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`J Neural Transm (1994) [Suppl] 43: 219-225
`© Springer-Verlag 1994
`
`Pharmacological evaluation of phenyl-carbamates as eNS-selective
`acetylcholinesterase inhibitors
`
`M. Weinstock\ M. Razin\ M. Chore~, and A. Enz3
`Departments of 1 Pharmacology and 2 Pharmaceutical Chemistry, School of Pharmacy,
`Hebrew University Hadassah Medical Centre, Jerusalem, Israel
`3 Department of Preclinical Research, Sandoz Pharma Ltd, Basle, Switzerland
`
`Summary. The pharmacological and clinical properties of a novel phenyl
`carbamate acetylcholinesterase (AChE) inhibitor, SDZ ENA 713 are
`described. In animals and human subjects this compound showed superior
`chemical stability, oral bioavailability and a longer duration of action than
`physostigmine. SDZ ENA 713 produced a lO-fold greater inhibition of
`AChE in the hippocampus and cortex than in the heart and skeletal muscle,
`which explains its relatively low toxicity and freedom from cholinergic side
`effects. The selective effect in the cortex and hippocampus may be due to its
`preferential inhibition of the G1 form of the enzyme, which is present in
`relatively higher concentrations in these brain areas. Evidence of a selective
`hippocampal action was obtained in normal human subjects in whom REM
`sleep density was increased at doses that had no effect on plasma cholin(cid:173)
`esterase. If memory impairments in AD are related to a lack of cholinergic
`activity in cortical and hippocampal brain areas, SDZ ENA 713 should
`produce significant symptomatic improvement.
`
`Introduction
`
`Alzheimer's dementia (AD) is a severe degenerative disorder in which there
`is selective damage to neurons in the forebrain and hippocampus (White(cid:173)
`house et al., 1981). Although this results in significant changes in several
`neurotransmitters, the reduction in the number of cholinergic neurons and
`in choline acetyl-transferase, correlates best with the memory impairment
`(Sims and Bowen, 1983). Anticholinergic drugs also cause memory dis(cid:173)
`turbances and produce some of the symptoms of early dementia in normal
`human subjects (Drachmann and Leavitt, 1974). Thus, in the absence of a
`more specific treatment for AD, acetylcholinesterase (AChE) inhibitors
`have been tested in this and other forms of dementia in the hope that they
`would increase cholinergic transmission in the affected brain areas and
`improve cognitive function. In one study, which reported some beneficial
`effect with physostigmine, the extent of the improvement was correlated
`
`1
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`220
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`M. Weinstock et al.
`
`with the degree of cholinesterase inhibition in the spinal fluid and thus to
`the amount of the drug reaching the CNS (ThaI et aI., 1983). However, the
`narrow therapeutic window, unpredictable oral bioavailability and high
`incidence of side effects of physostigmine, resulting mainly from peripheral
`cholinergic hyperactivity (Christie et aI., 1981), prompted the search for
`safer AChE inhibitors with a more selective effect in the CNS.
`
`Derivatives of N-N-dimethylamino-ethyl-phenyl carbamate
`
`For this purpose we prepared a series of N-monoalkyl and N,N-dialkyl
`derivatives of m-[1-(N,N-dimethylamino)-ethyl] phenyl carbamate, many of
`which are more lipid soluble and show greater chemical stability than
`physostigmine (Weinstock et aI., 1986). Their inhibitory activity was evalu(cid:173)
`ated in vitro on a purified preparation of human erythrocyte AChE, and on
`whole brain AChE after subcutaneous injection to mice. The concentration
`of each drug that would inhibit by 50% the activity of the enzyme prepara(cid:173)
`tion was determined by the method of Ellman et a1. (1961). From ex vivo
`measurements, the dose of each drug was also computed that would block
`total brain enzyme activity by 50% (ED so) , when this reached its peak after
`injection (Weinstock et aI., 1986, 1992). The most potent inhibitors of the
`human erythrocyte enzyme were the mono- and dimethyl derivatives.
`Increasing the length of the alkyl chain to ethyl resulted in an 80-fold
`reduction in inhibitory potency in both mono- and di-alkyl derivatives. This
`was gradually restored in the former, to values similar to that of the methyl
`derivative, as the chain lengthened from n-propyl, through n-butyl to n(cid:173)
`hexy1. In general, this relationship between chemical structure and anti(cid:173)
`AChE potency was maintained in the whole animal, the exception being the
`I-isomer of the methyl-ethyl derivative, SDZ ENA 713, (formerly RA7,
`Weinstock et aI., 1986) which was about 10 times more potent in vivo than
`would be expected from its activity on red cell AChE (Weinstock et aI.,
`"1 aG-"';
`t ~,' ,/ ."::'/ "
`
`~~ .
`
`Acute toxicity of phenylcarbamates
`
`Acute toxicity was assessed after sc. injection of the carbamates in mice.
