Note: Descriptions are shown in the official language in which they were submitted.
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Title: Improvements in or Relating to Protease Inhibitors
Introduction
With an ageing population in most Western countries the incidence of
Alzheimer's disease
(AD) is expected to increase into the next millennium (Brody 1985 Nature 315,
463-466).
Complementing research into cures for the disease, the development of drugs to
combat
the cognitive and behavioral effects of AD will become increasingly important
(Tarriot et
al, 1997 Postgrad. Medicine 101, 73-76).
A consistent feature of AD is reduced cholinergic activity in the brain,
largely as a result
of the reduction in forebrain cholinergic neurons (Whitehouse et al, 1982
Science 215,
1237-1239). Decreases in cholinergic activity have been associated with the
progression
and severity of AD (Bierer et al, 1995 J. Neurochem. 63, 749-760). Thus, the
use of
drugs that potentiate cholinergic function have been advocated as an aid to
improve deficits
in cognitive function in AD patients. Direct inhibitors of the enzyme
acetylcholinesterase
(AChE) are currently the most effective strategy in attaining cholinergic
potentiation.
Early cholinergic potentiators examined included physostigmine and tacrine,
both of which
caused severe side effects. The development of 'second generation'
cholinesterase
inhibitors, such as ENA 713, E2020 (Donepezil) and metrifonate (dichlorvos),
offers
greater therapeutic potential through greater selectivity for AChE with fewer
adverse
reactions.
Metrifonate is a prodrug for the organophosphorus ("OP") compound dichlorvos
(2,2'dichlorovinyl dimethyl phosphate; DDVP), which is formed by the slow, non-
enzymatic hydrolysis of the parent compound {Hint et al, 1996 Neurochem.
Research 21,
331-337). Metrifonate has been used for a long time as a treatment for
schistosomiasis,
and dichlorvos is a widely-used domestic and agricultural pesticide.
Metrifonate has a low
toxicity in warm-blooded animals with good selectivity, and long in vivo half
life and as
such is currently one of the most effective potential AD therapeutics.
Metrifonate has been
the subject of a double-blind placebo study (Becker et al, 1996 Alzheimer
Disease and
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Associated Disorders 10, 124-131) the results of which showed a slight but
significant
increase in cognitive function in the treated group compared to the control.
Subsequent
double blind trials confirmed these findings (Cummings et al, 1998 Neurology
50, 1214-
1221; Morris et al, 1998 Neurlogy 50, 1222-1230). The therapeutic level of
metrifonate
has been established at -- SO % inhibition of AChE in an open trial (Becker &
Giacobini,
1990 Drug Dev. Res. 19, 425-435).
The central tenet of the 'cholinergic hypothesis' of AD treatment has however
recently
been questioned. Improvements in shuttle box and Morris-maze escape behavior
in young-
adult rats were demonstrated at doses of metrifonate and dichlorvos
insufficient to cause
significant inhibition of brain AChE (van der Staay et al, 1996 Behavioural
Pharmacology
7, 56-64; van der Staay et al, 1996 J. Pharmacol. Exp. Therapeutics 278, 697-
708).
It appears that if the pharmacological spectrum of metrifonate compounds is to
be
broadened new molecular mechanisms of action have to be sought. The potential
for OP
compounds to react with a wide range of serine-hydrolases in the CNS has long
been
appreciated (O'Neill, 1981 Fundamental and Applied Toxicology l, 154-160;
Aldridge,
1964 Biochem. J. 93, 619-623). However, the possibility that previously
unrecognised
target-proteins can be significantly inhibited by OPs in vivo has not been
systematically
investigated.
Serine peptidases of the prolyl oligopeptidase family; N acylpeptide
hydrolase, (ACPH);
prolyl oligopeptidase (also known as post-proline cleaving enzyme, or PPCE);
and
dipeptidyl peptidase IV, (DPP IV), react with the organophosphorus compound,
diisopropylfluorophosphate (DFP) in vitro (Kato et al, 1980 J. Neurochem. 35,
527-535).
Mammalian (and especially, human) ACPH has been studied quite extensively
(see, for
example, Jones & Manning 1985 Biochem. Biophys. Res. Comm. 2, 933-940; Jones &
Manning 1986 Biochem. Biophys. Res. Comm. 1, 244-250; Jones & Manning 1988
Biochim. Biophys. Acta 953, 357-360; Jones et al, 1991 Proc. Natl. Acad. Sci.
USA 88,
2194-2198; Scaloni et al, 1994 269, 15076-15084; and Raphel et al, 1993
Biochimie 75,
891-897). The activity of ACPH is to cleave N-acylated amino acid residues in
peptides.
