WO1999008672A1

WO1999008672A1 - Use of cholinesterase inhibitor for treating diseases associated with proteolytic enzyme activity

Info

Publication number: WO1999008672A1
Application number: PCT/GB1998/002448
Authority: WO
Inventors: Ernir Snorrason; James Robert Murray
Original assignee: Shire International Licensing Bv
Priority date: 1997-08-15
Filing date: 1998-08-14
Publication date: 1999-02-25
Also published as: AU8742198A

Abstract

The invention relates to the use of a pharmaceutically acceptable cholinesterase inhibitor or a pro-drug therefor in the manufacture of a medicament for combatting diseases associated with proteolytic enzyme activity, such as psoriasis, osteoarthritis, rheumatoid arthritis, Crohn's disease and Ulcerative Colitis.

Description

USE OF CHOLINESTERASE INHIBITOR FOR TREAΗNG DISEASES ASSOCIATED WITH PROTEOLYΗC ENZYME ACTIVITY

The present invention relates to a method of therapy. In particular the invention relates to a method of combatting diseases associated with elevated levels of proteolytic enzymes.

Proteolytic enzymes have been found to play a role in a variety of diseases. These include skin diseases such as psoriasis, osteoarthritis, rheumatoid arthritis, other forms of arthritis, inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, chronic obstructive pulmonary diseases, amyloidosis, emphysema and certain cancers such as cancer of the pancreas, breast and colon. Despite what might be perceived as an extremely diverse range of symptoms exhibited by patients suffering from these diseases, they are all characterised by the underlying involvement of proteolytic enzymes. We have developed a new therapeutic method capable of combatting any of these diseases.

Thus we have surprisingly found that cholinesterase inhibitors such as galantamine can effectively combat diseases associated with proteolytic enzymes at the affected body site.

According to one aspect, the present invention provides a method of combatting diseases associated with proteolytic enzyme activity comprising administering to a subject a pharmaceutically acceptable cholinesterase inhibitor.

According to a related aspect, the invention provides the use of a pharmaceutically acceptable cholinesterase inhibitor in the manufacture of a medicament for combatting diseases associated with proteolytic enzyme activity.

As used herein the term 'combatting' includes both therapy and prophylaxis. Without wishing to be bound by theory, it is believed that the acetylcholinesterase inhibitors act to block or inhibit the protease activity. This belief is based on our findings that in joint diseases such as osteoarthritis and rheumatoid arthritis, where increased protease activity in synovial fluid is well established, there is an increase in acetylcholinesterase levels, and that treatment with acetylcholinesterase inhibitors such as galantamine has been found useful in counteracting the symptoms associated with joint diseases. It is believed that in such treatment of joint diseases the proteolytic activity is associated with acetylcholinesterase and that the acetylcholinesterase inhibitor is acting to block or inhibit proteolytic activity. It is further believed that the proteolytic activity in other diseases is associated with acetylcholinesterase and that such acetylcholinesterase inhibitors will likewise act to block or inhibit proteolytic activity and thereby counteract the symptoms of other diseases associated with proteolytic enzyme activity.

It is well established that proteolytic enzyme activity plays a role in diseases such as psoriasis, Crohn's disease and ulcerative colitis. Thus collagenase is known to be associated with the inflammatory bowel diseases ulcerative colitis and Crohn's disease, PMN protease is associated with psoriasis, elastase with psoriasis and ulcerative colitis, proteinase 3 and plasminogen activator with psoriasis.

Psoriasis is one example of a disease where proteolytic enzyme activity play a role. Psoriasis is an inflammatory skin disease which can effect all age groups. It is characterised by redness and scaly patches over the skin surface. The disfiguration which results can cause considerable distress to the sufferer, with implications on all aspects of daily life. Current therapeutic approaches aim to reduce epidermal cell turnover and thereby control the skin lesions without causing damage to the skin or to other organs. There is no known cure. Treatment by drugs generally involves topical application to the affected sites (s) . Topical steroids have been used extensively however long term use of steroids is limited by the skin atrophy which occurs and is not recommended. Other topical agents include tar and dithranol but these have the disadvantage that they cause staining of the skin and are cosmetically unacceptable. Systemic therapies generally used for severe psoriasis include the antimitotic agent methotrexate, which has serious side effects of liver damage, and retinoic acid, which is teratogenic . All existing therapeutic approaches are thus unsatisfactory and there is a need for a new method of combatting psoriasis. The present invention provides such a method.

Thus in a further aspect, the invention provides a method of combatting psoriasis comprising administering to a subject a pharmaceutically acceptable cholinesterase inhibitor.

According to a related aspect, the invention provides the use of a pharmaceutically acceptable cholinesterase inhibitor in the manufacture of a medicament for combatting psoriasis.

Arthritic diseases, such as osteoarthritis and rheumatoid arthritis are also examples of diseases where proteolytic enzyme activity plays a role, as are other rheumatoid diseases such as Juvenile Arthritis, Systemic

Lupus Erythematosis, Sjδgren's Syndrome, Progressive Systemic Sclerosis, Polymyositis, Dermatomyositis, Ankylosing Spondilitis, Reiter's Syndrome, Psoriatic Arthritis, Relapsing Polychondritis , Relapsing Panniculitis, Crohn's Disease, Ulcerative Colitis,

Hereditary Complement Deficiencies, Collagen Vascular Diseases, Felty's Syndrome, rheumatological manifestations associated with bacterial and viral endocarditis or myocarditis and other rheumatological manifestations such as anaemia of chronic disorders. The invention also relates to a novel method of treatment or prophylaxis of such diseases and manifestations. The method comprises administering a pharmaceutically acceptable cholinesterase inhibitor.

