GB2417682A - Histone deacetylse inhibitor for treating connective tissue disorders - Google Patents

Abstract

The use of a histone deacetylase inhibitor in the manufacture of a medicament for the treatment of a disease associated with the destruction of connective tissue mediated by metalloproteinases. The diseases may be selected from arthritis, multiple sclerosis, cardiovascular disease, ocular disease, or tumour invasion and metastasis. Examples of inhibitors include sodium butyrate, trichostatin A, depsipeptide and MS-275.

Description

24 1 7682 - 1 - HDAC Inhibitors and methods of use thereof

Field of the Invention

The present invention relates to novel strategies for treatment and/or prevention of disease associated with the destruction of the connective tissue mediated by metalloproteinases.

Background to the Invention

Arthritis is a major health problem. There has been intensive research aimed at developing treatments for the disease. Several forms of arthritis are known, including rheumatoid arthritis (RA) and osteoarthritis (OA). The currently available forms of treatment include, for example, COX-2 inhibitors or nonsteroidal anti-inflammatory drugs.

However, treatment with these drugs is associated with complications in the gastrointestinal tract. Most available therapies do not prevent destruction of the articular cartilage. Accordingly, there is a need to develop alternative methods of treatment of arthritis.

Articular cartilage is made up of two main extracellular matrix (ECM) macromolecules, type II collagen, and aggrecan (a large aggregating proteoglycan) (1, 2). The type II collagen scaffold endows the cartilage with its tensile strength, whilst the aggrecan, by virtue of its high negative charge, draws water into the tissue, swelling against the collagen network, and enabling the tissue to resist compression. Quantitatively more minor components (e.g. type IX, XI and VI collagens, biglycan, decorin, COMP etc.) also have important roles in controlling matrix structure and organization (2). - 2

Normal cartilage ECM is in a state of dynamic equilibrium, with a balance between synthesis and degradation. For the degradative process, the major players are metalloproteinases that degrade the ECM, and their inhibitors. Pathological cartilage destruction can therefore be viewed as a disruption of this balance, favouring proteolysis.

The matrix metalloproteinases (MMPs) are a family of currently 23 enzymes in man which facilitate turnover and breakdown of the ECM in both physiology and pathology. The MMP family contains the only mammalian proteinases that can specifically degrade the collagen triple helix at neutral pH. The 'classical' collagenases, MMP-1, -8 and -13, have differing substrate specificities for types I, II and III collagen, with MMP-13 showing a preference for type II collagen. More recently, MMP-2 and MMP-14 have also been shown to cleave the collagen triple helix, though with less catalytic efficiency than the classical collagenases, at least in vitro. The enzyme(s) responsible for cartilage collagen cleavage in the arthritides is open to debate, but the dogma has been that MMP-1, produced in the synovium, is the primary collagenase in rheumatoid arthritis (RA), whilst MMP-13, produced by the chondrocyte, is the foremost collagenase in osteoarthritis (OA). Several other members of the MMP family have been localised to cartilage or synovium in the arthritides (3).

A second group of metalloproteinases, the ADAMTS family (a disintegrin and metalloproteinase domain with thrombospondin motifs) contains 19 members, including the so-called 'aggrecanases'. These enzymes (ADAMTS-1, -4, -5, -9 and -15) are defined by their ability to degrade the interglobular domain separating G1 and G2 of aggrecan at a specific Glu373-Ala374 bond. Cleavage within this interglobular domain can also be mediated by MMPs (cleaving at Asn341-Phe342), and both activities can be detected in articular cartilage from OA and RA patients. Current data support the hypothesis that aggrecanases are active early in the disease process with later increases in AMP activity, however, the exact enzyme(s) responsible for cartilage aggrecan destruction at any stage in arthritis is unclear (3, 4).

A family of four specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) has been described.

These are endogenous inhibitors of MMPs and potentially ADAMTSs (5). The ability of TIMP-1 to -4 to inhibit active MMPs is largely promiscuous, though a number of functional differences have been uncovered. TIMP-3 appears to be the most potent inhibitor of ADAMTSs e.g. with subnanomolar Ki against ADAMTS-4 (3).

Metalloproteinase activity is regulated at multiple levels including gene transcription. However, the role of chromatin modification, and in particular acetylation, is little researched in the metalloproteinase arena. The packaging of eukaryotic DNA into chromatic plays an important role in regulating gene expression. The DNA is wound round a histone octamer consisting of two molecules each of histones H2A, H2B, H3 and H4 to form a nucleosome.

This unit is repeated at approximately 200bp intervals with histone H1 associating with the intervening DNA. - 4

Nucleosomes are generally repressive to transcription, hindering access of the transcriptional apparatus (6).

However, two major mechanisms exist which modulate chromatin structure to allow transcriptional activity: firstly, ATP- dependent nucleosome remodellers such as the Swi/Snf complex (7,8); secondly, the enzymatic modification of histones, via acetylation, methylation and phosphorylation (9-11).

Acetylation by histone acetyltransferases (HATs) occurs on specific lysine residues on the N-terminal tails of histone H3 and H4. This neutralization of positive charge leads to a loosening of the histone:DNA structure, allowing access of the transcriptional machinery; furthermore, the acetyl groups may associate with and recruit factors containing bromodomains (6). Many transcriptional activators or coactivators have (or recruit) HAT activity, giving a mechanism whereby acetylation can be targeted to specific gene promoters (10,11). Conversely, histone deacetylases (HDACs) have also been characterized.

Hypoacetylation of histones associates with transcriptional silence, and several transcriptional repressors and co- repressors have been identified which have (or recruit) HDAC activity (12- 14). Non-histone substrates of HAT s have also been described e.g. p53, E2F, NF-KB, Sp3 and c-dun (15, 16).

There are two families of HDACs, the NAD±dependent SIR2 family (sometimes called class III HDACs) and the classical HDAC family. The classical HDACs can be split into three classes (I, II and IV) based on phylogeny (17).