However, the exact function of ACPH is not firmly established, although a
possible rote
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is in the degradation of hormonal and neuropeptides (Mentlein, 1988 FEBS Lett.
234, 251-
256). Changes at the genetic and protein levels within members of the prolyl
oligopeptidase family are possibly associated with a number of disorders
including AD
(Mantle et al 1996, Clin Chem. Acta 249, 129-139) depression (Maes et al, 1994
Biol.
Psychiatry 35, 545-552), autoimmunity (Aoyagi et al, 1987 Biochem. Int. 18,
383-389)
and cancer (Scaloni et al, 1992 J. Lab. Clin. Med. 120, 546-552; Erlandsson et
al, 1991
Oncogene 6, 1293-1295).
Summary of the Invention
The present inventors have surprisingly found that cognitive enhancement noted
in
mammalian subjects upon consumption of certain substances is due, not to
inhibition of
acetyl cholinesterase (AChE) as is conventionally suggested, but due to
inhibition of a
different enzyme, acylpeptide hydrolase (ACPH).
Thus, in a first aspect the invention provides a method of manufacturing a
medicament for
use in providing cognitive enhancement in a mammalian subject, the method
comprising
mixing a substance which inhibits acylpeptide hydrolase (ACPH) with a
physiologically
acceptable carrier, excipient or diluent.
In a second aspect, the invention provides a medicament for use in providing
cognitive
enhancement in a mammalian subject, the medicament comprising a substance
which
inhibits ACPH, and a physiologically acceptable carrier, excipient or diluent.
Cognitive enhancement may be defined for present purposes as a measurable
improvement
in a cognitive ability of a mammalian subject. Methods and means of measuring
cognitive
abilities of experimental laboratory animals, such as rats, are well-known to
those skilled
in the art (e.g. shuttle boxes, Morris-mazes etc). Similarly, there are
methods of
measuring the cognitive abilities of human subjects (e.g. as employed by
Becker et al,
1996 Alzheimer Disease and Associated Disorders 10, 124-131) which are well
known to
those skilled in the art. A number of tests have been used to investigate the
cognitive
abilities of Alzheimer's Disease patients in clinical trials to assess the
effectiveness of drug
therapies. Examples include the "Alzheimer Disease Assessment Scale" (ADAS-
Cog)
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(Rosen et al, Am. J. Psychiatry 1984 141, 1356-1364) and the "Mini Mental
State
Examination" (MMSE) (Rosen et al, J. Psychiatric Res. 1975 12, 189-198).
Cognitive
enhancement is detected by a statistically significant improvement (e.g. p <
0.01) in the
test group receiving the drug compared to a control group, as measured by an
appropriate
statistical test (e.g. Student's T test).
There are numerous physiologically acceptable carriers, excipients or diluents
known to
those skilled in the art of formulating pharmaceuticals. The preferred
carrier, excipient
or diluent may depend on the route by which the medicament is to be
administered. A
preferred route of administration is the oral route, such that the medicament
may take the
form of a capsule or tablet. Alternatively, and less preferably, the
medicament may be
administered, for example, by injection infra-venously, sub-cutaneously, infra-
muscularly
or infra-peritoneally. When the medicament is to be administered by injection,
the
substance which inhibits ACPH will preferably be mixed with a liquid diluent,
such as
sterile saline or phosphate-buffered saline solution and the like.
ACPH activity can be measured by means of, for example, the in vitro assay
method
disclosed herein. An ACPH inhibitor suitable for use in the present invention
will
generally (but not necessarily) be a substance which is capable of causing at
least 50 %
reduction of in vitro ACPH activity at a concentration of less than 10
millimolar.
Preferably the inhibitor will be selective for ACPH (i.e. will exhibit greater
inhibitory
activity for ACPH than for AChE in particular and other mammalian enzymes in
general).
Desirably the inhibitor will cause at least 50% inhibition of in vivo ACPH
activity whilst
causing less than 50% inhibition of AChE (preferably less than 30%, most
preferably less
than 20% and most preferably less than I0% inhibition of AChE). More
preferably the
inhibitor will cause over 75 %, most preferably over 90 % inhibition of in
vivo ACPH
activity, whilst causing less than 10% inhibition of AChE.