As used herein, the term "rheumatoid" covers any of a variety of disorders marked by degeneration or metabolic derangement of the connective tissue structures of the body, especially the joints and related structures, including muscles, bursae (synovial membranes), tendons and fibrous tissue. They are attended by pain, stiffness, or limitation of motion of these parts. Rheumatoid Arthritis is a chronic, recurrent systemic inflammatory disease primarily of the joints, usually polyarticular, marked by inflammatory changes in the synovial membranes and articular structures and by atrophy and rarefaction of the bones. In late stages deformity and ankylosis develop. Extra- articular manifestations include vasculitis, atrophy of the skin and muscle, subcutaneous nodules, lymphadenopathy, splenomegaly, leukopaenia and often chronic anaemia. In one important embodiment of the invention, the arthritic disorder is osteoarthritis. Osteoarthritis is a chronic degenerative disease of skeletal joints, which affects specific joints, commonly knees, hips, hand joints and spine, in adults of all ages. Osteoarthritis is characterized by a number of the following manifestations including degeneration and thinning of the articular cartilage with associated development of "ulcers" or craters, osteophyte formation, hypertrophy of bone at the margins, and changes in the synovial membrane and enlargement of affected joints.

Furthermore, osteoarthritis is accompanied by pain and stiffness, particularly after prolonged activity. The method of the invention results in the treatment or prevention of one or several of the following symptoms or signs associated with the above- mentioned diseases: muscle pain, muscle weakness, muscle stiffness, joint stiffness, joint pain, joint or tissue swelling, inflammation, and extra-articular manifestation as mentioned above, including anaemia.

The invention is based on discoveries made in connection with the treatment of patients for other conditions, e.g. chronic fatigue syndrome.

Surprisingly, it was found that patients who, in addition to fibromyalgia or chronic fatigue syndrome, suffered from osteoarthritis or rheumatoid arthritis, had improvement of their arthritis symptoms. Thus, ten patients with rheumatoid arthritis were treated with galantamine. The purpose of the treatment was to treat their fatigue syndrome resulting from their rheumatoid arthritis. However, it was found that not only did the ten patients improve with respect to their fatigue syndrome, but also in the symptoms of their rheumatoid arthritis condition itself. They had less articular pain and the inflammatory process was less active (as evidenced by less swollen and less painful joints) while they were on treatment with galantamine. In other studies, reported in detail below, patients suffering from rheumatoid arthritis, osteoarthritis and other rheumatoid diseases, respectively, experienced marked improvements of their condition, e.g. with respect to tiredness, pain, stiffness and grip strength, upon treatment with galantamine .

It was also found by direct measurement that there is a considerable acetylcholinesterase activity in the synovial fluid of patients with rheumatological conditions. This finding is new and may shed light on the clinical observations with galantamine in rheumatological patients as this acetylcholinesterase activity is believed to exert a proteolytic activity in the synovial fluid of these patients and may be directly related to tissue damage seen in both rheumatoid diseases and in osteoarthritis. Patients with rheumatoid diseases or other chronic diseases very often suffer from anaemia of unknown pathophysiology . It is a normochromic, normocytic or hypochromic, non-progressive anaemia - the severity being related to the severity of the chronic disease. Both the serum iron and total iron binding capacity are reduced. The serum ferritin is normal or elevated and bone marrow storage iron is normal but erythroblast iron is reduced. The pathogenesis of this anaemia appears to be related to the decreased release of iron from macrophages, reduced cell lifespan and inadequate erythropoietin response to the anaemia. This anaemia is normally only corrected by successful treatment of the underlying disease and does not respond to iron therapy despite the low serum iron. Thus the present invention relates to the use of cholinesterase inhibitors for the preparation of a medicament for treatment or prophylaxis of anaemia associated with chronic disorders.

The present invention provides a method of treating or preventing arthritic disorders, including osteoarthritis, rheumatoid arthritis and other rheumatoid diseases such as Juvenile Arthritis, Systemic Lupus Erythematosis, Sjδgren's Syndrome, Progressive Systemic Sclerosis, Polymyositis, Dermatomyositis , Ankylosing Spondilitis, Reiter's Syndrome, Psoriatic

Arthritis, Relapsing Polychondritis, Relapsing Panniculitis, Crohn's Disease, Ulcerative Colitis, Hereditary Complement Deficiencies, Collagen Vascular Diseases, Felty's Syndrome, rheumatological manifestations associated with bacterial and viral endocarditis or myocarditis and other rheumatological manifestations such as anaemia of chronic disorders, said method comprising administering to a patient in need thereof an effective amount of a cholinesterase inhibitor.

The invention encompasses the use of any cholinesterase inhibitor, provided of course that it is pharmaceutically acceptable.

Examples of cholinesterase inhibitors which may be used according to the invention include, but are not limited to, physostigmine, tacrine and tacrine analogues, fasiculin, metrifonate, heptyl -physostigmine, norpyridostigmine, norneostigmine, huperazine, donepezil and pro-drugs of any of these in which the inhibitor is modified in accordance with principles of pro-drug construction known in the art. Examples of such modifications include the introduction of hydrophilic or lipophilic groups to enhance solubility, or penetration through cell membranes, respectively.

Preferred cholinesterase inhibitors for use according to the invention are acetylcholinesterase inhibitors particularly those which are capable of crossing the blood brain barrier.

Particularly preferred cholinesterase inhibitors for use according to the invention include galantamine, epigalantamine and norgalantamine, and analogues, salts and derivatives of any of these. Galantamine was previously known as galantamine. It is a tertiary alkaloid which can be extracted from various snowdrop bulbs e.g. the Caucasian snowdrop galanthus woronowii (Amaryllidaceae) and related species and daffodil bulbs or made by chemical synthesis. It has a high selectivity for acetylcholinesterase as opposed to butyrylcholinesterase . It is active substantially selectively at nicotinic receptor sites with substantially little effect on muscarinic receptor sites.