Class I HDACs (HDAC1, 2, 3 and 8) are related to yeast RPD3, and class II HDACs (HDAC4, 5, 6, 7, 9 and 10) are more closely related to yeast HDA1 (12). HDAC11 alone represents class IV and HDAC11-related proteins have been described in all eukaryotic organisms other than fungi (17) . HDAC inhibitors (HDACi) are thought to function by blocking access to the active site of HDAC. A number of different compounds with HDACi activity are known and the effects of different HDACi in inhibiting HDAC activity are considered the same (12). HDACi include trichostatin A (TSA) and sodium butyrate (NaBy) which have a broad spectrum of activity against the classical HDAC family (18, 19).

Addition of these reagents to cells should therefore block histone deacetylation and result in increased acetylation of histones on susceptible genes. The prediction would be that this would lead to an increase in gene expression, and this is largely borne out experimentally. However, there are many instances of HDAC inhibitors acting as repressors of gene expression (20-24).

HDAC inhibitors have potent anti-proliferative and pro apoptotic activities in cancer cells and this has led to the development of specific inhibitors for cancer chemotherapy.

Such compounds are currently in both preclinical development and clinical trials (25). A recent report demonstrates that HDAC inhibitors modulate gene expression in synovial cells (26). In an animal model of RA (adjuvant arthritis), tumour necrosis factor (TNFa) expression was inhibited and this led to a reduction in synovial hyperplasia and joint swelling with maintenance of joint integrity.

However, no study has looked at the effect of these inhibitors on cartilage. The inventors are the first to - 6 demonstrate that HDAC inhibitors repress the expression of several members of the metalloproteinase family in chondrocytes and block cartilage destruction. Hence, inhibition of HDAC activity offers a therapeutic strategy to prevent cartilage destruction in the arthritides and other diseases affecting the destruction of connective tissue.

Description of the Invention

In accordance with a first aspect of the invention there is provided a histone deacetylase inhibitor for the use in the treatment and/or prophylaxis of a disease associated with the destruction of the connective tissue mediated by metalloproteinases. In particular, the invention relates to the treatment of a disease associated with the destruction of cartilage. Accordingly, the invention relates to HDACi as chondroprotective agents.

In a preferred embodiment, the invention relates to the use of a histone deacetylase inhibitor in the treatment and/or prophylaxis of any joint disease where cartilage destruction is prominent. In one embodiment, the invention relates to non-inflammatory joint disease. In a another embodiment, the invention relates to inflammatory joint disease. In another preferred embodiment, the disease is arthritis. Preferably, the arthritis is selected from the group of osteoarthritis, reactive arthritis, gout/pseudogout arthritis, juvenile idiopathic arthritis or psoriatic arthritis. However, as will be appreciated by a person skilled in the art, the invention may also relate to other forms of destructive arthritis.

In another preferred embodiment, the invention relates to the use of HDACi in the treatment and/or prophylaxis of multiple sclerosis, tumour growth, invasion and metastasis, cardiovascular disease or ocular disease or other disease associated with the destruction of connective tissue.

It is known that the large number of well established compounds with HDAC inhibiting activity can broadly be divided into four general groups.

HDACi that fall within group 1 are hydroxamic acids or their derivatives or salts characterized by the general structure as shown in formula 1.

Formula 1 o -R- ' HDACi of this common general formula include trichostatin A (TSA), suberoyl anilide hydroxamic acid (SAHA), Mcarboxycinnamic acid bishydroxamide (CBHA), scriptaid, pyroxamide and oxamflatin.

However, a large number of compounds has been generated based on the structure of the compounds listed above. These include, but are not limited to, compounds related to TSA, such as TSA-like straight chain hydroxamates or TSA derived compounds wherein the cap substructure that interacts with amino acids has been altered. - 8

Other compounds are derived from SAHA, such as thiol based SAHA analogues or SAHA based non-hydroxamtes wherein the hydroxamic acid group has been replaced with another functional group, such as an N-formyl hydroxylamino group.

Other examples of the SAHA based non-hydroxamtes include bromoacetamides and semicarbazide.

HDACi of group 1 comprise a metal chelating element attached to an aromatic group via R. R can comprise a connection unit and a spacer. Preferably, the spacer is a hydrophobic unit.

The connection unit can be amide, sulphonamide, ketone, ether or aromatic heterocycles such as oxazole and thizole.

Many synthetic compounds are known wherein the linker has been altered and may, for example, contain a 1,4-phenylene carboxamide linker.

HDACi that fall within group 2 of well established HDACi are short fatty acid chains or salts thereof and are characterized by the general formula 2.

Formula 2 Rage l3H HDACi of group 2 includes valproic acid and pharmaceutically acceptable salts thereof, butyrate, for example sodium butyrate (NaBy), and phenylbutyrate.

These include but are not limited to the compounds listed above coupled to Zn2+ chelating motifs (hydroxamic acid and o-phenylenediamine). - 9

HDACi of the third group are cyclic tetrapeptides/epoxides characterized by the general formula 3. Some of these compounds of this group are products of fungi or bacteria, others have been chemically engineered.

Formula 3 0.' caret / C_ Of I.-C-R 11 \. / HDACi of the third group comprise trapoxin, HC-toxin, chlamydocin, depudesin, apicidine and depsipeptide (FK228).

HDACi of the fourth group are benzamindes of the general formula 4.

Formula 4 a Q__>ll Compounds of the general formula 4 comprise N acetyldinaldine and MS-275.

Further HDACi compounds include, for example, aroyl-pyrrolehydroxy-amides (ALPHA) and derivatives thereof, cyclic hydroxamic acid peptides (CHAPs) or sulphur-containing cyclic peptides (SCOPs). -

It is well known that, in addition to the classical HDACi of groups 1 to 4, a number of compounds showing HDAC inhibiting activity have been developed from those compounds, such as the APHAs described above (45 to 55). The skilled person will know that all known compounds with HDACi activity can be used according to the invention.

Accordingly, the invention relates to the use of HDACi selected from group 1, 2, 3 or 4 or other chemical classes with HDAC inhibitory activity. The list above showing compounds belonging to the separate groups merely shows examples of such compounds and should not be regarded as exhaustive.

The invention is exemplified in the experiments described herein using TSA and NaBy, two HDACi from chemically very different groups, to illustrate the invention. It will be understood by a person skilled in the art that the use of these two different inhibitors illustrates that HDACi from different classes can be used in the invention and that accordingly, all compounds with HDACi activity known in the art can be used according to the invention.

