WO1993007872A1

WO1993007872A1 - Lysosomal enzyme inhibitors for the treatment of neurodegenerative diseases

Info

Publication number: WO1993007872A1
Application number: PCT/GB1992/001902
Authority: WO
Inventors: Roland John Mayer
Original assignee: The University Of Nottingham
Priority date: 1991-10-17
Filing date: 1992-10-16
Publication date: 1993-04-29
Also published as: GB9122051D0

Abstract

The progression of neurodegenerative diseases is hindered by inhibiting lysosomal enzymes. Such enzymes may be inhibited outside the cell or inside the lysosome, for example with enzyme inhibitors such as bestatin or with antibodies directed to the enzyme(s). Active immunisation of the patient may be used to raise such antibodies in situ. Alternatively, the pH of the lysosome may be raised.

Description

LYSOSOMAL ENZYNE INHIBITORS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES

The present invention relates to the treatment of neurodegenerative diseases,

particularly when spongiform lesions occur. By "neurodegenerative diseases"

we mean diseases in which neurons die. Thus, we include stroke, where a

penumbra of cells dies around the initial region of death.

Such diseases also include Alzheimer's Disease (AD), diffuse Lewy Body

Disease, kuru, Creutzfeldt-Jakob Disease (CJD), Gerstmann-Straussler-

Scheinker Disease (GSS) and other dementias in humans, scrapie in sheep and

bovine spongiform encephalopathy (BSE) in cattle. These diseases are

characterised by the development of spongy changes in the brain together with

amyloid plaques. The cause of these diseases is unknown, although in some

cases modes of transmission are known.

The present invention provides a means of at least slowing the progression of

the disease in a patient.

One aspect of the invention provides an enzyme inhibitor for use in medicine,

the enzyme inhibitor being one which inhibits a lysosomal enzyme.

A preferred embodiment of the invention provides an enzyme inhibitor, the enzyme inhibitor being one which is adapted to inhibit a lysosomal enzyme

outside the lysosome.

Such enzyme inhibitors are believed to work, in the context of the present

invention, by inhibiting lysosomal enzymes when they are in the cytoplasm of

neurons and/or in an extracellular environment because of lysis ofthe lysosome

or lysis of the whole cell, respectively. We have found that development of

some of the pathological changes in neurodegenerative diseases, including

spongiform change, involves release of lysosomal enzymes from the lysosomes

and consequent destruction of normal tissue. Lysosomes contain a cocktail of

degradative enzymes, including proteases, upases, nucleases and glycanases,

for example those shown in Table 1.

TABLE 1: Enzymes of the Lysosomal Metabolic Pathways³

Proteolytic Glycanolytic Pathway Pathway

Cathepsin D Hyaluronidase

10

Cathepsin B Heparin endoglucuronidase Cathepsin H Heparan sulphate endoglycosidase

Cathepsin L Lysozyme^b

15

(Glycosidases and phospho Exonuclease (5'- -protein phosphatase also act on intact proteins)

20 α-L-Fucosidase α-Galactosidase iS-Galactosidase

Tripeptidyl peptidase α-Glucosidase Dipeptidyl peptidase I 0-Glucosidase

25 Dipeptidyl peptidase II α-N-Acetylgalactosaminidase Arginyl aminopeptidase α-N-Acetylglucosaminidase (cathepsin H) jS-N-Acetylglucosaminidase

Peptidyl dipeptidase C -Glucuronidase lysosomes) over, there are (cathepsin B) α-L-Iduronidase necessary Carboxypeptidase A α-Mannosidase 'activator' Carboxypeptidase B -Mannosidase proteins) Prolyl carboxypeptidase Neuraminidase Tyrosine carboxypeptidase (8-Aspartylglucosylaminase Dipeptidase I Chrondroitin 6-sulphatase Dipeptidase II Heparin sulphamatase

Iduronosulphatase

10 Sulphatases A and B

Major lysosomal enzymes are grouped here according to the principal classes of natural substrates on which they a Enzymes restricted to lysosomes of one or a very few cell types have mostly not been included here. As indicated f selected examples, enzymes listed in a given pathway participate in other degradation as well, Certain of the enzymes ha

15 broader specifications than their names imply (eg the triacylglycerol lipase probably hydrolyzes cholesterol esters as w as triglycerides) , For lUPAC-IUB Enzyme Commission numbers, consult Barrett A.J., and Heath M.F. (1977) Lysosomes: A Laboratory Handbook (2nd ed,)(J.T. Dingle, ed,), Elsevier: North-Holland, Amsterdam, pp. 22 ff. Ta from Barrett A.J. (1984) Trans. Biochem. Soc, 12: 899. Not widespread among cell types; present principally in phagocytes.