Particularly preferred cholinesterase inhibitors for use in the invention are galantamine and its derivatives of formula ( I )

wherein the broken line represents an optionally present double bond between carbon atoms 3 and 4, each R_x is independently selected from hydrogen, hydroxyl, straight or branched chain alkyl , hydroxyalkyl , carboxyalkyl amino, alkylamino, acyl, lower alkanoyl, cyano, sulfhydryl, C^alkoxy, alkylthio, aryloxy, arylthio, R₃- substituted aryloxy, R₃-substituted arylthio, aralkoxy, an optionally R₃- substituted aliphatic or aryl carbamyl group, aralkylthio, R₃-substituted aralkoxy, R₃- substituted aralkylthio, aryloxymethyl , R₃-substituted aryloxymethyl , alkanoyloxy, hydroxy-substituted alkanoyloxy, benzoyloxy, R₃-substituted benzoyloxy, aryloxycarbonyl and R₃-substituted aryloxycarbonyl ,

R₂ is selected from hydrogen, straight or branched chain

, alkenyl or alkaryl group, optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl , acylamino, heteroaryl, heteroaryl-alkyl, aryl, arylalkyl, cyano, amyl , aroyl , cycloalkylmethyl , allyl, phenyl, R₃-substituted phenyl, alkylphenyl, R₃-substituted alkylphenyl, heterocyclyl selected from α- or β-furyl, α- or β-thienyl, thenyl , pyridyl, pyrazinyl, and pyrimidyl, alkyl -heterocyclyl or R' -substituted heterocyclyl, where R' is alkyl or alkoxy, each R₃ is independently selected from hydrogen, hydroxyl, sulfhydryl, alkyl, hydroxyalkyl, aryl, aralkyl , alkoxy, mercaptoalkyl , aryloxy, thiaryloxy, alkaryloxy, mercaptoalkaryl , nitro, amino, N-alkylamino, N-arylamino, N-alkarylamino, fluoro, chloro, bromo, iodo, and trifluoromethyl , each R₄ is independently selected from hydrogen, halo, trifluoromethyl or C₁_₄-alkyl, each R₅ is independently selected from hydrogen or hydroxymethyl ,

R₆ is hydrogen or

or when R_λ at carbon atom 2 is hydroxyl, R₆ may be a moiety of formula I wherein R₆ is hydrogen and R_λ is a linking bond; or

R_λ at carbon atom 2 and R₆ may jointly form semicarbazone ,

X is oxygen or NR₃,

Y is nitrogen or phosphorus, and methylenedioxy derivatives thereof and pharmaceutically acceptable acid addition salts thereof.

Of the compounds of formula I which may be used in the method of the invention, preferred compounds are those in which the alkyl moieties contain 1 to 8 carbon atoms, halogen atoms are preferably fluorine, bromine, chlorine, aryl moieties are preferably phenyl, cycloalkyl groups are preferably 3- to 7-membered rings, especially cyclopropyl or cyclobutyl, acyl groups are preferably lower alkanoyl groups and heteroaryl moieties are preferably 5- to 8-membered rings, e.g., thienyl, furyl , pyridyl, pyrrolyl , or pyrizanyl.

Preferred compounds of formula I are the compounds of formula II

wherein R¹ and R² which may be the same or different each represents a hydrogen atom or an acyl group, such as a lower alkanoyl group, e.g. an acetyl group or a straight-chained or branched alkyl group, e.g. methyl, ethyl, propyl, or isopropyl;

R³ is a straight or branched chain alkyl, alkenyl or alkaryl group which is optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl , acylamino, heteroaryl, heteroarylalkyl , aroyl , aroylalkyl or cyano group; and

R⁴ represents a hydrogen or a halogen atom attached to at least one of the ring carbons of the tetracyclic skeleton, and pharmaceutically acceptable salts thereof, such as a hydrobromide , hydrochloride, methylsulphate or methiodide .

Formula II includes galantamine itself.

Particularly preferred is galantamine itself, and salts thereof such as halides for example galantamine hydrobromide and the use of these compounds in the manufacture of a medicament for combatting diseases associated with proteolytic entymes provides a further aspect of the invention.

Among these compounds are those described in EP-A- 236684 and WO88/08708, the disclosures of which are incorporated herein by reference. Galantamine and its derivatives of formula I and II may be prepared by the methods described in these publications.

The cholinesterase inhibitors for use in the invention include compounds which are functionally similar to galantamine. These are defined herein as compounds which possess an at least 10-fold selectivity, preferably an at least 20-fold selectivity, more preferably an at least 40-fold selectivity, and most preferably an at least 50 fold selectivity, for acetylcholinesterase as opposed to butyryl- cholinesterase, when measured by the in vitro method of Thomsen and Kewitz: Selective Inhibition of Human Acetylcholinesterase by Galantamine in vitro and in vivo, Life Sciences, Vol 46, pp. 1553-1558 (1990), and T. Thomsen, H. Kewitz and O. Pleul, J. Clin. Chem. Clin. Biochem. 26_. 469-475 (1988) . The in vitro test described by Thomsen and Kewitz in Life Sciences, Vol 46, pp 1553- 1558 (1990) is the one referred to herein whenever numeric (10-fold, 20-fold, 40-fold) reference to selectivity for acetylcholinesterase as opposed to butyrylcholinesterase is made. According to Thomsen and Kewitz, galantamine hydrobromide, when tested under the conditions described, shows a 50-fold selectivity; this selectivity value is taken as the "fix-point" whenever in vitro selectivities are discussed herein and could be used, for the purpose of determining the selectivities for other cholinesterase inhibitors, as a calibration value which is the one to establish with galantamine hydrobromide in any repetition of the experiment described by Thomsen and Kewitz. Thus, with reference to this determination method, a preferred acetylcholinesterase inhibitor is one which in the in vitro method described has an at least 10 -fold selectivity for acetylcholinesterase as opposed to butyrylcholinesterase, such as an at least 20-fold selectivity for acetylcholinesterase as opposed to butyrylcholinesterase, e.g. an at least 40-fold selectivity for acetylcholinesterase as opposed to butyrylcholinesterase. A selectivity test is commercially available (from Sigma Diagnostics) . For use in the method of the invention the cholinesterase inhibitor such as galantamine and derivatives and salts thereof may be formulated according to conventional methods of pharmacy, together where appropriate with one or more pharmaceutically acceptable carriers, excipients or diluents such as, for example, are described in Remingtons Pharmaceutical Sciences. Such formulations may for example take the form of tablets, capsules, solutions, or lozenges, pessaries, creams, suppositories or transdermal formulations such as patches, creams, ointments or lotions, depending upon the administration route to be used, which may include enterally or parenterally, including orally or injection via the intravenous, intramuscular or subcutaneous routes, or intrathecally by means of an implanted device.

