JP2002511407A - Improvements in or related to protease inhibitors - Google Patents

Abstract

(57) Abstract: A medicament for enhancing cognition in a mammalian subject, comprising an inhibitor substance that inhibits acylpeptide hydrolase (ACPH), a physiologically acceptable carrier, excipient, or diluent. A pharmaceutical comprising:

Description

DETAILED DESCRIPTION OF THE INVENTION

In the elderly population of most Western countries, the prevalence of Alzheimer's disease (AD) is expected to increase towards the next millennium (Brody 1985 Nature 315,463-466). The development of drugs that complement the study of treatments for this disease and eliminate the effects of AD on cognition and behavior is becoming increasingly important (Tarriot et al., 1997 Postgrad. Medicine 101, 73-76).
.

A consistent feature of AD is a decrease in cholinergic activity in the brain, mainly as a result of a decrease in forebrain cholinergic neurons (Whitehouse et al., 1982 Science).
215,1237-1239). Decreased cholinergic activity has been linked to the progression and severity of AD (Bierer et al., 1995 J. Neurochem. 63, 749-760). Thus, the use of drugs that enhance cholinergic function has been proposed as an adjunct to improve cognitive deficits in AD patients. At present, direct inhibitors of the enzyme acetylcholinesterase (AChE) are the most effective strategies in achieving cholinergic enhancement. The development of "second generation" cholinesterase inhibitors, such as ENA713, E2020 (donepezil), and metrifonate (dichlorvos), offers greater therapeutic potential due to their high selectivity for AChE and low adverse reactions.

[0003] Metrifonate is a prodrug of the organophosphorus ("OP") compound dichlorvos (2,2-dichlorovinyldimethylphosphate), which is formed by the slow non-enzymatic hydrolysis of its parent compound (Hinz et al., 1996 Neurochem.Resear
ch 21,331-337). Metriphonate has been used for the treatment of schistosomiasis for many years, and dichlorvos is a widely used household and agricultural pesticide.
Metriphonate is currently one of the most effective potential AD treatments because of its selectivity, low toxicity in warm-blooded animals and long half-life in vivo. Metrifonate has been included in a double-blind placebo study (Becker 1996 Alzheimer Dise
ase and Associated Disorders 10, 124-131), which resulted in a slight but significant increase in cognitive function in the treated group compared to the control group. Later double-blind studies confirmed these findings (Cummings et al., 1998 Neurology 50, 1).
214-1221; Morris et al., 1998 Neurology 50, 1222-1230). In one open trial, the therapeutic level of metrifonate was set at about 50% AChE inhibition (Becker and Gi
acobini 1990 Drug Dev. Res. 19,425-435).

However, the central view of the “cholinergic hypothesis” of treating AD has recently been questioned. Improvements in shuttle box and Morris maze escape behavior in young-adult rats have been demonstrated at doses of metrifonate and dichlorvos insufficient to cause significant inhibition of brain AChE (van der Staay et al., 1996 Behavio).
ural Pharmacology 7, 56-64; van der Staay et al., 1996 J. Pharmacol. Exp. Therapeu
tics 278, 697-708).

[0005] In order to broaden the pharmacological spectrum of metrifonate compounds, it seems necessary to seek out new molecular mechanisms of action. The potential of OP compounds to react with a wide range of serine hydrolases in the CNS has long been recognized (O'Neill, 1981).
Fundamental and Applied Toxicology 1,154-160; Aldridge, 1964 Biochem. J.9
3,619-623). However, the possibility that an unrecognized target protein is significantly inhibited in vivo by OPs has not been systematically studied.

[0006] Seryl peptidases of the prolyl oligopeptidase family, N-acyl peptide hydrolases (ACPH), prolyl oligopeptidases (also known as postproline degrading enzymes or PPCE) and dipeptidyl peptidase IV (DP
PIV) reacts in vitro with the organophosphorus compound diisopropylfluorophosphate (DFP) (Kato et al., 1980 J. Neurochem. 35, 527-535). Mammalian (especially human) ACPH has been extensively studied (eg, Jones and Manning 1985 Bioche
m. Biophys. Res. Comm. 2, 933-940; Jones and Manning 1986 Biochem. Biophys. Re.
s.Comm. 1,244-250; Jones and Manning 1988 Biochim. Biophys. Acta 953,357-3.
60; Jones et al., 1991 Proc. Natl. Acad. Sci. USA 88, 2194-2198; Scaloni et al., 1994 269.
, 15076-15084; Raphel et al., 1993 Biochemie 75, 891-897, etc.). AC
The activity of PH is to cleave N-acylated amino acid residues in the peptide. However, although the exact function of ACPH may play a role in the degradation of hormonal and neuropeptides, it has not been firmly confirmed (Mentlein
, 1988 FEBS Lett. 234, 251-256). Changes at the gene and protein levels within members of the prolyl oligopeptidase family are determined by AD (Mantle et al. 1
996, Clin Chem. Acta 249, 129-139), depression (Maes et al., 1994 Biol. Psychiatry 35).
545-552), autoimmune diseases (Aoyagi et al., 1987 Biochem. Int. 18, 383-389) and cancers (Scaloni et al., 1992 J. Lab. Clin. Med. 120, 546-552; Erlandsson et al., 1991 Oncogene.
6,1293-1295).