The substance which inhibits ACPH may be any substance which, when
administered to
the subject, inhibits ACPH whilst at a sufficiently low concentration to avoid
severe
adverse reaction. For example, the inhibitor may be an organophosphorus (OP)
compound
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such as metrifonate (or its metabolite, dichlorvos). Where the inhibitor is
metrifonate, the
medicament will preferably comprise metrifonate at a lower concentration than
has been
used in the clinical trial reported by Becker et al, such that administration
of suitable doses
of the medicament will lead to a concentration of dichlorvos in the subject
which inhibits
ACPH whilst causing less than 50 % (preferably less than 30 % , more
preferably less than
20% and most preferably less than 10%) inhibition of AChE. Typically an OP-
based
ACPH inhibitor compound will conform to the general formula:
(R10), (R20) P (O) X
wherein R1 and RZ are each, independently, substituted or unsubstituted C1-C6
alkyl
(preferably, C1, or C2 or C3) and where X is a relatively electronegative
leaving group.
Conveniently X contains one or more halides. Examples of suitable leaving
groups for
X include 3,5,6-trichloro-2-pyridinyl (Cf. chlorpyrifos) and 2,2-dichloroethyl
(Cf.
dichlorvos. The inventors consider that the degree of electronegativity of the
leaving
group primarily determines, or at least influences, the potency of the
inhibitor, whilst the
overall structure of the inhibitor compound determines its selectivity.
Alternatively, the inhibitor may be other than an OP compound, e.g. comprises
a peptide
or polypeptide. In particular, the inhibitor may comprise an analogue of the
ACPH
substrate, such as an acylpeptide. Peptides with N-acetyl alanine, methionine,
or serine
residues represent suitable examples. Of these, N-acetyl alanine is preferred,
as
methionine residues are prone to oxidation and the -OH group of serine is
liable to
modification during synthesis of the ACPH inhibitor. Such substrate analogues
may
comprise 1 to 50 amino acids, but shorter peptides are more convenient, being
cheaper
and easier to make. Typically the substrate analogue will comprise less than
30 amino
acid residues, and preferably less than 10 residues. Those skilled in the art
will appreciate
that any amino acid residue may be positioned adjacent to the N-acetyl
residue. In
addition, the substrate analogue may, and preferably will, comprise non-
peptide moieties,
for example to reduce toxicity, and/or to increase selectivity and/or potency
of ACPH
inhibition.
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Substrate analogues, whilst readily synthesised and potentially very
selective, are not ideal
for therapeutic use in that they bind to ACPH in a non-covalent manner.
Accordingly,
their ability to inhibit ACPH is very concentration-dependent. Preferred
inhibitors may
therefore act as irreversible inhibitors of ACPH and bind covalently thereto,
such
inhibition being essentially [inhibitor]-independent. Examples include well-
known
peptidase inhibitors, such as Chloro-methyl-ketones. Such compounds probably
possess
too much toxicity to be used therapeutically, but those skilled in the art can
readily screen
other compounds, based on common general knowledge and with the benefit of the
present
disclosure, to find alternative ACPH inhibitors with acceptably low toxicity
(e.g. which
inhibit ACPH effectively without causing too much inhibition of AChE). In a
particular
embodiment, the inhibitor will comprise an N-acyl (especially N-acetyl)
peptide substrate
analogue portion, and an irreversible peptidase inhibitor portion; such as an
N-acetyl-
alanine-containing portion covalently linked to: chloromethyl-ketone; or an
aldehyde; or
a phosphate/phosphonate group; or boronic acid.
The medicament will conveniently be such that a suitable dose provides in the
range 0.5-
50 mgs of ACPH inhibitor per Kg body weight of subject, per day. Preferably,
the
amount of ACPH inhibitor is in the range 1-10 mgs per Kg body weight per day,
although
the precise dose will clearly depend, inter alia, on the efficacy and toxic
effects (if any)
of the ACPH inhibitor employed, and the route of administration.
The medicament of the invention may find particular usefulness in prevention
and/or
treatment of neurodegenerative diseases which affect cognitive function.
Alzheimer's
Disease (AD) is a well-known example of such a disease, and there is a great
unfilled need
for a suitable prophylactic and/or therapeutic drug for AD. It is known that
there is a
genetic factor involved in pre-disposition to AD, so the medicament could be
given to
individuals with a family history of the condition.
Alternatively the medicament could be given to individuals, particularly those
involved in
mental exercise, who wish to obtain cognitive enhancement. In particular, the
evidence
available from studies on metrifonate/dichlorvos, suggests that learning
processes can be
accelerated, so individuals undergoing tuition may benefit from taking the
medicament.
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Preferably the medicament of the invention is administered to a human subject.
