IMPROVEMENTS IN OR RELATING TO PROTEASE INHIBITORS

Abstract: Disclosed is a medicament for use in providing cognitive enhancement in a mammalian subject, the medicament comprising an inhibitor substance which inhibits acylpeptide hydrolase (ACPH), and a physiologically acceptable carrier, excipient or diluent.

Description: 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 EN A 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 (Hinz 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 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 - 50% 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 St ay 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 1, 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 role 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), autoirnmunity (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) (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 intra-venously, sub-cutaneously, intra-muscularly or ntra-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 10% 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 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: wherein R j and R 2 are each, independently, substituted or unsubstituted C j -C 6 alkyl (preferably, C j , or C 2 or C 3 ) 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. 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. 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 and/or 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. Examples Example 1 Materials and Methods Dichlorvos was obtained from ChemServ (PO Box 3108, West Chester, PA 19381-29941 USA), α-melanocyte stimulating hormone (α-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 4mg/kg; the same concentration was used for subsequent dosing experiments. Rats were killed with an over-dose of anaesthetic after 1, 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/HCl, pH 7.4 or 0.1M 2[Ν-morpholino] ethanesulphonic acid (MES) pH 6.0. For peptidase assays ImM 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 20μl of this diluted homogenate was added to a solution of 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 ACPH activity, 30μl of the 10% homogenate was added to 1 ml of 4mM N- acetyl-alanyl-p-nitroanilide (ΝAAΝA) 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, 30μl of 10% homogenate was added to 1 ml of 0.5mM Gly-Pro- p-nitroanilide in 50mM Tris/HCl pH 7.4, lmM 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 50mM 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 (IT) is defined as the amount of enzyme required to hydrolyse lμ 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, K obs , for the individual reactions was calculated from the slope of graphs of In % activity remaining plotted against time. Overall second-order rate constants, K j , were calculated from the slope of graphs of K b s against 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 NaCl in lOmM citrate, 0.5mM 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 NaCl, 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 (5μl, 0.11 Units) was added to an equal volume of 4rnM dichlorvos in 0.1 M sodium phosphate pH 7.0 for 15 min. at 37°C to give 80%o inhibition of activity. The resulting solution was diluted 200 fold in 0.1 M sodium phosphate pH 7.0 at 37°C and aliquots removed at various time intervals and assayed for ACPH activity. Results were plotted as In % remaining inhibition vs time. Hydrolysis of α-MSH by ACPH α-MSH was dissolved in 0.2 M potassium phosphate, 0.2 M NaCl, pH 7.4. Purified ACPH was added and the mixture incubated at 37°C, aliquots (50μl) were removed at various time intervals and assayed for the release of free amino groups using the fluram assay (Jones & Manning 1985 Biochem. Biophys. Res. Comm. 126, 933-940). Inhibition of ACPH activity by α-MSH α-MSH (55μM, final concentration) was added to various concentrations of NAANA in 0.2 M Tris/HCl, 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 α-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, ACPH > AChE »PPCE » DPP IV. Table 1. In vitro reactivity of porcine-brain peptidases with dichlorvos. 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). Furthermore, the reaction remained pseudo-first order to ~ 97% inhibition, indicating that ACPH is substantially the only enzyme in the brain capable of hydro lysing 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 mg/kg 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. Table 2. In vivo inhibition of peptidases. Rats dosed with dichlorvos at 4 mg /kg i.p. and killed after 1 hour. * (p<l 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 (Hinz et al, 1996, 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 ± 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 ± 0.0047 min "1 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 1, 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 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 ± 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 Ν- acetylated neuropeptides such as α-MSH. Incubation of α-MSH (0.155 mM) with purified ACPH gave only 23% hydrolysis after 20 hours incubation at 37°C (data not shown). Inhibitor studies with α-MSH revealed no significant changes in the k m or V m of ACPH in the presence of 55μM α-MSH. We conclude that full-length α-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. Table 3. The reaction of ACPH and AChE with anti-cholinesterase compounds. 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 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/HCl.pH 8.0. Aliquots of homogenate were warmed to 37°C and 3 H-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 μM DFP) at different concentrations (0.01-10.91μM) of DFP for labelled polypeptides of various molecular weights (27-154 KDa). Incubation was for 20 minutes other than the samples incubated with 10.91μM DFP. The inventors found that very low concentrations of DFP (0.01 and 0.04 μM) 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 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, 89-97) 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 (Hinz 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 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. 50mM Tris/HCl, ImM dithiotheitol, pH 7.4). In a suitable assay, 30μl of the brain homogenate is added to 1 μl of 4mM N-acetyl-alanyl-p-nitroanilide (Sigma) in 0.2 M Tris/HCl, ImM 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 IC 50 . Inhibition of acetylcholinesterase can be determined in a similar way using the standard Ellman assay for measuring cholinesterase activity. Thus, 20μl of a 1% (w/v) brain homogenate may be added to 3 ml of DTΝB (2.6 x lO "4 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 Ν-acetyl amino acids, for example Ν-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.