The present inventors have surprisingly found that cognitive enhancement observed in mammalian subjects upon consumption of certain substances, as previously suggested, inhibits acetylcholinesterase (AChE). But not by the inhibition of a different enzyme, acyl peptide hydrolase (ACPH).

Accordingly, in a first aspect, the present invention is directed to a method of producing a medicament for providing cognitive enhancement in a mammalian subject, comprising a method for producing an acylpeptide hydrolase (ACPH)
Provided by mixing a substance that inhibits the above with a physiologically acceptable carrier, excipient or diluent.

In another aspect, the invention relates to a medicament for providing cognitive enhancement in a mammalian subject, comprising a substance that inhibits ACPH, a physiologically acceptable carrier, excipient, or diluent. And a medicament comprising:

Here, cognitive enhancement can be defined as a measurable improvement in the cognitive ability of a mammalian subject. Methods and means for measuring the cognitive abilities of laboratory animals such as rats are well known to those skilled in the art (eg, shuttle boxes, Morris maze, etc.). Similarly, well known to those skilled in the art (eg, Becker et al., 1996 Alzheimer Disease and Associated Dis
There are also methods for measuring the cognitive abilities of human subjects (as used in orders 10,124-131). In clinical trials to evaluate the efficacy of drug therapy, several studies have been used to examine the cognitive abilities of Alzheimer's disease patients. "ADAS-Cog (Al
zheimer Disease Assessment Scale ”(Rosen et al., A
mJPsychiatry 1984 141,1356-1364) and “MMSE (Mini Mental State Examinati
on simple mental status test) ”(Rosen et al., J. Psychiatric Res. 1975 12,189-198)
This is an example. Cognitive enhancement is detected by a statistically significant improvement (eg, p <0.01) in the test group receiving the drug, as measured by an appropriate statistical test (eg, Student's T test), as compared to the control group. You.

[0011] Numerous physiologically acceptable carriers, excipients or diluents are known to those skilled in the pharmaceutical arts. The preferred carrier, excipient or diluent will depend on the route by which the medicament is to be administered. The preferred route of administration is oral, so that the medicament can be taken in capsule or tablet form. Alternatively, but less preferably, the medicament can be administered, for example, by intravenous, subcutaneous, intramuscular or intraperitoneal injection. ACPH when the medicament is administered by injection
Will preferably be mixed with a liquid diluent such as sterile saline or phosphate buffered saline.

[0012] ACPH activity can be measured, for example, using the in vitro assays disclosed herein. ACPH inhibitors suitable for use in the present invention will generally (although not necessarily) be substances that are capable of reducing in vitro ACPH activity by at least 50% at concentrations below 10 mM.

[0013] The inhibitor will preferably be selective for ACPH (ie, a higher inhibition of ACPH, especially as compared to AChE and compared to other mammalian enzymes in general). Activity). The inhibitor desirably causes at least 50% inhibition of in vivo ACPH activity while causing less than 50% AChE inhibition (preferably less than 30%, more preferably less than 20%, most preferably less than 10% AChE inhibition). Will cause. The inhibitors more preferably cause in vivo ACPH greater than 75% (most preferably greater than 90%) while causing less than 10% AChE inhibition.
Will cause activity inhibition.

