METHOD - 011

The present invention relates to methods for the treatment of osteoarthritis and compounds for use in the treatment thereof.

Osteoarthritis is a non-inflammatory degenerative joint disease seen mainly in older persons, characterized by degeneration of the articular cartilage, hypertrophy of bone at the margins, and changes in the synovial membrane. It is accompanied by pain, usually after prolonged activity, and stiffness, particularly in the morning or with inactivity. It is also called degenerative arthritis, hypertrophic arthritis, and degenerative joint disease

Available therapy for the treatment of osteoarthritis comprises use of simple analgesics, opioid-containing analgesics, nonsteroidal antiinflammatory drugs (NSAIDs, local analgesics, intra-articular corticosteroid injections, intra-articular injections of hyaluronic acid like products).

Abnormal expression and activity of matrix metalloproteinases (MMPs) has been linked to the pathological processes underlying osteoarthritis. MMP inhibitors are currently being developed.

The need exists for further therapeutic methods and agents for treating osteoarthritis.

In a first aspect of the invention we now provide a method for the prophylaxis and/or therapy of osteoarthritis (OA) which method comprises administering to a mammalian subject in need of such prophylaxis and/or therapy, a pharmaceutically effective amount of a glucokinase activator.

Whilst we don't wish to be bound by theoretical consideration the glucokinase activator may act to decrease the activity of one or more catabolic proteases and/or cytokines. Specifically our studies have shown that the glucokinase activator may act in one or more of ways, independently selected from:

1. to downregulate MMP 1 ;

2. to downregulate MMP 13;

3. to downregulate VEGF;

4. to downregulate Osteopontin; 5. to downregulate Interleukin-6

6. to downregulate Interleukin-8

7. to downregulate CTX-2

or any combination thereof.

In a further aspect of the invention we provide the use of a pharmaceutically effective amount of a glucokinase activator in the prophylaxis and/or therapy of osteoarthritis in a mammalian subject. In a further aspect of the invention we provide a pharmaceutically effective amount of a glucokinase activator for use in the prophylaxis and/or therapy of osteoarthritis in a mammalian subject.

In a further aspect of the invention we provide the use of a glucokinase activator in the manufacture of a medicament for use in the treatment of osteoarthritis in a mammalian subject.

In a further aspect of the invention the glucokinase activator is used to prevent and/or treat cartilage destruction in the intra-articular joint of a mammalian subject.

In a further aspect of the invention the glucokinase activator is used to prevent or treat bone joint remodelling associated with osteoartritis. In a further aspect of the invention we provide the glucokinase activator is used to prevent or treat collagen degradation in the intra-articular cartilage of a mammalian subject.

Any convenient GLK activator can be used in the methods of this invention. See for example B. Leighton et al, Biochemical Society Transactions (2005), VoI 33, Part 2, pages 371-374 and the references comprised therein. See also Sarabu, R. and Grimsby, J. (2005) Curr Opin.Drug Discov.Devel. 8, 631-637 and the references comprised therein.

Other convenient GLK activators include those disclosed by KJ. Brocklehurst et al.(2004) Diabetes 53, 535-541, GJ. Coope et al (2006) Br.J.Pharmacol. 149, 328-335, D. McKerrecher et al (2005) Bioorg.Med.Chem.Lett. 15, 2103-2106, and D. McKerrecher et al (2006) Bioorg.Med.Chem.Lett. 16, 2705-2709.

Further convenient GLK activators are disclosed by J. Grimsby et al (2003) Science, 301,

370-373.

Further convenient GLK activators are disclosed by A.M.Efanov et al (2005) Endocrinology 146, 3696-3701.

Further convenient GLK activators are disclosed by M.Futamura et al (2006) J.Biol.Chem. 281, 37668-37674 and K.Kamata et al (2004) Structure. 12, 429-438.

Further convenient GLK activators are disclosed by Castelhano et al (2005) Bioorg.Med.Chem.Lett. 15, 1501-1504 and M.C. Fyfe et al (2007) Diabetologia. 50, 1277- 1287.

Further convenient GLK activators include compounds Rl 440 (Ro-4389620) and Rl 511 (GK3) being developed by F. Hoffmann La Roche.

