A novel peptide

Field of the Invention

The present invention relates to novel peptides inhibiting tumour growth and reducing cancer cell motility, growth and spread. In particular, the present invention relates to novel peptides, which are useful as inhibitors of MTl-MMP activity and/or are capable of targeting to a cell or cells having enhanced MTl-MMP activity. Further, the present invention concerns pharmaceutical and diagnostic compositions comprising these peptides and the use of the peptides for pharmaceutical and diagnostic imaging preparations. Still further, the present invention relates to the use of these peptides as medicaments and diagnostic imaging agents, for the preparation of medicaments and diagnostic imaging agents and use in biochemical isolation and purification procedures as well as in cellular and molecular biology experimental research.

Description of Related Art

In cancerous tissue the cells have a higher proliferation rate than normally making the cell cycle a common target for conventional anticancer treatment by drugs and radiation therapies. Though cancer cells are more susceptible to these therapies many unwanted side effects are obvious. For decades researchers and physicians have been aware that cancer progression is not just about cell division but solid malignant tumours are capable of degrading surrounding tissues and forming new vascular structures to supply their spatial and nutritional needs (Folkman J et all 971, Poole AR 1970). In this invasion process also proteolytic enzymes play an important role allowing non-coherent cancer cells to colonize the surrounding tissues (Christofori 2006). This remodelling and destruction of the tumour surrounding stroma has been of interest for anticancer drug development.

Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases known to cleave almost all extracellular and basement membrane proteins (Visse and Nagase 2003). Enhanced production and activation of several MMPs has been associated with different cancer types (Vihinen and Kahari 2002). Synthetic MMP inhibitors have been developed during the last 20 years, few of which have been in clinical trials for treatment of cancer (Whittaker et all 999, Peterson 2004). However, most synthetic MMP inhibitors

have been unsuccessful due to lack of selectivity and unwanted side-effects. We have previously developed small molecular weight peptides, which can be used to selectively inhibit proteolytic activity of gelatinases MMP-2 and -9 (Koivunen et al. 1999 and EP 1 071 704). We have also showed that these antigelatino lytic peptides (CTTl and CTT2) have a potential to inhibit gelatinase-dependent cancer cell migration and invasion in vitro as well as tumour growth in vivo (Heikkila et al. 2006). However, CTTl and CTT2 do not inhibit membrane type-1 matrix metalloproteinase (MTl-MMP, MMP- 14) or other MMPs (Koivunen et al. 1999).

MMP inhibitors have been described in several patent publications: US 7,060,248 discloses MMP inhibitors, including some specific inhibitors intended for MMP- 14 inhibition. The publication describes diagnostic agents comprising a diagnostic metal and a compound, wherein the compound comprises: 1-10 targeting moieties; a chelator; and 0-1 linking groups between the targeting moiety and chelator; wherein the targeting moiety is a matrix metalloproteinase inhibitor; and wherein the chelator is capable of conjugating to the diagnostic metal. The publication also provides novel compositions of the compounds of the invention, kits, and their uses in diagnosis of diseases associated with MMPs. The patent publication US 6,989,139 describes novel compounds comprising 1-10 targeting moieties; a chelator; and 0-1 linking groups between the targeting moiety and chelator; wherein the targeting moiety is a matrix metalloproteinase inhibitor; and wherein the chelator is capable of conjugating to a cytotoxic radioisotope. The disclosure provides novel compositions of the compounds of the invention, kits and their uses in treatment of diseases associated with MMPs. US 6,989,139 contains a specific reference to selective inhibition of MMP- 14: Preferred pharmaceuticals of the publication are comprised of inhibitors and compounds, which exhibit selectivity for MMP-I, MMP-2, MMP-3, MMP- 9, or MMP-14 alone or in combination over the other MMPs. In US 6,946,264 a novel metalloproteinase inhibitor, analogues thereof, polynucleotides encoding the same, and methods of production, are disclosed. Pharmaceutical compositions and methods of treating disorders caused by excessive amounts of metalloproteinase are also disclosed. US 7,067,670 provides a sulfamato hydroxamic acid compound that inhibits matrix metalloproteinase activity and a process for preparing the same, intermediate compounds, and a treatment process that comprises administering a sulfamato hydroxamic acid compound to a host having a condition associated with pathological matrix metalloproteinase activity. US 6,924,276 provides dicarboxylic acid-substituted heteroaryl

derivatives of a specific formula or a pharmaceutically acceptable salt thereof. The compounds are inhibitors of matrix metalloproteinase enzymes, including MMP-13. This publication also provides pharmaceutical compositions and methods of treating diseases mediated by MMP-13, including arthritis, asthma, heart disease, atherosclerosis, and osteoporosis, or a pharmaceutically acceptable salt thereof. Only US Patents Numbers 7,060,248 and 6,989,139 address MMP-14 inhibition. MTl-MMP is also classified as MMP-14.

Although several MMP inhibitors are available, there are only a few available for MTl- MMP. These are generally derived from a recombinant production process from human genes, such as TIMP-4. These inhibitors are manufactured versions of natural peptides.

However, these inhibitors inhibit also other MMPs and are thus not specific to MTl-MMP.