The
invention thus provides for use of an ACPH inhibitor to provide cognitive
enhancement
in a human subject and, in particular, a method of preventing and/or treating
Alzheimer's
disease in a human subject, the method comprising administering an ACPH
inhibitor
(typically as the medicament of the invention defined above).
The invention also provides a method of a method of screening substances for
use as
active ingredients in a medicament for causing cognitive enhancement in a
human subject,
the method 'comprising the steps of: testing the substance for ability to
inhibit ACPH;
testing the substance for ability to inhibit AChE; and selecting those
substances which
exhibit greater inhibitory activity for ACPH than for AChE. In particular, the
screening
method may comprise: mixing ACPH with the compound to be screened, adding a
substrate of ACPH which, upon digestion with ACPH gives rise to a detectable
signal;
measuring the signal; and thus determining the amount of ACPH activity.
Typically the
substrate will be such that a colour change takes place upon its digestion
with ACPH,
enabling the signal to be measured spectrophotometrically. Suitable assays of
ACPH
activity are known to those skilled in the art, and one such assay is
disclosed herein. Such
assays have not hitherto been used for the purpose of screening a compound for
use in a
medicament to provide cognitive enhancement.
In a further aspect the invention provides for the use of an ACPH inhibitor,
especially a
selective serine peptidase/hydrolase inhibitor, in a medicament for causing
cognitive
enhancement in a human subject or preventing andlor treating Alzheimer's
disease.
The invention will now be further described by way of illustrative examples
and with
reference to the accompanying drawings, in which:
Figure 1 is a graph of enzyme {ACPH or AChE) activity against time; and
Figure 2 is a bar chart showing % labelling of various polypeptides in rat
brain
homogenates at different concentrations of DFP.
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ExamQes
Example 1 '
Materials and Methods
Dichlorvos was obtained from ChemServ (PO Box 3108, West Chester, PA 19381-
29941
USA). a-melanocytestimulatinghormone(a-MSH), N acetylalanyl-p-nitroanilide,
glycine-
proline-AMC and fluorescamine, were obtained from Sigma (Poole, Dorset U.K.).
Z-
glycine-proline-AMC was obtained from Bachem (AMC = amino methyl coumarin; Z =
benzyloxycarbonyl).
In vivo Inhibition
Male, Fisher 344 rats ( 180-220g) were used. The animals were housed under
standard
conditions. Rats were given i.p. doses of water or dichlorvos dissolved in
water at 0.1
ml/100 g body weight. Initial dosing experiments showed a reduction of brain
AChE
activity of --50% with an i.p. dose of 4mglkg; the same concentration was used
for
subsequent dosing experiments. Rats were killed with an over-dose of
anaesthetic after
l, 4, 8, 24, 48 and 120 hours and the brains dissected out onto ice and assays
conducted
within 4 hours of death.
Brain homogenates were prepared in 10 volumes of 10 mM Tris/HCI, pH 7.4 or
O.1M
2[N-morpholino] ethanesulphonic acid (MES) pH 6Ø For peptidase assays 1mM
DTT
was added to the homogenate. AChE activity was measured using the Ellman assay
(Ellman et al, 1961 Biochem. Pharm. 7, 88-95). The brain homogenate was
further diluted
x 10 in water, and 20~c1 of this diluted homogenate was added to a solution of
DTNB
(0.375 mIvl) and ACTI (0.583 mM), total volume 1 ml. The change in absorbance
at 405
nm was recorded at 37°C.
To assay ACPH activity, 30.1 of the 10% homogenate was added to 1 ml of 4mM N
acetyl-alanyl-p-nitroanilide (NAANA) in 0.2M Tris/HCl pH 7.4, 1 mM DTT and the
change in absorbance at 405 nm recorded at 37°C.
To assay DPP IV activity, 30.1 of 10% homogenate was added to 1 ml of O.SmM
Gly-Pro-
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p-nitroanilide in SOmM Tris/HCl pH 7.4, 1mM DTT and the change in absorbance
at 405
nm recorded at 37°C.
PCCE activity was measured using 0.25mM Z-Gly-Pro-AMC in SOmM Tris/HCl pH 7.4,
1 mM DTT and the change in fluorescence recorded at an excitation wavelength
of 383 nm
and an emission wavelength of 455 nm at 37°C.
One unit of enzyme activity (U) is defined as the amount of enzyme required to
hydrolyse
l~c mole of substrate/min.
Total protein was measured using the Bio-Rad DC assay.