Oral and transdermal administration routes are preferred.

Precise dosage rates and regimes will depend upon the individual patient and may be determined by the medical practitioner based on individual circumstances. For oral administration doses may be within the range of 5-100 mg per day, such as 2 to 70 mg per day eg. 10 to 30 mg. For transdermal administration galanthamine may be delivered in equivalent daily doses. For parenteral administration, dosages may be in the range of 0.1 to 100 mg per day, such as 5 to 100 mg per day, e.g. 10 to 50 mg per day, including 5 to 30 mg per day; lower dosages are often preferred.

Galantamine and its acid addition salts form crystals. They are generally only sparingly soluble in water at room temperature; therefore, injectable compositions are normally in the form of an aqueous suspension. If necessary, pharmaceutically-acceptable suspension aids may be employed. Typically, such a suspension will be employed at a concentration of 0.1-50 mg/ml, such as 1-50 mg/ml, more commonly 5-40 mg/ml, for example, 5-30 mg/ml or 10-40 mg/ml, such as 10-30 mg/ml, especially 20-30 mg/ml of galantamine. Cholinesterase inhibitors such as galantamine and salts thereof may be used as the sole drug in the management of diseases associated with proteolytic enzyme activity, or may be used together with other agents useful in managing such diseases . The invention will now be described with reference to the following non-limiting examples.

Example 1

Formulation of tablets containing galantamine

Composition of 1 tablet containing 1 mg galantamine

Galantamine hydrobromide 0.001 g

Calcium phosphate 0.032 g Lactose 0.005 g

Wheat Starch 0.0056 g

Microcrystalline Cellulose 0.015 g

Talc 0.0007 g

Magnesium Stearate 0.0007 g

Composition of 1 tablet containing 5 mg galantamine

Galantamine hydrobromide 0 . . 005 g

Calcium phosphate 0 . . 024 g Lactose 0 . . 004 g

Wheat Starch 0 . . 004 g

Microcrystalline Cellulose 0 , . 04 g

Talc 0 . 002 g

Magnesium Stearate 0 . 001 g Composition of 1 tablet containing 10 mg galantamine

Galantamine hydrobromide 0.010 g

Lactose 0.040 g

Wheat Starch 0.0234 g

Microcrystalline Cellulose 0.0374 g

Talc 0.0036 g

Magnesium Stearate 0.0012 g

Gelatin 0.0044 g

Preparation

All the tablets are prepared according to routine tabletting procedures.

Example 2

Topical Formulations of Galantamine

(i)

Wool Fat BP 50g Hard Paraffin BP 50g

Cetostearyl Alcohol BP 50g

White Soft Paraffin BP 850g

Galantamine lOg

Mix the ingredients, heat gently with stirring until homogeneous and stir until cold. Fill into tubes containing 60g of ointment.

(ii) Cetostearyl Alcohol BP 80g

Sodium Lauryl Sulphate BP lOg

White Soft Paraffin BP 150g

Liquid Paraffin BP 60g

Phenoxyethanol BP lOg Purified Water BP 690g

Galantamine lOg Melt the Cetostearyl Alcohol and heat to approximately 95°C. Add the Sodium Lauryl Sulphate, mix, add approximately 1 ml Purified Water, heat to 115°C and maintain at this temperature, stirring vigorously, until frothing ceases and the product is translucent. Cool quickly while adding the White Soft Paraffin and Liquid Paraffin and stir until the product reaches room temperature .

Dissolve the Phenoxyethanol in the remaining Purified

Water with gentle heating. Melt the ointment base from the previous step, add the galantamine and the Phenoxyethanol solution and stir gently until cold.

Fill into tubes containing 60g of cream.

Example 3

Treatment of patients suffering from psoriasis

8 patients with unequivocal recalcitrant plaque type psoriasis are evaluated clinically, and given

15 - 30 mg galantamine daily. At 1 , 2 and 4 weeks, the patients are examined and biopsies taken, ultrasound measurements made and a photographic record kept.

Example 4

Test for cholinesterase activity in blood samples

Method

SIGMA DIAGNOSTICS^® CHOLINESTERASE (PTC) kit, available from Sigma Diagnostics, can be used for determining the activity and selectivity of cholinesterase inhibitors. In the following, it is illustrated how the kit is used for the determination of the activity and selectivity of galantamine hydrobromide.

Reactions involved in the cholinesterase assay are as follows : esterase Propionylthiocholine + H₂0 ^→ Propionic Acid +

Thiocholine

Thiocholine + 5 , 5 ' -Dithiobis-2-Nitrobenzoic Acid - 5-Thio-2-Nitrobenzoic Acid

5-Thio-2-Nitrobenzoic Acid is assessed by measuring the absorbance at 405 nm. The rate of change in absorbance at 405 nm is directly proportional to cholinesterase activity.

The activity of erythrocyte cholinesterase may be calculated on the basis of the measurement of butyrylcholinesterase (pseudocholinesterase) in serum and cholinesterase in haemolyzed whole blood (haemolysate) , both measured simultaneously by the method described above, and evaluated according to the haematocrit value according to the formula

HChE = (EChE x Hct* ) + (PChE x ( 1 -Hct* .

HChE- ( PChE x ( 1 -Hct* ) Therefore , EChE =

Hct*

* Haematocrit value expressed as decimal equivalent (i.e. , 44% = 0.44. In the above formulae, EChE is erythrocyte cholinesterase activity, PChE is plasma cholinesterase activity, HChE is haemolysate cholinesterase activity and Hct is haematocrit value of the sample.

Another way of assessing the cholinesterase activity is to measure the plasma cholinesterase and the cholinesterase in purified haemolyzed erythrocytes . By doing this, the values are obtained directly. Blood samples from 3 patients were tested with the Sigma test. The tests were carried out with samples where no galantamine was added and with samples where 1.25 μg/ml galantamine and 2.5 μg/ml were added in vitro. The results are shown below in table 1.