This lack of specificity can cause unwanted side effects. There is currently a relative lack of suitable MTl-MMP inhibitors that could be developed into clinical treatments or diagnosis for various cancers.

Summary

It is an aim of the present invention to eliminate at least some of the problems of the prior art. In particular, it is an aim of this invention to develop an inhibitor, which has anticancer potential.

It is an aim of the present invention to provide novel inhibitors against matrix metalloproteinases, in particular against membrane type-1 matrix metalloproteinase called also MTl-MMP or MMP-14.

More specifically, it is an aim of the present invention to provide novel peptides for inhibiting selectively MT 1 -MMP .

Furthermore, it is an aim of the present invention to provide novel peptides for targeting to a cell or cells having enhanced (increased) MTl-MMP activity compared to normal cells.

These and other aims, together with the advantages thereof over known peptides and processes, are achieved by the present invention as hereinafter described and claimed. The invention is based on the finding that specific peptides inhibit MTl-MMP activity. The membrane type-1 matrix metalloproteinase has been recognized as one of the most

important MMP in terms of both development and tumour invasion (Etoh et al. 2000, Ohashi et al. 2000, Seiki 2003). There are very few specific inhibitors against this enzyme. The present invention provides novel and selective MTl-MMP inhibitor-peptides exhibiting also anticancer potential in vitro and in vivo. Thus, according to the invention, novel isolated peptides are provided comprising the amino acid sequence of Formula I

C Xi X ₂ X ₃ X ₄ X ₅ X ₆ C (I) (SEQ ID NO: 1)

wherein C is cysteine, Xi is Phenylalanine, X ₂ is Serine, X ₃ is Isoleusine, X ₄ is Alanine, X ₅ is Histidine, X ₆ is Glutamic acid, or a conservative analogue of any or each of the amino acids.

Furthermore, novel isolated peptides are provided comprising the amino acid sequence of Formula II

X ₇ X ₈ C Xi X ₂ X ₃ X ₄ X ₅ X ₆ C X ₉ Xio (H) (SEQ ID NO:2)

wherein C is cysteine, Xi is Phenylalanine, X ₂ is Serine, X ₃ is Isoleusine, X ₄ is Alanine, X ₅ is Histidine, X ₆ is Glutamic acid, X ₇ is Glycine, Xg is Alanine, X ₉ is Glycine, X _1O is Alanine, or a conservative analogue of any or each of the amino acids.

More specifically, a composition comprising a peptide according to the present invention and a pharmaceutically acceptable carrier and/or labelling substance is mainly characterized by what is stated in claim 1.

A composition comprising a peptide according to the present invention and a diagnostically acceptable carrier and/or labelling substance is mainly characterized by what is stated in claim 2.

A peptide exhibiting the selective inhibition of MTl-MMP activity and/or exhibiting the capability of targeting to a cell or cells having enhanced (increased) MTl-MMP activity compared to normal cells in mammal is mainly characterized by what is stated in claim 3.

An MTl-MMP inhibitor comprising the peptide is mainly characterized by what is stated in claim 8.

Use and methods of using the peptides are characterized by what is stated in the characterizing parts of claims 9, 10, 12, 15 and 18

The present invention includes the use of the peptides and compositions of the present invention for inhibiting invasion and migration of cancer cells from different origin. The peptides and compositions of the present invention are capable of reducing tumour growth in various tissues in vitro and in vivo.

The present invention includes also the use of the peptides and compositions of the present invention for targeting to a cell or cells having enhanced MTl-MMP activity compared to normal cells. By the term "enhanced" activity is in connection of this invention meant "increased" activity.

Furthermore, the present invention relates to pharmaceutical and/or diagnostic compositions comprising an amount effective to inhibit the activity/function of MTl-MMP or an amount effective to target to a cell or cells having enhanced MTl-MMP activity compared to normal cells.

The present invention relates also to the use of the peptides and compositions of the present invention for the manufacture of pharmaceutical compositions for the treatment or diagnosis of any condition causing enhanced MTl-MMP activity in a cell or cells compared to normal cells.

The present invention relates also to peptides for inhibiting MMTl-MMP activity in a cell or cells in mammals and/or for targeting to a cell or cells having enhanced MTl-MMP activity in mammals, and for finding the presence and/or location of a cell or cells exhibiting enhanced MMTl-MMP activity in mammals.

Peptides for use in these methods are characterized by what is stated in the characterizing parts of claims 6 and 7, 14 and 17.

Next, the invention will be described in more detail with the aid of a detailed description and by making reference to the attached drawings.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Effects of the novel MTl-MMP peptide inhibitor (MTl-I) on cancer cell motility was studied using Matrigel invasion and Transwell migration assays. The MTl-I (at a concentration of lOOμM) inhibited HTl 080 fibrosarcoma cell migration on fibronectin (A), C8161 melanoma cell migration on fibronectin (B) and laminin-5 (C). The inhibitory effect of MTl-I was also tested (at concentrations of 20, 100, 250 μM) on C8161 melanoma cell Matrigel invasion (D). The MTl-I peptide (at concentrations of 20, 50 and 100 μM) also inhibited HSC-3 tongue carcinoma cell Matrigel invasion (E).