In vitro inhibition of peptidase activity
Portions of brain homogenate, prepared as described above, were incubated with
different
concentrations of the relevant inhibitor at 37°C and aliquots removed
and assayed for
residual peptidase activity as above. The pseudo-first order rate, Kobs, for
the individual
reactions was calculated from the slope of graphs of In % activity remaining
plotted against
time. Overall second-order rate constants, Ki, were calculated from the slope
of graphs of
Kobs ag~nst inhibitor concentration.
Purification of ACPH
ACPH was purified essentially as described previously (Jones & Manning, 1985
Biochem.
Biophys. Res. Comm. 126, 933-940), except porcine brain was used as the tissue
source.
A final purification by Mono-Q FPLC was performed using a linear gradient of 0-
0.7M
NaCI in IOmM citrate, O.SmM DTT, pH 6.0, 30 minutes at a flow rate of 0.5
ml/min.
ACPH activity eluted as a single peak at 0.53-0.60 M NaCI, which corresponded
to single
uv-absorbing peak on the elution profile. Aliquots were stored at -20°C
until required.
Reactivation of ACPH activity following inhibition by dichlorvos
Purified ACPH (5~,1, 0.11 Units) was added to an equal volume of 4mM
dichlorvos in 0.1
M sodium phosphate pH 7.0 for 15 min. at 37°C to give 80% inhibition of
activity. The
resulting solution was diluted 200 fold in 0.1 M sodium phosphate pH 7.0 at
37°C and
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aliquots removed at various time intervals and assayed for ACPH activity.
Results were
plotted as In % remaining inhibition vs time.
Hydrolysis of a-MSH by ACPH
a-MSH was dissolved in 0.2 M potassium phosphate, 0.2 M NaCI, pH 7.4. Purified
ACPH
was added and the mixture incubated at 37°C, aliquots (SO~cI) were
removed at various time
intervals and assayed for the release of free amino groups using the fluram
assay (Tones &
Manning 1985 Biochem. Biophys. Res. Comm. 126, 933-940).
Inhibition of ACPH activity by a-MSH
a-MSH (SSp,M, final concentration) was added to various concentrations of
NAANA in 0.2
M Tris/HCI, pH 7.4. Purified ACPH was added and hydrolysis of the substrate
monitored
as described above. Control reactions were also performed in the absence of a-
MSH.
Kinetic constants for the hydrolysis of NAANA by ACPH were determined in the
presence
and absence of inhibitor.
Results
The active product from metrifonate hydrolysis, dichlorvos, was chosen to
screen in vitro
reactivities of the various enzymes examined in this study. This allows direct
comparison
of rate constants without having to consider pro-drug activation, facilitating
direct and
accurate kinetic comparisons. Metrifonate has been shown to induce cholinergic
inhibition
exclusively via slow release of dichlorvos.
In vitro screening of brain homogenates with dichlorvos revealed large
differences in rate
constants of inhibition with four enzymes tested (Table 1). Thus, A CPH > AChE
>>PPCF
» DPP IV.
Table 1. In vitro reactivity of porcine-brain peptidases with dichlorvos.
Enzyme k, (M' miri /10' ) Rate re ative to
that of AChE (%).
A p 7.4 .44 ~ .014 n= 10
A PH pH7.4 2.92 ~ 0.24 n=S) 60
A P p .0 2.12 + p, not done
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The results show that within the same family of serine peptidases/hydrolases
large
variations in the rate of reaction exist for a particular inhibitor.
Furthermore, the rate of
reaction of ACPH with dichlorvos is over six times that of AChE. The result is
surprising
as dichlorvos is considered a specific inhibitor of cholinesterase and does
not have the
adverse effects of early AD therapeutics, such as hepatotoxicity.
Using the substrate NAANA inhibition of ACPH by dichlorvos displayed simple
pseudo-
first order kinetics (Table 1). Furthernnore, the reaction remained pseudo-
first order to
97% inhibition, indicating that ACPH is substantially the only enzyme in the
brain capable
of hydrolysing NAANA. This strongly suggests that N-acetyl (especially N-
acetyl-alanyl)
substrate analogues will be extremely specific inhibitors for ACPH. Reaction
of ACPH
with metrifonate at pH 6.0 and pH 7.4 revealed inhibition only at the higher
pH (data not
shown), which is known to favour conversion of metrifonate to dichlorvos.
Conversion of
metrifonate to dichlorvos is therefore required for inhibitory potential of
metrifonate
towards ACPH in the same manner as for inhibition of AChE. Comparison of the
rates
of reaction of dichlorvos with ACPH at pH 6.0 and 7.4 revealed little
difference in the
rates of phosphorylation (demonstrating that conversion of metrifonate to
dichlorvos is the
rate-limiting step).
