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Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase
Neuroscience Letters, 100 (1989) 227-230
227
Elsevier Scientific Publishers Ireland Ltd.
NSL 06044
Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase R a y m o n d M. D a w s o n Defence Science and Technology Organization, Materials Research Laboratory. Ascot Vale, Vic. 3032. (Australia) (Received 19 August 1988; Revised version received 11 January 1989; Accepted 11 January 1989)
Key words: Tacrine; Sarin; Acetylcholinesterase; Reactivation; Ageing; Oxime Bovine erythrocyte acetylcholinesterase was inhibited by the organophosphate sarin, and the rate of ageing (the time-dependent decrease in the ability of an oxime to reactivate the enzyme) was studied. At pH 7.0 and 37°C, I0 5 M or 10 -6 M tacrine (tetrahydroaminoacridine) decreased the rate of ageing in low ionic strength buffer. Tacrine at 10 5 M also significantly decreased the rate of ageing in 150 mM NaC1. The results indirectly demonstrated that the inhibition of substrate hydrolysis by tacrine is reversible, and that tacrine does not prevent reactivation of sarin-inhibited acetylcholinesterase. Both these observations, which were also made for rat brain acetylcholinesterase, are in contrast with reports in the literature.
Tacrine [l,2,3,4-tetrahydro-9-aminoacridine; THA) is a potent inhibitor of cholinesterases and is currently of interest because of its potential to alleviate the symptoms of Alzheimer's disease [9]. It also shows promise as a prophylactic drug against the lethal effects of chemical warfare agents of the organophosphate class [I]. Patocka et al. reported that the inhibition of acetylcholinesterase (ACHE) by tacrine is irreversible, and purely non-competitive in nature [7]. This is unusual for a nonacylating inhibitor of ACHE. The same authors deduced that tacrine binds to a hydrophobic, allosteric site on ACHE, the same site as that which binds the allosteric effector gallamine [7]. AChE used in these studies [7] was from bovine erythrocytes, human erythrocytes and rat brain. Recently Patocka and Bajgar found that whereas Triton-solubilized rat brain AChE that had been inhibited with the organophosphate sarin could be reactivated by the oxime trimedoxime (TMB-4), negligible reactivation by TMB-4 occurred in the presence of 10 -5 M tacrine [6]. They concluded that tacrine interfered with the reactivation process. An alternative explanation is that tacrine accelerated the rate of ageing of satin-inhibited ACHE. Ageing is the acid-catalyzed loss of the isopropyl group which renders the inhibited AChE incapable of reactivation. Gallamine is known to influence the rate of ageing of sarin-inhibited AChE [2], and since tacrine Correspondence: R.M. Dawson, Defence Science and Technology Organization, Materials Research Laboratory, P.O. Box 50, Ascot Vale, Vic. 3032, Australia. 0304-3940/89/$ 03.50 (t~ 1989 Elsevier Scientific Publishers Ireland Ltd.
228
is reputed to bind to the same allosteric site as gallamine (see above; ref. 7), it is plausible that tacrine might also affect the ageing process. The experiments described below were performed to explore this possibility. The experimental conditions were as in previous studies [2]. Thus bovine erythrocyte acetylcholinesterase (Sigma) was inhibited > 99% with 0.2 ~M satin (synthesized in-house; purity > 95%) for 30 rain at 37°C. Sarin was then separated from the inhibited enzyme by chromatography on a Sephadex P D I 0 column (Pharmacia). Aliquots of satin-inhibited AChE were incubated in 8.2 ml 2 m M KH2PO4 pH 7.0 at 37'C. At various times, 1.0 ml aliquots of the solution were taken and incubated 30 rain at 37°C with 0.5 m M 2-pyridine aldoxime methiodide (2-PAM) to reactivate unaged enzyme. Since 2-PAM interferes with the enzyme assay, it was removed by chromatography on P D I 0 columns. Free enzyme activity was then assayed in the presence of 0.5 mM acetylthiocholine and 0.15 mM 5,5'-dithiobis (2-nitrobenzoic acid) by following the rate of increase of absorbance at 412 nm at 25'~C using a Unicam SP1750 spectrophotometer. The rate constant (k) for ageing was calculated as the gradient of a plot of In (reactivated AChE activity) vs