Figure 2. MMP cleavage assay

MTl-I peptide inhibits proteolytic activity of MTl-MMP. MTl-I peptide inhibited MTl- MMP mediated proteolysis of the Laminin-5 (Ln-5) γ2-chain at 100 μM (Fig.2A lane 1 : Laminin-5 γ2-chain alone, lane 2: Laminin-5 γ2-chain and MTl-MMP, lane 3: Laminin-5 γ2-chain and MTl-MMP and MTl-I peptide). MTl-I peptide inhibited MTl-MMP mediated proteolysis of β-casein at 50 μM (Fig.2B. lanel : β-casein alone; lane 2: β-casein + MTl-MMP; lanes 3-6: β-casein + MTl-MMP + MTl-I at concentrations of 10 μM, 50 μM, 100 μM and 500 μM, respectively; lane 7: β-casein + MTl-MMP + Control peptide 500 μM; lane 8. β-casein + MTl-MMP + MMPI 10 μM). The non-relevant Control peptide did not have any effect on MTl-MMP activity (Fig.2B. lane 7.).

The inhibition of other MMP mediated proteolysis by MTl-I peptide was also studied

(Fig.2C. lane 1 : MW standard; lane 2: β-casein alone; lane 3: β-casein and MMP-2; lanes 4. - 6: β-casein + MMP-2 + MMP-2-inhibitor peptide at concentrations of 50 μM, 100 μM and 250 μM, respectively; lanes 7. - 9: β-casein + MMP-2 + MTl-I at concentrations of 50 μM, 100 μM and 250 μM, respectively; lane 10: β-casein + MMP-2 + MMPI 10 μM). MTl-I did not inhibit MMP-2 mediated proteolysis of β-casein. In comparison, the MMP2 inhibiting peptide at concentration of 250 μM inhibited β-casein proteolysis by MMP-2 as expected (Fig.2C. lane 6).

Figure 3. Cell viability was unaffected by MTl-I peptide. MTl-I peptide at 500 μM did not affect C8161 melanoma cell adhesion (A) as determined by using the MTT reagent or cell proliferation as determined by BrdU incorporation ELISA assay (B). Fig. A: Bar 1. no

inhibitor; bar 2. 100 μM antigelatinolytic peptide ; bar 3. lOOμM CTRL-peptide; bar 4. 100 μM MTI-I. Fig. B: Bar 1. no inhibitor; bar 2. 100 μM antigelatinolytic peptide; bar 3. 250 μM CTRL-peptide; bar 4. 20 μM MTl-I; bar 5. 100 μM MTl-I; bar 6. 250 μM MTl-I.

Figure 4. MTl-I peptide reduced migration of both MTl-MMP transfected (Fig. 4A. lane 1 : no inhibitor; lane 2: 100 μM control peptide; lane 3: 100 μM MTl-I peptide) as well as vector (Fig.4B. lane 1 : no inhibitor; lane 2: control peptide at 100 μM; lane 3: MTl-I peptide at 100 μM) transfected SCC25 oral carcinoma cells on Laminin-5 coated Transwell-migration chambers.

Figure 5. Dose-dependent inhibition of MTl-MMP transfected SCC25 oral carcinoma cell migration over Laminin-5 coated Transwell-migration chambers by MTl-I peptide (Fig.5. lane 1 : no coating; lane 2: no inhibitor; lane 3: control peptide at 100 μM; lane 4: MTl-I peptide at 100 μM; lane 5: MTl-I peptide at 500 μM).

Figure 6. Mean tumour volumes on day 10. HSC-3 tumours were inoculated on the backs of nude mice and the mice then treated with MTl-I peptide or its irrelevant controls. Tumours in MTl-I peptide treated mice grew slower as compared to the controls. Mean tumour volumes (mm ³ ) on day 10 when all mice were still alive. MTl-I peptide treated mice having a tumour volume /4-1/3 of the controls. (Fig.6. Lane 1 : 0.9% NaCl; lane 2: C- control peptide; lane 3: Scrambled control peptide; lane 4: MTl-I-peptide).

Figure 7. MTl-I peptide treated HSC-3 tumour bearing mice survived longer. Kaplan- Meier survival function of mice treated with MTl-I peptide (dash line) or controls (solid line) where the MTl-I peptide group was surviving significantly longer as compared to the saline, SCR-peptide and C-peptide controls (LogRank p=0.0013).

Figure 8. MTl-I peptide inhibits MTl-MMP proteolytic activity in QuantiZyme™ -assay whereas scrambled version (SCR) have no major inhibitory effect on MTl-MMP activity. MTl-I-peptide reduced the MTl-MMP enzyme activity at 43μM, 129μM and 430μM to 48%, 41% and 36% from the original (SD: ±2,7%, 3,6% and 4,5%). NNGH represents kit positive control inhibitor (Fig. 8. lane 1 : no inhibitor; lane 2: NNGH; lane 3: SCR at 43 μM; lane 4: SCR at 86 μM; lane 5: SCR at 129 μM; lane 6: MTl-I peptide at 43 μM; lane 7: MTl-I peptide at 86 μM; lane 8: MTl-I peptide at 129 μM).