20

Suitable inhibitors of these enzymes include actinonin, amastatin, antipain (to

inhibit cathepsin B), peptide aldehydes (for serine and cysteine proteases),

arphamenine A, arphameinine B, bestatins, chymostatin (for cathepsin B and

D), diprotin A, E-64 ([N-(L-3-frørc5-carboxyoxiran-2-carbonyl)-L-leucyl]-

amido(4-guanidine)butane]) and other peptide epoxides such as Ep-479 and Ep-

460 (for cysteine proteinases), ebelactone A (for esterase and lipase),

ebelactone B (ditto), leupeptin (for cathepsin B), pepstatin A (for cathepsin D),

phosphoramidon and cystatins such as cystatin A, cystatin B, cystatin C,

kininogen and ovocystatin. Other inhibitors are disclosed in Barrett & Salvesen

(1986) "Proteinase Inhibitors", Elsevier, Amsterdam. Many of these inhibitors

are available from Peptide Institute (Osaka, Japan), Boehringer, Cambridge

Research Biochemicals, Novobiochem, Peninsula Laboratories and Sigma.

Modified forms of the enzymes' natural substrates may also be used, for

example modified forms of L-alanine-4-methylcoumaryl-7-amide (Ala-MCA)

or Leu-MCA (for aminopeptidase), Arg-MCA (for cathepsin H), Gly-Pro-MCA

or glycyl-L-proline-p-nitroanilide (for X-prolyl dipeptidylaminopeptidase), Lys-

Ala-MCA (for dipeptidylaminopeptidase II), Phe-MCA (for aminopeptidase),

Z-Arg-Arg-MCA (for cathepsin B), fluoride or tartarate (for acid phosphatase).

Pepstatin, leupeptin, E-64, cystatins, bestatins and mixtures thereof are

particularly preferred. It is generally preferred for the inhibition to be

irreversible or have a K^ < 10^"10 M. Many of these inhibitors, when applied extracellularly, will specifically enter

the lysosome by endocytosis and will thus inhibit lysosomal enzymes in the

lysosome, which may be undesirable unless they are rapidly degraded in the

lysosome. Therefore, they are preferably conjugated with a larger molecule to

prevent endocytosis, as is described in more detail below.

The enzyme inhibitors in this preferred embodiment of the invention are

effective at the neutral pH ofthe cytoplasm or extracellular environments. The

enzyme inhibitors used in this preferred embodiment of the invention should be

selective for the lysosomal enzymes when such enzymes are outside the

lysosome since otherwise the normal function of the lysosomes will be

impaired- Such specificity can be imparted in different ways. Firstly, the

inhibitor may be ineffective at the acid pH of the lysosome. A preferred class

of inhibitors are active at pH 6.0-8.0 but inactive at pH 5.0 and below.

Secondly, the inhibitor can be one which is incapable of gaining entry to the

lysosome; this is most easily achieved by preventing it from gaining access to

the cell at all. Thirdly, the inhibitor can be one which is itself inhibited or

degraded in the lysosome; a limited period of inhibition of a lysosomal enzyme

may be tolerable. Thus, when we refer to the inhibitor not inhibiting the

lysosomal enzyme, we allow for a non-harmful level of inhibition in the

lysosome. Some of these inhibitors have already been proposed for the treatment of HIV

infections, for reasons which are completely different from those which

underlie the present invention, and thus the present invention provides a second

or further medical use of such compounds.

Inhibitor compounds which cannot gain access to the cell or lysosome may be

too large or too hydrophilic and have no transport mechanism (eg endocytosis)

by which they may gain access to the cell or lysosome. Smaller molecules may

be attached to larger ones to form conjugates retaining the inhibitory properties

of the smaller molecule but no longer able to enter the cell or lysosome, for

example inhibitors linked to insoluble matrices. Thus, the compounds indicated

above may be conjugated to polymers for which there is little cell or lysosomal

transmembrane importation mechanism. For example, microspheres of

commercially available resins such as Affi-gel 10 and Affi-gel 15 (Biorad

Laboratories) may be prepared and enzyme inhibitors, especially peptides,

attached thereto very easily. Other suitable matrices from which microspheres

can be prepared include albumin and Sepharose (Regd. T.M., Pharmacia).

The inhibitors used in the invention are suitable for administration to the brain:

if they are not to be injected on the brain side of the blood-brain barrier (BBB),

then they are capable of crossing the BBB in the patient. The inhibitors may be formulated for delivery in known ways and administered

as determined by the clinician. It may be particularly desirable to provide

delayed release formulations using, for example, a sub-cutaneous depot of a

suitable matrix.

In a particular embodiment of the invention, antibodies are raised to at least one

lysosomal enzyme and are administered to the patient, or the patient is himself

immunised with one or more lysosomal enzymes, preferably a cocktail of

lysosomal enzymes. In the former case the antibodies may be polyclonal or

monoclonal and will be selected for inhibitory properties. Genetically

engineered antibody fragments can be small enough to cross the blood-brain

barrier (BBB) and these form a further aspect ofthe invention but, in any case,

in many dementias the BBB becomes chronically more permeable and may

permit whole antibodies to gain entry.