Further convenient GLK activators include compound PSNOlO being developed by Prosidion/Lilly.

Further convenient GLK activators include compounds NN9101 (TTP355) and

TTP399 being developed by Novo-Nordisk/TransTech Pharma.

Further convenient GLK activators include those disclosed for example in any one of our published patent applications nos. WO-03/000267, WO-03/015774, WO-03/000262, WO-04/046139, WO-04/045614, WO-05/054233, WO-05/056530, WO-05/080359, WO- 05/080360, WO-05/121110, WO-06/040529, WO-06/040528, WO-06/125958, WO- 06/124972, WO-07/007041, WO-07/007042, WO-07/007040, WO-07/017649 and WO- 07/031739.

Glucokinase activators have been used in the treatment or prevention of a disease or medical condition mediated through glucokinase (GLK or GK), leading to a decreased glucose threshold for insulin secretion. In addition, glucokinase activators are predicted to lower blood glucose by increasing hepatic glucose uptake. Such compounds have been proposed for use in the treatment of Type 2 diabetes and obesity.

The pharmaceutically effective amount of the glucokinase activator may be determined by routine experimentation. Whilst we don't wish to be bound by theoretical considerations the effective amount of the glucokinase activator may be less than the dose required to lower blood glucose levels in a clinical context, for example as used in the treatment of type 2 diabetes. By way of non-limiting example the effective amount may be no more than 80%, such as no more than 75% or 65% of the dose required to lower blood

glucose levels in a clinical context. The effective amount may be no more than 50% such as no more than 40% or 30% of the said dose.

The mammalian subject may be a human or a non-human animal such as a horse, dog and cat. In a further aspect of the invention we provide the combination of a glucokinase activator and an antihistamine compound for use in the treatment of osteoarthritis. Convenient antihistamine compounds will be apparent to the scientist of ordinary skill.

The invention will now be illustrated but not limited by reference to the following specific description, Examples and Figures wherein: Figure 1 shows that OA chondrocytes express GLK. (A) PCR expression of GLK in 6 different OA chondrocyte donors and (B) Western blot for GLK. OA chondrocytes express a protein band at the same molecular weight as recombinant GLK.

Figure 2 shows representative gene-expression profile of OA cartilage treated with 10 x E. C.50 glucokinase activator AZ2 for 96-hours. X-axis represents log fold change in gene-expression normalised to housekeeping gene.

Figure 3 shows that GLK activators reduce release of biologically relevant endpoints from OA cartilage explants (10 x E. C.50 AZ2 for 96-hours). Each coloured bar represents a different cartilage donor.

Figure 4 shows that GLK activator AZ2 reduces MMPl release from OA chondrocytes (A) and OA cartilage (B) under both normo- (5.5mM) and hyperglycaemic (1OmM) conditions.

Figure 5 shows that the GLK inhibitor 5-thioglucose (5TG) increasesd MMPl release from OA cartilage. GLK activators reduced this increase in MMPl release. GLK activator alone reduced release of MMPl to below basal levels. Figure 6 shows that a GLK activator from a different chemical series (Roche 2) reduced release of MMP 13 and MMPl from unstimulated OA cartilage explants.

Figure 7 shows that a GLK activator from a different chemical series (Roche 2) reduced MMP 13 and MMPl release from ILl β / oncostatin M stimulated OA cartilage explants. Figure 8 shows several different GLK activators (at 3uM) reducing ILIb /

Oncostatin M mediated MMP 13 release from OA chondrocytes.

Figure 9 shows that a GLK activator (AZ2) reduces ILl β / Oncostatin M mediated VEGF release from OA chondrocytes.

Figure 10 shows that a GLK activator (AZ2) reduces ILl β / Oncostatin M mediated MMP 13 release from OA cartilage explants. Figure 11 shows that a GLK activator (AZ2) reduces ILl β / Oncostatin M mediated MMPl release from PM cartilage explants in a dose-dependent manner.

Figure 12 shows that cartilage MMP 13 is increased in fatty rats over lean controls. There was a tendency for this to be reduced on dosing with a GLK 10 mg / kg activator (AZ3). Figure 13 shows that plasma CTXII was increased in fatty rats over lean controls.