Figure 9. MT 1 -MMP binding to MT 1 -I peptide variants .

Binding of MTl-MMP to MTl-I peptide and its' variants was analyzed using pepspot membrane (Haartman institute, PeproLab). In the membrane effect of each amino acid was analyzed by replacement or alanine scan mutagenesis. Recombinant MTl-MMP containing a his-tag sequence was incubated on top of the membrane. After elution of unbound MTl- MMP the formed peptide-MMP -complexes were detected using antihis-tag-probes

(Santacruz) and ECL protocol (Amersham Pharmacia Biotech). The most dramatic effect on MMP-binding was detected when both of the cysteines were replaced. Also changing any amino acid between the cysteines reduced MMP-binding; the serine being most effective. Exocyclin amino acids had less effect on MMP-binding.

Figure 10. MTl-I peptide inhibits MTl-MMP mediated breakdown of β-casein. Lane 1 denotes β -casein. Lane 2 denotes β-casein + MTl-I peptide 250 μM + MTl-MMP. Lanes 3 - 13 denote β-casein + MTl-I peptide 250 μM + MMP-2, -3, -7, -8, -9, -10, -11, -13, -15, -17 and -20.

Detailed description of the invention

Definitions

"Inhibition" means here an inhibitory action on the MTl-MMP activity and/or function. The inhibition of the peptides can be compared by MTl-MMP mediated cleavage of natural or synthetic substrates. The inhibitory activity of a peptide on the MTl-MMP activity and/or function can be studied for example by Quantizyme™ fluorescent assay measuring its effect to the proteolytic activity of MMP- 14. Anti-MMP effect of the MTl-I -peptide can be also studied by incubating peptide, recombinant enzymes and laminin or β- casein as a substrate. The capability of a peptide to inhibit cancer cell migration and invasion can be studied in vitro by using Transwell- and Matrigel assays as antimigratory and -invasive potential. The inhibition of the peptides can also be studied by using cultured cells expressing the cell membrane -bound MTl-MMP, which is the form in which MTl-MMP is mostly found in vivo.

The term inhibition includes also inhibition and/or down-regulation reducing the activities, activations, functions and/or expression of MTl-MMP.

A "selective inhibition" comprises an at least 20% inhibition, preferably at least 30% inhibition compared to a control and "significant inhibition" stands for an at least 40% inhibition compared to a control without inhibitor.

In particular, by "selective inhibition" is here meant selective inhibition of MTl-MMP activity/function and not other MMPs.

According to one preferred embodiment of the invention the peptide of the invention is capable of targeting to a cell or cells having enhanced MTl-MMP activity compared to normal cells.

The present invention includes pharmaceutical and/or diagnostic compositions comprising an amount effective to inhibit the activity/function of MTl-MMP and/or an amount effective to target to a cell or cells having enhanced MTl-MMP activity compared to normal cells.

By "mediated through" the present invention refers to any biological events that result from the proteolytic activity of MTl-MMP, such as, for example, result from MTl-MMP mediated growth or that result from an aberration in the expression or activity of MTl- MMP.

The present invention relates also to the use of the peptides and compositions of the present invention for the manufacture of pharmaceutical compositions for the treatment and/or diagnosis of any condition causing enhanced MTl-MMP activity in a cell or cells compared to normal cells. Such conditions include but are not limited to cancer and metastasis progression in various tissues/cancer including breast, large intestine, melanoma, oral cancer and inflammatory diseases of both chronic and acute character such as wounds, burns, lesions, fractions, ulcers.

The present invention relates also to a process for the preparation of the peptides and compositions of the present invention. The process comprises standard solid-phase Merrifield peptide synthesis.

The present invention relates also to a method for isolating and purifying matrix metalloproteinases, in particular MTl-MMP with the aid of the peptides and compositions of the present invention.

According to the present disclosure conditions causing enhanced MTl-MMP activity in a cell or cells compared to normal cells can be prevented or treated with the peptides and compositions of the present invention. The peptides and compositions of the present invention can be used in combination with other substances, such as drugs or medicaments, which are normally used to treat the conditions. Such substances include for example molecules or chemicals homing or carrying to the sites of tumours or metastases, for example integrin-binding peptides (Arap et al. 1998).

The amount of the peptides of the present invention to be used in pharmaceutical or diagnostic compositions depends on the disease to be treated, the patient, the conditions of the patient and the administration route.

The present invention includes a method for therapeutic or prophylactic treatment of conditions causing enhanced MTl-MMP activity in a cell or cells compared to normal cells in mammals by administering an amount effective for inhibiting or blocking the actions, activation and formation of MTl-MMP.