In the latter case, preparations of lysosomal enzymes (active or attenuated) are

injected intramuscularly and subcutaneously in adjuvant preparations.

Enzymically deglycosylated preparations may also be used to diminish their

removal from serum by cellular scavenging pathways (eg by liver Kupfer cells).

Lysosomal glycoproteins are known to rapidly enter lysosomes of antigen-

processing cells to provoke the immune response. All immunogens may be used for cerebrospinal immunization to bypass the

blood brain barrier and provoke the brain immune system.

Monoclonal antibodies may be prepared generally by the techniques of Zola,

H. (1988) "Monoclonal Antibodies: a Manual of Techniques ", C.R.C. Press

which is incorporated herein by reference. Antibody fragments such as F_ab

fragments may be prepared therefrom in known ways. The antibodies may be

humanized in known ways. Antibody-like molecules may be prepared using the

recombinant DNA techniques of WO 84/03712. All such antibodies may be

screened in an enzymatic assay to ensure that they inhibit the enzyme, rather

than just binding to it in a non-inhibitory way.

The art of "antibody engineering" is advancing rapidly, as is described in Tan,

L.K. and Morrison, S.L. (1988) Adv. Drug Deliv. Rev. 2, 129-142, Williams,

G. (1988) Tibtech 6, 36-42 and Neuberger, M.S. et al (1988) 8th International

Biotechnology Symposium Part 2, 792-799 (all of which are incorporated herein

by reference), and is well suited to preparing suitable inhibitors.

Active immunisation of the patient is preferred. In this approach, one or more

lysosomal enzymes are prepared in an immunogenic formulation containing

suitable adjuvants and carriers and administered to the patient in known ways.

Suitable adjuvants include Freund's complete or incomplete adjuvant, muramyl dipeptide, the "Iscorns" of EP 109 942, EP 180 564 and EP 231 039,

aluminium hydroxide, saponin, DEAE-dextran, neutral oils (such as migiyol),

vegetable oils (such as arachis oil), liposomes, Pluronic polyols or the Ribi

adjuvant system (see, for example GB-A-2 189 141). "Pluronic" is a

Registered Trade Mark.

It may be advantageous to use an enzyme from a species other than the one

being treated, in order to provide for a greater immunogenic effect. Another

compound can be used instead of the whole enzyme in order to produce

inhibitory antibodies in the patient. Such other compounds include fragments

and analogues of the enzymes, especially peptides corresponding to

predominantly hydrophilic regions of the enzyme and peptides adjoining the

catalytic site of the enzyme. Such compounds may be screened as above to

ensure that they are capable of producing inhibitory antibodies in the patient.

The sequence of peptides useful in this aspect of the invention may be predicted

from the corresponding gene or cDNA sequences encoding the aforementioned

lysosomal enzymes.

Peptides in which one or more of the amino acid residues are chemically

modified, before or after the peptide is synthesised, may be used providing that

the function of he peptide, namely the production of specific antibodies in vivo, remains substantially unchanged. Such modifications include forming salts with

acids or bases, especially physiologically acceptable organic or inorganic acids

and bases, forming an ester or amide of a terminal carboxyl group, and

attaching amino acid protecting groups such as N-t-butoxycarbonyl. Such

modifications may protect the peptide from in vivo metabolism. The peptides

may be present as single copies or as multiples, for example tandem repeats.

Such tandem or multiple repeats may be sufficiently antigenic themselves to

obviate the use of a carrier. It may be advantageous for the peptide to be

formed as a loop, with the N-terminal and C-terminal ends joined together, or

to add one or more Cys residues to an end to increase antigenicity and/or to

allow disulphide bonds to be formed. If the peptide is covalently linked to a

carrier, preferably a polypeptide, then the arrangement is preferably such that

the peptide of the invention forms a loop.

According to current immunological theories, a carrier function should be

present in any immunogenic formulation in order to stimulate, or enhance

stimulation of, the immune system. It is thought that the best carriers embody

(or, together with the antigen, create) a T-cell epitope. The peptides may be

associated, for example by cross-linking, with a separate carrier, such as serum

albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.

More recently developed carriers which induce T-cell help in the immune

response include the hepatitis-B core antigen (also called the nucleocapsid protein), presumed T-cell epϊtopes such as Thr-Ala-Ser-Gly-Val-Aia-Glu-Thr-

Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-l . The

latter compound may variously be regarded as a carrier or as an adjuvant or as

both. Alternatively, several copies of the same or different peptides of the

invention may be cross-linked to one another; in this situation there is no

separate carrier as such, but a carrier function may be provided by such cross-

linking. Suitable cross-linking agents include those listed as such in the Sigma

and Pierce catalogues, for example glutaraldehyde, carbodiimide and

succinimidyl 4-(N-maIeimidomethyl)cyclohexane- 1 -carboxy late, the latter agent

exploiting the -SH group on the C-terminal cysteine residue (if present).