The results show a significant reduction of the haemolysate cholinesterase activity with increased concentration of galantamine hydrobromide, whereas the data for the serum activity do not show any statistically significant changes as a response to the addition of the galantamine hydrobromide, which is an indication of a high selectivity of the galantamine hydrobromide with respect to acetylcholinesterase as opposed to butyrylcholinesterase.

Selectivity for acetylcholinesterase in erythrocytes opposed to butyrylcholinesterase is contemplated to reflect the selectivity for acetylcholinesterase at nicotinic receptor sites opposed to the acetylcholinesterase at muscarinic receptor sites .

This test may be used as a screening for candidate cholinesterase inhibitors with respect to their selectivity. However, other tests may be used.

Example 5

Acetylcholinesterase activity in synovial fluid:

Acetylcholinesterase (AcChE) activity was determined in synovial fluid from 8 patients with osteoarthritis (OA) and 5 subjects with different Rheumatoid conditions. In each patient synovial fluid was taken from an affected joint by arthrocentesis . Table 2 shows mean values of up to six samples from individual subjects. A broad spectrum of values was seen between these patients, but with a high degree of correspondence between samples taken on different days from the same patient. Samples with high values show bands with AcChE activity on isoelectric focusing gels. It seems that this acetylcholinesterase activity is from free proteins rather than membrane bound simply because of the parsity of lymphocytes seen in the microliter samples.

Patients numbers 1, 3 and 11 are males, the remaining patients are females.

In patient numbers 1, 6, 7, 11 and 14 samples were taken from the left knee. As for the remaining patients, samples were taken from the right knee.

AcChE = Acetylcholinesterase activity

N = number of measurements

SEM = Standard Error of the Means OA = Osteoarthritis

RA = Rheumatoid Arthritis

Menisc = Lesion of the meniscus

Hydrops = Swollen knee

Example 6

Treatment of patients with rheumatoid disease

Ten patients with rheumatoid arthritis were treated with galantamine 10 mg 3 times per day. The purpose of the treatment was to treat their fatigue syndrome resulting from their rheumatoid arthritis. However, it was found that not only did the ten patients improve with respect to their fatigue syndrome, but also in respect of the symptoms of their rheumatoid arthritis condition itself. They spontaneously reported less articular pain and the inflammatory process of their affected joints was less active (as evidenced by less swollen and less painful joints) while they were on treatment with galantamine. Most of the patients have experienced relapse of their symptoms when treatment was interrupted.

Example 7

Treatment of patients with osteoarthritis

In a bioavailability study of galantamine in 24 elderly volunteers, 2 volunteers asked to remain on galantamine at the end of the trial .

The first volunteer, who was already acknowledged by the General Practitioner to be suffering from osteoarthritis, received 10 mg galantamine hydrobromide per day (in the form of 2 x 5 mg doses orally) . After seven days, the patient spontaneously reported an improvement in osteoarthritis symptoms. This improvement was maintained over a 31 day period, during which the galantamine hydrobromide dose was raised to 30 mg per day. Symptoms were reported to recur when medication was discontinued.

The second patient, who had been experiencing joint pain for several years, but had not had osteoarthritis diagnosed, also received 10 mg per day galantamine hydrobromide in 2 x 5 mg tablet doses. This patient spontaneously reported an improvement in symptoms after three days of treatment with galantamine.

Example 8 Treatment of a patient with anaemia of chronic disorder

A 61 years old woman with Rheumatoid Arthritis had for 3 years suffered from anaemia which did not respond to conventional therapy. The patient received 15 mg galantamine hydrobromide per day (in the form of 3 x 5 mg doses orally) . After 14 days, the laboratory blood tests reflecting anaemia showed significant improvement as shown in Table 3. The patient was treated in the period between 3 January 1997 and 20 January 1997.

Two weeks' treatment with galantamine 15 mg per day apparently raised haemoglobin to 124, the highest value in three years, and also improved considerably other clinical signs and symptoms. * = abnormal values

Example 9

Treatment of patients with rheumatoid diseases

13 patients suffering from different arthritic disorders of rheumatoid origin received galantamine tablets 2.5 mg 3 times per day for 1 week followed by 5 mg 3 times per day for the next four weeks. After the 5 weeks of treatment the patients reported the following differences in symptoms as shown in Table 4.

Patient numbers 4 and 12 are males, the remaining patients are females. Age is measured in years, age in brackets reflect approximate age.

OA = Osteoarthritis

RA = Rheumatoid Arthritis

Post Inf. a. = Arthritis after infection SLE = Systematic Lupus Erythematosis

Spond = Ankylosing Spondilitis

Poly = Polyarthritis

Pso. a. = Arthritis associated with psoriasis u = unaffected d = decreased i = increased (corresponding to at least 50% improvement ) Stiffness: Joint stiffness is a complaint covering sensations which range from slight resistance to all movements through the normal range, to blocking of certain movements due to fixed anatomical changes. Certain types of stiffness are highly characteristic, such as early morning stiffness.

Early morning stiffness occurs primarily in rheumatoid arthritis, but also in other arthritic diseases. The patient awakes with distressing, painful stiffness of all affected joints. This is gradually

"worked off" by activity over one half to three hours or more. This "limbering up time" provides a reliable measure of inflammatory activity. In osteoarthritis stiffness tends to come on later in the day and is preceded by activity.

In one of the examples given of rheumatoid patients on galantamine, the parameters evaluated were tiredness, sleep, pain and stiffness. In one RA patient (patient number 1) the grip strength was evaluated and proved to augment over 50%. All these parameters were evaluated with a questionnaire and VAS (Visual Analogue Scale) which refer to the patients' reporting as regards the severity of the symptoms, represented by a 0-100 percentage line. As appears from Table 4, 50% of the patients

(patient numbers 1, 2, 3, 5, 9, 10 and 12) reported a significant improvement in their symptoms.

Claims

CLAIMS :

1. The use of a pharmaceutically acceptable cholinesterase inhibitor or a pro-drug therefor in the manufacture of a medicament for combatting diseases associated with proteolytic enzyme activity.