The term "amino acid" means here natural amino acids (e.g., L-amino acids), modified and unnatural amino acids (e.g. β-alanine), as well as amino acids which are known to occur biologically in free or combined form but usually do not occur in proteins. Included within this term are modified and unusual amino acids, such as those disclosed in, for example, Roberts and Vellaccio, 1983, the teaching of which is hereby incorporated by reference. Genetically coded, "natural" amino acids occurring in proteins include, but are not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tyrosine, tryptophan, proline, and valine. Natural non-protein amino acids include, but are not limited to arginosuccinic acid, citrulline, cysteine sulfuric acid, 3,4-dihydroxyphenyl- alanine, homocysteine, homoserine, ornithine, 3-monoiodotyrosine, 3,5-diiodotryosine, 3,5,5'-triiodothyronine, and 3,3',5,5'-tetraiodothyronine. Modified or unusual amino acids which can be used to practice the invention include, but are not limited to, D-amino acids, hydroxy Iy sine, 4-hydroxyproline, an N-Cbz-protected amino acid, 2,4-diaminobutyric acid, homoarginine, norleucine, N-methylaminobutyric acid, naphthylalanine, phenyl-

glycine, 9-phenylproline, tert-leucine, 4-aminocyclohexylalanine, N-methyl-norleucine, 3,4-dehydroproline, N,N-dimethyl-aminoglycine, N-methylaminoglycine, 4-amino- piperidine-4-carboxylic acid, 6-amino-caproic acid, trans-4-(aminomethyl)-cyclohexane- carboxylic acid, 2-, 3-, and 4-(amino-methyl)-benzoic acid, 1-aminocyclopentane- carboxylic acid, 1-aminocyclopropane-carboxylic acid, and 2-benzyl-5-aminopentanoic acid.

Generally, "peptide" stands for a strand of several amino acids bonded together by amide bonds to form a peptide backbone. The term "peptide", as used herein, includes compounds containing both peptide and non-peptide components, such as pseudopeptide or other non- amino acid components. Such a compound containing both peptide and non-peptide components may also be referred to as a "peptide analogue".

The present invention includes also "peptidyl analogues" or "conservative substitution- analogues", which are chemical derivatives of the peptides based on the modification of the peptides by various chemical reactions, such as cycloadditions, condensation reactions and nucleophilic additions.

The present invention includes also "peptidomimetic compounds", which are compounds, which resemble the original peptides mentioned above. They are generally built up of different chemical building blocks than the amino acids, which form the original peptides. For example, non-peptidyl compounds like benzolactam or piperazine based analogues based on the primary sequence of the original peptides can be used (Houghten et al. 1999 and Nargund et al. 1998). The resemblance between the peptidomimetic compounds and the original peptides is based on structural and functional similarities. Thus, the peptidomimetic compounds mimic the bioactive conformation of the original peptides and, for the purpose of the present application, their capability of inhibiting and/or targeting to MTl-MMP is similar to that of the peptide they resemble. The peptidomimetic compounds can be made up of amino acids, such as D-amino acids, which do not appear in the original peptides, or they can be made from other compounds forming amide bonds or ester bonds.

The peptides, polypeptides, non-peptides and peptidomimetics optionally bearing a linking group, or a fragment of the linking group, can be synthesized using standard synthetic methods known to those skilled in the art. Generally, peptides, polypeptides, and

peptidomimetics are elongated by deprotecting the alpha-amine of the C-terminal residue and coupling the next suitably protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained. This coupling can be performed with the constituent amino acids in a stepwise fashion, or condensation of fragments (two to several amino acids), or combination of both processes, or by solid phase peptide synthesis according to the method originally described by Merrifield (Merrifield 1963), the disclosure of which is hereby incorporated by reference. The peptides, polypeptides and peptidomimetics may also be synthesized using automated synthesizing equipment. In addition, procedures for peptide, polypeptide and peptidomimetic synthesis are described in Gross, Meienhofer, Udenfriend (1980-1987), Bodanszky et al. (1984) and Stewart and Young (1984), and the disclosures of which are hereby incorporated by reference.

The present peptides preferably comprise molecules with a molecular weight lower than 10 kDa, i.e. containing about 50 amino acids or less. Peptides can be designated as "small peptides" when they consist of about 6 - 30 amino acid units. The present peptides may be linear or they may comprise one or more cross-links formed by disulfide bonding between cysteine units. If the peptides contain several pairs of cysteines, there can be a multiple number of such cross-links. In addition to disulfide bonds, there can be other cross-links within the peptides as well.

The terms "conservative substitution" and "conservative substitutes" as used herein denote the replacement of an amino acid residue by another, biologically similar residue with respect to hydrophobicity, hydrophilicity, cationic charge, anionic charge, shape, polarity and the like. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. Neutral hydrophilic amino acids, which can be substituted for one another, include asparagine, glutamine, serine and threonine. The term "conservative substitution" also includes the use of a substituted or modified amino acid in place of an unsubstituted parent amino acid provided that the substituted peptide reacts with MTl-MMP. By "substituted" or

"modified" the present invention includes those amino acids that have been altered or modified from naturally occurring amino acids.

As such, it should be understood that in the context of the present invention, a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Examples of conservative substitutions are set out in the Table 1 below:

Table 1. Conservative Substitutions I

Alternatively, conservative amino acids can be grouped as described in Lehninger, 1975, as set out in Table 2, immediately below.

Table 2. Conservative Substitutions II

As still another alternative, exemplary conservative substitutions are set out in Table 3, immediately below.