If the peptide is prepared by expression of a suitable nucleotide sequence in a

suitable host, then it may be advantageous to express the peptide as a fusion

product with a peptide sequence which acts as a carrier. Kabigen's "Ecosec"

system is an example of such an arrangement.

The peptide of the invention may be linked to other antigens to provide a dual

effect.

Peptides (ie enzyme fragments for immunisation) may be synthesised by the

Fmoc-polyamide mode of solid-phase peptide synthesis. Temporary N-amino

group protection is afforded by the 9-fluoreny methyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is

effected using 20% piperidine in N,N-dimethylformamide. Side-chain

functionalities may be protected as their butyl ethers (in the case of serine

threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic

acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl

derivative (in the case of cysteine) and 4-methoxy-2,3,6-

trimethylbenzenesulphonyl derivative (in the case of arginine). Where

glutamine or asparagine are C-terminal residues, use is made of the 4,4'-

dimethoxybenzhydryl group for protection of the side chain amido

functionalities. The solid-phase support is based on a polydimethyl-acrylamide

polymer constituted from the three monomers dimethylacrylamide (backbone-

monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine

methyl ester (functionalising agent). The peptide-to-resin cleavable linked agent

used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All

amino acid derivatives are added as their preformed symmetrical anhydride

derivatives with the exception of asparagine and glutamine, which are added

using a reversed N,N-dicyclohexyl-carbodiimide/l-hydroxybenzotriazole

mediated coupling procedure. All coupling and deprotection reactions are

monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test

procedures. Upon completion of synthesis, peptides are cleaved from the resin

support with concomitant removal of side-chain protecting groups by treatment

with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithioi, phenol, anisole and water, the exact choice

depending on the constituent amino acids of the peptide being synthesised.

Trifluoroacetic acid is removed by evaporation in vacuo. with subsequent

trituration with diethyi ether affording the crude peptide. Any scavengers

present are removed by a simple extraction procedure which on lyophilisation

of the aqueous phase affords the crude peptide free of scavengers. Purification

may be effected by any one, or a combination of, techniques such as size

exclusion chromatography, ion-exchange chromatography and (principally)

reverse-phase high performance liquid chromatography. Analysis of peptides

may be carried out using thin layer chromatography, reverse-phase high

performance liquid chromatography, amino-acid analysis after acid hydrolysis

and by fast atom bombardment (FAB) mass spectrometric analysis.

The peptides and adjuvants and/or carriers may be formulated in any suitable

way which may be devised by the man skilled in the art using known or yet-to-

be-discovered delivery vehicles and criteria. In particular, the formulations

may be based on biodegradable polymers such as lactide glycolide copolymers,

such as those described in EP-A-58581 (ICI).

The small peptides and antigenic compositions of the invention will usually be

administered intravenously, sub-cutaneously or intra-muscularly although intra-

nasal, transdermal, oral and rectal routes may be suitable for the same formulations of the invention. The formulation will normally be sterile and (for

parenteral use) non-pyrogenic and a unit dose will typically include 1 to 1000

μg of the small peptide of the invention, typically 10 to 500 μg, preferably

about 50 μg or less. One or more repeat immunisations may be advantageous,

as is known in the art of immunology. The formulations may generally be

prepared and/or administered by a physician or veterinary surgeon according

to his skill and expertise.

A further aspect of the invention provides a process for preparing one of the

enzyme fragments mentioned above, by known methods of peptide synthesis or

by appropriate cleavage of the native enzyme molecule. Peptide synthesis may

be achieved according to the general method of Stewart et al, described in

"Solid Phase Peptide Synthesis" (W.H. Freeman, San Francisco, 1969) or by

the methods described by Marglin and Merrifield in Annual Reviews of

Biochemistry 39, 841-866 at 862 (1970), and subsequent articles. Established

methods of peptide synthesis by solid phase and similar techniques are usually

not suitable for large scale production (although they may become so in the

future) and thus commercial production of the peptides would normally be by

cultivation of a suitable organism transformed with a polynucleotide sequence

encoding the desired peptide. Thus, further aspects of the invention include

such polynucleotides, transformation and expression vectors carrying such

polynucleotides, organisms transformed therewith and processes for cultivating such organisms.

In certain circumstances, it may be preferably to use the inhibitory compounds

to inhibit lysosomal enzymes within the lysosome. So, in a further preferred

embodiment an enzyme inhibitor is one which is adapted to inhibit a lysosomal

enzyme within the lysosome. In this preferred embodiment, inhibitors of the

enzymes listed in Table 1 are, preferably, active at pH5.0 or less, ie in the acid

pH of the lysosome; can enter the lysosome in ways described below; and

preferably are not degraded, or at least not quickly, in the lysosome.

