Reversibility of the inhibition of acetylcholinesterase by tacrine

Neuroscience Letters, 118 (1990) 85-87 Elsevier Scientific Publishers Ireland Ltd.

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NSL 07188

Reversibility of the inhibition of acetylcholinesterase by tacrine R a y m o n d M. D a w s o n Materials Research Laboratory, Defence Science and Technology Organisation, Ascot Vale, Vic. (Australia) (Received 7 May 1990; Accepted 4 June 1990)

Key wordsv Tacrine; Acetylcholinesterase; Reversibility; Organophosphate; Kinetics Inhibition of bovine erythrocyte acetylcholinesterase (ACHE) by 1,2,3,4-tetrahydro-9-acridinamine (tacrine) was independent of time of incubation and was partially reversed by dilution and by increased substrate concentration. It was fully reversed by dialysis. Similar results were obtained with AChE from other sources. The results are consistent with some reports in the literature, but not with others; none of these reports examined all four criteria of reversibility. The results do not explain the prolonged inhibition of AChE in vivo or the ability of tacrine to protect animals against the lethal effects of organophosphate anticholinesterases.

Tacrine (l,2,3,4-tetrahydro-9-acridinamine) is a potent inhibitor of cholinesterases, and this may underlie its reported ability to alleviate the symptoms of Alzheimer's disease [14]. Some workers have reported that the nature of the inhibition of acetylcholinesterase (ACHE) is mixed competitive/non-competitive, i.e. tacrine affects both K m and Vmax[8, 11, 13], but others have claimed that the inhibition is purely non-competitive, i.e. effect on Vmax only [9, 12, 15, 17]. Pure reversible noncompetitive inhibition by species other than protons and heavy-metal ions is rare [5] but one possible explanation for such unusual behaviour is that tacrine is an irreversible inhibitor of acetylcholinesterase (ACHE), i.e. it permanently inactivates a portion of the enzyme without affecting the affinity of the remaining free enzyme for substrate. Such a property of tacrine would help explain its prolonged inhibition of AChE in vivo [7, 10] and its ability to protect laboratory animals against the lethal effects oforganophosphate anticholinesterases, when tacrine is given prophylactically [1]. Berry and Davies had reported earlier that acylating ('irreversible') inhibitors of ACHE, e.g. carbamates, were effective prophylactically against the organophosphate compounds, whereas fully reversible, competitive inhibitors were not [3]. Tacrine does not have the ability to acylate ACHE, but other irreversible non-acylating inhibitors of AChE are known, e.g. l-anilino-8-naphthalene-sulfonic acid [4]. It is important to establish the nature of the inhibition of AChE by tacrine because of its current clinical interest

Correspondence: R.M. Dawson, Materials Research Laboratory, P.O. Box 50, Ascot Vale, Vic. 3032, Australia. 0304-3940/90/$ 03.50 ((" 1990 Elsevier Scientific Publishers Ireland Ltd.

[14] and its efficacy against organophosphate anticholinesterases. A truly reversible inhibitor, with at least some competitive aspect with respect to substrate hydrolysis, has the following characteristics: inhibition is reversed by dilution, is independent of time, decreases with increasing substrate concentration and is reversed by dialysis. Few reports in the literature have addressed any of these points for tacrine/AChE, and none has addressed all four. Heilbronn observed that inhibition of human erythrocyte AChE by tacrine was independent of time, and that the inhibition was reversed by dialysis, although it should be noted that recovery of enzymic activity was incomplete after 48 h dialysis [8]. Benveniste et al. examined three of the above four criteria for inhibition of human serum cholinesterase (butyrylcholinesterase, BuChE) by tacrine, and obtained results consistent with reversible inhibition, but they did not study AChE [2]. The interaction of tacrine with AChE may well be different from that with BuChE [12]. Hunter et al. reported that inhibition of rat brain AChE by tacrine was independent of time and reversed by dilution, consistent with reversible inhibition [9]. Preliminary results in our laboratory provided evidence in favour of reversible inhibition [6]. In contrast to the above studies, Patocka et al. claimed that the inhibition of human erythrocyte AChE and rat brain AChE by tacrine was irreversible, based on the lack of recovery of activity on dialysis and the lack of effect of dilution on the extent of the inhibition [12]. Notwithstanding these results, the amount of inhibition was independent of time of incubation before assay. We have assessed for the first time the reversibility of

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Fig. 1. Time dependence of the inhibition of bovine erythrocyte AChE by tacrine in 2 mM KH2PO4pH 7.0 at 25°C. Tacrine and AChE were incubated for the times shown before addition of ASCh for spectrophotometric assay. For the assay at zero-time, tacrine was added to AChE after addition of ASCh. Data are shown as mean + S,E.M. from 4 experiments, Concentration of tacrine=4 × 10-8 M (O) or 1 x 10-7 M(Q).