time. The amount of reactivated AChE was determined by subtracting an appropriate blank assay (in which water replaced 2-PAM) from the observed activity. Four experiments were conducted simultaneously with the concentration oftacrine being 0 (control), 10- 7 M, 10 6 M and 10 5 M respectively. The same amount of sarin-inhibited AChE was present in each reactivation flask. Tacrine hydrochloride was obtained from the Institute of Drug Technology, Melbourne, Australia; its identity and purity were confirmed by UV spectrometry [8], I~C-NMR of the free base [4] and H P L C [5]. In preliminary experiments it was established that tacrine was a mixed inhibitor of the hydrolysis of acetylthiocholine in 2 m M KHzPO4 pH 7.0, 25°C with Ki (competitive inhibition constant) = 10.5 nM and Ki t = 5 9 nM. In buffer containing 150 mM NaC1, K i = 8 6 nM, Ki 1= 191 nM. The results of a typical ageing experiment are shown in Fig. l, where it can be seen that tacrine in fact decreased the rate of ageing at concentrations of 10 6 M and 10 5 M. The actual rate constants (mean + S.E.M. from 4 experiments) were found to be 0.425 + 0.028 (control), 0.406 _+0.019 (10- v M tacrine), 0.278 _+0.024 ( I 0-6 M tacfine) and 0.158 +0.020 (10 5 M tacrine) h 1. The last two values are significantly different from the control value, P < 0.05. Similar results were obtained when TMB-4 replaced 2-PAM as the oxime. The experiments were repeated in buffer of physiological ionic strength, i.e. with 150 m M NaC1, Rate constants for tacrine at concentrations of 0, 10 -7 M, l0 (' M and 10- 5 M were then found to be 0.291 -+0.007, 0.309-+0.013, 0.268-+0.014 and 0.165-+0.013 h J ( n = 5 ) respectively. Only the last value (i.e. for 10 -5 M tacrine) was significantly different from the control value. The experiments were then repeated at low ionic strength using Triton-solubilized rat brain AChE (prepared exactly as described by Patocka et al. [6, 7]) instead of bovine erythrocyte ACHE, and with TMB-4 as the oxime. The results were qualitatively the same as shown in Fig. 1 for bovine erythrocyte ACHE. Tacrine at 10 5 M significantly decreased the rate of ageing 2.5-fold (k=0.131_+0.023 h i vs control k=0.332 -+0.025 h I n = 5 ) . Further experiments with the rat brain enzyme were
229
5.(
4.0
= I
I
2
I
rime (hours)
Fig. 1. Ageing of sarin-inhibited ACHE, 2 m M KH2PO4, pH 7.0, 25°C. At various times as shown, aliquots of the ageing inhibited enzyme were incubated with 0.5 m M 2-PAM for 30 min at 37°C to determine the a m o u n t of inhibited enzyme that was capable of reactivation. The activity of reactivated A C h E is expressed as 103 A abs/5 min at 412 nm. The oxime 2-PAM was separated from the various forms of the enzyme by chromatography on Pharmacia PD 10 columns prior to assay. Concentration of tacrine = 0 ( © ) , 1 0 - 7 M ( O ) , 10 6 M ( A ) o r l 0 5(A).
done at pH 8.0 (5 mM HEPES), with reactivation by 0.1 mM TMB-4 for 5 min at 25°C. In this case, only the initial stage of the ageing experiment (i.e. at t = 0 in Fig. 1) was performed; this is essentially the reactivation experiment described by Patocka and Bajgar [6]. In 3 experiments, each in triplicate, there was no significant difference between the assay results in the presence and absence of 10 -5 M tacrine, after separating the oxime from AChE by chromatography. It is tempting to speculate that tacrine, given as a pretreatment against the highly toxic nerve agent sarin, might exert part of its beneficial effect by slowing the rate of ageing of the inhibited acetylcholinesterase and thereby enhancing the effect of subsequent administration of an oxime to reactivate unaged enzyme and restore normal cholinergic function. However, tacrine at the concentration required (10 -5 M) would produce > 98 % reversible inhibition of acetyicholinesterase, based on the inhibition constants quoted above, and this would lead to toxic effects by itself. The concentration of tacrine in vivo that has been used in the treatment of Alzheimer's disease is in the range 10 - 7 M to 10 - 6 M [3]. The present results suggest that the results of Patocka and Bajgar [6] are not likely to be due to tacrine accelerating the rate of ageing. Two further very significant conclusions can be drawn from the results. An inspection of Fig. 1 reveals that all 4 regression lines have essentially the same intercept on the y-axis, i.e. enzyme activity is restored to the same level at zero time after oxime-induced reactivation and chromatography, regardless of the concentration of tacrine. This indicates firstly that sizeexclusion chromatography of the enzyme-oxime-tacrine mixture has separated enzyme not only from oxime (as it was designed to do), but also from tacrine. Had