[0014] The substance that inhibits ACPH can be any compound that inhibits ACPH, when administered to a subject, at a concentration low enough to avoid serious adverse reactions. For example, the inhibitor can be an organophosphorus (OP) compound such as metrifonate (or its metabolite dichlorvos). When the inhibitor is metrifonate, administration of an appropriate dose of the medicament causes ACPH inhibition while causing less than 50% (preferably less than 30%, more preferably less than 20%, and most preferably less than 10%) AChE inhibition. The drug will preferably contain a lower concentration of metrifonate than the concentration used in the clinical trials reported by Becker et al. So as to provide an inhibitory dichlorvos concentration in the subject. Typically, OP-based ACPH inhibitor compounds will follow the general formula: (R ₁ O), (R ₂ O) P (O) X, wherein R ₁ and R ₂ are each independently substituted or unsubstituted. Substituted C ₁ -C ₆ alkyl (preferably C ₁ or C ₂ or C ₃ ), and X is a relatively electronegative leaving group]. X is 1
Conveniently one or more halides are included. Examples of suitable leaving groups for X are 3,5,6
There are -trichloro-2-pyridinyl (see chlorpyrifos) and 2,2-dichloroethyl (see dichlorvos). We consider that the electronegativity of the leaving group primarily determines, or at least affects, the potency of the inhibitor, while the overall structure of the inhibitor compound determines its selectivity. I do.

[0015] Alternatively, the inhibitor may be a compound other than the OP compound,
For example, peptides or polypeptides. Specifically, the inhibitor is
An analog of an ACPH substrate, such as an acyl peptide, may be included. Peptides having N-acetylalanine, N-acetylmethionine or N-acetylserine residues represent suitable examples. Of these, N-acetylalanine is preferred because methionine is susceptible to oxidation and the -OH group of serine is susceptible to modification during the synthesis of ACPH inhibitors. Such substrate analogs may contain 1 to 50 amino acids, but shorter peptides are more convenient and cheaper to produce. Typically, a substrate analog will consist of less than 30 amino acid residues, preferably less than 10 residues. N-
It will be understood by those skilled in the art that any amino acid residue can be placed next to an acetyl residue. Also, the substrate analog may, for example, reduce toxicity and / or
Non-peptidic moieties may be included, and may be preferred, to improve selectivity and / or ACPH inhibitory potency.

[0016] While substrate analogs are readily synthesized and potentially very selective,
It is not ideal for therapeutic use in that it binds non-covalently to PH. Therefore, their ability to inhibit ACPH is very concentration dependent. Thus, preferred inhibitors will act as irreversible inhibitors of ACPH and will be covalently linked thereto. Such inhibition is basically [inhibitor] independent. Examples include well-known peptidase inhibitors such as chloromethyl ketones. Although such compounds are likely to be too toxic to be used therapeutically, those skilled in the art will be able to screen for other compounds using the disclosure herein based on well-known general knowledge. Alternative ACPH inhibitors can be readily found that have an acceptably low toxicity (ie, effectively inhibit ACPH without over-inhibiting AChE). In one specific embodiment, the inhibitor will comprise an N-acyl (particularly N-acetyl) peptide substrate analog moiety and an irreversible peptidase inhibitor moiety. For example, an N-acetylalanine-containing moiety covalently linked to a chloromethyl ketone or aldehyde or a phosphate / phosphonic acid group or a boronic acid.

The medicament may be administered at an appropriate dose of 0.5 to 0.5 kg of ACPH inhibitor per 1 kg of subject body weight per day.
A range of 50mg would be useful. While it will be clear that the precise dosage will depend, inter alia, on the potency and toxicity (if any) of the ACPH inhibitor used and the route of administration, the amount of ACPH inhibitor will preferably be 1 kg body weight per day 1 to 1 per
The range is 0 mg.

[0018] The medicament of the present invention may be particularly useful for the prevention and / or treatment of neurodegenerative diseases affecting cognitive function. Alzheimer's disease (AD) is a well-known example of such a disease, and there is a great need for a suitable preventive and / or therapeutic drug for AD, but that need is not yet met. Individuals with a family history of this condition could be given the drug, as there are known genetic factors involved in predisposition to AD.

As another option, the medicament could be given to individuals who want to improve their cognition, especially those who have received intellectual training. Specifically,
Evidence from studies on metrifonate / dichlorvos suggests that the learning process may be accelerated, so that people taking classes will benefit from taking the drug.

The medicament of the present invention is preferably administered to a human subject. Therefore, the present invention
Use of an ACPH inhibitor to produce cognitive enhancement in a human subject, specifically a method of preventing and / or treating Alzheimer's disease in a human subject, comprising the use of an ACPH inhibitor (typically as defined herein above) (As a medicament).