2. The use as claimed in claim 1 wherein the cholinesterase inhibitor blocks or inhibits protease activity or protease production.

3. The use as claimed in claim 1 or claim 2 wherein the disease is psoriasis.

4. The use as claimed in claim 1 or claim 2 wherein the disease is an inflammatory bowel disease.

5. The use as claimed in claim 4 wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

6. The use as claimed in claim 1 or claim 2 wherein the disease is selected from osteoarthritis, rheumatoid arthritis and other forms of arthritis.

7. The use as claimed in any one of the preceding claims wherein the cholinesterase inhibitor is an acetyl cholinesterase inhibitor.

8. The use as claimed in any one of the preceding claims wherein the cholinesterase inhibitor is active substantially selectively at nicotinic receptor sites.

9. The use as claimed in any one of the preceding claims wherein the cholinesterase inhibitor is capable of crossing the blood brain barrier.

10. The use as claimed in any one of claims 1 to 7 wherein the cholinesterase inhibitor is selected from physostigmine, tacrine and tacrine analogues, fasiculin, metrifonate, heptyl -physostigmine, norpyridostigmine, norneostigmine, huperazine, donepezil and pro-drugs of any of these.

11. The use is claimed in any one of claims 1 to 9 wherein the cholinesterase inhibitor is selected from glantamine, epigalantamine and norgalantamine, and analogues, salts and derivatives of any of these.

12. The use as claimed in any of the preceding claims wherein the cholinesterase inhibitor is selected from galantamine and its derivatives of formula (I) :

wherein the broken line represents an optionally present double bond between carbon atoms 3 and 4, each R_┬▒ is independently selected from hydrogen, hydroxyl, straight or branched chain alkyl, hydroxyalkyl, carboxyalkyl amino, alkylamino, acyl, lower alkanoyl, cyano, sulfhydryl, C^alkoxy, alkylthio, aryloxy, arylthio, R₃- substituted aryloxy, R₃-substituted arylthio, aralkoxy, an optionally R₃-substituted aliphatic or aryl carbamyl group, aralkylthio, R₃-substituted aralkoxy, R₃- substituted aralkylthio, aryloxymethyl, R₃-substituted aryloxymethyl, alkanoyloxy, hydroxy- substituted alkanoyloxy, benzoyloxy, R₃-substituted benzoyloxy, aryloxycarbonyl and R₃-substituted aryloxycarbonyl,

R₂ is selected from hydrogen, straight or branched chain C_halky1 , alkenyl or alkaryl group, optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl , acylamino, heteroaryl, heteroaryl -alkyl, aryl, arylalkyl, cyano, amyl , aroyl , cycloalkylmethyl , allyl, phenyl, R₃-substituted phenyl, alkylphenyl, R₃-substituted alkylphenyl, heterocyclyl selected from ╬▒- or ╬▓-furyl, ╬▒- or ╬▓-thienyl, thenyl , pyridyl, pyrazinyl, and pyrimidyl, alkyl -heterocyclyl or R' -substituted heterocyclyl, where R' is alkyl or alkoxy, each R₃ is independently selected from hydrogen, hydroxyl, sulfhydryl, alkyl, hydroxyalkyl, aryl, aralkyl, alkoxy, mercaptoalkyl, aryloxy, thiaryloxy, alkaryloxy, mercaptoalkaryl, nitro, amino, N-alkylamino, N-arylamino, N-alkarylamino, fluoro, chloro, bromo, iodo, and trifluoromethyl , each R₄ is independently selected from hydrogen, halo, trifluoromethyl or C₁_₄-alkyl, each R₅ is independently selected from hydrogen or hydroxymethyl ,

R₆ is hydrogen or C^alkyl, or when R_x at carbon atom 2 is hydroxyl, R₆ may be a moiety of formula I wherein R₆ is hydrogen and R_: is a linking bond; or

R_x at carbon atom 2 and R₆ may jointly form semicarbazone ,

X is oxygen or NR₃, Y is nitrogen or phosphorus, and methylenedioxy derivatives thereof and pharmaceutically acceptable acid addition salts thereof.

13. The use as claimed in any one of the preceding claims wherein the cholinesterase inhibitor is selected from compounds of formula II

R³ is a straight or branched chain alkyl, alkenyl or alkaryl group which is optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl , acylamino, heteroaryl, heteroarylalkyl, aroyl , aroylalkyl or cyano group; and R⁴ represents a hydrogen or a halogen atom attached to at least one of the ring carbons of the tetracyclic skeleton, and pharmaceutically acceptable salts thereof, such as a hydrobromide, hydrochloride, methylsulphate or methiodide .

14. The use according to any one of the preceding claims where the cholinesterase inhibitor is galantamine or a salt thereof.

15. The use of galantamine or a derivative thereof of formula I :

wherein the broken line represents an optionally present double bond between carbon atoms 3 and 4, each R_x is independently selected from hydrogen, hydroxyl, straight or branched chain alkyl, hydroxyalkyl, carboxyalkyl amino, alkylamino, acyl, lower alkanoyl, cyano, sulfhydryl, C^alkoxy, alkylthio, aryloxy, arylthio, R₃- substituted aryloxy, R₃-substituted arylthio, aralkoxy, an optionally R₃-substituted aliphatic or aryl carbamyl group, aralkylthio, R₃-substituted aralkoxy, R₃- substituted aralkylthio, aryloxymethyl, R₃-substituted aryloxymethyl, alkanoyloxy, hydroxy-substituted alkanoyloxy, benzoyloxy, R₃-substituted benzoyloxy, aryloxycarbonyl and R₃-substituted aryloxycarbonyl, R₂ is selected from hydrogen, straight or branched chain C^alkyl , alkenyl or alkaryl group, optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl , acylamino, heteroaryl, heteroaryl -alkyl , aryl, arylalkyl , cyano, amyl , aroyl , cycloalkylmethyl, allyl, phenyl, R₃-substituted phenyl, alkylphenyl, R₃-substituted alkylphenyl, heterocyclyl selected from - or ╬▓-furyl, ╬▒- or ╬▓-thienyl, thenyl , pyridyl, pyrazinyl, and pyrimidyl, alkyl-heterocyclyl or R' -substituted heterocyclyl, where R' is alkyl or alkoxy, each R₃ is independently selected from hydrogen, hydroxyl, sulfhydryl, alkyl, hydroxyalkyl, aryl, aralkyl, alkoxy, mercaptoalkyl , aryloxy, thiaryloxy, alkaryloxy, mercaptoalkaryl, nitro, ammo, N-alkylammo, N-arylammo, N-alkarylam o, fluoro, chloro, bromo, lodo, and trifluoromethyl , each R₄ is independently selected from hydrogen, halo, trifluoromethyl or C₁_₄-alkyl, each R₅ is independently selected from hydrogen or hydroxymethyl ,