References to compounds of the general formulae 1 to 4 particularly with regard to therapeutic use, will be understood to also encompass pharmaceutically acceptable salts of such compounds. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids, as would be well known to persons skilled in the art.

Many suitable inorganic and organic bases are known in the art.

The scope of the invention also extends to derivatives of the compounds of general formula 1, 2, 3 or 4 that retain the desired activity of HDAC inhibition. Derivatives that retain substantially the same activity as the starting material, or more preferably exhibit improved activity, may be produced according to standard principles of medicinal chemistry, which are well known in the art. Such derivatives may exhibit a lesser degree of activity than the starting material, so long as they retain sufficient activity to be therapeutically effective. Derivatives may exhibit improvements in other properties that are desirable in pharmaceutically active agents such as, for example, improved Volubility, reduced toxicity, enhanced uptake, etc. The invention also encompasses pharmaceutical compositions comprising HDACi, or pharmaceutically acceptable salts or derivatives thereof, formulated into pharmaceutical dosage forms, together with suitable pharmaceutically acceptable carriers, such as diluents, fillers, salts, buffers, stabilizers, solubilizers, etc. The dosage form may contain other pharmaceutically acceptable excipients for modifying conditions such as pH, osmolarity, taste, viscosity, sterility, lipophilicity, Volubility etc. The choice of diluents, carriers or excipients will depend on the desired dosage form, which may in turn be dependent on the intended route of administration to a patient.

Suitable dosage forms include, but are not limited to, solid dosage forms, for example tablets, capsules, powders, - 12 dispersible granules, cachets and suppositories, including sustained release and delayed release formulations. Powders and tablets will generally comprise from about 5\ to about 70% active ingredient. Suitable solid carriers and excipients are generally known in the art and include, e.g. magnesium carbonate, magnesium stearate, talc, sugar, lactose, etc. Tablets, powders, cachets and capsules are all suitable dosage forms for oral administration.

Liquid dosage forms include solutions, suspensions and emulsions. Liquid form preparations may be administered by intravenous, intracerebral, intraperitoneal, parenteral or intramuscular injection or infusion. Sterile injectable formulations may comprise a sterile solution or suspension of the active agent in a non-toxic, pharmaceutically acceptable diluent or solvent. Liquid dosage forms also include solutions or sprays for intranasal, buccal or sublingual administration.

Also encompassed are dosage forms for transdermal administration, including creams, lotions, aerosols and/or emulsions. These dosage forms may be included in transdermal patches of the matrix or reservoir type, which are generally known in the art.

Pharmaceutical preparations may be conveniently prepared in unit dosage form, according to standard procedures of pharmaceutical formulation. The quantity of active compound per unit dose may be varied according to the nature of the active compound and the intended dosage regime.

The active agents are to be administered to human subjects in "therapeutically effective amounts", which is taken to mean a dosage sufficient to provide a medically desirable result in the patient. The exact dosage and frequency of administration of a "therapeutically effective amount" of active agent will vary, depending on the condition which it is desired to treat, the stage and severity of disease, and such factors as the nature of the active substance, the dosage form and route of administration. A typical dosage range for compounds of general formula l(a) and/or formula l(b) is about O.1-lOmg of compound per kg of mammal by weight, however this is given by way of example only and is not intended to limit the invention to this dosage range.

The appropriate dosage regime for a given patient will generally be determined by a medical practitioner having regard to such factors as the severity of disease, and the age, weight and general physical condition of the patient, and the intended duration of treatment, as would be appreciated by those skilled in the art.

According to the invention, the inhibitors - and/or pharmaceutical compositions containing one or more of these inhibitors - may be used for the prevention (e.g. prophylaxis) and/or treatment of a disease associated with the destruction of connective tissue mediated by metalloproteinases(which for the purposes herein in its broadest sense also includes preventing, treating and/or alleviating the symptoms and/or complications of such disease).

HDAC inhibitors are currently being developed as cancer therapeutics largely by virtue of their impact upon cell cycle and apoptosis (25) in transformed cells. However, it is clear that such compounds have pleiotropic effects on gene expression. Conceptually, the action of HDACi leading to an increase in histone acetylation should induce expression of susceptible genes, but in fact, many instances of a repression of gene expression have been reported (20- 24). Genome-wide studies in yeast have shown both the deletion of HDACs (e.g. rpd3 and sin3) and the addition of TSA to wild-type strains leads to an increase in cellular histone acetylation (20), however, in both cases a number of transcripts are down-regulated. The deletion mutants cannot differentiate between genes that are direct targets of HDAC- mediated activation and indirect effects (e.g. secondarily via altered expression of activators/repressors/signalling molecules). However, the ability of TSA to down-regulate some genes very rapidly (within 15 minutes of exposure) does suggest that HDACs may function as direct transcriptional activators in some instances.

The combination of interleukin-1 and oncostatin M potently induces both cartilage aggrecan and collagen degradation in vitro and in vivo (34, 35). We have previously shown that IL-1/OSM induces the expression of a number of metalloproteinase genes in chondrocyte cell lines (37). The addition of HDAC inhibitors to cartilage explant cultures blocks IL-l/OSM-induced cartilage catabolism.

Measurement of collagenolytic activity in the conditioned culture medium of these explants showed a decrease in both the proportion of collagenase which was active and also the total amount of collagenase. TSA or NaBy themselves do not directly inhibit collagenase activity, and it therefore seemed likely that they were altering expression of genes - 15 encoding the metalloproteinases or their inhibitors.

Gelatin zymography showed a similar repression of IL-1/OSM- induced gelatinolytic activity by TSA. Using SW1353 chondrosarcoma cells which are known to respond to IL-1/OSM (33), real-time RT-PCR gene profiling showed that the expression of a number of MMP and ADAMTS genes was robustly induced by IL-1/OSM and repressed by HDACi. Of the MMPs, these were MMP1, MMP3, MMP7, MMP8, MMP10, MMP12, MMP13: interestingly these genes all cluster on chromosome llq22.