The cellular lysosome system is amenable to drug targeting yja the endocytic

pathway. The inhibitory compounds of the invention may be conjugated to

olϊgosaccharides, or to glycoproteins, or to fragments thereof which are known

to enter the lysosomes yja the endocytic route. Such an example of a

glycoprotein known to enter the endocytic route is the mannose 6-phosphate

receptor. Some inhibition may enter the lysosomes without further conjugation.

It is preferable to inject such inhibitory compounds either directly into the brain

behind the BBB, or into the cerebrospinal fluid, so that non-specific lysosomal

targeting of the said compounds is limited.

Slow release formulations may be based on biopolymers that target to, and are degraded in, the lysosomes.

Of course, the features and uses of the inhibitory compounds disclosed in the

first preferred embodiment of the invention may be common to those of the

second preferred embodiment, except in this second embodiment the inhibitory

compounds are able to enter the lysosomes where they are active in inhibiting

lysosomal enzymes, whereas in the first embodiment the inhibitory compounds

are not able to enter the lysosomes, at least to any great extent, but can inhibit

lysosomal enzymes outside of the lysosome.

A further aspect of the invention is the use of a weak organic base which

increases the pH of the lysosome by at least 0.1 pH units when the said base

enters the lysosome for the manufacture of a medicament for use in treating

neurodegenerative disease.

At least some weak organic bases act to increase the pH of the lysosomal

compartments and thereby inhibit the activity of at least some lysosomal

enzymes, and may lead to the export of lysosomal enzymes from the cell rather

than to the lysosomes. Such alterations in enzyme activities within the

lysosome, and the subsequent changes in protein processing by the lysosome,

particularly those associated with the development of amyloid plaques, are

beneficial in ameliorating neurodegenerative disease. The increase in pH by at least 0-1 pH units, preferably at least 0.5 pH units,

for example 1.0, 1.5, 2.0 or 2.5 units, but preferably no more than 3.5 pH

units, more preferably no more than 3.0 pH units, brought about by the weak

organic base, may be monitored by partitioning lϊpophilic, pH-sensitive dyes

across the lysosomal membrane; and observing the respective absorption signals

within isolated lysosomes and in the cytoplasm.

In a preferred embodiment ofthe invention, 4-aminoquinoIine or its derivatives

are used.

In further preference, chloroquine, hydroxychloroquine or amodiaquine or their

pharmaceutically acceptable salts are used. These particular compounds are

well known in medicine for use in the prevention or treatment of malarial

infection.

Chloroquine and the other weak bases of the invention are able to locate to the

lysosome and are effective without being conjugated to a specific lysosome

targeting moiety.

In this further medical use, chloroquine, and its pharmaceutically acceptable

salts, are used in the treatment of neurodegenerative disease- Chloroquine

phosphate may be administered orally as tablets each containing between 1 mg and 1 g of chloroquine. It is preferable to use between 10 mg and 750 mg, and

is more preferable to use between 250 mg and 500 mg. Chloroquine

hydrochloride may be injected intramuscularly in a formulation with a sterile,

non-pyrogenic medium, such as sterile distilled water. Each injection may

contain between 1 mg and 1 g of chloroquine. It is preferable to use between

10 mg and 750 mg and is more preferable to use between 250 mg and 500 mg.

It may be preferable to combine chloroquine with cetylsteryl alcohol to protect

it from the leaching effect of high humidity.

The invention will now be described in further detail, by way of example.

EXAMPLE 1

E-64 is made up as an 0.1 % aqueous solution in sterile, non-pyrogenic

isoosmotic saline and a 10-500 mg dose is administered daily by intravenous

injection for a period of 3 months.

EXAMPLE 2

Lysosome and endosome preparations from human brain are obtained following

brain homogenisation and centrifugation on isoosmotic inert polymer gradients of Nycodenz or Percoll. Extracts of lysosomes and endosomes are then

prepared by mild detergent fractionation.

A lysosomal enzyme mixture (from human brain lysosomes) is inactivated by

heat treatment or chemical modification and then prepared in an immunogenic

formulation as follows:

Enzyme mixture 1 mg in 0.5 mi

Freund's incomplete adjuvant 0.5 ml

The 1 ml dose is injected sub-cutaneously in the left buttock of a patient,

followed three weeks later by a similar booster dose of 1 ml, in order to

generate anti-enzyme antibodies.

The immumzation schedule is started when early stages of Alzheimer or Prion-

related disorders are detected, for example, when Alzheimer precursor protein

fragments or Prion proteins are found in cerebrospinal fluid or seruπu

EXAMPLE 3: Production of antibodies which inhibit lysosomal enzymes

using inactivated enzymes as immunogen.