This was significantly reduced on dosing with 10 mg / kg GLK activator (AZ3).

Figure 14 shows that plasma CTXII was increased in heterozygous GLK knockout mice over wild-type controls. This was significantly reduced on dosing with both 2.5 and 5 mg / kg GLK activator (AZl). Figure 15 shows knee joints from lean and fatty Zucker rats. Glycosaminoglycan content of the articular cartilage is stained with Toluidine blue. As shown in Figure 15(a) the joint of the lean rat shows normal articular cartilage which is in contrast to results shown in Figure 15(b) using fatty Zucker rats where areas of glycosaminoglycan loss are seen (b, arrowhead). In addition, preliminary findings in the fatty Zucker rats suggest the cells "lined-up" in columns in addition to an apparent thickening of the area of mineralised cartilage (B, double-headed arrow). Figure 15(c). shows the glycosaminoglycan content of Zucker rat cartilage (normalised to the wet weight of the cartilage). The fatty rats can be seen to have a large amount of extractable glycosaminoglycan. Treatment with AZl brings the glycosaminoglycan content of the cartilage to the levels seen in lean animals, suggesting a more functional matrix of correctly incorporated glycosaminoglycan.

Figure 16 shows toluidine blue stained knee joints from heterozygous GLK knockout mice (from study represented in Figure 14.). In Figure 16(a) vehicle treated animals show areas of glycosaminoglycan loss from the articular cartilage (arrow) which is reduced as shown in Figure 16(b) in those animals treated with AZl (b; arrow). Table 1 shows mean plasma CTXII in pg / ml from four different studies carried out in Zucker rats with two different AstraZeneca Glucokinase activator compounds.

Specific description

We now provide evidence of:

• The role of GLK activation in chondrocytes (gene changes and supporting protein data).

• Target specific effects with the use of GLK inhibitors to reverse compound effects and the use of structurally distinct compounds.

• Disease linkage, GLK activation can reverse IL lβ mediated catabolic effects in OA cartilage.

• In vivo cartilage biomarker modulation.

Materials and Methods:

GLK activators used in our studies include: Roche 1:

Chi

Roche 2:

Chiral

Prosidion 1:

Chiral

AZl:

Cartilage Explant Culture

Human articular cartilage samples from osteoarthritic (OA) and post-mortem (PM) donors (normal, no history of OA) were obtained after prior ethical approval. Cartilage was carved from all parts of the knee and pooled. Discs were taken and put into individual wells of a 96-well plate. 250ul culture media was added and cartilage explants were rested for 48-hours. After this time, explants were treated with 10 x E. C.50 of glucokinase activator compound for 96-hours. Additional treatments were the catabolic drivers IL lβ and Oncostatin M (R&D Systems). Starting concentrations of 5 ng / ml and 50 ng / ml respectively were used. Where stated dilutions were ten- fold from these starting concentrations. Media supernatants were collected for ELISA assay and cartilage discs were collected, pooled and stored at -80 ⁰ C prior to RNA extraction.

Western Blot

Total protein was isolated from primary chondrocytes using RIPA buffer (0.15 M NaCl, 0.05 M Tris-HCl pH 7.4, ImM EDTA, 1% v/v Triton XlOO, 0.1% w/v SDS, 1% deoxycholate) containing 1:100 dilution of protease inhibitor cocktail (Sigma). Twenty micrograms was run on a 4-12% Tris-Glycine gel (Invitrogen). Blotting, onto nitrocellulose membrane, was using the iBlot system (Invitrogen). After blocking in 5% Marvel in Tris buffered saline (TBS) + 0.05% Tween-20, blots were incubated in primary antibody overnight at 4°C. Primary antibody was an AstraZeneca rabbit anti-GLK antibody and recombinant GLK (AstraZeneca) was used as a positive control. Detection was using HRP-conjugated anti-rabbit antibody followed by ECL+ visualisation according to manufacturers instructions (GE Healthcare).