230 tacrine not been separated from the enzyme, it would have inhibited bovine erythrocyte A C h E activity by 67% at l0 7 M, a n d 99% at 10 --5 M. C o n t r o l experiments showed that rat brain A C h E activity was also practically completely inhibited by 10 5 M tacrine. C o n s e q u e n t l y , tacrine c a n n o t be an irreversible i n h i b i t o r of ACHE, because if it were it would not have been separated from A C h E by c h r o m a t o g r a p h y . Separate experiments showed that the reversibility is not d e p e n d e n t on the presence of oxime. Secondly, tacrine did n o t prevent reactivation of sarin-inhibited A C h E by 2 - P A M or T M B-4. The results do n o t preclude some slowing of the rate of reactivation, and effects on this rate may have been observed had the reactivation period not been as long as 30 rain. However, a p r o n o u n c e d i n h i b i t i o n by tacrine of the rate of reactivation would have p r o d u c e d a lesser y-intercept than was observed (Fig. 1). Further, no retardation of reactivation by 0.1 m M T M B - 4 was observed when rat brain A C h E was studied in the presence of l0 5 M tacrine with a reactivation time of only 5 rain, a n d a temperature of 25 ° rather than 3T~C. Patocka and Bajgar reported that reactivation by T M B - 4 was practically abolished by 10 5 M tacrine u n d e r the same experimental c o n d i t i o n s and with an identical p r e p a r a t i o n of rat brain A C h E [6]. O u r observed lack of a p r o n o u n c e d i n h i b i t o r y effect on reactivation, and the reversibility of the t a c r i n e - A C h E complex, are therefore c o n t r a r y to the reports of Patocka et al. [6, 7]. The reasons for the discrepancies are u n k n o w n at this stage. I a m grateful to Mr. D. A m o s for synthesis o f sarin. I Bajgar, J., Patocka, J., Fusek, J. and ttrdina, V., Some possibilitiesof protection against acetylcholinesterase inhibition by organophosphates in vivo, Sb. Ved. Pr. Lek. Fak. Univ. Karlovy Hradci Kralove, 27 (1984) 425 435. 2 Dawson, R.M., Crone, HD., Bladen, M.P. and Poretski, M., A comparison of the effects of ionic strength on three preparations of acetylcholinesterasein the presence and absence of gallamine, Neurochem. Int., 3 (1981) 335 341. 3 Drukarch, B., Leysen, J.E. and Stool, J.C., Further analysis of the neuropharmacological profile of 9-amino-l,2,3,4-tetrahydroacridine (THA), an alleged drug for the treatment of Alzheimer's Disease, Life Sci., 42 (1988) 1011 1018. 4 Faurc, R., Giovannangeli, G., Gaby, J-P., Soyfer, J-C., Vincent, E-J. and Barbe, J., Etude par resonance magndtique nucl6aire du carbone 13 de quelques tetrahydro-l,2,3,4 acridines substitu6es en position 9, J. (;him. Phys., 78 (1981) 527 530. 5 Hsieh, J.Y.K., Yang, R.K. and Davis, K.L., High-performance liquid chromatographic determination of tetrahydroaminoacridine in human and rat tissue using a rapid Sep-Pak C~8extraction, J. Chromatogr., 274 (1983) 388 392. 6 Patocka, J. and Bajgar, J., The reactivation of o-isopropylmethylphosphonylated acetylcholinesterase and its modification by some non-acylating ligands, Sb. Ved. Pr. Lek. Fak. Univ. Karlovy Hradei Kralove, 27 (1984) 477 480. 7 Patocka, J., Bajgar, J., Bielavsky,J. and Fusek, J., Kinetics of inhibition of cholinesterases by 1,2,3,4tetrahydro-9-amino-acridine in vitro, Coll. Czech. Chem. Commun., 41 (1976) 816-824. 8 Steinberg, G.M., Mednick, M.L., Maddox, J. and Rice, R., A hydrophobic binding site in acetylcholinesterase, J. Med. Chem., 18 (1975) 1056 1061. 9 Summers, W.K., Majovski, L.V., Marsh, G.M., Tachiki, K. and Kling, A., Oral tetrahydroaminoacridine in long-term treatment of seniledementia, Alzheimer type, N. Engl. J. Med., 315 (1986) 1241-1245.