Whether this chromosomal localization allows them to be co- regulated at the level of chromatin structure is currently unknown, or potentially they are derived from a recent gene duplication and retain similar promoter elements. In SW1353 cells, MMP2 is not induced by IL- 1/OSM nor altered by HDACi; MMP9 is weakly induced by IL-1/OSM and this induction is repressed by HDACi. In primary chondrocytes, MMP2 expression is induced approximately 2-4-fold by IL-1/OSM, but not then repressed by HDACi. This is in marked contrast to the zymography data from cartilage explants and suggests a role for cell-matrix interactions in mediating the effects of IL-1/OSM on these gelatinase genes.

Previous studies demonstrate that TSA represses MMP2 expression in mouse 3T3 fibroblasts, but not human HT1080 fibrosarcoma cells (38, 39) showing that the effects of HDACi on MMP expression are cell-type specific. In primary chondrocytes, the effects of HDACi on the collagenases (MMP1, MMP8, MMP13) mirrored that seen in the SW1353 cell line; however, MMP3, though strongly induced by IL-1/OSM in primary chondrocytes, was not significantly repressed by HDACi. In primary chondrocytes, the aggrecanases ADAMTS4, ADAMTS5 and ADAMTS9 were also highly induced by IL1/OSM, and repressed by HDACi. The ability of HDACi to repress AMP expression at the mRNA level is reiterated at the protein level, as we have shown for MMP-1 and -13.

Since almost all metalloproteinase genes which are robustly induced by IL1/OSM are then repressed by the further addition of HDACi, a likely explanation is the ability of HDACi to interfere with IL-l/OSM signalling.

Since these cytokines are proinflammatory mediators, action via NFKB is one possibility, however, the literature shows that TSA actually potentiates signalling through this pathway (40, 41). OSM, an interleukin6 family cytokine, signals through the STAT pathway; recent reports show that HDAC activity plays an essential role in at least STAT1 signalling, and that TSA can therefore abrogate STAT1- induced gene expression (42, 43). The inventors have previously reported that at least STATS signalling indirectly mediates the ability of IL- 1/OSM to induce MMP1 gene expression (44).

A previous report using the rat adjuvant arthritis model of rheumatoid arthritis, demonstrated that HDACi (TSA and phenylbutyrate) block proliferation of cultured rat synovial fibroblasts with accompanying upregulation of cell cycle inhibitors (pl6IN4 and p21CiPl). In viva, this was mirrored with inhibition of synovial hyperplasia and pannus formation leading to abrogation of cartilage destruction in the model. Interestingly, the HDACi also repressed expression of TNFa in synovial tissue. The authors suggest of that report that HDACi may represent a new class of compounds for treatment of rheumatoid arthritis (26).

The inventors have now shown that HDACi can also function as potent repressors of metalloproteinase expression in cartilage and chondrocytes. Accordingly, HDACi can also be used in the treatment of any joint disease wherein cartilage destruction is prominent, including non-inflammatory and inflammatory joint disease. Moreover, they can also have wider therapeutic use outside of just the arthritides, as protective agents in diseases associated with the destruction of connective tissue.

The invention will be further understood by reference to the following experimental examples, together with the accompanying Figures, in which: Figure 1 illustrates that histone deacetylase inhibitors block IL-la/OSMinduced cartilage glycosaminoglycan and collagen loss. Bovine nasal cartilage discs were cultured in the presence or absence of IL-la/OSM (I/O) and histone deacetylase inhibitors (A, leg/ml IL-la, 10ng/ml OSM, trichostatin A (TSA); B. 0.2ng/ml IL-la, 2ng/ml OSM, sodium butyrate (NaBy). Cartilage was incubated until day 7 and supernates were harvested and replaced with fresh reagents until day 14. Proteoglycan release is shown at day 7, assayed using the dimethylmethylene blue method for glycosaminoglycan whilst collagen release is shown at day 14 measured using an assay for hydroxyproline. Viability was assessed by measurement of lactate dehydrogenase in the conditioned medium. Assays were performed at least twice using quadruplicate samples; means +/- standard deviations are shown. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2 illustrates that histone deacetylase inhibitors decrease total collagenolytic and gelatinolytic activity secreted by bovine nasal explants and block collagenase activation. Conditioned media from cartilage assays (day 14) as in Figure 1A were assayed for (A) collagenase activity in the presence or absence of 0.67mM APMA (mean +/- s.e.m. shown) and (B) gelatinase activity using gelatin zymography. Figure 2A shows an assay for collagenase activity in the conditioned medium from the explant assay above at day 14 in the absence or presence of TSA.

Treatment with IL-1 and OSM increases collagenase activity in the medium, and all collagenases are in the active form.

The additional presence of TSA at the lowest dose (50ng/ml) decreases the level of active collagenase, whilst total collagenase is unchanged, i.e. the percentage of collagenase which is active is decreased. With increasing dose, TSA decreases the level of both active and total collagenase, i.e. the total amount of collagenase in the medium is decreased and the percentage of this enzyme(s) which is activated also decreases. Similar data are, for example, obtained using sodium butyrate.

Figure 2B shows a gelatin zymogram of the day 14 cartilage explant conditioned medium in the absence or presence of TSA. Unstimulated explants produce a low constitutive level of gelatinolytic activity which is likely proMMP-2. The addition of IL-1a and OSM induces three major gelatinolytic activities which run as poorly resolved doublets (all activities were shown blocked by metalloproteinase inhibitors, see Methods). The largest of these likely equates to bovine MMP-9 (pro- and active); there is an induction and activation of MMP-2 and an induction of a - 19 lower molecular weight activity which may represent collagenases MMP-1 and -13, but could potentially include other MMPs, many of which have at least some activity against gelatin. Both the collagenases, and particularly MMP-13, have gelatinolytic activity, and this would fit with the induction of collagenase activity shown in Figure 2. At the lowest dose (50ng/ml), TSA causes a marked reduction in the lowest molecular weight activity, whilst with increasing dose, the activities of all gelatinolytic enzymes are

reduced to background.

