Use of a novel radiometric method to assess the inhibitory effect of donepezil on acetylcholinesterase activity in minimally diluted  tissue samples.
OBJECTIVE
Cholinesterase inhibitors have been widely used for the treatment of patients with dementia. Monitoring of the cholinesterase activity in the blood is used as an indicator of the effect of the cholinesterase inhibitors in the brain. The selective measurement of cholinesterase with low tissue dilution is preferred for accurate monitoring; however, the methods have not been established. Here, we investigated the effect of tissue dilution on the action of cholinesterase inhibitors using a novel radiometric method with selective substrates, N-[(14)C]methylpiperidin-4-yl acetate ([(14)C]MP4A) and (R)-N- [(14)C]methylpiperidin-3-yl butyrate ([(14)C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively.


METHODS
We investigated the kinetics of hydrolysis of [(14)C]-MP4A and [(14)C]-MP3B_R by cholinesterases, and evaluated the selectivity of [(14)C]MP4A and [(14)C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC(50) values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [(14)C]MP4A and [(14)C]MP3B_R.


RESULTS
AChE and BChE activities were selectively measured as the first-order hydrolysis rates of [(14)C]-MP4A and [(14)C]MP3B_R respectively. The AChE selectivity of [(14)C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC(50) values of specific AChE and BChE inhibitors, donepezil and ethopropazine, in 1.2-fold diluted human whole blood were much higher than those in 120-fold diluted blood. In addition, the IC(50) values of donepezil in monkey brain were dramatically decreased as the tissue was diluted.


CONCLUSIONS
This method would effectively monitor the activity of cholinesterase inhibitors used for therapeutics, pesticides and chemical warfare agents.
Kikuchi
Tatsuya
T
Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, Japan. kiku@nirs.go.jp <kiku@nirs.go.jp>
Okamura
Toshimitsu
T
Arai
Takuya
T
Obata
Takayuki
T
Fukushi
Kiyoshi
K
Irie
Toshiaki
T
Shiraishi
Tetsuya
T
D000110:Acetylcholinesterase / Q000187:drug effects* / Q000378:metabolism; D000328:Adult; D002091:Butyrylcholinesterase / Q000187:drug effects / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D000077265:Donepezil; D006801:Humans; D006868:Hydrolysis; D007189:Indans / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D010880:Piperidines / Q000378:metabolism / Q000494:pharmacology*; D011874:Radiometry / Q000379:methods*
D002800:Cholinesterase Inhibitors; D007189:Indans; C471399:N-methylpiperidin-4-yl acetate; D010880:Piperidines; D000077265:Donepezil; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase
Ann Neurol. 2000 Sep;48(3):391-5
10976649
J Clin Psychopharmacol. 2002 Dec;22(6):615-20
12454562
J Neurosci. 1983 Apr;3(4):871-6
6834109
Int J Clin Pharmacol Ther Toxicol. 1993 May;31(5):223-9
8314652
Biochem J. 2001 Aug 1;357(Pt 3):795-802
11463350
Fundam Appl Toxicol. 1993 Aug;21(2):196-203
8405782
J Nucl Med. 2004 Dec;45(12):2032-9
15585478
J Pharmacokinet Biopharm. 1977 Oct;5(5):513-31
925885
Anal Chim Acta. 2008 Nov 23;629(1-2):145-57
18940331
Eur J Pharmacol. 1999 Dec 10;386(1):7-13
10611458
J Cereb Blood Flow Metab. 2001 Feb;21(2):132-43
11176278
J Nucl Med. 1996 Apr;37(4):649-55
8691261
Toxicol Appl Pharmacol. 2007 Mar;219(2-3):202-9
17197007
Neuropsychopharmacology. 2005 Dec;30(12):2154-61
15920507
Anal Biochem. 1974 Feb;57(2):588-92
4856500
Br J Clin Pharmacol. 1998 Nov;46 Suppl 1:13-8
9839760
J Pharm Pharm Sci. 1999 Jan-Apr;2(1):23-9
10951659
Biochem Pharmacol. 1983 Sep 1;32(17):2565-70
6137223
Br J Clin Pharmacol. 2004 Nov;58 Suppl 1:18-24
15496219
J Med Chem. 2008 Jun 26;51(12):3588-98
18517184
Clin Chim Acta. 1999 Oct;288(1-2):73-90
10529460
J Pharm Pharm Sci. 1999 May-Aug;2(2):39-46
10952768
Nucl Med Biol. 2003 Jul;30(5):491-500
12831986
Eur J Pharmacol. 1994 Apr 4;270(2-3):189-93
8039548
Eur J Pharmacol. 2000 Feb 18;389(2-3):173-9
10688981
Food Cosmet Toxicol. 1969 Mar;7(2):149-51
5822870
Alzheimer Dis Assoc Disord. 2000 Oct-Dec;14(4):216-27
11186600
Jpn J Pharmacol. 2002 May;89(1):7-20
12083745
Biochem Pharmacol. 1961 Jul;7:88-95
13726518
Bioorg Med Chem Lett. 2004 Apr 19;14(8):1927-30
15050629
Biol Pharm Bull. 1995 Aug;18(8):1145-7
8535413
Methods Find Exp Clin Pharmacol. 2000 Oct;22(8):609-13
11256231
Biochem J. 1997 Nov 1;327 ( Pt 3):747-57
9581552
Biochem J. 1966 Oct;101(1):127-34
5971773
Lancet. 1997 Jun 21;349(9068):1805-9
9269216
Arch Intern Med. 1998 May 11;158(9):1021-31
9588436
J Neurochem. 1988 Oct;51(4):1227-31
2901462
J Med Chem. 2005 Apr 7;48(7):2577-83
15801847
J Neurol Neurosurg Psychiatry. 2005 Mar;76(3):315-9
15716518
Chem Biol Interact. 2008 Sep 25;175(1-3):298-302
18555980
Br J Pharmacol Chemother. 1959 Dec;14:559-66
13821613