227
Elsevier Scientific Publishers Ireland Ltd.
NSL 06044
Tacrine slows the rate of ageing of sarin-inhibited acetylcholinesterase R a y m o n d M. D a w s o n Defence Science and Technology Organization, Materials Research Laboratory. Ascot Vale, Vic. 3032. (Australia) (Received 19 August 1988; Revised version received 11 January 1989; Accepted 11 January 1989)
Key words: Tacrine; Sarin; Acetylcholinesterase; Reactivation; Ageing; Oxime Bovine erythrocyte acetylcholinesterase was inhibited by the organophosphate sarin, and the rate of ageing (the time-dependent decrease in the ability of an oxime to reactivate the enzyme) was studied. At pH 7.0 and 37°C, I0 5 M or 10 -6 M tacrine (tetrahydroaminoacridine) decreased the rate of ageing in low ionic strength buffer. Tacrine at 10 5 M also significantly decreased the rate of ageing in 150 mM NaC1. The results indirectly demonstrated that the inhibition of substrate hydrolysis by tacrine is reversible, and that tacrine does not prevent reactivation of sarin-inhibited acetylcholinesterase. Both these observations, which were also made for rat brain acetylcholinesterase, are in contrast with reports in the literature.
Tacrine [l,2,3,4-tetrahydro-9-aminoacridine; THA) is a potent inhibitor of cholinesterases and is currently of interest because of its potential to alleviate the symptoms of Alzheimer's disease [9]. It also shows promise as a prophylactic drug against the lethal effects of chemical warfare agents of the organophosphate class [I]. Patocka et al. reported that the inhibition of acetylcholinesterase (ACHE) by tacrine is irreversible, and purely non-competitive in nature [7]. This is unusual for a nonacylating inhibitor of ACHE. The same authors deduced that tacrine binds to a hydrophobic, allosteric site on ACHE, the same site as that which binds the allosteric effector gallamine [7]. AChE used in these studies [7] was from bovine erythrocytes, human erythrocytes and rat brain. Recently Patocka and Bajgar found that whereas Triton-solubilized rat brain AChE that had been inhibited with the organophosphate sarin could be reactivated by the oxime trimedoxime (TMB-4), negligible reactivation by TMB-4 occurred in the presence of 10 -5 M tacrine [6]. They concluded that tacrine interfered with the reactivation process. An alternative explanation is that tacrine accelerated the rate of ageing of satin-inhibited ACHE. Ageing is the acid-catalyzed loss of the isopropyl group which renders the inhibited AChE incapable of reactivation. Gallamine is known to influence the rate of ageing of sarin-inhibited AChE [2], and since tacrine Correspondence: R.M. Dawson, Defence Science and Technology Organization, Materials Research Laboratory, P.O. Box 50, Ascot Vale, Vic. 3032, Australia. 0304-3940/89/$ 03.50 (t~ 1989 Elsevier Scientific Publishers Ireland Ltd.
228
is reputed to bind to the same allosteric site as gallamine (see above; ref. 7), it is plausible that tacrine might also affect the ageing process. The experiments described below were performed to explore this possibility. The experimental conditions were as in previous studies [2]. Thus bovine erythrocyte acetylcholinesterase (Sigma) was inhibited > 99% with 0.2 ~M satin (synthesized in-house; purity > 95%) for 30 rain at 37°C. Sarin was then separated from the inhibited enzyme by chromatography on a Sephadex P D I 0 column (Pharmacia). Aliquots of satin-inhibited AChE were incubated in 8.2 ml 2 m M KH2PO4 pH 7.0 at 37'C. At various times, 1.0 ml aliquots of the solution were taken and incubated 30 rain at 37°C with 0.5 m M 2-pyridine aldoxime methiodide (2-PAM) to reactivate unaged enzyme. Since 2-PAM interferes with the enzyme assay, it was removed by chromatography on P D I 0 columns. Free enzyme activity was then assayed in the presence of 0.5 mM acetylthiocholine and 0.15 mM 5,5'-dithiobis (2-nitrobenzoic acid) by following the rate of increase of absorbance at 412 nm at 25'~C using a Unicam SP1750 spectrophotometer. The rate constant (k) for ageing was calculated as the gradient of a plot of In (reactivated AChE activity) vs time. The amount of reactivated AChE was determined by subtracting an appropriate