With the relatively large differential in pseudo-first order rate constant
between ACPH and
AChE, the former would be expected to be a potential in vivo target at the
therapeutic
level. To test this, rats were dosed rats at 4 mglkg i.p. and killed after 1
hour (within the
range of optimum AChE inhibition). The selection of dose correlates to the
degree of brain
AChE inhibition expected from therapeutic levels of metrifonate
administration. The dosed
rats showed an average of 47% inhibition of AChE activity whereas in the same
rats 93%
inhibition of ACPH activity was observed. PPCE and DPP IV were not
significantly
different from control activities (Table 2). These in vivo experiments
confirmed the in
vitro results and as expected the sensitivity of ACPH was greater than that of
AChE
towards dichlorvos.
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Table 2. In vivo inhibition of peptidases. Rats dosed with dichlorvos at 4 mg
/kg i.p.
and killed after 1 hour.
nzyme Control activity 4 mg /kg i.p. ichlorvoso inhibition
U g'' brain wet wt U g' brain wet wt
A 1 . X0.24 (n= ) 5. 8 ~ . n= ) 4
A PH 2.97 t 0.16 (n=9) 0.21 t 0.08 (n=5) 93*
t 0. n= ) . 8 t 0. ~ (n=5) 0
P 0.310 t~~l~ (n=5) .3 t . 7 n=5
* (p<1 x 0.001 compared to controls: one tailed Student's T-test)
AChE activity is known spontaneously to reactivate following inhibition with
dimethylphosphates, such as dichlorvos. Thus, 80-90% of brain and plasma AChE
activity
regenerates within 24 hours of a single dose of metrifonate or dichlorvos
(Hint et al, 1995,
Neurochemical Research 21, 339-345).
Reactivation experiments performed with dichlorvos-inhibited ACPH in vitro
showed the
slope of In % remaining inhibition vs. time was not significantly different
from zero (100
f 1 % over 80 minutes; p> 0.5). ACPH appeared to reactivate spontaneously only
very
slowly, if at all. In contrast, AChE inhibited by dichlorvos has an apparent
rate constant
of reactivation of 0.0113 f 0.0047 mini ~ at 25°C, as reported by
Clother et al, (1981
Biochim. Biophys. Acta 660, 306-316).
To confirm the in vitro data, rats were dosed with dichlorvos and the relative
inhibition
of AChE and ACPH observed after l, 4, 24, 48 and 120 hours post dose. The
results are
shown in Figure 1, which is a graph of ACPH (square symbols) or AChE (round
symbols)
activity (units/gm brain) against time post-dose (in hours). It was found that
AChE
activity rapidly recovers and is not significantly different from control
levels after 24h post
i.p. dichlorvos dose. However, ACPH activity remains depressed for the entire
time
course. Furthermore, the gradual reactivation appears linear with time
suggesting the
recovered activity results exclusively from de novo synthesis, with an
apparent half life
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for re-synthesis of 5 days.
The exact biological function of ACPH remains unknown. Preference for
substrates with
N acetyl-methionine, alanine and serine (common N terminal residues for
cytosolic
proteins) has led to the suggestion that the enzyme is important in protein
breakdown.
Analysis of the concentration of total brain protein over a 5 day time course
revealed no
significant differences between dosed and control rats (p < 0.5) with little
inter-individual
variation; 116 t 0.71 (SE; n=24) mg protein/g tissue wet wt. Clearly no gross
changes
in protein levels occur as a result of ACPH inhibition.
Suggestions have been made that ACPH may have a role in the degradation of N-
acetylated neuropeptides such as a-MSH. Incubation of a-MSH (0.155 mM) with
purified
ACPH gave only 23% hydrolysis after 20 hours incubation at 37°C (data
not shown).
Inhibitor studies with a-MSH revealed no significant changes in the km or Vm
of ACPH in
the presence of SS~cM a-MSH. We conclude that full-length a-MSH is a poor
substrate for
ACPH.
Example 2
To extend the findings described in Example 1 above, the inventors proceeded
to
investigate the relative reaction rates of ACPH and AChE with a large number
of other
known anti-cholinesterase compounds. The in vitro assays were performed, using
the
relevant test compound, as described above. The results are shown in Table 3.
It is
apparent that, whilst some compounds are potent inhibitors of AChE, they have
no
significant inhibitory effect on ACPH. Conversely, some compounds show good
selectivity
for inhibition of ACPH relative to AChE. These latter compounds include DFP,
dichlorvos, chlorpyrifos methyl oxon, mipafox (and acephate, a competitive
inhibitor of
ACPH). DFP and mipafox could not be used clinically, as they display
neuropathic side-
effects, but it may be possible to use safer variants of these compounds.