Lysosomal-endosomal enzymes are prepared as described in Example 2. The enzymes are assayed with substrates which generate fluorescent products after

cleavage such as coumarin derivatives of each of proteins, peptides,

sphingoiipids and glycoproteins. Immunogenic preparations of the lysosomal

and endosomal enzymes are prepared using standard techniques and are used

to immunize mice. Hybridoma cells secreting monoclonal antibodies are made

in the usual way. Each of the monoclonal antibodies so produced are screened

in assays containing the lysosomal and endosomal enzyme mixture and each of

the coumarin conjugates. The inhibitory effect of each monoclonal antibody is

determined by comparison with equivalent assay mixtures lacking the

monoclonal antibody.

EXAMPLE 4: Peptides for use as immunogens.

Three peptide sequences were chosen for each human lysosomal enzyme for

which a sequence is available. In most cases, at least one of the peptide spans

an active site region of the enzyme. Each peptide sequence was synthesised,

using standard Fmoc chemistry, with an additional N-terminal cysteine residue

and each with an additional C-terminal cysteine residue, and each with no

additional cysteine residue.

Sequences have 12 residues (or 13 with the additional cysteine) given in single

letter code. 3-gIucuronidase which hydrolyses glycosaminoglycans

Sequences corresponding to residues

141-152 EHEGGYLPFEAD;(C) EHEGGYLPFEAD; EHEGGYLPFEAD (C)

445-456 MWSVANEPASHL;(C) MWSVANEPASHL; MWSVANEPASHL (C)

568-580 YHLGLDQKRRKY;(C) YHLGLDQKRRKY; YHLGLDQKRRKY (C)

Cathepsin B which hydrolyses proteins

Sequences corresponding to residues

102-113 QGSCGSCWAFGA;(C) QGSCGSCWAFGA; QGSCGSCWAFGA(C)

269-280 HVTGEMMGGHAI;(C) HVTGEMMGGHAI; HVTGEMMGGHAI(C)

289-300 NGTPYWLVANSW;(C)NGTPYWLVANSW; NGTP YWLVANSW(C)

Cathepsin D which hydrolyses proteins

Sequences corresponding to residues

89-100 PQCFTVVFDTGS;(C) PQCFTVVFDTGS; PQCFTVVFDTGS (C)

21-232 QKLVDQNIFSFY;(C) QKLVDQNIFSFY; QKLVDONIFSFY (C)

89-300 GCEAIVDTGTSL;(C) GCEAIVDTGTSL; GCEAIVDTGTSL (C) Cathepsin H which hydrolyses proteins and has aminopeptidase activity

Sequences corresponding to residues

135-146 QGACGSCWTFST;(C) QGACGSCWTFST; QGACGSCWTFST (C)

275-286 TPDKVNHAVLAV ;(C) TPDKVNHAVLAV; TPDKVNHAVLAV (C)

295-306 PYWIVKNSWGPQ; (C) PYWIVKNSWGPQ; PYWIVKNSWGPQ (C)

Cathepsin L which hydrolyses proteins

Sequences corresponding to residues

133-144 GQCGSCWAFSAT;(C) GQCGSCWAFSAT;GQCGSCWAFSAT (C)

271-282 SEDMDHGVLVVG;(C) SEDMDHGVLVVG;SEDMDHGVLVVG(C)

295-306 YWLVKNSWGEE;(C) YWLVKNSWGEE; YWLVKNSWGEE (C)

β-hexosaminidase which hydrolyses gangliosides

Sequences corresponding to residues

121-132 NDDQCLLLSETV;(C) NDDQCLLLSETV; NDDQCLLLSETV (C)

321-332 GDEVDFTCWKSN;(C) GDEVDFTCWKSN; GDEVDFTCWKSN (C)

471-482 PRLWPRAGAVER ;(C) PRLWPRAGAVER; PRLWPRAGAVER (Q

The additional N- and C-terminal cysteines are shown in parentheses. EXAMPLE 5: Conjugation to ovalbumin.

3.0mg of peptide is dissolved in 300μi of dimethyl formamide. 150μl of

lOmg/ml ovalbumin in Dulbecco's phosphate buffered saline (PBS) is added

and thoroughly mixed. 250μl of freshly prepared 0.04M glutaraldehyde is

added slowly, with stirring, over a period of 10 minutes then left at room

temperature for a further 60 minutes with continuous mixing (SPIRAMIX,

Deiύey Instruments). 1.0ml of PBS is added and followed by a further lOOμl

of 0.04M glutaraldehyde as above. This is left for 60 minutes at room

temperature before being dialysed overnight at +4°C against PBS.

EXAMPLE 6: Adjuvants & Administration - 'Freunds'.

After dialysis, the volumes of the preparation of Example 5 may be made up

to 4.5ml with PBS and 'water-in-oil' emulsions prepared using two volumes of

Freund's Complete Adjuvant (FCA) (Difco or Sigma). This may be achieved

by sonication in the cold or using a Potter-Elvehjen homogeniser. Emulsions

may be tested by dispersion (or absence) on a water surface. The formulation

is administered subcutaneously.