Claims: Claims 1. A medicament for use in providing cognitive enhancement in a mammalian subject, the medicament comprising an inhibitor substance which inhibits acylpeptide hydrolase (ACPH), and a physiologically acceptable carrier, excipient or diluent. 2. A medicament according to claim 1, wherein the inhibitor inhibits human ACPH and the medicament is formulated for use in a human subject. 3. A medicament according to claim 1 or 2, wherein the inhibitor comprises an organophosphorus (OP) compound. 4. A medicament according to any one of the preceding claims, wherein the inhibitor is a compound other than metrifonate or dichlorvos. 5. A medicament according to any one of the preceding claims, wherein the inhibitor conforms to the general formula (R j O), (R 2 O) P (O)X, wherein R j and R 2 are independently C j -Cg substituted or unsubstituted alkyl, and X comprises an electronegative leaving group. 6. A medicament according to claim 5 wherein R, = R 2 . 7. A medicament according to claim 5 or 6, wherein X comprises one or more halides. 8. A medicament according to any one of claim 1 or 2, wherein the inhibitor comprises an acyl-peptide substrate analogue. 9. A medicament according to any one of claims 1, 2 or 8, wherein the inhibitor comprises an N-acetyl alanine, methionine or serine residue. 10. A medicament according to any one of claims 1, 2, 8 or 9, wherein the inhibitor comprises one of the following: chloromethyl ketone; an aldehyde; a phosphate or phosphonate group; or boronic acid. 11. A medicament according to any one of the preceding claims in unitary dose form, each dose, when consumed by an average human adult, providing a concentration of inhibitor sufficient to cause 50% or more inhibition of ACPH whilst causing less than 50% inhibition of acetylcholinesterase. 12. A medicament according to claim 11, wherein each dose provides a concentration of inhibitor sufficient to cause 50% or more inhibition of ACPH whilst causing less than 30% inhibition of acetylcholinesterase. 13. A medicament according to claim 11, wherein each dose provides a concentration of inhibitor sufficient to cause 50% or more inhibition of ACPH whilst causing less than 20% inhibition of acetylcholinesterase. 14. A medicament according to claim 11, wherein each dose provides a concentration of inhibitor sufficient to cause 50% or more inhibition of ACPH whilst causing less than 10% inhibition of acetylcholinesterase. 15. 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. 16. A method according to claim 15, for making a medicament according to any one of claims 1-10. 17. A method of causing cognitive enhancement in a human subject, the method comprising administering to the subject a medicament in accordance with any one of claims 1-14. 18. A method of preventing and/or treating Alzheimer's disease in a human subject, the method comprising administering to the subject a medicament in accordance with any one of claims 1-14. 19. Use of an inhibitor of ACPH in a medicament for causing cognitive enhancement in a human subject. 20. Use of an inhibitor of ACPH in a medicament according to any one of claims 1-14 for causing cognitive enhancement in a human subject. 21. Use of an inhibitor of ACPH in a medicament for preventing and/or treating Alzheimer's disease in a human subject. 22. A use according to any one of claims 19-21, where the inhibitor is a selective serine peptidase/hydrolase inhibitor. 23. 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. 24. A medicament substantially as hereinbefore described. 25. A method of making a medicament substantially as hereinbefore described.

Inventors: JOHNSON MARTIN, RAY DAVID, RICHARDS PAUL, JOHNSON, MARTIN, RAY, DAVID, RICHARDS, PAUL
Applicants: BAYER AG, JOHNSON MARTIN, RAY DAVID, RICHARDS PAUL, JOHNSON, MARTIN, RAY, DAVID, RICHARDS, PAUL
Application year: 1999
Main classification: A61K31
IPC codes: A61K31, A61K45, A61P25, A61P43