`Therapeutic ratios (LDso/EDso) were derived, where LDso is the dose that
`is lethal in 50% of mice. The monomethyl, dimethyl and methyl-ethyl der(cid:173)
`ivatives were the least toxic. Increasing the chain length, or introduction of
`a branched chain, e.g. isopropyl or sec-butyl resulted in greater toxicity
`(Weinstock et aI., 1992).
`Death from overdose of anti-AChE is due to respiratory arrest, which
`itself results from a combination of excessive stimulation of muscarinic
`receptors in the respiratory centre in the brainstem and paralysis of the
`respiratory muscles (depolarization blockade via nicotinic receptors)
`(Machne and Unna, 1963). The former can be prevented by the admini-
`
`I.,
`
`1 y
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`SDZ ENA-713, a CNS-selective acetylcholinesterase inhibitor
`
`221
`
`ATROPINE PROTECTION IN MICE
`
`12
`
`9
`
`6
`
`ENA 713
`
`I:::=J monosub.
`mm branched
`
`_ disub.
`
`C1 C2 C3 C4 C6 C3 C4 C6 C1 C2 C3 C4
`
`LENGT H OF SIDE CHAIN
`
`Fig. 1. Increase in LDso of SDZ ENA 713 by pretreatment of mice with atropine.
`Ordinate: Ratio of LDso in the presence and absence of atropine (S mg/kg) Abcissa:
`No. of C atoms in alkyl substituent on carbamate. Open columns: monosubstitued;
`filled columns: disubstituted; crossed hatched columns; branched side chain. * SDZ
`ENA 713
`
`stration of a centrally-active muscanmc antagonist such as atropine or
`scopolamine. On the other hand, muscle paralysis cannot be antagonised
`by drugs. The relative contributions of excess central muscarinic receptor
`stimulation and muscle paralysis towards the lethal effects of anti-AChE
`were assessed by comparing the acute toxicity of the phenyl carbamates
`with and without pretreatment by atropine (5 mg/kg). Muscarinic receptor
`blockade resulted in a 2-3.5-fold increase in LD50 in physostigmine and in
`all the mono-alkyl and branched-chain derivatives. This rose to 5-6-fold
`in the dimethyl, methyl-propyl and methyl-butyl derivatives and to 12-fold
`in the methyl-ethyl ~Omp0Ui.id, SDZ ENA 713 (Fig. 1). This finding sug(cid:173)
`gested that SDZ ENZ 713 inhibited brain AChE at much lower dose levels
`that those needed to paralyse the respiratory muscles and was therefore a
`potentially much safer drug than the other derivatives or physostigmine.
`
`AChE inhibition in different regions of rat brain
`
`After oral administration to rats, SDZ ENA 713 caused a preferential
`inhibition of AchE in the cortex and hippocampus, in comparison with
`other brain regions. This is shown in Fig. 2. The relatively weak inhibition
`in the heart and skeletal muscle, that was suggested by the atropine pro(cid:173)
`tection experiment, can readiliy be seen in the same animals (Table 1). In
`contrast, physostigmine inhibited the enzyme in all brain areas and in the
`periphery at similar doses. Direct measurements of the concentrations of
`ACh produced in the CNS by increasing doses of SDZ ENA 713, also
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`M. Weinstock et al.
`
`ACh LEVELS IN RAT BRAIN
`AFTER ENA 7 13 P.O.
`
`60
`
`50
`
`-l
`
`W >
`~
`..c o «
`z
`
`t75 « w a:
`~
`
`-er- Cortex
`
`·--0--- Hippocampus
`
`--w-- StriatLm
`
`DOSE (umoles/kg p.o.)
`
`Fig. 2. Inhibition of AChE in different regions of rat brain by SDZ ENA 713. Brains
`were removed 30 min after oral administration of the drug or saline. Inhibition of
`AChE represented as % of value in saline treated controls
`
`EX VIVO AChE INHIBITION
`IN RAT BRAIN REGIONS
`
`w
`<5 «
`LL o
`Z o
`i=
`ill
`I
`z
`
`100
`
`80
`
`60
`
`40
`
`20
`
`-er- Cortex
`
`---0--- Hippocampus
`
`--"'-- Pons/medJlla
`
`--w-- StriatLm
`
`OL---'---'.....-=:~...J-~~~ ......... _~
`0.1
`10
`
`DOSE (umoles/kg P.o)
`
`Fig. 3. Increase in acetylcholine levels in different brain regions by SDZ ENA 713.
`Measurements were made 30 min after oral administration of the drug or saline. Levels
`of acetylcholine expressed as a % of saline control
`
`confirmed a preferential increase in the cortex and hippocampus, which
`correlated well with the AChE inhibition (Fig. 3).