The present invention also provides a method of screening a substance for use as an active ingredient in a medicament for causing cognitive enhancement in a human subject, the method comprising the steps of: It is tested for its ability to inhibit ACPH, and the substance is tested for its ability to inhibit AChE. Specifically, the screening method involves mixing ACPH with a compound to be screened, adding an ACPH substrate that produces a detectable signal when digested with ACPH,
Measuring the signal and thus determining the amount of ACPH activity. Typically, the substrate will undergo a color change when it is digested with ACPH, allowing the signal to be measured spectroscopically. Suitable assays for ACPH activity are known to those of skill in the art, and one such assay is disclosed herein. These assays have not been used before to screen compounds used in medicine to produce cognitive enhancement.

In yet another aspect, the invention relates to the use of ACPH inhibitors (particularly selective serine peptidase / hydrolase inhibitors) in medicaments for providing cognitive enhancement in human subjects and / or for treating Alzheimer's disease. Prepare for use.

The invention will now be described in more detail by way of illustrative examples and with reference to the accompanying drawings, in which:

[0024]

【Example】

Example 1 Materials and Methods Dichlorvos was obtained from ChemServ (P.O. Box 3108, Westchester, PA, 19381-29941, USA). α-melanocyte stimulating hormone (α-MSH
), N-acetylalanine-p-nitroanilide, glycine-proline-AMC and fluorescamine were obtained from Sigma (Pool, Dorset, UK). Z-glycine-proline-AMC was obtained from Bachem (AMC = aminomethylcoumarin; Z = benzyloxycarbonyl).

In Vivo Inhibition Male Fischer 344 rats (180-220 g) were used. These animals were housed under standard conditions. Rats were intraperitoneally administered water or dichlorvos dissolved in water at a concentration of 0.1 ml / 100 g body weight. Initial dosing experiments were performed at about 50 mg intraperitoneally at 4 mg / kg.
% Of brain AChE activity was used, and this concentration was used in subsequent dosing experiments. After 1, 4, 8, 24, 48 and 120 hours the rats were sacrificed with an overdose of anesthetic, the brains were removed on ice and the assay was performed within 4 hours of death.

Brain homogenates were prepared in 10 volumes of 10 mM Tris / HCl (pH 7.4) or 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES) (pH 6.0). 1 mM DTT was added to the homogenate for the peptidase assay. AChE activity was measured by the Ellman method (Ellman et al., 1961 Biochem. Pharm. 7, 88-95). The brain homogenate was further diluted 10-fold with water, and 20 μl of the diluted homogenate was added to DTNB (0.375
mM) and ACTI (0.583 mM) (total volume 1 ml). The change in absorbance at 405 nm was recorded at 37 ° C.

To assay for ACPH activity, 30 μl of 10% homogenate was added to 0.2 M Tris / HC
l (pH 7.4), 4 mM N-acetyl-alanyl-p-nitroanilide (NAANA) in 1 mM DTT
In addition to 1 ml, the change in absorbance at 405 nm was recorded at 37 ° C.

To assay for DPP IV activity, 30 μl of 10% homogenate was added to 50 mM Tris /
HCl (pH 7.4), 0.5 mM Gly-Pro-p-nitroanilide in 1 mM DTT
The change in absorbance at was recorded at 37 ° C.

The PCCE activity was measured using 0.25 mM Z-Gly-Pro-AMC in 50 mM Tris / HCl (pH 7.4), 1 mM DTT, and the change in fluorescence was measured at an excitation wavelength of 383 nm and an emission wavelength of 455 nm. Recorded in ° C.

One unit (U) of enzymatic activity is defined as the amount of enzyme required to hydrolyze 1 micromole of substrate per minute.

[0031] Total protein was measured using the BioRad DC assay. In vitro inhibition of peptidase activity Portions of the brain homogenate prepared as described above were incubated with various concentrations of the inhibitor at 37 ° C. and residual peptidase activity was assayed as described above. From the slope of the graph of In [residual activity (%)] plotted against time, the pseudo first order rate K _obs was calculated for each reaction. The overall second-order rate constant K _i was calculated from the slope of the K _obs graph versus inhibitor concentration.

Purification of ACPH ACPH was purified essentially as described above except that porcine brain was used as a tissue source (Jones and Manning, 1985 Biochem. Biophys. Res. Comm.
.126,933-940). Final purification by Mono-Q FPLC is 10 mM citric acid, 0.5 mM DTT
This was performed using a linear gradient of 0-0.7 M NaCl in (pH 6.0) (30 min at a flow rate of 0.5 ml / min). ACPH activity eluted as a single peak at 0.53-0.60 M NaCl, which corresponded to a single UV absorption peak on the elution profile. Each aliquot was stored at -20 C until needed.