R₆ is hydrogen or C_halky1 , or when R_: at carbon atom 2 is hydroxyl, R₆ may be a moiety of formula I wherein R₆ is hydrogen and R_╬╗ is a linking bond; or

R_╬╗ at carbon atom 2 and R₆ may jointly form semicarbazone ,

X is oxygen or NR₃,

Y is nitrogen or phosphorus, and methylenedioxy derivatives thereof and pharmaceutically acceptable acid addition salts thereof in the manufacture of a medicament for combatting diseases associated with proteolytic enzyme activity.

16. The use of galantamine or a derivative thereof of formula II

wherein R¹ and R² which may be the same or different each represents a hydrogen atom or an acyl group, such as a lower alkanoyl group, e.g. an acetyl group or a straight-chained or branched alkyl group, e.g. methyl, ethyl, propyl , or isopropyl; R³ is a straight or branched chain alkyl, alkenyl or alkaryl group which is optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl, acylamino, heteroaryl, heteroarylalkyl, aroyl , aroylalkyl or cyano group; and

R⁴ represents a hydrogen or a halogen atom attached to at least one of the ring carbons of the tetracyclic skeleton, and pharmaceutically acceptable salts thereof, such as a hydrobromide, hydrochloride, methylsulphate or methiodide in the manufacture of a medicament for combatting diseases associated with proteolytic enzyme activity.

17. The use of galantamine or a salt thereof in the manufacture of a medicament for combatting diseases associated with proteolytic enzyme activity.

18. A method of combatting diseases associated with proteolytic enzyme activity comprising administering a pharmaceutically acceptable cholinesterase inhibitor or a pro-drug therefor.

19. A method as claimed in claim 18 wherein the disorder is as defined in any one of claims 2 to 6.

20. A method as claimed in claim 18 or claim 19 wherein the cholinesterase inhibitor is as defined in any one of claims 7 to 14.

21. The use of a pharmaceutically acceptable cholinesterase inhibitor or a prodrug therefor in the manufacture of a medicament for combatting psoriasis.

22. The use as claimed in claim 21 wherein the cholinesterase inhibitor is defined in any one of claims 7 to 14.

23. The use of galantamine or a derivative thereof of formula I :

wherein the broken line represents an optionally present double bond between carbon atoms 3 and 4, each R_╬╗ is independently selected from hydrogen, hydroxyl, straight or branched chain alkyl , hydroxyalkyl , carboxyalkyl amino, alkylamino, acyl, lower alkanoyl, cyano, sulfhydryl, C^alkoxy, alkylthio, aryloxy, arylthio, R₃- substituted aryloxy, R₃-substituted arylthio, aralkoxy, an optionally R₃-substituted aliphatic or aryl carbamyl group, aralkylthio, R₃-substituted aralkoxy, R₃- substituted aralkylthio, aryloxymethyl, R₃-substituted aryloxymethyl, alkanoyloxy, hydroxy-substituted alkanoyloxy, benzoyloxy, R₃-substituted benzoyloxy, aryloxycarbonyl and R₃-substituted aryloxycarbonyl,

R₂ is selected from hydrogen, straight or branched chain

alkenyl or alkaryl group, optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl, acylamino, heteroaryl, heteroaryl-alkyl , aryl, arylalkyl, cyano, amyl , aroyl, cycloalkylmethyl , allyl, phenyl, R₃-substituted phenyl, alkylphenyl, R₃- substituted alkylphenyl, heterocyclyl selected from ╬▒- or ╬▓-furyl, - or ╬▓-thienyl, thenyl , pyridyl, pyrazinyl, and pyrimidyl, alkyl -heterocyclyl or R' -substituted heterocyclyl, where R' is alkyl or alkoxy, each R₃ is independently selected from hydrogen, hydroxyl, sulfhydryl, alkyl, hydroxyalkyl, aryl, aralkyl, alkoxy, mercaptoalkyl , aryloxy, thiaryloxy, alkaryloxy, mercaptoalkaryl, nitro, amino, N-alkylamino, N-arylamino, N-alkarylamino, fluoro, chloro, bromo, iodo, and trifluoromethyl , each R₄ is independently selected from hydrogen, halo, trifluoromethyl or C₁_₄-alkyl, each R₅ is independently selected from hydrogen or hydroxymethyl ,

R₆ is hydrogen or C^alkyl, or when R_x at carbon atom 2 is hydroxyl, R₆ may be a moiety of formula I wherein R₆ is hydrogen and R_x is a linking bond; or

R_x at carbon atom 2 and R₆ may jointly form semicarbazone,

X is oxygen or NR₃,

Y is nitrogen or phosphorus, and methylenedioxy derivatives thereof and pharmaceutically acceptable acid addition salts thereof in the manufacture of a medicament for combatting psoriasis .

24. The use of galantamine or a derivative thereof of formula II

wherein R¹ and R² which may be the same or different each represents a hydrogen atom or an acyl group, such as a lower alkanoyl group, e.g. an acetyl group or a straight -chained or branched alkyl group, e.g. methyl, ethyl, propyl, or isopropyl;

R³ is a straight or branched chain alkyl, alkenyl or alkaryl group which is optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl, acylamino, heteroaryl, heteroarylalkyl, aroyl, aroylalkyl or cyano group; and

R⁴ represents a hydrogen or a halogen atom attached to at least one of the ring carbons of the tetracyclic skeleton, and pharmaceutically acceptable salts thereof, such as a hydrobromide, hydrochloride, methylsulphate or methiodide in the manufacture of a medicament for combatting psoriasis.

