoic acid (DTNB, Ellman s reagent), dioxane, all of p.a. quality, and AChE from electric eel (preparation type VI-S) were obtained from Sigma-Aldrich (Praha, Czech Republic). Twelve cholinesterase inhibitors, the structures of which are shown in Fig. 1, were used for IC 50 determination. Seven carbamates (sevin, C1 C6), of which three (sevin, C1, C2) had one, three (C3, C4, C5) had two, and one (C6) had three carbamate groups, and two imidazole derivatives (I1, I2) were synthesized in the Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic. 1 H and 13 C NMR spectra, melt-

3 M. Kovářová et al. IC 50 of Cholinesterase Inhibitors 135 ing points, and elemental analyses (unpublished results) confirmed their structure and a purity of > 99%. The carbamate rivastigmine in the form of the drug Exelon 6 mg (Novartis Europafarm Ltd, Horsham, West Sussex, GB) and two nitro genous heterocyclic compounds, galanthamine hydrobromide in the form of the drug Reminyl 12 mg (Janssen Pharmaceutica N.V., Beerse, Belgium) and donepezil hydrochloride in the form of the drug Donepezil 10 mg (Mylan Company, Potters Bar, Herfordshire, GB), were used. We have no information on the stereochemistry of galanthamine and donepezil used in these drugs. The inhibitors were dissolved in dioxane p.a. at 0.01 M and then diluted as necessary with demineralized water. Results and Discussion The IC 50 (pi 50 ) values of the 12 inhibitors (Fig. 1) were determined for hydrolysis by AChE of 1) ATCh by the ph(t) and Ellman s methods, respectively, and 2) ACh by the ph(t) and ph-stat methods, respectively. All measurements were performed under the conditions described above. The ph value was kept constant at ph 8 only in Ellman s and the ph-stat method, respectively. Ellman s method delivered the absorbance A (at 412 nm) as a function of time, and the phstat method gave [HAc] as a function of time, both for the uninhibited and inhibited reaction. From these functions the initial reaction rates v 0 and v were calculated for three to four inhibitor concentrations in the range of 20, 40, 60, 80, and 120 μm. The IC 50 values were then determined using the equation (2). Two typical examples of the [H + ](t) functions obtained by the described ph(t) method for inhibitors C1 and C5 are presented in Figs. 2a (substrate, ACh) and 2b (substrate, ATCh). The ratios (v 0 /v) a as a function of [C5] and [C1], respectively, calculated from the data in Figs. 2a and 2b, are shown in Figs. 2c and 2 d. Table I summarizes the IC 50 and pi 50 values of all 12 inhibitors obtained by the different methods and additionally lists the values of the correlation coefficient R and the residual standard deviation б of (v 0 /v) a as a function of [I]. The average values for all tested inhibitors are R = and , and б = and for ACh and ATCh, respectively. The limits of the 95% confidence intervals of the IC 50 values calculated for (v 0 /v) a = 2 are presented as L. Table I reveals that the IC 50 values determined for ACh and ATCh hydrolysis, respectively, by two independent procedures are fully comparable. When (IC 50 ) ph(t) is plotted against (IC 50 ) ph-stat in the case of ACh hydrolysis or (IC 50 ) ph(t) against (IC 50 ) Ellman s for ATCh, linear regression yields straight lines with the intercept near to zero (ACh, 7.574; ATCh, ), slope (ACh, 0.949; ATCh, 0.943) and correlation coefficients (ACh, ; ATCh, ) near to one, as expected in positive case. So, the two methods are compatible with a probability (expressed as explained variability) of 98.23% and 98.35% for the inhibition of the hydrolysis of ACh and ATCh, respectively. The ph(t) method is simpler and more universally applicable because it is suitable for both ACh and ATCh. The ph-stat method is suitable only for ACh, and Ellman s method only for ATCh hydrolysis, respectively. The change of the ph value of the reaction mixture shifts the activity of AChE during the uninhibited and inhibited hydrolysis of ACh and ATCh in the same way. Some values of pi 50 in Table I, determined by Ellman s method, can be compared with those published: sevin, pi 50 = 4.33 (Kulhavý et al., 2002) and 4.11 (Pavlová, 1998); rivastigmine (Exelon), 3.3 (Zdražilová et al., 2004, 2006b); galanthamine (Reminyl), 3.26 (Štěpánková et al., 2007); imidazoles I1, 4.56, and I2, 4.61 (Kovářová et al., 2010). The [H + ] value of the reaction mixture increases during the hydrolysis followed by the ph(t) method by about two orders of magnitude ( ph 7 5), whereas in the ph-stat or Ellman s method, respectively, it is kept constant. All time courses of [H + ] in Figs. 2a and 2b show initial sigmoidal behaviour, which may be due to the autocatalytic enhancement of the hydrolytic step by the protons resulting from the dissociation of HAc. Conclusion A new, simpler, and more universally applicable method is described for the determination of the IC 50 values of inhibitors of the enzymatic hydrolysis of ACh or ATCh by AChE. This method requires only a ph meter with a suitable ph measuring cell and a small thermostated and stirred batch reactor. The values obtained by this method are comparable to those obtained by other, more expensive, methods.