Twenty eight or thirty five days after the primary immunisation a second,

similar immunisation may be completed using freshly prepared antigen conjugated in the same way but emulsified into Freund's Incomplete Adjuvant

(FIA, Difco or Sigma). Subsequent boosts may be given at further intervals

of twenty one and fifty three days at one-half and one-quarter of the original

dose.

EXAMPLE 7: Conjugation to Keyhole Limpet Haemocyanin (KLH).

Peptides may be conjugated in the same way as described above, but using

KLH as the carrier instead of ovalbumin. A lOmg/ml solution of KLH is

prepared in PBS by rolling overnight with borosilicate glass beads (5mm

diameter). After dialysis the volumes of the peptide + conjugate preparations

are made up to 4.5ml with PBS and water-in-oil emulsions prepared using two

volumes of Freund's Complete Adjuvant (Sigma). This may be achieved by

sonication after cooling the well shaken oil 4- aqueous peptide mix to 0°C and

pre-cooling the probe of the sonicator. Two short (5 seconds) bursts at full

power (SONIPREP 150, Gallenkamp, Loughborough, England) should produce

a stable emulsion ready to use.

The peptide so prepared may be administered subcutaneously to give 500μg

peptide per patient. A second immunisation using a similar, fresh, preparation

may be given 28 days later in Freund's Incomplete Adjuvant. EXAMPLE 8: Assaying compounds for their inhibitory effect on

Alzheimer precursor protein (APP) and prion protein processing.

In order to assay compounds for their anti-lysosomal activity, including their

ability to inhibit the processing of APP and prion proteins, human tissue culture

fibroblasts, kidney and neuroblastoma cells were transfected with the cDNA for

APP and independently with the cDNA for prion protein using methods known

in the art.

The generation from APP of β/A4 amyloidogenic fragments in transfected cells

was detected using antibodies which specifically bind to these fragments.

Similarly, pulse-chase labelling of cells with radioactive amino acids (eg ³H-

amino acids), followed by SDS-PAGRE electrophoresis and fluorography

revealed the amyloidogenic fragments in the case of APP cDNA transfected

cells, or prion protein-related fragments in the case of prion protein cDNA-

transfected cells. Those compounds preventing the formation of either j8/A4

peptides or prion protein fragment, or slow their synthesis, were potential

lysosomal inhibitors. Such compounds were then evaluated for their effects on

cell morphology and the structure and function of the lysosome system. EXAMPLE 9: Assaying compounds for their anti-spongiform effects

Compounds are evaluated in animals infected with vacuolating strains of

scrapie. Mice and hamsters are infected with scrapie using methods known in

the art such as those taught by Carlson et al (1986) Cell 46, 503-511 and

citations contained therein are incorporated in this Example by way of

reference. The compounds of Example 3 are injected into the brain of scrapie-

infected mice and scrapie-infected hamsters and over a period of weeks the

effect of these compounds on the size and number of spongiform vacuoles is

assessed. Those compounds which lead to a reduction in either the size, or

number, or both, of such vacuoles are evaluated further.

Claims

1. An enzyme inhibitor for use in medicine, the inhibitor being one that

inhibits a lysosomal enzyme.

2. An enzyme inhibitor according to Claim 1, the inhibitor being one

which is adapted to inhibit a lysosomal enzyme outside the lysosome.

3. An enzyme inhibitor according to Claim 1, the inhibitor being one

which is adapted to inhibit a lysosomal enzyme within the lysosome.

4. An enzyme inhibitor according to Claim 1 which is pepstatin,

leupeptin, E-64, a cystatin or a bestatin.

5. The use of a weak organic base which increases the pH of the

lysosome by at least 0.1 pH units when the said base enters the

lysosome, in the manufacture of a medicament for use in treating

neurodegenerative disease.

6. The use of 4-aminoquinolιne or a derivative thereof in the manufacture

of a medicament for use in treating neurodegenerative disease.

7. The use of chloroquine, hydroxychloroquine, amodiaquine or a

pharmaceutically acceptable salt of any of these in the manufacture of

a medicament for use in treating neurodegenerative disease.

8. A method of treating neurodegenerative disease comprising

administering to a patient having the disease a weak organic base which

increases the pH of the lysosome by at least 0.1 pH unit when the said

base enters the lysosome.

9. An antibody specific for a lysosomal enzyme wherein the antibody,

when bound to the enzyme, inhibits the enzymatic function of the

enzyme.

10. An antibody specific for a lysosomal enzyme according to Claim 9

wherein the antibody binds to the active site of the enzyme.

11. An antibody according to Claim 9 or 10 wherein the said antibody is

a monoclonal antibody.

12. An enzyme inhibitor according to Claim 2 wherein a portion is adapted

to inhibit a lysosomal enzyme and a portion is adapted to prevent entry

of the molecule into the lysosome of a mammalian cell.

13. An enzyme inhibitor according to Claim 3 wherein a portion is adapted

to inhibit a lysosomal enzyme and a portion is adapted to enhance

uptake into the lysosome of a mammalian cell.