PCR

OA and PM chondrocyte RNA was purified using Qiagen RNeasy minicolumn (Qiagen) methodology, according to the manufacturers instructions. After quantification using an Agilent Bioanalyser 2100 with the RNA NanoόOOO chip, each test RNA was diluted to a concentration of 5 ng / ul. One-step PCR was carried out using 25ng RNA under the following conditions: 30 mins at 50 ⁰ C, 15 sees at 95°C, 30 sees at 94°C, 30 sees at 55°C, 30 sees at 72°C (40 cycles) and 10 minutes at 72°C. At the end of the run samples were run on 2% agarose gels (with ethidium bromide) for product detection.

Cartilage RNA Extraction

Cartilage was snap frozen and ground under liquid nitrogen using a Glen Creston Spex mill. RNA was extracted from the ground cartilage using a standard TRIzol extraction method (Invitrogen) following manufacturer's protocols. The RNA was purified using a Qiagen RNeasy minicolumn (Qiagen) and treated with DNase. RNA was quantified using an Agilent Bioanalyser 2100 with the RNA NanoόOOO chip.

Tag man™ Methodology Taqman RT-PCR using Applied Biosystems Low Density Arrays (Taqman

Micro fluidics) was performed on the ABI Prism 7900. Transcriptional effects were measured against 48 primer / probe sets (Applied Biosystems Assays on Demand) plated

on a custom designed Low Density array. Each test RNA sample was added to Quantitect probe RT-PCR mastermix (Qiagen) at a concentration of 1 ng / ul and applied to the card according to the manufacturer's instructions. One step RT-PCR was carried out with an initial reverse transcription reaction at 50 ⁰ C for 30 mins, 94.5 ⁰ C for 15 mins, followed by 35 cycles at 97 ⁰ C for 30 sees and 59.7 ⁰ C for 1 min. Data analysis was carried out using the Applied Biosystems SDS 2.1 software and transcriptional changes represented as fold change (2 ^"δδCt ) with respect to the untreated sample. Data were normalised to the housekeeping gene,18S.

ELISA Protocols

All ELISA protocols are for 384-well plates and are 2-day protocols. MMPl

All antibodies were obtained from R&D systems. Anti-human MMPl monoclonal antibody was coated onto plates in phosphate buffered saline (PBS) overnight at 4°C. After blocking the plate in PBS + 1% Bovine serum albumin (BSA), samples were added at the appropriate dilution (in PBS + 0.1 % BSA). A 7-point standard curve was used (recombinant human MMPl, R&D systems) with the top standard being 25 ng / ml. After incubation at room temperature detection was using a biotinylated anti-human MMPl polyclonal antibody (R&D Systems), streptavidin-HRP (Biosource) and TMB substrate (Sigma). The plate was read at 45OnM after stopping the development reaction with 2M sulphuric acid.

MMP13

Plates were coated overnight at 4°C with anti-human MMP 13 monoclonal antibody (AbCam) in phosphate buffer. After blocking the plates, samples were added at the appropriate dilution in PBS + 0.5 % BSA + 0.1 % Tween-20. A 7-point standard curve was used (human MMP13, R&D Systems) with the top standard being 8,000 pg / ml. Detection was using rabbit anti-human MMP 13 polyclonal antibody (AbCam), anti-rabbit HRP (Amersham Bioscience) and TMB substrate (Sigma). The plate was read at 45OnM after stopping the development reaction with 2M sulphuric acid.

MMP3, IL6 and IL8

These ELISAs were carried out using Duoset antibody pairs (Biosource) according to the manufacturers recommendations. Osteopontin Plates were coated overnight at 4°C with mouse anti-human osteopontin monoclonal (R&D systems) in PBS. After blocking the plate in PBS + 1% Bovine serum albumin (BSA), samples were added at the appropriate dilution (in PBS + 0.1 % BSA). A 7-point standard curve was used (recombinant human osteopontin, R&D systems) with the top standard being 40 ng / ml. After incubation at room temperature detection was using a biotinylated anti-human osteopontin polyclonal antibody (R&D Systems), streptavidin- HRP (Biosource) and TMB substrate (Sigma). The plate was read at 45OnM after stopping the development reaction with 2M sulphuric acid.