Figure 3 illustrates that histone deacetylase inhibitors abrogate ILla/OSM-induced expression of key metalloproteinase genes. Cells were serum-starved for 24 hours prior to stimulation with IL-la (5ng/ml) and OSM (lOng/ml) (I/O) for 6 hours in the absence or presence of TSA (A, as shown; B and C 50Ong/ml) or NaBy (A, as shown, B and C, 500ng/ml). Total RNA was isolated and subjected to quantitative RT-PCR for MMP1, MMP13 (Aand B), ADAMTS4, ADAMTS5 and ADAMTS9 (B) gene expression; data was normalized to the 18S rRNA housekeeping gene; mean and range are plotted. Absolute numbers are primer/probe set dependent and so cannot be compared between genes. A. SW1353 chondrosarcoma cells; B. and C. primary human chondrocytes; B (inset). expression of COL2A1 and aggrecan by conventional RT-PCR.

Figure 4 illustrates that histone deacetylase inhibitors repress MMP protein expression and activity. Cells were serum-starved for 24 hours prior to stimulation with IL-la (5ng/ml) and OSM (lOng/ml) (I/O) for 24 hours in the absence - 20 or presence of TSA (500ng/ml)or NaBy (500ng/ml).

Conditioned media were subjected to western blot analysis using a rabbit anti-(human MMP-1) antibody or a sheep anti- (human MMP-13) antibody or gelatin zymography as described

the examples.

EXAMPLE 1

Cell culture. SW1353 human chondrosarcoma cells were routinely cultured in Dulbecco's modified Eagle medium (DMEM, Invitrogen) containing 10% foetal bovine serum (FBS, Invitrogen), 2mM glutamine, lOOIU/ml penicillin and lOOg/ml streptomycin. Serum-free conditions used identical medium without FBS. For assays, cells were grown to confluence, then serum-starved for 24 hours prior to the addition of IL 1 (R&D Systems, 5ng/ml) and oncostatin M (OSM, R&D Systems lOng/ml) in the absence or presence of HDAC inhibitors (trichostatin A, TSA, and sodium butyrate, NaBy, Calbiochem). Experiments were performed in 6-well plates with all conditions in duplicate or triplicate. To obtain primary human chondrocytes, fresh human articular cartilage samples were digested overnight in DMEM containing 2mg/ml of collagenase Type 1A (Sigma). The resulting cells were washed with PBS, resuspended in DMEM containing 10% FCS and antibiotics as above and then plated at 1x106 cells in 75cm2 flasks. At confluence, cells were passaged and replated at 1:2 dilution.

RNA isolation and synthesis of cDNA. RNA was isolated from monolayer cultures using Trizol reagent (Invitrogen). cDNA was synthesised from lg of total RNA using Superscript II reverse transcriptase (Invitrogen) and random hexamers in a - 21 total volume of 201 according to manufacturers instructions. cDNA was stored at -20 C until used in downstream PCR.

RT-PCR. For quantitative real-time PCR, sequences and validation for AMP and TIMP primers and probes are as described by Nuttall et al. 2003 (27) and ADAMTS primers and probes are as described by (28). In order to control against amplification of genomic DNA, primers were placed within different exons close to an intron/exon boundary with the probe spanning two neighbouring exons where possible.

BLAST searches for all the primer and probe sequences were also conducted to ensure gene specificity. The 18S ribosomal RNA gene was used as an endogenous control to normalise for differences in the amount of total RNA present in each sample; 18S rRNA primers and probe were purchased from PE Applied Biosystems.

Relative quantification of genes was performed using the ABI Prism 7700 sequence detection system (PE Applied Biosystems) in accordance with the manufacturer's protocol.

PCR reactions contained 5ng of reverse transcribed RNA (1 ng for 18S analyses), 50% TaqMan 2X Master Mix (PE Applied Biosystems), lOOnM of each primer and 200nM of probe in a total volume of 251. Conditions for the PCR reaction were 2 minutes at 50 C, 10 minutes at 95 C, then 40 cycles each consisting of 15 seconds at 95 C and 1 minute at 60 C.

Conventional RT-PCR for collagen and aggrecan expression was as previously described (29).

Results Using the SW1353 chondrosarcoma cell line as a model in which to look at the regulation of metalloproteinase and TIMP gene expression, the expression of all Amps, ADAMTSs and TIMPs in cells stimulated with IL-1 and OSM in the absence or presence of HDACi was profiled at the doses used for the cartilage explant experiments (see example 1, 2 Figure 3A).

A number of genes, MMP1, MMP3, MMP7, MMP8, MMP10, MMP12, MMP13, ADAMTS4 and ADAMTS9 are robustly induced by the combination of IL-1a and OSM (though both ADAMTS4 and ADAMTS5 are only expressed at low levels in this cell line).

Of these induced genes, all but ADAMTS4 show repression by both TSA and NaBy. ADAMTS4, whilst strongly induced by IL 1 and OSM is not repressed by either HDACi in this cell line. The expression of a number of genes (MMP2, MAPS, MMP16 and MMPl9, ADAMTS1, ADAMTS2, ADAMTS7, ADAMTS12, ADAMTS13 and ADAMTS20, TIMP3) was unaffected by the HDACi.

The expression of several genes was induced by HDACi alone (MMP17, MMP23, MMP28, ADAMTS15 and ADAMTS17, TIMP2).

In order to verify that the effects of HDACi were not specific to the SW1353 cell line, a similar experiment on a subset of genes was undertaken using primary articular chondrocytes isolated from both knee and hip joint (Figure 3B) MMP1 and MMP13, the two major specific collagenases, are strongly induced by IL-1a and OSM and this induction is repressed by both TSA and NaBy. MMP8 was expressed at much lower levels in these cells but followed the same pattern of responses (data not shown). The IL-1a and OSM induction of MMP3 gene expression was only poorly repressed by HDACi in the primary chondrocytes (data not shown). In these cells, ADAMTS4, ADAMTS5 and ADAMTS9 were all induced by IL-1a and OSM and this was repressed by HDACi (Figure 3C). These - 23 primary chondrocytes, although grown in monolayer culture, still express type II collagen and aggrecan at the passage at which this experiment was performed.