blank assay (in which water replaced 2-PAM) from the observed activity. Four experiments were conducted simultaneously with the concentration oftacrine being 0 (control), 10- 7 M, 10 6 M and 10 5 M respectively. The same amount of sarin-inhibited AChE was present in each reactivation flask. Tacrine hydrochloride was obtained from the Institute of Drug Technology, Melbourne, Australia; its identity and purity were confirmed by UV spectrometry [8], I~C-NMR of the free base [4] and H P L C [5]. In preliminary experiments it was established that tacrine was a mixed inhibitor of the hydrolysis of acetylthiocholine in 2 m M KHzPO4 pH 7.0, 25°C with Ki (competitive inhibition constant) = 10.5 nM and Ki t = 5 9 nM. In buffer containing 150 mM NaC1, K i = 8 6 nM, Ki 1= 191 nM. The results of a typical ageing experiment are shown in Fig. l, where it can be seen that tacrine in fact decreased the rate of ageing at concentrations of 10 6 M and 10 5 M. The actual rate constants (mean + S.E.M. from 4 experiments) were found to be 0.425 + 0.028 (control), 0.406 _+0.019 (10- v M tacrine), 0.278 _+0.024 ( I 0-6 M tacfine) and 0.158 +0.020 (10 5 M tacrine) h 1. The last two values are significantly different from the control value, P < 0.05. Similar results were obtained when TMB-4 replaced 2-PAM as the oxime. The experiments were repeated in buffer of physiological ionic strength, i.e. with 150 m M NaC1, Rate constants for tacrine at concentrations of 0, 10 -7 M, l0 (' M and 10- 5 M were then found to be 0.291 -+0.007, 0.309-+0.013, 0.268-+0.014 and 0.165-+0.013 h J ( n = 5 ) respectively. Only the last value (i.e. for 10 -5 M tacrine) was significantly different from the control value. The experiments were then repeated at low ionic strength using Triton-solubilized rat brain AChE (prepared exactly as described by Patocka et al. [6, 7]) instead of bovine erythrocyte ACHE, and with TMB-4 as the oxime. The results were qualitatively the same as shown in Fig. 1 for bovine erythrocyte ACHE. Tacrine at 10 5 M significantly decreased the rate of ageing 2.5-fold (k=0.131_+0.023 h i vs control k=0.332 -+0.025 h I n = 5 ) . Further experiments with the rat brain enzyme were
229
5.(
4.0
= I
I
2
I
rime (hours)
Fig. 1. Ageing of sarin-inhibited ACHE, 2 m M KH2PO4, pH 7.0, 25°C. At various times as shown, aliquots of the ageing inhibited enzyme were incubated with 0.5 m M 2-PAM for 30 min at 37°C to determine the a m o u n t of inhibited enzyme that was capable of reactivation. The activity of reactivated A C h E is expressed as 103 A abs/5 min at 412 nm. The oxime 2-PAM was separated from the various forms of the enzyme by chromatography on Pharmacia PD 10 columns prior to assay. Concentration of tacrine = 0 ( © ) , 1 0 - 7 M ( O ) , 10 6 M ( A ) o r l 0 5(A).
done at pH 8.0 (5 mM HEPES), with reactivation by 0.1 mM TMB-4 for 5 min at 25°C. In this case, only the initial stage of the ageing experiment (i.e. at t = 0 in Fig. 1) was performed; this is essentially the reactivation experiment described by Patocka and Bajgar [6]. In 3 experiments, each in triplicate, there was no significant difference between the assay results in the presence and absence of 10 -5 M tacrine, after separating the oxime from AChE by chromatography. It is tempting to speculate that tacrine, given as a pretreatment against the highly toxic nerve agent sarin, might exert part of its beneficial effect by slowing the rate of ageing of the inhibited acetylcholinesterase and thereby enhancing the effect of subsequent administration of an oxime to reactivate unaged enzyme and restore normal cholinergic function. However, tacrine at the concentration required (10 -5 M) would produce > 98 % reversible inhibition of acetyicholinesterase, based on the inhibition constants quoted above, and this would lead to toxic effects by itself. The concentration of tacrine in vivo that has been used in the treatment of Alzheimer's disease is in the range 10 - 7 M to 10 - 6 M [3]. The present results suggest that the results of Patocka and Bajgar [6] are not likely to be due to tacrine accelerating the rate of ageing. Two further very significant conclusions can be drawn from the results. An inspection of Fig. 1 reveals that all 4 regression lines have essentially the same intercept on the y-axis, i.e. enzyme activity is restored to the same level at zero time after oxime-induced reactivation and chromatography, regardless of the concentration of tacrine. This indicates firstly that sizeexclusion chromatography of the enzyme-oxime-tacrine mixture has separated enzyme not only from oxime (as it was designed to do), but also from tacrine. Had