In Vivo GLK Activator Study Male Zucker rats were treated once a day for 1 -month with GLK activator (AZ3) at

10mg/kg, which gave glucose lowering for 12 hours. Vehicle treated lean rats were used as controls. Cartilage samples were taken and extracted in 200 μl high-salt buffer (25 mM Tris-HCL, 500 mM sodium chloride, pH 7.5) overnight at 4°C and MMP 13 protein was measured by ELISA. Plasma samples were also taken and CTX-II (type II collagen telopeptide cleavage marker) was measured (as a marker of protease activity). CTX-II was measured using Nordic Biosciences pre-clinical cartilaps kit with no deviations from the manufacturers instructions.

In Vivo Study: GLK Knockout Mice Male C57BL6 and GLKdel/wt mice (heterozygous GLK knockout mice) were treated once daily for 28 days with GLK activator (AZl) at 2.5 mg / kg and 5 mg / kg, which gave glucose lowering for 12 hours. Vehicle treated C57BL6 & GLKdel / wt mice were used as controls. Plasma samples were taken and CTX-II was measured. CTX-II was measured using Nordic Biosciences pre-clinical cartilaps kit with no deviations from the manufacturers instructions.

Toluidine Blue Staining Methodology

Left hind knees were fixed in 10% buffered formalin for 2 weeks following which they were decalcified in EDTA solution at 50 ⁰ C for 2-3 weeks.

The samples were trimmed and blocked in paraffin wax ready for sectioning using a microtome. 5 μm sections were taken every 100 μm throughout the joint. They were rehydrated through Xylene (x3), absolute alcohol (x2), industrial methylated spirit (IMS) (x2) followed by deionised water. 0.1 % toluidine blue stain was applied to each slide for 20 seconds and washed away with distilled water. The sections were dehydrated (above in reverse), mounted using Histomount mounting medium and cover-slipped.

Measurement of Glvcosaminoglycan in Rat Cartilage (DMMB Method)

Articular cartilage was removed from the joint and placed into a high salt buffer (HSB). Hylauronidase (Sigma, 110 units/ mL) was added and incubated overnight at 37 ⁰ C, the samples were then digested using papain (Sigma, 125 μg/ mL) for 6 hours at 60 ⁰ C, and the supernatant was removed for dimethylmethylene blue (DMMB) analysis. Samples were diluted in deionized water and mixed with a DMMB solution. The plate was read immediately at 532 nm and 620 nm and data expressed as 532 / 620 nm. The sGAG content was calculated from standard curve (range 25-200 ng) generated using chondroitin sulphate (Sigma).

High Salt Buffer

Tris 25mM

NaCl 50OmM (pH to 7.5 with HCl)

DMMB

16 mg DMMB (Polysciences Inc, 03610) 1.52 g Glycine (Fisher, G/P460/53) 1.19 g NaCl (Fisher, S/3160/53) 4.75 mL IM HCl MiIIiQ water to 500 mL

Hyaluronidase buffer

0.12 mL 1OX Tris acetate, 0.4 mL 3M Sodium acetate, MiIIiQ water to 12 mL

Papain buffer

37.22 mg EDTA (Sigma, E5134), 32.64 mg N-Acetyl-L-cysteine (Sigma A9165), 1.33 mL 3M sodium acetate, 8.67 mL 0.05M Phosphate buffer pH 7.

RESULTS

GLK Activation Reduces Expression of Biologically Relevant OA Genes

GLK was detected in OA chondrocytes at both mRNA and protein level (Figure 1). Cartilage explants from four OA donors were exposed to 10 x E. C.50 GLK activator AZ2 for 96 hours. Responses were examined by RT-PCR (methodology above). Genes which showed consistent, significant downregulation, were the MMPs (MMPl and MMP 13) and loss of differentiation genes (type I collagen, osteopontin, alkaline phosphatase, type X collagen and IL8). Gene changes from a representative OA donor are shown in Figure 2.

ELISAs on explant supernatants from compound treated OA cartilage donors showed decreased levels of the biochemical readouts MMPl, MMP 13, MMP3, osteopontin, IL6, IL8 and PGE2. Data showing four of these biologically relevant endpoints, on four OA cartilage donors, are shown in Figure 3. OA chondrocytes (n=4) treated with GLK activator AZ2, at 5.5 mM

(normoglycaemic) and 10 mM (hyperglycaemic) media glucose concentrations (to reproduce the situation in a diabetic OA patient) showed similar efficacy under both conditions. MMPl reduction was reproduced in OA cartilage explants (n=l) under normo- and hyperglycaemic conditions (Figure 4).