Table 3. Conservative Substitutions III

According to a preferred embodiment of the invention, the novel peptide has at least 8 amino acids bonded together to form a peptide backbone and including the amino acids

Cysteine, Phenylalanine, Serine, Isoleusine, Alanine, Histidine, and Glutamic acid, or a conservative analogue of any or each of the amino acids.

In addition, the peptide may comprise 1 to 10, preferably 2 to 8 amino acids at one or both ends of the peptide. According to one preferred embodiment of the invention the amino acids may comprise Glycine and Alanine or a conservative analogue of these amino acids.

Particularly preferred peptides comprise the amino acid sequence CFSIAHEC (SEQ ID NO:3). Furthermore, preferred peptides comprise the amino acid sequence GACFSIAHECGA (SEQ ID NO :4).

Inhibition can further be strengthened by incorporating spacer residues around the motifs.

If desired, the peptides of the invention can be modified, for instance, by glycosylation, amidation, carboxylation, pegylation, or phosphorylation, or by the creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention. The peptides also can be modified to create peptide derivatives by forming covalent or non-covalent complexes with other moieties. Covalently bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N- or C-terminus.

The binding and inhibiting peptides of the invention will be used as therapeutic compositions either alone or in combination with other therapeutic agents.

The present invention also relates to a pharmaceutical composition comprising an amount of the novel peptides. The pharmaceutical composition comprising the peptides according to the invention may be used systemically, locally and/or topically, and may be administered e.g. parenterally, intravenously, subcutaneously, intramuscularly, intranasally, by pulmonary aerosol or in depot form. The compositions may also include all potential combinations of the peptides with labelling reagents, imaging reagents, drugs and other chemicals/-molecules. For example pharmaceutical compositions suitable for intravenous infusion or injection comprise the active component in a concentration of, generally, about 0.1 to 500 g/1, preferably about 1 to 250 g/1. It is preferred to have somewhat higher concentrations (e.g. about 20 to 200 g/1), to allow for administration

without causing excessive volume load to the patients. The pH of the solution product is in the range of about 5 to about 8, preferably close to physiological pH. The osmolality of the solution can be adjusted using sodium chloride and/or sugars, polyols or amino acids or similar components. The compositions can further contain pharmaceutically acceptable stabilizers and excipients, such as albumin, sugars and various polyols. The amounts of these components can vary broadly within a range of about 0 to 50 wt-% of the active component. The preparation may be lyophilized and reconstituted before administration or it may be stored as a solution ready for administration.

Exemples of cytotoxic agents are for example radioisotopes, any known anti-neoplastic drug or pro-drug that can be administered and converted to a drug in vivo; and the like. The peptides of the present invention will be used for the therapeutic intervention of the disorders discussed herein above as well as any other disorders that are mediated through the proteolytic activity of MTl-MMP. These therapies will be particularly useful as anti- metastatic and/or anti-angiogenic treatments, however it is contemplated that the instant invention is not limited to these beneficial effects. Formulations would be selected based on the route of administration and purpose including, but not limited to, classic pharmaceutical preparations and for example liposomal formulations.

In general, per oral dosage forms for the therapeutic delivery of peptides is ineffective because in order for such a formulation to the efficacious, the peptide must be protected from the enzymatic environment of the gastrointestinal tract. Additionally, the peptide must be formulated such that it is readily absorbed by the intestinal epithelial cell barrier in sufficient concentrations to effect a therapeutic outcome. The peptides of the present invention may be formulated with uptake- or absorption-enhancers to increase their efficacy. Such enhancers include for example, salicylate, glycocholate/linoleate, glycholate, aprotinin, bacitracin, SDS caprate and the like.

The amounts of peptides in a given dosage will vary according to the size of the individual to whom the therapy is being administered as well as the characteristics of the disorder being treated. In exemplary treatments, it may be necessary to administer about 50 mg/day, 75 mg/day, 100 mg/day, 150 mg/day, 200 mg/day or 250 mg/day. These concentrations may be administered as a single dosage form or as multiple doses.

An additional use for the peptides of the present invention is in tissue imaging to determine whether a cancer tissue is expressing MTl-MMP. The use of such diagnostic imaging is particularly suitable in obtaining an image of, for example, a tumour mass or the neovascularizarion near a tumour mass. The peptides are potentially useful as radioactively, lanthanide or magnetic particle labeled derivates for tumour imaging with magnetic resonance imaging (MRI) or with single photon emission computed tomography/positron emission tomography (SPECT/PET) or they may be conjugated with cytotoxic drugs and/or antibodies to be used for targeted killing of the tumor cells.

The peptides of the present invention may be coupled either covalently or noncovalently to a suitable supramagnetic, paramagnetic, electron-dense, echogenic or radioactive agent to produce a targeted imaging agent. In such embodiments, the peptide imaging agent will localize to the receptor and the area of localization can be imaged using the above referenced techniques.