Reactivation of ACPH activity after inhibition by dichlorvos Purified ACPH (5 μl, 0.11 units) is added to an equal volume of 4 mM dichlorvos in 0.1 M sodium phosphate, pH 7.0 for 15 minutes at 37 ° C. Inhibited the activity by 80%. The resulting solution was diluted 200-fold in 0.1 M sodium phosphate (pH 7.0) at 37 ° C., and aliquots were removed at various time intervals and assayed for ACPH activity. The results were plotted as ln [% residual inhibition] versus time.

Hydrolysis of α-MSH by ACPH α-MSH was dissolved in 0.2 M potassium phosphate and 0.2 M NaCl (pH 7.4). Purified ACPH was added and the mixture was incubated at 37 ° C., and at various time intervals portions (50 μl
) And the fluram method (Jones and Manning 1985 Biochem. Biophys. Res. Comm. 1)
26,933-940) for the release of free amino groups.

Inhibition of ACPH activity by α-MSH α-MSH (final concentration 55 μM) was added to various concentrations of NAANA in 0.2 M Tris / HCl (pH 7.4)
Was added. Purified ACPH was added and substrate hydrolysis was monitored as described above. α-MSH
A control reaction was also performed in the absence of. The rate constants for the hydrolysis of NAANA by ACPH were determined in the presence and absence of the inhibitor.

Results The active product dichlorvos obtained from metrifonate hydrolysis was selected to screen the in vitro reactivity of the various enzymes examined in this study. This allows direct comparison of rate constants without considering prodrug activation and facilitates direct and accurate kinetic comparisons. Metriphonate has been shown to induce cholinergic inhibition exclusively through sustained release of dichlorvos.

In vitro screening of brain homogenates with dichlorvos revealed significant differences in the rate constants of inhibition for the four enzymes tested (Table 1). That is, ACPH> AChE >> PPCE >> DPP IV.

[0038]

[Table 1]

[0039] These results indicate that within the same serine peptidase / hydrolase family, there is a large variation in reaction rates for certain inhibitors. Furthermore, the reaction rate of ACPH with dichlorvos is more than 6 times that of AChE. This result is surprising since dichlorvos is considered to be a specific inhibitor of cholinesterase and has no early AD therapeutic side effects such as hepatotoxicity.

Using the substrate NAANA, inhibition of ACPH by dichlorvos showed simple pseudo-first-order kinetics (Table 1). Furthermore, the response remained pseudo-primary up to about 97% inhibition, indicating that ACPH was virtually the only brain enzyme capable of hydrolyzing NAANA. This strongly suggests that N-acetyl (especially N-acetyl-alanyl) substrate analogs will be very specific inhibitors for ACPH. Reaction of ACPH with metrifonate at pH 6.0 and pH 7.4 showed inhibition only at the higher pH, which is known to favor the conversion of metrifonate to dichlorvos (data not shown). Thus, the ability of metrifonate to inhibit ACPH, as well as AChE inhibition, requires the conversion of metrifonate to dichlorvos. Comparing the reaction rates of dichlorvos with ACPH at pH 6.0 and 7.4, there was little difference in the rate of phosphorylation (this proves that the conversion of metrifonate to dichlorvos is the rate-limiting step. There).

The relatively large difference in pseudo-first-order rate constants between ACPH and AChE suggests that at therapeutic levels, the former is a potential in vivo target. To verify this, rats were dosed intraperitoneally at a dose of 4 mg / kg and sacrificed 1 hour later (within the range of optimal AChE inhibition). The choice of dose correlates with the degree of brain AChE inhibition expected from treatment level administration of metrifonate. Administered rats exhibited an average of 47% inhibition of AChE activity, while 93% inhibition of ACPH activity was observed in the same rats. PPCE and DPP IV did not differ significantly from control activity (Table 2). These in vivo experiments confirmed the in vitro results, and, as expected, the sensitivity of ACPH to dichlorvos was higher than that of AChE.

[0042]

[Table 2]

AChE activity is known to reactivate spontaneously after inhibition by dimethyl phosphates such as dichlorvos. For example, 80-90% of brain and plasma AChE activity
Regenerates within 24 hours after a single dose of metrifonate or dichlorvos (Hi
nz et al., 1996, Neurochemical Research 21,339-345).

In a reactivation experiment performed in vitro with the dichlorvos-inhibiting ACPH, ln [
Residual inhibition rate (%)]. It was found that the slope of time versus time was not significantly different from 0 (8
100 ± 1% at 0 min; p> 0.5). ACPH seemed to play very slowly, even if spontaneously. In contrast, AChE inhibited by dichlorvos was reported by Clother et al. (1981 Biochim. Biophys. Acta 660, 306-316).
), With an apparent rate constant for reactivation of 0.0113 ± 0.0047 min ⁻¹ at 25 ° C.