Alternatively
chlorpyrifos and dichlorvos could be used which are less toxic and are thus
potentially
useful in the treatment of AD and/or useful as cognition enhancers.
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Table 3. The reaction of ACPH and AChE with anti-cholinesterase compounds.
Compound Bimolecular rate
constant (M-' min'I)
ACPH AChE
Chlorpyrifos-methyl 1.89+0.22x106 3.2+0.05x105
oxon
DFP 1. 54+0.02x 106 1.45+0.04x 1 O5
Dichlorvos 2.92+0.24x 1 O5 4.40+0.14x 104
Diethyl dichiorvos 9.66x105 1.25x105
Dipropyl dichlorvos 1.54x106 1.67x105
Dibutyl dichlorvos 2.58x106 2.69x106
Dipentyl dichlorvos 7.80x105 1.78x105
Dihexyl dichlorvos 3.80x105 2.28x105
Chlorfenvinphos 2. S Ox 1 OS 5 . 20+0. 80x 105
Diazonon-oxon 2.50+0.24x104 1.29+0. I9x105
Mipafox 1.15+0.01 x 104 519+64
Paraoxon 9.10+1.30x103 8.29+0.69x105
Methyl-paraoxon 447+41 1.47+0.37x105
Carbaryl <50 2.20+0.16x104
Dicrotophos 325+3 5 4.26+0.17x 103
isoOMPA 60+13 15.9+1.3
Eserine <SO I .28+0.49x105
+Methamidophos 51 + 19 1. 34+0.3 7x 103
-Methamidophos <20 4.79+0.79x 103
Demeton-S-methyl <50 8.29x 105
Crotoxyphos 26+10 5.54+1.18x 104
Malaoxon 24+6 1.17+0.3 7x I OS
The above results also show a surprising effect, as illustrated by the various
derivatives of
dichlorvos. The inhibitory potential of the different dichlorvos variants
increased with
increasing chain length of the alkyl groups until n = 4 (i.e. dibutyl
dichlorvos), whereafter
the inhibitory potential decreased. In terms of selectivity for ACPH vs. AChE,
diethyl and
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dipropyl dichlorvos proved to be optimum, with reaction rates over 9 times
faster with
ACPH than with AChE. In practice, these longer chain variants of dichlorvos
are not
themselves clinically useful, due to slow-developing neuropathic effects, but
it should prove
possible to devise less toxic variants. The findings about optimum alkyl chain
length
should be equally applicable to other OP-based ACPH inhibitors.
Example 3 - Titration of DFP-sensitive sites
DFP is commercially available as a radiolabelled (tritiated) compound. The
inventors used
tritiated DFP in an in vitro system to titrate DFP-binding sites in rat brain
homogenates.
In outline, the method was as follows:-
Fresh rat brain tissue was homogenised in nine volumes of 50 mM Tris/HCI.pH
8Ø
Aliquots of homogenate were warmed to 37°C and 3H-DFP (DuPont, NEN) was
added to
give the desired final concentration. After the required length of time an
equal volume of
SDS sample buffer was added, the samples boiled for three minutes and the
proteins
resolved on a 10% polyacrylamide gel (Laemlli, U.K. Nature, 277, 680 ( 1970)).
Following
electrophoresis the proteins were blotted and tritium detected as described
previously
(Richards et al Chemico-Biological Interactions, 1999).
The results are illustrated in Figure 2, which is a bar chart showing %
labelling with DFP
(relative to amount of labelling with 60 minute incubation at a concentration
of 10.9 ~.M
DFP) at different concentrations (0.01-10.9I~,M) of DFP for labelled
polypeptides of
various molecular weights (27-154 KDa). Incubation was for 20 minutes other
than the
samples incubated with 10.91 pM DFP.
The inventors found that very low concentrations of DFP (0.01 and 0.04 ~,M)
resulted in
labelling of only two polypeptides, with molecular weights of 85KDa (major
band) and 77
KDa (minor band). The 85KDa band was determined to be ACPH. The 77KDa
polypeptide was not unambiguously identified, but might represent
butrylcholinesterase.
The low concentrations of DFP are safe cognition-enhancing levels, which would
cause
only about a 10% inhibition of AChE. Thus ACPH represents a major, highly-
sensitive
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16
target for DFP at low-dose potentially cognition enhancing levels.