In particular, it is anticipated that the aforementioned peptides can be conjugated to a reporter group, including, but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes a colorimetric or fluorometric reaction), a substrate, a solid matrix, or a carrier (e.g., biotin or avidin) and an antibody. The invention accordingly provides a molecule comprising a peptide inhibiting MTl-MMP, wherein the molecule preferably further comprises a reporter group selected from the group consisting of a radiolabel, a fluorescent label, an enzyme, a substrate, a solid matrix, and a carrier and an antibody. Such labels are well known to those of skill in the art, e.g., biotin labels are particularly contemplated. Any of the peptides of the present invention may comprise one, two, or more of any of these labels.

The peptides of the present invention may be labeled according to techniques well known to those of skill in the art. For example, the peptides can be iodinated by contacting the peptide with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite or an enzymatic oxidant such as lactoperoxidase. Peptides may be labeled with """technetium by ligand exchange, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the peptide to the column. Peptides may be coupled to different chelates, like DTPA or DOTA. Several chelate molecules may be coupled to one peptide in order to increase the

sensitivity. Chelates may be labeled with technetium for imaging or indium for therapy. These and other techniques for labeling proteins and peptides are well known to those of skill in the art.

Many appropriate imaging agents are known in the art, as are methods of attaching the labeling agents to the peptides of the invention as described for example in U.S. Pat. Numbers 4,472,509, 4,965,392, 5,037,630, and 5,201,236 incorporated herein by reference). The labeled peptides are administered to a subject in a pharmaceutically acceptable carrier, and allowed to accumulate at a target site containing MTl-MMP. This peptide imaging agent then serves as a contrast reagent for X-ray, magnetic resonance, sonographic or scintigraphic imaging of the target site.

Paramagnetic ions useful in the imaging agents of the present invention include for example chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II) copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III). Ions useful for X-ray imaging include but are not limited to lanthanum (II), gold (III), lead (II) and particularly bismuth (III). Radioisotopes for diagnostic applications include for example, ²¹¹ astatine, ¹⁴ carbon, ⁵¹ chromium, ³⁶ chlorine, "cobalt, ⁶⁷ copper, ¹⁵² Eu, ⁶⁷ gallium, ³ hydrogen, ¹²³ iodine, ¹²⁵ iodine, ^lu indium, ⁵⁹ iron, ³² phosphorus, ¹⁸⁶ rhenium, ⁷⁵ selenium, ³⁵ sulphur, ^99m technicium and ⁹⁰ yttrium.

Antigelatino lytic peptides CTTl and -2 have been demonstrated to exert high penetrance in cancer tissue and thus be eventually useful in imaging and drug targeting (Medina et al. 2005). MTI-I peptide can be considered to exert corresponding potential when targeting high MTl-MMP expression tumours.

High MTl-MMP (MMP- 14) activity is linked to several cancer types (Ohashi et al. 2000, Etoh 2000) and there is strong evidence that it also correlates with poorer prognosis of certain cancer types (Vihinen and Kahari 2002, Wiegand et al. 2005). Selectively inhibiting MTl-MMP activity has been quite complicated; this invention provides a novel peptide inhibiting the proteolytic activity of MTl-MMP. We have also demonstrated that this MTl-MMP targeting peptide can inhibit invasion and migration in vitro of cancer cells from different origin. The peptide also reduces tumour growth in various tissues. This has

been shown for example by an experiment, where HSC-3 squamous cell carcinoma xenograft tumour growth was reduced in vivo prolonging the survival of the inoculated mice significantly.

Though strong clinical evidence exists regarding the positive association of MMPs to cancer aggressiveness their role in metastatic tumour invasion may be dualistic. Deryugina et al. (2005) reported that by using in vivo models different HT- 1080 fibrosarcoma cells treated with MMP-9 and MMP- 14 inhibitors showed similar or even more aggressive behaviour depending on the cell line dissemination profile. One reason for the differences may be explained by hypoxia: low invasive breast cancer cell lines MDA-MB-435, also used in our experiments, seems to have almost four times higher invasion potential under hypoxic conditions and also overexpresses MMP-2 and -14 (Munoz-Najar 2006). Cancer cell lines also differ in their protease profile. MTl-I peptide effectively inhibited the growth of HSC-3 tumours in our recent study but had little effect on MDA-MB-435 tumours in vivo (Koivunen 1999). On the other hand the MDA-MB-435 cells show almost no invasive potential in the Matrigel invasion assay whereas HSC-3 cells perform high invasive rates.

Invasive potential is a key factor when evaluating cancer aggressiveness, however, evaluation of the metastatic potential and process is far more complicated. Patients with solid tumours can seed thousands to hundred thousand cancer cells to their circulation and still only a few of them manage to develop macrometastasis. Circuculating micro metastases are often found in patients with epithelial tumors with the amount of seeded cancer cells not necessarily affecting patient survival (Marth 2002). Peptides such as CTT2 (Heikkila et al. 2006) and the MTl-I peptide are effective inhibitors of proteolytic activity. The peptides effectively reduce cancer cell invasion potential in vitro and tumour progression in vivo. In the future antiproteolytic peptides may have a role in cancer therapy if more proper MMP -pro filing of the solid tumours is noted.