4 136 M. Kovářová et al. IC 50 of Cholinesterase Inhibitors Acknowledgement This work is a part of research project MSM supported by the Czech Ministry of Education, Youth and Sport. We are very grateful to Prof. Miloš Sedlák, head of the Institute of Organic Chemistry and Technology, University of Pardubice, Pardubice, Czech Republic for the gift of the tested carbamates. Sevin Donepezil C1 Rivastigmine C2 Galanthamine C3 H 3 CO O H OH C4 N C5 CH 3 C6 I1 I2 Fig. 1. Chemical structures of the used inhibitors.

5 M. Kovářová et al. IC 50 of Cholinesterase Inhibitors 137 [H + ] [ M] (v0/v)a a) ACh, C5 [C5] = 0 [ M] [ M] Reaction time t [min] c) ACh, C [C5] [ M] [H + ] [ M] (v0/v)a 80 [C1] = 0 [ M] b) ATCh, C Reaction time t [min] d) ATCh, C [C1] [ M] Fig. 2. Time course of [H + ] during hydrolysis of (a) ACh and (b) ATCh catalyzed by AChE in the presence or absence of C5 and C1. Linear regression of (v 0 /v) a as a function of (c) [C5] and (d) [C1], respectively, according to equation (2). The parameters of the straight lines are: (c) (v 0 /v) a = 3149 [C5] [ACh], and (d) (v 0 /v) a = [C1] [ATCh]. From these relations, the values of IC 50 = 317 (C5) and 36.7 μm (C1) (i.e. pi 50 = 3.50 and 4.44) were calculated.

6 138 M. Kovářová et al. IC 50 of Cholinesterase Inhibitors Table I. The IC 50 and pi 50 values of 6 carbamates, 2 imidazoles, and 3 drugs inhibiting the hydrolysis of ACh and ATCh catalyzed by AChE, obtained by the ph(t) method described here, and, for comparison, by the ph-stat method for ACh and Ellman s method for ATCh hydrolysis, respectively. R and б are the correlation coefficient and residual standard deviation, respectively, of (v 0 /v) a as a function of inhibitor concentration [I]. L is the limit of the 95% confidence interval of the IC 50 value calculated for (v 0 /v) a = 2. The asterisks indicate IC 50 values approximated by extrapolation which was necessitated by the limited solubility of an inhibitor that did not allow to reach the ratio (v 0 /v) a = 2. Inhibitor R 2 б 10 2 IC 50 [μm] L [μm] pi 50 IC 50 [μm] pi 50 ACh ph(t) ph-stat Sevin ( ) C ( ) C ( ) C * C * C * C ( ) Rivastigmine ( ) I ( ) I ( ) Galanthamine ( ) Donepezil ( ) ATCh ph(t) Ellman s Sevin ( ) C ( ) C ( ) C * C ( ) C * C ( ) Rivastigmine ( ) I ( ) I ( ) Galanthamine ( ) Donepezil ( ) Atkins P. W. (1992), Physical Chemistry, 4th ed. Oxford University Press, Oxford, UK, p Ellman G. L., Courtney K. D., Andres V. Jr., and Featherstone R. M. (1961), A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, Kovářová M., Komers K., Štěpánková Š., and Čegan A. (2010), Inhibition of acetylcholinesterase by 14 achiral and five chiral imidazole derivates. Bioresour. Technol. 101, Kulhavý D., Čegan A., Komers K., and Mindl J. (2002), Inhibition of enzymatic reactions. A rapid method to determine the index pi 50. Z. Naturforsch. 57c, Pavlová V. (1998), Inhibition of cholinesterases by carbamates. Diploma thesis, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic (in Czech). Purich D. L. (2010), Enzyme Kinetics: Catalysis and Control. Academic Press, London, UK, p Štěpánková Š., Komers K., Komersová A., Masopustová M., and Čegan A. (2007), Inhibition of cholinesterases by dialkylcarbamates. Z. Naturforsch. 62c, Zdražilová P., Štěpánková Š., Komers K., Ventura K., and Čegan A. (2004), Half inhibition concentration of new cholinesterase inhibitors. Z. Naturforsch. 59c, Zdražilová P., Štěpánková Š., Komersová A., Vránová M., Komers K., and Čegan A. (2006a), Kinetics of 13 new cholinesterase inhibitors. Z. Naturforsch. 61c, Zdražilová P., Štěpánková Š., Vránová M., Komers K., Komersová A., and Čegan A. (2006b), Kinetics of total enzymatic hydrolysis of acetylcholine and acetylthiocholine. Z. Naturforsch. 61c,