To support this in vitro data, rats were administered dichlorvos and AC
Relative inhibition of hE and ACPH was observed 1, 4, 24, 48 and 120 hours after administration. FIG. 1 shows the results. Figure 1 shows ACPH activity or AC against time (unit: hours) after administration.
It is a graph of hE activity (unit / g- brain). AChE activity was found to recover rapidly and was not significantly different from control levels 24 hours after intraperitoneal dichlorvos administration. However, ACPH activity remains depressed throughout. In addition, the gradual reactivation appears linear with time, suggesting that the restored activity is solely the result of de novo synthesis, with an apparent half-life for resynthesis of 5 days.

The exact biological function of ACPH is not yet known. N-acetyl-methionine, N
Substrate selectivity for -acetylalanine and N-acetylserine (the N-terminal residue common to cytoplasmic proteins) suggests that this enzyme is important for proteolysis. Analysis of total brain protein concentration over 5 days showed no significant differences between treated and control rats and little interindividual variability; 116 ± 0.71 (SE
N = 24) mg-protein / g-tissue wet weight. It is clear that there is no global change in protein levels as a result of ACPH inhibition.

It has been proposed that ACPH may play a role in the degradation of N-acetylated neuropeptides such as α-MSH. Incubation of α-MSH (0.155 mM) with purified ACPH resulted in only 23% hydrolysis after 20 hours incubation at 37 ° C. (data not shown). In inhibitor studies using α-MSH, 55 μM α-MSH
Significantly in k _m or V _m of ACPH in the presence of MSH changes were not observed. We conclude that full-length α-MSH is not a good substrate for ACPH.

Example 2 Extending the findings described in Example 1 above, the researchers decided to investigate the relative kinetics of ACPH and AChE with a number of other known anticholinesterase compounds.
In vitro assays were performed as described above using the appropriate test compounds. Table 3 shows the results. Some compounds are potent inhibitors of AChE, but they
ACPH does not appear to have a significant inhibitory effect. Conversely, some compounds have AChE
Shows good selectivity for inhibition of ACPH compared to These latter compounds include DFP, dichlorvos, chlorpyrifosmethyloxone, mipafox (and acephate, a competitive inhibitor of ACPH). DFP and mipafox will not be clinically usable because they exhibit neuropathic side effects, but safer variants of these compounds may be used. Another option is to use low toxicity and thus potentially useful in the treatment of AD and / or cognitive enhancers (c
Chlorpyrifos and dichlorvos, which are useful as ognition enhancers, could also be used.

[0049]

[Table 3]

The above results show a surprising effect, as exemplified by various derivatives of dichlorvos. The inhibitory potency of the various dichlorvos variants increased with increasing chain length of the alkyl group, up to n = 4 (ie dibutyldichlorvos), after which the inhibitory potency decreased. Regarding the selectivity for ACPH over AChE, diethyl dichlorvos and dipropyl dichlorvos are optimal, and the reaction rate with ACPH is AC
It was found that the reaction speed with hE was more than 9 times faster. Indeed, it should be found that longer dichlorovos variants are not clinically useful per se due to their late-onset neuropathic effect, but are capable of devising less toxic variants. These findings regarding optimal 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 radiolabeled (tritiated) compound. We titrated the DFP binding site in rat brain homogenate using tritiated DFP in an in vitro system. At a high level, the method is as follows.

[0052] Fresh rat brain tissue was homogenized in 9 volumes of 50 mM Tris / HCl (pH 8.0). A portion of the homogenate was warmed to 37 ° C. and ³ H-DFP (DuPont NEN) was added to reach the desired final concentration. After the required time, an equal volume of SDS sample buffer was added, the sample was boiled for 3 minutes, and the proteins were separated on a 10% polyacrylamide gel (Laemlli, UK).
Nature, 277, 680 (1970)). After electrophoresis, proteins were blotted and tritium was detected as previously described (Richards et al., Chemico-Biological In
teractions, 1999).

FIG. 2 shows the results. FIG. 2 shows the labeling rate (%) by DFP at various DFP concentrations (0.01 to 10.91 μM) for labeled polypeptides of various molecular weights (27 to 154 KDa).
FIG. 9 is a bar graph showing (percentage of labeling by incubation at a DFP concentration of 10.9 μM for 60 minutes). Incubation was for 20 minutes except for samples incubated with 10.91 μM DFP.