Example 2

Cartilage degradation assay. Bovine nasal cartilage was cultured as previously described (30). Briefly, discs (approximately lmm3) were punched from bovine nasal septum cartilage; three discs per well in a 24-well plate were incubated overnight in control, serum-free medium (DMEM containing 25mM HEPES, 2mM glutamine, 100pg/ml streptomycin, IU/ml penicillin, 2.5pg/ml gentamicin and 40u/ml nystatin). Fresh control medium with or without test reagents (each condition in quadruplicate) was then added (day 0). Cartilage was incubated until day 7 and supernates were harvested and replaced with fresh medium containing the same test reagents as day 0. On day 14, supernates were harvested and the remaining cartilage digested with papain.

Viability of cartilage explants was assessed by measurement of lactate dehydrogenase (LDH) in the conditioned medium (CytoTox 96 assay, Promega). Hydroxyproline release was assayed as a measure of collagen degradation whilst glycosaminoglycan release was assayed as a measure of proteoglycan degradation (30). Collagenase activity was determined by the 3H-acetylated collagen diffuse fibril assay using a 96-well pate modification (31); one unit of collagenase activity degraded log of collagen per minute at 37 C. Statistical analysis was performed using Student's t- test.

Results - 24 The combination of IL-1a and OSM has previously been shown to induce cartilage proteoglycan and collagen proteolysis both in vitro and in vivo (34, 35). The addition of TSA or NaBy to bovine nasal cartilage explant culture stimulated to resorb with IL-1 and OSM causes a dose-dependent inhibition of both proteoglycan and collagen release (at day 7 and 14 respectively) (Figure 1). TSA is reported to have an ICso in the nM range (50ng/ml = 165nM), but this does vary depending upon the HDAC and assay used (e.g.(36)); sodium butyrate is reported to have an IC50 in the mM range. The need for TSA, a hydroxamate, to penetrate the highly negatively charged cartilage matrix, will also raise the effective IC50 in the cartilage explant assay. The time points of media collection, day 7 and 14, represent those at which proteoglycan and collagen release respectively are reproducibly close to 100%; again, maximum sensitivity of the assay to inhibition may be achieved at earlier time points. As a measure of toxicity, lactate dehydrogenase release is no greater in the presence of TSA or NaBy than in the comparator control cultures (i.e. either no addition or IL-1/OSM treated).

Example 3

Gelatin demography. Samples were electrophoresed under non reducing conditions by SDS-PAGE in 10% polyacrylamide gels copolymerised with 1% gelatin. Gels were washed vigorously twice for 15 minutes in 2.5% Triton X-100 to remove SDS, then incubated overnight in 50mM Tris-HCl, pH7.5, 5mM CaCl2 at 37 C. Gels were then stained with Coomassie Brilliant Blue. Parallel gels were incubated in buffers containing either 5mM EDTA or 2mM 1, 10-phenanthroline to show that lysis of gelatin was due to metalloproteinase activity. -

Gelatin zymography shows some induction of MMP-9, as well as multiple bands at around the Mr of the collagenases that are induced by IL-1/OSM and repressed by the additional presence of HDACi in this system.

Example 4

Western blotting. In order to ascertain if changes at the level of steadystate mRNA are mirrored at the protein level, western blots were performed on the conditioned medium of SW1353 cells at a 24 hour time point. Samples of conditioned culture medium were precipitated with an equal volume of ice-cold 10% w/v trichloroacetic acid.

Precipitates were resuspened in loading buffer and electrophoresed under reducing conditions by SDS-PAGE in 10% polyacrylamide gels. Proteins were then transferred to PVDF membrane and probed with either rabbit anti(human MMP-1), (32), sheep anti-(human MMP-1) (33), or sheep anti-(human MMP-13) (33).

Both MMP-1 and MMP-13 proteins are potently induced by treatment with IL1 and OSM and that this is repressed by both TSA and NaBy in the same manner as the mRNA (Figure 4).

Two different anti-MMP-1 antibodies (one raised in rabbit (32) and one raised in sheep (33)) cross-react with a protein of slightly lower Mr that MMP-1 in the SW1353 conditioned medium. The identity of this protein is unknown, but its expression has been previously documented in (33), it is unaltered by the stimuli used, and it is not present in conditioned medium from primary chondrocytes. - 26

CITED REFERENCES

(1). Eyre D. Collagen of articular cartilage. Arthritis Res 2002j4:30-5.

(2). Roughley PJ. Articular cartilage and changes in arthritis: noncollagenous proteins and proteoglycans in the extracellular matrix of cartilage. Arthritis Res 2001j3:342-7.

(3). Clark IM, Parker AK. Metalloproteinases: their role in arthritis and potential as therapeutic targets. Expert Opin Ther Targets 2003j7:19-34.

(4). Porter S. Clark IM, Kevorkian L, Edwards DR. The human ADAMTS gene family. Biochemical Journal 2004;In press.

(5). Baker AH, Edwards DR, Murphy G. Metalloproteinase inhibitors:biological actions and therapeutic opportunities. J Cell Sci 2002 j115: 3719-27.

(6). Wolffe AP, Guschin D. Review: chromatin structural features and targets that regulate transcription. J Struct Biol 2000j129:102-22.

(7). Narlikar GJ, Fan HY, Kingston RE. Cooperation between complexes that regulate chromatin structure and transcription. Cell 2002j108:475-87.

(8). Sudarsanam P. Winston F. The Swi/Snf family nucleosome remodeling complexes and transcriptional control.

Trends Genet 2000j16:345-51.

(9). Berger SL. Histone modifications in transcriptional regulation. Curr Opin Genet Dev 2002j12:142-8.

(10). Clayton AL, Rose S. Barratt MJ, Mahadevan LC.

Phosphoacetylation of histone H3 on c- fos- and c-j un associated nucleosomes upon gene activation. EMBO J 2000j19:3714-26. - 27

(ll). Kouzarides T. Acetylation: a regulatory modification to rival phosphorylation? EMBO J 2000j19:1176-9.

(12). de Ruijter AJ, van Gennip AH, Caron HE, Kemp S. van Kuilenburg AB. Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem J 2003;370:737-49.