Fig. 2. Effect of substrate concentration on the inhibition of bovine erythrocyte AChE by 2 x l0 -7 M tacrine. The assay for 0,025 mM ASCh was recorded first in the presence and absence respectively of tacrine, and additional ASCh was added in stages to give the concentrations shown. Data are shown as mean + S.E.M. from 4 experiments in 2 mM KH:PO4pH 7.0 at 25°C.

the inhibition of AChE by tacrine according to the four criteria outlined above, and present our results below. Bovine erythrocyte ACHE, type XII, was obtained from Sigma, U.S.A. The sample of tacrine was that used previously [6]. All assays were performed spectrophotometrically in 3.3 ml buffer containing 0.15 m M 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) and 0.2 m M acetylthiocholine (ASCh) at 25°C [6] unless indicated otherwise, using a Pye Unicam PU 8610 or Shimadzu U V I 6 0 spectrophotometer. All experiments and assays were performed in 2 m M KH2PO4 p H 7.0 and 25°C unless indicated otherwise. D a t a are quoted as mean ± S.E.M. from 4-6 experiments. Time dependence. Tacrine (4 x 10-8 M) was incubated with A C h E for 60, 40, 20 and 10 min respectively before addition of substrate. Another sample of A C h E received tacrine after substrate (zero-time sample), and a sixth sample was assayed in the absence o f tacrine and served as a control. All 6 assays were run simultaneously. The experiment was repeated 3 times, and was also conducted 4 times with tacrine present at 1 x 10 - 7 M. The results are shown in Fig. l, where it can be seen that inhibition was independent of time at both concentrations of tacrine. An analysis of variance [16] indicated no significant differences between any of the time points at either concentration o f tacrine. Dilution effects. The rate o f hydrolysis o f ASCh was measured in the presence a n d absence of 1 x 10 - 6 M tacrine, respectively. The assays were run simultaneously for 2 rain, and the rate o f change of absorbance for the control was 0.33 m i n - k A 0.3 ml aliquot o f each solution was then added to 3.0 ml buffer-DTNB-ASCh, and the rate of change o f absorbance o f each diluted

reaction was measured for a further 6 min. The activity of the sample containing tacrine was 6 . 4 _ 0.29% of control before dilution, and 35.2±0.98% o f control after dilution (n = 4). Dilution therefore substantially reduced the extent of inhibition. Substrate effects. The activity of A C h E in the presence and absence o f 2 × t0 -7 M tacrine respectively was recorded in 3.0 ml buffer with a concentration o f ASCh of 0.025 m M . F o u r successive 0.02 ml aliquots o f ASCh were then added to each reaction at 6 minute intervals such that the final concentration of ASCh was 0.05, 0.1. 0.2 and 0.4 m M respectively. The rate of change o f absorbance was measured at each substrate concentration. The results are shown in Fig. 2, where it can be seen that the ability of tacrine to inhibit AChE reduced progressively with increasing concentration of ASCh. A simultaneous multiple comparison o f results o f all 5 concentrations of ASCh [16] showed that there were significant differences at the 95% confidence level for all pairs of points in Fig. 2 except 0.025 vs 0.05 m M ASCh, and 0.05 vs 0.1 m M ASCh. Dialysis. A solution o f A C h E and tacrine (7 x 10 - 7 M ) was prepared in 10 ml buffer. 3.0 ml was assayed by addition of ASCh and D T N B , and the remaining 7.0 mi was dialysed against 1 liter buffer at 2°C for 24 h, with one change of buffer during this time. A controlsohition (no tacrine) was treated similarly. Before dialysis, the activity of the tacrine solution was 8.9±0.46% of control; after dialysis the corresponding figure was ~107+1.6% (n = 6). Recovery of activity in the control solution after 24 h dialysis was 74 _+ 10.0%. Dialysis therefore completely restored enzyme activity that had been inhibited by > 90% by tacrine. Instability of A C h E over 24 h is the