Inhibitors of the Glycolytic Pathway Reverse the GLK Mediated Effect

The GLK inhibitors, 5-thioglucose and mannoheptulose were tested on OA cartilage explants to demonstrate GLK pathway effects in cartilage. Protocols for cartilage stimulation are given above. These are glucose analogues that are not phosphorylated by

GLK and inhibit its activity because they are not able to be phosphorylated hence blocking the glycolytic pathway.

• Both 1OmM 5-thioglucose (n=3) and 1OmM mannoheptulose (n=l) cause a significant increase in the release of IL6, IL8, MMPl, MMP3 and VEGF from OA cartilage (Figure 5).

• The changes in MMP 1 , MMP 13 , VEGF, IL6 and IL8 induced by the GLK inhibitors were reversed by 10 x E. C.50 GLK activator AZ2 (Figure 5).

GLK Activator from a Different Chemical Series Results in Similar Effects In order to demonstrate GLK mediated effects, a GLK activator from a distinct chemical series (Roche 2) was tested in both OA chondrocytes and OA explants. Both compounds were tested 1O x EC50. The efficacy of glucokinase activators in reducing CTXII was seen in 4 studies. Two different activator compounds were used, AZ3 and AZl (Table 1).

• In unstimulated cartilage (n=3) the competitor compound decreased basal levels of MMP 1 and MMP 13 (Figure 6).

• In IL lβ / Oncostatin M stimulated OA cartilage (n=2) the competitor compound decreased MMP 13, VEGF and IL6 in both donors and MMPl, MMP3 and IL8 in one of the two donors (Figure 7).

• Additionally, in IL lβ / Oncostatin M stimulated OA chondrocyes, several GLK activators (Rochel, Roche2, Prosidionl, AZl and AZ2 all at 3 μM) decrease

MMP 13 release (Figure 8).

GLK Activation Reduces ILlβ Mediated Effects on Cartilage Explants

IL lβ is a culprit catabolic driver of OA. • OA chondrocytes were treated with three doses of ILlβ / Oncostatin M (n=2), all of which caused an increase in a panel of biochemical readouts (IL6, IL8, MMP13 and VEGF). Glucokinase activator (AZ2) inhibited these effects (Figure 9).

• ILl β upregulates release of all of our biochemical readouts from OA cartilage explants (n=4). GLK activator (AZ2) at 10 x EC50 reduced this increase (Figure 10).

• IL lβ upregulated release of MMPl and MMP 13 from PM cartilage explants. GLK activator (AZ2) reduced MMPl and MMP 13 release in a dose-dependent manner (Figure 11).

In Vivo Study: Zucker Rats

Analysis of samples obtained from an in vivo compound study were carried out.

• MMP 13 was detectable in the cartilage of all animals. The vehicle treated rats had significantly increased MMP 13 over the lean vehicle control animals. Animals treated with glucokinase activator (AZ3) had a tendency towards decreased MMP13 in the cartilage, however, this failed to reach significance (Figure 12).

• Vehicle treated Zucker rats had significantly increased plasma CTX-II over glucokinase activator (AZ3) treated and lean vehicle control animals (Figure 13).

In Vivo Study: GLK Knockout Mice Analysis of samples obtained from an in vivo compound study were carried out.

• Vehicle treated GLK knockout mice had increased plasma CTX-II over glucokinase activator (AZl) treated and wild-type control animals (Figure 14).

The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intra-articular or intramuscular dosing or as a suppository for rectal dosing). Dosage forms suitable for oral use are preferred.

The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.

Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p_-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal tract, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art. Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p_-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame). Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as

beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present. The pharmaceutical compositions of the invention may also be in the form of oil-in- water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.

The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.

Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.

For further information on formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

The size of the dose for therapeutic or prophylactic purposes of a compound of Formula (I) will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.

In using a compound of Formula (I) for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred. Table 1.

Study Details:

1. AZ3, 1 -month study, 12-hour cover

2. AZ3, 1 -month study, 12-hour cover

3. AZl, 2-week study, 8-hour cover 4. AZl, 2-week study, 24-hour cover