14. A peptide, wherein the peptide constitutes the active site of a lysosomal

enzyme.

15. The use of a peptide according to Claim 14 to raise antibodies.

16. An antibody raised according to Claim 14.

17. A formulation suitable for injection into a mammal, the formulation

comprising a lysosomal enzyme inhibitor.

18. An immunogenic formulation suitable for administration to a mammal,

the formulation comprising (1) a lysosomal enzyme or an active site

peptide thereof or other molecule adapted to produce, in the mammal,

inhibitory antibodies specific for the enzyme and (2) a suitable adjuvant

or earner.

19. An immunogenic formulation according to Claim 18 for use in

medicine-

20. The use of an antibody according to any of Claims 9, 10, 1 1 and 16 in

ameliorating neurodegenerative disease.

21. A method of ameliorating or hindering the progression of a

neurodegenerative disease, the method comprising administering to a

mammalian patient suffering from a said disease a non-toxic, disease-

ameliorating amount of a lysosomal enzyme inhibitor.

22. A method of ameliorating or hindering the progression of a

neurodegenerative disease, the method comprising administering to a

mammalian patient suffering from a said disease an immunogenic

formulation according to Claim 18.

AMEMDED CLAIMS

[received by the International Bureau on 7 April 1993 (07.04.93) original claims 1-22 replaced by amended claims 1-19 (3 pages⁾ ]

1. The use of an enzyme inhibitor, the inhibitor being one that inhibits a lysosomal enzyme, in the manufacture ofa medicament for use in treating a neurodegenerative disease, provided that (1) the inhibitor is not leupeptin, or (2) if the disease is Alzheimer's disease the inhibitor is not pepstatin or (3) the lysosomal enzyme is not phospholipase A₂.

2. The use of an enzyme inhibitor, the inhibitor being one that is adapted to inhibit a lysosomal enzyme outside the lysosome, in the manufacture of a medicament for use in treating neurodegenerative disease, provided that the lysosomal enzyme is not phospholipase A₂.

3. The use of an enzyme inhibitor according to Claim 1, the inhibitor being one which is adapted to inhibit a lysosomal enzyme within the lysosome.

4. The use of an enzyme inhibitor according to Claim 1 or 2 which is E-64, a cystatin or a bestatin.

5. The use of an enzyme inhibitor according to Claim 2 wherein the inhibitor is an antibody specific for a lysosomal enzyme which, when bound to the enzyme, inhibits the enzymatic function of the enzyme.

6. The use of an enzyme inhibitor according to Claim 5 wherein the antibody binds to the active site of the enzyme. 7. The use of an enzyme inhibitor according to Claim 6 wherein the antibody is a monoclonal antibody.

8. The use of an enzyme inhibitor according to Claim 2 wherein a portion is adapted to inhibit a lysosomal enzyme and a portion is adapted to prevent entry of the molecule into the lysosome of a mammalian cell.

9. The use of an enzyme inhibitor according to Claim 3 wherein a portion is adapted to inhibit a lysosomal enzyme and a portion is adapted to enhance uptake into the lysosome of a mammalian cell.

10. The use of hydroxychloroquine, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in treating neurodegenerative disease.

11. A method of treating neurodegenerative disease comprising administering to a patient having the disease hydroxychloroquine or a pharmaceutically acceptable salt thereof.

12. A peptide, wherein the peptide constitutes the active site of a lysosomal enzyme provided that the enzyme is not phospholipase A₂, or cathepsins B, L or D.

13. The use of a peptide according to Claim 12 to raise or screen antibodies.

14. An antibody raised or screened according to Claim 13. 15. A formulation suitable for injection into a mammal, the formulation comprising a lysosomal enzyme inhibitor as defined in any one of Claims 1 to 9.

16. An immunogenic formulation suitable for administration to a mammal, the formulation comprising (1) a lysosomal enzyme or an active site peptide thereof or other molecule adapted to produce, in the mammal, inhibitory antibodies specific for the enzyme and (2) a suitable adjuvant or carrier provided that the lysosomal enzyme is not phospholipase A₂ and the active site peptide is not derived from phospholipase A₂, or cathepsins B, L or D.

17. An immunogenic formulation suitable for administration to a mammal, the formulation comprising (I) a lysosomal enzyme or an active site peptide thereof or other molecule adapted to produce, in the mammal, inhibitory antibodies for the enzyme and (2) a suitable adjuvant or carrier for use in medicine.

18. A method of ameliorating or hindering the progression of a neurodegenerative disease, the method comprising administering to a mammalian patient suffering from a said disease a non-toxic, disease ameliorating or disease-progression-hindering amount of - a medicament prepared according to any one of Claims 1 to 9.

19. A method of ameliorating or hindering the progression of a neurodegenerative disease, the method comprising administering to a mammalian patient suffering from a said disease an immunogenic formulation according to Claim 17.