(13). Gao L, Cueto MA, Asselbergs F. Atadja P. Cloning and functional characterization of HDAC11, a novel member of the human histone deacetylase family. J Biol Chem 2002;277:25748-55.

(14). Ng HH, Bird A. Histone deacetylases: silencers for hire. Trends Biochem Sci 2000j25:121-6.

(15). Braun H. Koop R. Ertmer A, Nacht S. Suske G. Transcription factor Sp3 is regulated by acetylation.

Nucleic Acids Res 2001j29:4994-5000.

(16). Vries RG, Prudenziati M, Zwartjes C, Verlaan M, Kalkhoven E, Zantema A. A specific lysine in c-dun is required for transcriptional repression by ElA and is acetylated by p300. EMBO J 2001j20:6095-103.

(17). Gregoretti IV, Lee YM, Goodson HV. Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis. J Mol Biol 2004j338:17-31.

(18). Yoshida M, Kijima M, Akita M, Beppu T. Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J Biol Chem 1990j265:17174-9.

(19). Kruh J. Effects of sodium butyrate, a new pharmacological agent, on cells in culture. Mol Cell Biochem 1982j42:65-82. - 28

(20). Bernstein BE, Tong JK, Schreiber SL. Genomewide studies of histone deacetylase function in yeast. Proc Natl Acad Sci U S A 2000;97:13708-13.

(21). Mulholland NM, Soeth E, Smith CL. Inhibition of MMTV transcription by HDAC inhibitors occurs independent of changes in chromatin remodeling and increased histone acetylation. Oncogene 2003j22:4807 18.

(22). Nair AR, Boersma LJ, Schiltz L, Chaudhry MA, Muschel RJ, Chaudry A. Paradoxical effects of trichostatin A: inhibition of NF-Y-associated histone acetyltransferase activity, phosphorylation of hGCN5 and downregulation of cyclin A and B1 mRNA. Cancer Lett 2001;166:55-64.

(23). Pujuguet P. Radisky D, Levy D, Lacza C, Bissell MJ. Trichostatin A inhibits beta-casein expression in mammary epithelial cells. J Cell Biochem 2001 j 83:660 70.

(24). Saunders N. Dicker A, Popa C, Jones S. Dahler A. Histone deacetylase inhibitors as potential anti-skin cancer agents. Cancer Res 1999 j 59:399-404.

(25). Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 2002j1:287-99.

(26). Chung YL, Lee MY, Wang AJ, Yao LF. A therapeutic strategy uses histone deacetylase inhibitors to modulate the expression of genes involved in the pathogenesis of rheumatoid arthritis. Mol Ther 2003j8:707-17.

(27). Nuttall RK, Pennington CJ, Taplin J. Wheal A, Yong VW, Forsyth PA, et al. Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. Mol Cancer Res 2003j1:333-45.

(28). Porter S. Scott SD, Sassoon EM, Williams MR, Jones JL, Girling AC, et al. Dysregulated expression of adamalysin-thrombospondin genes in human breast carcinoma. Clin Cancer Res 2004;10:2429-40.

(29). Robbins JR, Thomas B. Tan L, Choy B. Arbiser JL, Berenbaum F. et al. Immortalized human adult articular chondrocytes maintain cartilagespecific phenotype and responses to interleukin-lbeta. Arthritis Rheum 2000j43:2189-201.

(30). Billington CJ. Cartilage proteoglycan release assay. Methods Mol Biol 2001j151:451-6.

(31). Koshy PJ, Rowan AD, Life PF, Cawston TE. 96-Well plate assays for measuring collagenase activity using (3)H-acetylated collagen. Anal Biochem 1999j275:202-7.

(32). Clark IM, Wright JK, Cawston TE, Hazleman BL.

Polyclonal antibodies against human fibroblast collagenase and the design of an enzyme-linked immunosorbent assay to measure TIMP-collagenase complex. Matrix 1992j12:108-15.

(33). Cowell S. Knauper V, Stewart ML, D'Ortho MP, Stanton H. Hembry RM, et al. Induction of matrix metalloproteinase activation cascades based on membrane-type 1 matrix metalloproteinase: associated activation of gelatinase A, gelatinase B and collagenase 3. Biochem J 1998j331 ( Pt 2) :453-8.

(34). Cawston TE, Curry VA, Summers CA, Clark IM, Riley GP, Life PF, et al. The role of oncostatin M in animal and human connective tissue collagen turnover and its localization within the rheumatoid joint. Arthritis Rheum 1998j41:1760-71. -

(35). Rowan AD, Hui W. Cawston TE, Richards CD.

Adenoviral gene transfer of interleukin-1 in combination with oncostatin M induces significant joint damage in a murine model. Am J Pathol 2003; 162:1975-84.

(36). Furumai R. Komatsu Y. Nishino N. Khochbin S. Yoshida M, Horinouchi S. Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin. Proc Natl Acad Sci U S A 2001;98:87-92.

(37). Koshy PJ, Lundy CJ, Rowan AD, Porter S. Edwards DR, Hogan A, et al. The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: a time course study using real-time quantitative reverse transcription-polymerase chain reaction. Arthritis Rheum 2002;46:961-7.

(38). Ailenberg M, Silverman M. Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy. Biochem Biophys Res Commun 2003;302:181-5.

(39). Ailenberg M, Silverman M. Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression. Biochem Biophys Res Commun 2002;298:110-5.

(40). Chen LF, Fischle W. Verdin E, Green WC. Duration of NFKB action regulated by reversible acetylation.

Science 2001;293:1653-7.

(41). AshEurner BP, Westerheide SD, Baldwin AS Jr. The p65 (RelA) subunit of NFKB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to - 31 negatively regulate gene expression. Mol Cell Biol 2001;21:7065-77.

(42). Nusinzon I, Horvath CM. Interferon-stimulated transcription and innate antiviral immunity require deacetylase activity and histone deacetylase 1. Proc Natl Acad Sci U S A 2003j100:14742-7.

(43). Klampfer L, Huang J. Swaby L-A, Augenlicht L. Requirement of histone deacetylase activity for signaling by STAT1. J Biol Chem 2004; In Press.