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probable cause of the recovery of AChE being less than 100% and highly variable. Tacrine, when still present, probably protected AChE against the denaturation, and therefore gave an apparent relative activity > 100%. Other buffers and sources of enzyme. Similar results were obtained for bovine erythrocyte AChE in 2 mM KHzPO4-150 mM NaCI pH 7.0, and in 5 mM Tris, pH 8.0. Similar results were also obtained using Triton-solubilized rat brain AChE [6], eel AChE (Sigma) and membrane-bound human erythrocyte AChE (Sigma) in 2 mM KH2PO4 pH 7.0 and 2 mM KH2PO~150 mM NaC1, pH 7.0. Buffers included 0.1% gelatine in the studies with eel, and 0.01% Triton X-100 in the studies with rat brain ACHE. Conclusions. Overall, our results are consistent with reversible inhibition of AChE by tacrine for a variety of sources of ACHE, and in buffers of low and physiological ionic strength. The results confirm and extend those of us and others [6, 8, 9]. It is not clear why Patocka et al. obtained contrary results [12], and why Heilbronn observed incomplete recovery of activity on prolonged dialysis [8]. It should be noted that Patocka et al. reported similar irreversible inhibition of horse plasma BuChE to that they observed for ACHE, even though the inhibition was mixed competitive/non-competitive for BuChE [12]. This result is at variance with that of Benveniste et al. who found tacrine to be a reversible inhibitor of human serum BuChE [2]. Further work is necessary to establish the reasons for the prolonged inhibition of AChE by tacrine in vivo [7, 10] and the efficacy of tacrine as a prophylactic drug against the organophosphate anticholinesterases [1]. I am grateful to Mr. M. Poretski and Miss K.J. Lewis for technical assistance.

1 Bajgar, J., Patocka, J., Fusek, J. and Hrdina, V., Some possibilities of protection against acetylcholinesterase inhibition by organophosphates in vivo, Sb. Ved. Pr. Lek. Fak. Univ. Karlovy Hradci Kralove, 27 (1984) 425~435.

2 Benveniste, D., Hemmingsen~ L. and Juul, P., Tacrine inhibition of serum cholinesterase and prolonged succinylcholine action. Acta Anaesth. Scand., 11 (1967) 297 309. 3 Berry, W.K. and Davies, D.R., The use ofcarbamates and atropine in the protection of animals against poisoning by 1,2,2-trimethylpropylmethyl-phosphonofluoridate, Biochem. Pharmacol.~ 19 (1970) 927 934. 4 Christian, S.T. and Janetzko, R., A comparative study of the interactions of the nonpolar fluorescent ligand, 1-anilino-8-naphthalene sulfonic acid with butyryl and acetylcholinesterase, Arch. Biochem. Biophys.,145 (1971) 169 178. 5 Cornish-Bowden, A.J., Principles of Enzyme Kinetics, Butterworths~ London, 1976, Chap. 4. 6 Dawson, R.M., Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase, Neurosci. Lett., 100 (1989) 227 230. 7 Hallack, M. and Giacobini, E., Physostigmine, tacrine and metrifonate: The effect of multiple doses on acetylcholine metabolism in rat brain, Neuropharmacology, 28 (1989) 199 206. 8 Heilbronn, E., Inhibition of cholinesterases by tetrahydroaminacrin, Acta Chem. Scand., 15 (1961) 1386-1390. 9 Hunter, A.J., Murray, T.K., Jones, J.A., Cross, A.J. and Green, A.R., The cholinergic pharmacology of tetrahydroaminoacridine in vivo and in vitro, Br. J. Pharmacol., 98 (1989) 79 86. 10 Kumar, V. and Becker, R.E.~ Clinical pharmacology of tetrahydroaminoacridine: a possible therapeutic agent for Alzheimer's disease, Int. J. Clin. Pharmac. Ther. Toxicol., 27 (1989)478 485. 11 Nielsen, J.A., Mena, E.E., Williams, I.H., Nocerini, M.R. and Liston, D., Correlation of brain levels of 9-amino-l,2,3,4-tetrahydroacridine (THA) with neurochemical and behavioral changes, Eur. J. Pharmacol., 173 (1989) 53 64. 12 Patocka, J., Bajgar, J., Bielavsky, J. and Fusek, J., Kinetics of inhibition of cholinesterases by 1,2,3,4-tetrahydro-9-aminoacridine in vitro, Coll. Czech. Chem. Commun., 41 (1976) 816 824. 13 Steinberg, G.M., Mednick, M.L., Maddox, J,, Rice, R. and Cramer, J., A hydrophobic binding site in acetytcholinesterase, J. Med. Chem., 18 (1975) 1056-1061. 14 Summers, W.K., Majovski, L.V., Marsh, G.M., Tachiki, K. and Kling, A., Oral tetrahydroaminoacridine in long-term treatment of senile dementia, Alzheimer type, New Engl. J. Med., 315 (1986) 1241 1245. 15 Tonkopii, V.D., Prozorovskii, V.B. and Suslova, I.M.~ Interaction of reversible inhibitors with catalytic centres and allosteric sites of cholinesterases, Byull. Eksp. Biol. Med., 82(1976)947 950. 16 Wallenstein, S., Zucker, C.L. and Fleiss, J.L., Some statistical methods useful in circulation research, Circ. Res., 47 (1980) 1 9. 17 Wu, C.S. and Yang, J.T., Tacrine protection ofacetylcholinesterase from inactivation by diisopropylfluorophosphate: a circular dichroism study, Mol. Pharmacol., 35 (1989) 85 92.