25. The use of galantamine or a salt thereof in the manufacture of a medicament for combatting psoriasis.

26. A method of combatting psoriasis comprising administering a pharmaceutically acceptable cholinesterase inhibitor or a pro-drug therefor.

27. A method as claimed in claim 26 wherein the cholinesterase inhibitor is as defined in any one of claims 7 to 14.

28. The use of a pharmaceutically acceptable cholinesterase inhibitor or a prodrug therefor in the manufacture of a medicament for combatting osteoarthritis, rheumatoid arthritis or other forms of arthritis .

29. The use as claimed in claim 28 wherein the cholinesterase inhibitor is defined in any one of claims 7 to 14.

30. The use of galantamine or a derivative thereof of formula^* I :

wherein the broken line represents an optionally present double bond between carbon atoms 3 and 4, each R_╬╗ is independently selected from hydrogen, hydroxyl, straight or branched chain alkyl, hydroxyalkyl, carboxyalkyl amino, alkylamino, acyl, lower alkanoyl, cyano, sulfhydryl, C^alkoxy, alkylthio, aryloxy, arylthio, R₃- substituted aryloxy, R₃-substituted arylthio, aralkoxy, an optionally R₃-substituted aliphatic or aryl carbamyl group, aralkylthio, R₃-substituted aralkoxy, R₃- substituted aralkylthio, aryloxymethyl, R₃-substituted aryloxymethyl, alkanoyloxy, hydroxy-substituted alkanoyloxy, benzoyloxy, R₃-substituted benzoyloxy, aryloxycarbonyl and R₃-substituted aryloxycarbonyl,

R₂ is selected from hydrogen, straight or branched chain C_h lky1 , alkenyl or alkaryl group, optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl, acylamino, heteroaryl, heteroaryl -alkyl, aryl, arylalkyl, cyano, amyl , aroyl, cycloalkylmethyl, allyl, phenyl, R₃-substituted phenyl, alkylphenyl, R₃-substituted alkylphenyl, heterocyclyl selected from - or ╬▓-furyl, ╬▒- or ╬▓-thienyl, thenyl , pyridyl, pyrazinyl, and pyrimidyl, alkyl -heterocyclyl or R' -substituted heterocyclyl, where R' is alkyl or alkoxy, each R₃ is independently selected from hydrogen, hydroxyl, sulfhydryl, alkyl, hydroxyalkyl, aryl, aralkyl, alkoxy, mercaptoalkyl , aryloxy, thiaryloxy, alkaryloxy, mercaptoalkaryl, nitro, ammo, N-alkylammo, N-arylammo, N-alkarylammo, fluoro, chloro, bromo, lodo, and trifluoromethyl , each R₄ is independently selected from hydrogen, halo, trifluoromethyl or C^ -alkyl, each R₅ is independently selected from hydrogen or hydroxymethyl ,

R₆ is hydrogen or

or when R_x at carbon atom 2 is hydroxyl, R₆ may be a moiety of formula I wherein R₆ is hydrogen and R_x is a linking bond; or

R_╬╗ at carbon atom 2 and R₆ may jointly form semicarbazone,

X is oxygen or NR₃,

Y is nitrogen or phosphorus, and methylenedioxy derivatives thereof and pharmaceutically acceptable acid addition salts thereof m the manufacture of a medicament for combatting osteoarthritis, rheumatoid arthritis or other forms of arthritis .

31. The use of galantamine or a derivative thereof of formula II

wherein R¹ and R² which may be the same or different each represents a hydrogen atom or an acyl group, such as a lower alkanoyl group, e.g. an acetyl group or a straight-chained or branched alkyl group, e.g. methyl, ethyl, propyl, or isopropyl; R³ is a straight or branched chain alkyl, alkenyl or alkaryl group which is optionally substituted by a halogen atom or a cycloalkyl, hydroxy, alkoxy, nitro, amino, aminoalkyl, acylamino, heteroaryl, heteroarylalkyl, aroyl, aroylalkyl or cyano group; and R⁴ represents a hydrogen or a halogen atom attached to at least one of the ring carbons of the tetracyclic skeleton, and pharmaceutically acceptable salts thereof, such as a hydrobromide, hydrochloride, methylsulphate or methiodide in the manufacture of a medicament for combatting osteoarthritis, rheumatoid arthritis or other forms of arthritis.

32. The use of galantamine or a salt thereof in the manufacture of a medicament for combatting osteoarthritis, rheumatoid arthritis or other forms of arthritis .

33. The use of a pharmaceutically acceptable cholinesterase inhibitor or a prodrug therefor in the manufacture of a medicament for the treatment of rheumatoid diseases.

34. The use of a pharmaceutically acceptable cholinesterase inhibitor or a prodrug therefor in the manufacture of a medicament for the treatment of rheumatoid diseases such as Juvenile Arthritis, Systemic Lupus Erythematosis, Sj╧îgren's Syndrome, Progressive Systemic Sclerosis, Polymyositis , Dermatomyositis, Ankylosing Spondilitis, Reiter's Syndrome, Psoriatic Arthritis, Relapsing Polychondritis , Relapsing Panniculitis, Crohn's Disease, Ulcerative Colitis, Hereditary Complement Deficiencies, Collagen Vascular Diseases, Felty's Syndrome, rheumatological manifestations associated with bacterial and viral endocarditis or myocarditis and other rheumatological manifestations such as anaemia of chronic disorders.

35. The use of a pharmaceutically acceptable cholinesterase inhibitor or a prodrug therefor in the manufacture of a medicament for the treatment of anaemia associated with chronic disorders.

36. The use as claimed in any one of claims 33 to 35 wherein the cholinesterase inhibitor is as defined in any one of claims 7 to 14.