(44). Catterall JB, Carrere S. Koshy PJT, Degnan BA, Shingleton WD, Brinckerhoff CE, et al. Synergistic induction of matrix metalloproteinase 1 by interleukin la and oncostatin M in human chondrocytes involves signal transducer and activator of transcription and activator protein 1 transcription factors via a novel mechanism. Arthritis Rheum 2001j44:2296310.

(45). Dai, Y., Guo, Y., Curtin, ML., Li, J., Pease, LJ., Guo, J., Marcotte, PA., Glaser, KB., Davidsen, SK., Michaelides, MR. A novel series of histone deacetylase inhibitors incorporating hetero aromatic ring systems as connection units. Bioorganic & Med. Chem Let 2003; 13: 3817-3820.

(46) Kim, DK., Lee, JY., Kim, JS., Ryo, JH. Et al. Synthesis and biological evaluation of 3-(4 substituted-phenylO-N-hydroxy-2propenamides, a new class of histone deacetylase inhibitors. J/ Med. Chem. 2003; 46:5745-5751.

(47). Vasudevan, A., Ji, Z., Frey, RR., Wada, CK et al. heterocyclic ketones as inhibitors of histone deacetylase. Bioorganic & Med. Chem Let 2003; 13:3909 3913.

(48). Wu, TYH., Hassig, C., Wu, Y., Ding, S., Schultz, PG. Design, synthesis and activity of histone deacetylase inhibitors with a N-formyl hydroxylamine head group. Bioorganic & Med. Chem Let 2004; 14: 449 453.

(49). Suzuki, T., Kouketsu, A., Matsuura, A. et al. Thiol-based SAHA analogues as potent histone deacetylase inhibitors. Bioorganic & Med. Chem Let 2004; 14: 3313-3317.

(50). Suzuki, T., nagaono, Y., Mat suura, A. et al. Novel histone deacetylase inhibitors: design, synthesis, enzyme inhibition and binding mode study of SAHA-based non-hydroxamates. Bioorganic & Med. Chem Let 2003; 13: 4321-4326.

(51). Lu, Q., yang, YT., Chen, CS., Davis, M., Byrd, J., Etherton, MR., Umar, A., Chen, CS. Zn2+ chelating motif-tethered short-chain fatty acids as novel class of histone deacetylase inhibitors. J. Med. Chem. 2004; 47:467-474.

(52). Nishino, N., Jose, B., Okamura, S., Ebisusaki, S., Kato, T., Sumida, Y., Yoshida, M. Cyclic tetrapeptides bearing a sulfhydryl group potently inhibit histone deacetylase. Organic letters. 2003; 26:5079-5082. - 33

(53). Nishino, N., Yoshikawa, D., Wtanabe, LA., Kato, T., Jose, B., Komatsu, Y., Sumida, Y., Yoshida, M. Synthesis and histone deacetylase inhibitory activity of cyclic tetrapeptides containing a retrohydroxamate as zinc ligand. Bioorganic & Med. Chem Let 2004; 14: 2427-2431.

(54). Mai, A., Massa, S., Cerbara, I., Valente, S., Ragno, R., Bottoni, P. , Scatena, R., Loidl, P., Brosch, G. 3-(4-Aroyl-l-methyl-lH-2-pyrrolyl)-Nhydroxy-2 propenamides as new class of synthetic histone deacetylase inhibitors. Effect of pyrrole-C2 and/or C4 substitutions on biological activity. J. Med. Chem. 2004; 47: 1098-1109.

(55). Mai, A., Massa, S., Cerbara, I., Valente, S., Jesacher, F., Bottoni, P., Scatena, R., Loidl, P., Brosch, G. 3-(4-Aroyl-l-methyl-lH-2-pyrrolyl) -N hydroxy-2-propenamides as new class of synthetic histone deacetylase inhibitors. Discovery of novel lead compiounds through structure-based drug design. J. Med. Chem. 2004; 47: 1351-1359 - 34

Claims (17)

1. Claims: 1. The use of a histone deacetylase inhibitor in the manufacture

   of a medicament for the treatment of a disease associated with the destruction of connective tissue mediated by metalloproteinases.
2. 2. The use according to in claim 1 wherein the connective tissue is cartilage.
3. 3. The use according to claim 1 or claim 2 wherein the disease is a joint disease wherein cartilage destruction is prominent.
4. 4. The use according to claim 3 wherein the disease is a non-inflammatory joint disease.
5. 5. The use according to any of the preceding claims wherein the disease to be treated is arthritis.
6. 6. The use according to claim 5 wherein the arthritis is selected from the group of osteoarthritis, reactive arthritis, gout/pseudogout arthritis, juvenile idiopathic arthritis or psoriatic arthritis.
7. 7. The use according to claim 1 wherein the disease to be treated is selected from the group of multiple sclerosis, cardiovascular disease or ocular disease.
8. 8. The use according to claim 1 for the treatment of tumour invasion and metastasis.
9. 9. The use according to any of the preceding claims wherein HDACi is a compound having the formula 1 or a pharmaceutically acceptable salt thereof. o :9 1

   H
10. 10. The use according to claim 9 wherein the compound is trichostatin, suberoyl anilide hydroxamic acid, M- carboxycinnamic acid bishydroxamide, scriptaid, pyroxamide or oxamflatin.
11. ll. The use according to any of claims 1 to 8 wherein HDACi is a compound having the formula 2 or a pharmaceutically acceptable salt thereof. o RCon

    OH
12. 12. The use according to claim 11 wherein the compound is butyrate, phenylbutyrate or valporoic acid.
13. 13. The use according to claim 12 wherein the compound is sodium butyrate. 36
14. 14. The use according to any of claims 1 to 8 wherein HDACi is a compound having the formula 3.

    R O c-c/

    0=t e' / a' - /=0 / for A_ c' ._c_e AN /
15. 15. The use according to claim 14 wherein the compound is trapoxin, HC- toxin, chlamydocin, depudesin, apicidine or depsipeptide.
16. 16. The use according to any of claims 1 to 8 wherein HDACi is a compound having the formula 4. o

    R-ERIC-No

    HNH
17. 17. The use according to claim 16 wherein the compound is N acetyldinaldine or MS-275.

    571683, BF; BF