230 tacrine not been separated from the enzyme, it would have inhibited bovine erythrocyte A C h E activity by 67% at l0 7 M, a n d 99% at 10 --5 M. C o n t r o l experiments showed that rat brain A C h E activity was also practically completely inhibited by 10 5 M tacrine. C o n s e q u e n t l y , tacrine c a n n o t be an irreversible i n h i b i t o r of ACHE, because if it were it would not have been separated from A C h E by c h r o m a t o g r a p h y . Separate experiments showed that the reversibility is not d e p e n d e n t on the presence of oxime. Secondly, tacrine did n o t prevent reactivation of sarin-inhibited A C h E by 2 - P A M or T M B-4. The results do n o t preclude some slowing of the rate of reactivation, and effects on this rate may have been observed had the reactivation period not been as long as 30 rain. However, a p r o n o u n c e d i n h i b i t i o n by tacrine of the rate of reactivation would have p r o d u c e d a lesser y-intercept than was observed (Fig. 1). Further, no retardation of reactivation by 0.1 m M T M B - 4 was observed when rat brain A C h E was studied in the presence of l0 5 M tacrine with a reactivation time of only 5 rain, a n d a temperature of 25 ° rather than 3T~C. Patocka and Bajgar reported that reactivation by T M B - 4 was practically abolished by 10 5 M tacrine u n d e r the same experimental c o n d i t i o n s and with an identical p r e p a r a t i o n of rat brain A C h E [6]. O u r observed lack of a p r o n o u n c e d i n h i b i t o r y effect on reactivation, and the reversibility of the t a c r i n e - A C h E complex, are therefore c o n t r a r y to the reports of Patocka et al. [6, 7]. The reasons for the discrepancies are u n k n o w n at this stage. I a m grateful to Mr. D. A m o s for synthesis o f sarin. I Bajgar, J., Patocka, J., Fusek, J. and ttrdina, V., Some possibilitiesof protection against acetylcholinesterase inhibition by organophosphates in vivo, Sb. Ved. Pr. Lek. Fak. Univ. Karlovy Hradci Kralove, 27 (1984) 425 435. 2 Dawson, R.M., Crone, HD., Bladen, M.P. and Poretski, M., A comparison of the effects of ionic strength on three preparations of acetylcholinesterasein the presence and absence of gallamine, Neurochem. Int., 3 (1981) 335 341. 3 Drukarch, B., Leysen, J.E. and Stool, J.C., Further analysis of the neuropharmacological profile of 9-amino-l,2,3,4-tetrahydroacridine (THA), an alleged drug for the treatment of Alzheimer's Disease, Life Sci., 42 (1988) 1011 1018. 4 Faurc, R., Giovannangeli, G., Gaby, J-P., Soyfer, J-C., Vincent, E-J. and Barbe, J., Etude par resonance magndtique nucl6aire du carbone 13 de quelques tetrahydro-l,2,3,4 acridines substitu6es en position 9, J. (;him. Phys., 78 (1981) 527 530. 5 Hsieh, J.Y.K., Yang, R.K. and Davis, K.L., High-performance liquid chromatographic determination of tetrahydroaminoacridine in human and rat tissue using a rapid Sep-Pak C~8extraction, J. Chromatogr., 274 (1983) 388 392. 6 Patocka, J. and Bajgar, J., The reactivation of o-isopropylmethylphosphonylated acetylcholinesterase and its modification by some non-acylating ligands, Sb. Ved. Pr. Lek. Fak. Univ. Karlovy Hradei Kralove, 27 (1984) 477 480. 7 Patocka, J., Bajgar, J., Bielavsky,J. and Fusek, J., Kinetics of inhibition of cholinesterases by 1,2,3,4tetrahydro-9-amino-acridine in vitro, Coll. Czech. Chem. Commun., 41 (1976) 816-824. 8 Steinberg, G.M., Mednick, M.L., Maddox, J. and Rice, R., A hydrophobic binding site in acetylcholinesterase, J. Med. Chem., 18 (1975) 1056 1061. 9 Summers, W.K., Majovski, L.V., Marsh, G.M., Tachiki, K. and Kling, A., Oral tetrahydroaminoacridine in long-term treatment of seniledementia, Alzheimer type, N. Engl. J. Med., 315 (1986) 1241-1245.