We have demonstrated that the OP compound, dichlorvos, is a potent inhibitor
of ACPH
both in vitro and in vivo. This is the first in vivo demonstration of the
inhibition of a brain
peptidase activity by an organophosphorus pesticide/drug. We have shown that
at the
therapeutic level of metrifonate/dichlorvos administration ACPH activity would
be
depressed to < 10% of normal activities. Deletion of the gene for ACPH is
found in some
small cell lung and kidney cell carcinomas. Cell lines lacking the gene have a
low
endogenous ACPH activity and the balance between ACPH and acylase (the enzyme
that
releases the acyl-group from the released amino acid after the action of
ACPH), appears
to be critical for a particular cell type.
Mantle et al (1997 Clin. Chim. Acta 262, $9-97) have shown reduction in the
activity of
liver proteases after dosing rats with the OP-pesticide pirimiphos-methyl. The
effect was
not simply a result of binding to the active serine, as the activity of
cysteine and metallo-
protease was also decreased. However, Mantle et al failed to show any
inhibition of brain
proteases but instead found increased levels of activity of tripeptidyl
aminopeptidase, PPCE,
cathepsin L, DDP I and cathepsin H. We have shown large differences in the
rates of
reaction of members of the same peptidase family across a range of OP
compounds. It is
therefore possible that other OPs will react differentially with
peptidases/proteases.
Improvement in cognitive function at potentially sub-cholinergic levels of
dichlorvos and
metrifonate administration have been reported. The level of biogenic amines in
rats dosed
with dichlorvos is not significantly different from control animals.
Furthermore,
pharmacological investigations showed no binding of either dichlorvos or
metrifonate to
receptors in the brain (Hint et al, 1996 Drug Develop. Res. 38, 31-42).
Improvements in
cognitive function have been observed with metrifonate, dichlorvos and DFP. No
effects
were observed with the OP compound paraoxon, or the carbamate, eserine.
Interestingly
ACPH is inhibited by dichlorvos and DFP at a far greater rate than AChE,
conversely
paraoxon and eserine are both good inhibitors of AChE and poor inhibitors of
ACPH
(Table 3). Table 3 also reinforces the concept that large differential
activities toward a
particular OP can be observed even within the same family of enzymes. We are
currently
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17
extending the study to look at the structure-activity relationships of prolyl
oligopeptidases,
and other proteases with a range OP compounds.
Example 4 - Screening for Inhibitors of ACPH
Potential ACPH inhibitors can be screened by comparing activity against
acetylcholinesterase with activity against ACPH. The source of enzyme (ACPH)
may be
either a purified preparation or, for example, a sample of tissue (brain)
homogenate (10%
w/v) in a suitable buffer (e.g. SOmM Tris/HCI, 1mM dithiotheitol, pH 7.4). In
a suitable
assay, 30,1 of the brain homogenate is added to 1 ~1 of 4mM N acetyl-alanyl p-
nitroanilide
(Sigma) in 0.2 M TrisBCl, 1mM dithiothreitol, pH 7.4. The change in absorbance
at 405
nm is recorded at 37°C. To test for inhibition of activity the enzyme
sample can be pre-
incubated with a sample of inhibitor (at a range of concentrations) and the
activity
compared to that of the control. For example, a plot of Log (activity of
sample in presence
of inhibitor/activity of sample without inhibitor) vs. inhibitor concentration
will define a
straight line where the x-value at Log 0.5 will be equal to the ICsa.
Inhibition of
acetylcholinesterase can be determined in a similar way using the standard
Ellman assay
for measuring cholinesterase activity. Thus, 20p1 of a 1% (w/v) brain
homogenate may be
added to 3 ml of DTNB (2.6 x 10'~ M in 50 mM phosphate buffer pH 7.4), warmed
to
37°C and 0.1 ml of 0.156M acetylthiocholine iodide solution (in water)
added. The
activity is determined from the change in absorbance at 405 nM. Preferably,
potentially
useful therapeutic compounds will inhibit ACPH selectively relative to AChE.
Inhibitors could be based on structural analogues of dichlorvos, for example
by conversion
of the dimethyl ester to longer chain phosphonate analogues. Alternatively
inhibitors could
be based on the structure of an existing organophosphorus compound. Inhibitors
could also
be devised based on N-acetyl amino acids, for example N-acetyl-alanine. Here,
boronic
acid, chloromethyl ketone, aldehyde or phosphate/phosphonates may be examples
of
structural forms to be assayed for inhibitory potential.
Finally inhibitors can be screened for in vivo reactivity, and the usual
toxicological tests
to select for therapeutic potential.