The present inventors have found that at very low concentrations of DFP (0.01 and 0.04 μM), only two polypeptides with molecular weights of 85 KDa (main band) and 77 KDa (subband) are labeled. . This 85KDa band was determined to be ACPH. The 77KDa polypeptide was not clearly identified, but may correspond to butyrylcholinesterase.

[0055] These low DFP concentrations are safe cognitive enhancement levels that cause only 10% inhibition of AChE. Therefore, ACPH can be used at low doses to potentially increase cognitive levels of DFP
For, it represents a major sensitive target.

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 inhibition of brain peptidase activity by organophosphate pesticides / drugs. We have shown that administration of therapeutic levels of metrifonate / dichlorvos will push ACPH activity to <10% of normal activity. Several small cell lung and renal cell carcinomas have ACPH gene deletions. Cell lines lacking this gene have low endogenous ACPH activity,
The balance between ACPH and acylase, an enzyme that dissociates acyl groups from amino acids released after the action of ACPH, appears to be important for certain cell types.

(1997 Clin. Chim. Acta 262, 89-97) show a decrease in hepatic protease activity following administration of the OP pesticide pirimiphosmethyl to rats. This effect was not simply the result of binding to active serine, since the activity of cysteine and metalloproteases was also reduced. However, Mantle et al. Failed to show inhibition of brain protease, but rather found increased levels of tripeptidyl aminopeptidase, PPCE, cathepsin L, DDP I and cathepsin H activity.
We have shown that there are large differences in the kinetics of the same peptidase family members across a range of OP compounds. Thus, other OPs may react differentially with the peptidase / protease.

[0058] Improvements in cognitive function have been reported with administration of dichlorvos and metrifonate at potentially below cholinergic levels. Biogenic amine levels in rats receiving dichlorvos are not significantly different from control animals. In addition, in pharmacological research,
Neither dichlorvos nor metrifonate binding to brain receptors has been shown (Hinz et al., 1996 Drug Develop. Res. 38, 31-42). Improvements in cognitive function have been observed with metrifonate, dichlorvos, and DFP. No effect was observed with the OP compound paraoxon or the carbamate ezerin. Interestingly, ACPH is inhibited by dichlorvos and DFP at a much faster rate than AChE, whereas paraoxon and ezerin are both good inhibitors of AChE and not good inhibitors of ACPH (Table 3). . Table 3 also reinforces the concept that differential activity can be observed for a particular OP even within the same enzyme family. We are currently expanding this work to examine the structure-activity relationships of prolyl oligopeptidases and other proteases with a range of OP compounds.

Example 4-Screening for Inhibitors of ACPH Potential ACPH inhibitors can be screened by comparing their activity on acetylcholinesterase to their activity on ACPH. The source of the enzyme (ACPH)
It may be a purified product or a sample (10% w / v) of a tissue (brain) homogenate in an appropriate buffer (eg, 50 mM Tris / HCl, 1 mM dithiothreitol, pH 7.4). In a preferred assay, 30 μl of brain homogenate was added to 4 mM N-acetyl-alanyl-p-nitroanilide (Sigma) in 0.2 M Tris / HCl, 1 mM dithiothreitol (pH 7.4).
Add to 1 μl. The change in absorbance at 405 nm is recorded at 37 ° C. To test for inhibition of activity, a sample of the enzyme is pre-incubated with a sample of the inhibitor (at a range of concentrations) and its activity compared to that of a control. For example, a plot of Log [activity of the sample in the presence of inhibitor / activity of the sample in the absence of inhibitor] versus inhibitor concentration is linear, and the x value at Log _0.5 is equal to the IC50. Would. Inhibition of acetylcholinesterase can be similarly determined by measuring cholinesterase activity by the standard Ellman method. For example, add 20 μl of 1% (w / v) brain homogenate to 3 ml
Add DTNB (2.6 × 10 ⁻⁴ M in 50 mM phosphate buffer (pH 7.4)), warm to 37 ° C., add 0.1 ml
A 0.156 M acetylthiocholine iodide solution (aqueous solution) can be added. The activity is
Determined from the change in absorbance at 405 nm. Potentially useful therapeutic compounds will preferably inhibit ACPH selectively against AChE.

Inhibitors could be based on structural analogs of dichlorvos, such as by converting dimethyl esters to longer chain phosphonate analogs. Alternatively, the inhibitor could be based on the structure of an existing organophosphorus compound. Inhibitors could also be conceived based on N-acetyl amino acids (eg N-acetyl-alanine). Here, examples of structural types to be assayed for inhibitory capacity include boronic acid, chloromethyl ketone, aldehyde or phosphate / phosphonate.