`The reason for the relatively greater inhibition of AChE in the cortex
`and hippocampus than in the striatum or medulla is not yet clear. However,
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`SDZ ENA-713, a CNS-selective acetylcholinesterase inhibitor
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`Table 1. AChE inhibition ex vivo in rat by physostigmine
`and SDZ ENA 713
`
`Brain region or organ
`
`Physostigmine
`ICso (J.1moles/kg)
`
`SDZ ENA 713
`ICso (J.1moles/kg)
`
`Cortex
`Hippocampus
`Pons/medulla
`Striatum
`Heart
`Skeletal muscle
`
`1.20
`1.31
`1.44
`1.25
`1.23
`1.26
`
`2.69
`3.89
`7.21
`7.12
`38.4
`32.7
`
`Table 2. Inhibition of AChE forms in human cortex
`
`Inhibitor
`
`G4 from
`ICsoJ.1M
`
`G1 from
`ICsoJ.1M
`
`G4/G1
`
`SDZENA 713
`Physostigmine
`Tacrine
`
`13.3 ± 1.7
`0.041 ± 0.003
`0.97 ± 0.07
`
`3.15 ± 0.42
`0.037 ± 0.004
`1.02 ± 0.13
`
`4.2
`1.1
`0.9
`
`it is known that AChE exists in multiple molecular forms, which have
`similar catalytic activity (Massoulie and Bon, 1982). The globular tetrameric
`from (G4) and the monomeric form (G1), which sediment in a sucrose
`gradient with coefficients of approximately lOS and 4S respectively, are the
`predominant forms in human and rat brain. The proportion of these two
`forms varies in different brain areas. In human brain, G1 makes up less
`than 1 % of the total AChE in the caudate nucleus, but is about 17% in the
`hippocampus and nearly 50% in different parts of the cortex (Atack et aI.,
`1986). In contrast to physostigmine and tacrine, which block both forms
`;;dth equal efficiency, SDZ ENA 713 is 6 times more potent in inhibiting the
`G1 than the G4 form in human brain (Table 2) (Enz et aI., 1992). This
`selective effect on G1 could explain why SDZ ENA 713 is more effective in
`inhibiting AChE in the cortex and hippocampus than in the striatum. In the
`brains of Alzheimer patients there is a decrease in the G4 form in the cortex
`and hippocampus, with little or no change in the G1 form (Atack et aI.,
`1983; Siek et aI., 1990). A preferential inhibition of the G1 form in AD may
`therefore be advantageous.
`Further evidence in favour of a selective effect in the hippocampus
`was based on the finding that SDZ ENA 713, in doses ranging from
`0.08-0.8 mg/kg, also induced synchronization of hippocampal theta wave
`activity in the rat. This resulted from increased muscarinic activity in that
`area, consequent to AChE blockade (Enz et aI., 1989).
`
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`Selective action of SDZ ENA 713 in the eNS of human subjects
`
`The preferential effect of SDZ ENA 713 in those brain areas most affected
`in AD, together with its high oral bioavailability and relatively long duration
`of action (Weinstock et aI., 1986), led to its evaluation in human subjects.
`Studies in normal volunteers showed that in oral doses below 1.5 mg, SDZ
`ENA 713 had no significant effect on plasma butyryl cholinesterase. At
`higher ENA doses, it caused a dose-dependent inhibition, with a peak of
`30% after 1-2 hours, at a dose of 4.6mg, which lasted for about 10 hours
`(Enz et aI., 1991).
`SDZ ENA 713 was also found to have a selective effect in the human
`hippocampus. At a dose of 1.3 mg, that did not inhibit plasma cholinester(cid:173)
`ase, or produce signs of cholinergic hyperactivity, it increased REM sleep
`density by about 50% (Holsboer-Trachsler et aI., 1993). The rise in REM
`sleep density is believed to be due a selective action in the hippocampus and
`areas associated with memory consolidation (Smith and Lapp, 1991) and it
`has been suggested that a reduction in REM sleep density may occur in
`relation to cognitive impairment (Feinberg, 1969).
`So far, studies in laboratory animals and healthy human subjects have
`demonstrated that SDZ ENA 713 may possess several advantages over
`existing AChE inhibitors, which have already been tested for their ability to
`improve memory and cognitive impairments in AD patients or prevent their
`further deterioration (Davis and Mohs, 1982; Davis et aI., 1992). It is well
`absorbed after oral administration and produces consistent, selective, long(cid:173)
`lasting inhibition of AChE in those brain areas which show the greatest
`decrements in cholinergic neurones in AD. This is achieved at doses which
`produce minimal inhibition of peripheral cholinesterase and a low incidence
`of cholinergic side effects. If the lack of cholinergic transmission in cortical
`and hippocampal brain areas is responsible for the cognitive impariments in
`AD, one might expect this drug to produce significant symptomatic im(cid:173)
`provement. It is currently undergoing clinical evaluation in patients with
`Alzheimer's disease.
`
`References
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`acetylcholinesterase and butyrylcholinesterase in the aged human central nervous
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`Christie JE, She ring A, Fergusen 1, Glen AIM (1981) Physostigmine and arecholine:
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`Davis KL, Mohs RC (1982) Enhancement of memory processes in Alzheimer's disease
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`Drachman DA, Leavitt 1 (1974) Human memory and the cholinergic system. Arch
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`¢
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`Authors' address: Dr. M. Weinstock, Department of Pharmacology, School of
`Pharmacy, Hebrew University Hadassah Medical Centre, Jerusalem, Israel.
`
`~
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