Finally, inhibitors can be screened for in vivo reactivity and selected for therapeutic potential in routine toxicity studies.

[Brief description of the drawings]

FIG. 1 is a graph of enzyme (ACPH or AChE) activity over time.

FIG. 2 is a bar graph showing the labeling percentage (%) of various polypeptides in rat brain homogenate at various DFP concentrations.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] June 8, 2000 (2000.6.8)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Bayerwrk, Leverkusen, BRD ( 72) Inventor Ray, David United Kingdom, EL23 YD Leicester, Kingsmead Road, 58F term (reference) 4C084 AA02 AA17 BA22 DC32 DC50 ZA162 ZC202 4C086 AA01 AA02 DA34 MA10 ZA16 ZC20

Claims (25)

[Claims] 1. A medicament for providing cognitive enhancement in a mammalian subject, comprising:
A medicament comprising an inhibitor substance that inhibits acyl peptide hydrolase (ACPH) and a physiologically acceptable carrier, excipient or diluent. 2. The medicament according to claim 1, wherein the inhibitor inhibits human ACPH, and the medicament is formulated for a human subject. 3. The medicament according to claim 1, wherein the inhibitor comprises an organic phosphorus (OP) compound. 4. The medicament according to claim 1, wherein the inhibitor is a compound other than metrifonate or dichlorvos. 5. The method according to claim 1, wherein the inhibitor has the general formula (R ₁ O), (R ₂ O) P (O) X, wherein R ₁ and R ₂ are independently a C ₁ -C ₆ substituted or unsubstituted alkyl And X comprises an electronegative leaving group]. 6. The medicament according to claim 5, wherein R ₁ = R ₂ . 7. The medicament according to claim 5, wherein X contains one or more halides. 8. The method of claim 1, wherein said inhibitor comprises an acyl-peptide substrate analog. 9. The medicament according to claim 1, wherein the inhibitor comprises N-acetylalanine, N-acetylmethionine or N-acetylserine residue. 10. The method of claim 1, wherein said inhibitor comprises any one of a chloromethyl ketone, an aldehyde, a phosphate or phosphonate group, or a boronic acid.
The medicament according to any one of 2, 8 or 9. 11. Each dose, when consumed by the average adult, is 50%
A medicament according to any one of the preceding claims, in unit dosage form, which causes less than acetylcholinesterase inhibition and at the same time a sufficient inhibitor concentration to cause more than 50% ACPH inhibition. 12. The medicament of claim 11, wherein each dose results in less than 30% acetylcholinesterase inhibition and at the same time an inhibitor concentration sufficient to cause 50% or more ACPH inhibition. 13. The medicament of claim 11, wherein each dose results in less than 20% acetylcholinesterase inhibition and at the same time an inhibitor concentration sufficient to cause 50% or more ACPH inhibition. 14. The medicament of claim 11, wherein each dose causes less than 10% acetylcholinesterase inhibition and at the same time an inhibitor concentration sufficient to cause 50% or more ACPH inhibition. 15. A method of producing a medicament for providing cognitive enhancement in a mammalian subject, comprising administering a substance that inhibits acylpeptide hydrolase (ACPH) to a physiologically acceptable carrier, excipient or diluent. And mixing. 16. A method according to claim 15 for producing a medicament according to any one of claims 1 to 10. 17. A method of causing cognitive enhancement in a human subject, comprising administering to the subject a medicament according to any one of claims 1 to 14. 18. A method for preventing and / or treating Alzheimer's disease in a human subject, comprising administering to the subject a medicament according to any one of claims 1 to 14. 19. AC in a medicament for causing cognitive enhancement in a human subject
Use of PH inhibitors. 20. A method for causing cognitive enhancement in a human subject.
Use of an inhibitor of ACPH in the medicament according to any one of the above. 21. Use of an inhibitor of ACPH in a medicament for preventing and / or treating Alzheimer's disease in a human subject. 22. The use according to any one of claims 19 to 21, wherein said inhibitor is a selective serine peptidase / hydrolase inhibitor. 23. A method for screening a substance for use as an active ingredient in a medicament for causing cognitive enhancement in a human subject, the substance comprising ACPH
A method comprising the steps of testing for inhibitory ability, testing the substance for AChE inhibitory ability, and selecting a substance that exhibits a higher inhibitory activity on ACPH than AChE. 24. A medicament substantially as defined in the preceding claim. 25. A method for the manufacture of a medicament substantially as defined in the above claim.