EXAMPLES

METHODS

Peptides

All the peptides used were custom made (Neosystem, Strasbourg, France). GACFSIAHECGA (SEQ ID NO:4) cycliced between the two cysteines, abbreviated MTl-

I; as controls being scrambled version (SCR-peptide) and CERGGLETSC (SEQ ID NO:5).

(C-peptide or CTRL-peptide) both cyclic.

Cell cultures

C8161 melanoma, HT 1080 fibrosarcoma were cultured as previously described (Koivunen et al. 1999). MTl-MMP transfected SCC25 oral carcinoma cells (Aznavoorian et al,

Cancer Res 2001) were cultured in 1 :1 of DMEM (Calcium free) and Ham's F 12 culture medium supplemented with 10% fetal bovine serum, 0.65mg/ml G418, 100U/ml penicillin and ImM CaCl ₂ . Human tongue squamous cell carcinoma cell line HSC-3 was cultured as described (Heikkila et al. 2006). MDA-MB-435 breast carcinoma cells were cultured in 1 :1 (Calcium free) and Ham's F 12 culture medium supplemented with 10% fetal bovine serum and L-glutamine (Koivunen et al. 1999).

Enzyme activity assay

Effect of MTl-I peptide on MMP- 14 enzyme protease activity was studied using QuantiZyme™ -assay (Biomol, PA) according to the manufacturer's instructions. Concentration of the recombinant MMP- 14 being 60mU/μl, concentration of the peptides being from 43 μM to 129μM, and the assay performed in 96-well black flat bottom micro plates. MMP- 14 substrate fluorescence was recorded at 1 min time intervals for 15 minutes using Tecan Spectrafluor plus fluorometer using filters for excitation 340nm and emission 394nm.

Cytotoxicity, Gelatin zymography and western-blotting

Cytotoxicity and cell adhesion was studied using the MTT -reagent as described (Koivunen et al. 1999) and cell proliferation by BrdU incorporation ELISA assay (Roche) according to the manufacturers instructions. MTl-I peptide at raising concentrations was incubated with recombinant MMPs and β-casein or the Laminin-5 γ2-chain was added as substrates.

Substrate proteolysis was characterized by SDS-PAGE (for β -casein) or Western blotting (for the Laminin-5 γ2-chain) as described (Heikkila et al. 2006, Pirila et al. 2003).

In vitro cell invasion and migration

Cell invasion was studied using 8.0 micron pore size Matrigel™ 24-well -plate invasion chambers and control inserts (BD Biosciences, Bedford, MA). Inserts were rehydrated with serum containing culture medium 2 h before use. Lyophilized peptides were diluted to lmg/ml in dH ₂ O and desired concentration series were prepared to inserts using serum free culture medium. Cells (IxIO ⁵ / well) and peptides were preincubated for 2 h at 37 ⁰ C humidified 5% CO ₂ atmosphere. Serum containing culture medium was then added to the lower compartment of the chamber to function as a chemoattractant and the incubation was continued for 48 h. Cells were fixed with methanol, washed, and stained with 1% toluidine blue. Non-migrated cells were removed from the upper surface of the insert with a cotton swab. Quantitative analysis of the migrated cells was done using Bio-Rad GS-700 Imaging Densitometer. Random cell migration was also studied using Transwell 8.0 μm filters (Costar, MA) in serum- free medium. Filters were coated with either rat laminin-5 (Ln-5) or bovine fibronectin. Migration analysis was performed as above.

Animal experiments

The animal experiments were approved by the ethics committee for the animal experiments at the University of Helsinki. The HSC-3 tumours were initiated by injecting s.c. 2xlO ⁶ HSC-3-cells in 200μls of serum- free medium to both flanks of 6-8 weeks old athymic nude nu -female (Harlan, Holland). After the inoculation on the days 4-8 the mice were treated daily with MTl-I peptide and its controls (2 mg/ml in lOOμls of 0.9% NaCl/dH ₂ O i.v. (intravenously) to the tail). Criteria for euthanasia: significant weight loss observed or the tumour diameter >10mm. Tumour volumes were calculated with formula (II/6)xAxBxB, where A is the length and the B is the width of the tumour.

Statistical significances between the groups at the end of the experiment were studied using Kaplan-Meier survival analysis.

Immunohistochemistry. The vascular densities of xenograft tumor samples were analyzed as decribed by Heikkila et al. 2006 . Briefly, resected tumors of euthanized animals were embedded in OCT Compound (Sakura Tissue -T ek, Zoeterwounde, Netherlands), frozen and stored at -70 ⁰ C. Ten μm acetone-fixed frozen sections were incubated in 0.6% H ₂ O ₂

in methanol to reduce endogenous peroxidase activity, blocked with normal serum, and stained with vascular endothelial recognizing factor VIII antibody (diluted 1 :1000) using 3-amino-9-ethyl-carbazole as a chromogen (ABC Elite kit, Vector Labs, CA, USA). Eventually, sections were counterstained with hematoxylin and eosin to identify cancer stroma and endothelial cells, and their vessels were identified from four of the highest vessel density containing areas excluding the ones adjacent to necrosis.

MMP cleavage assay

MMP-2 ja MTl-MMP were incubated for 60 min with 1 mM of an organomercurical activator of MMPs and thereafter for 30 min with indicated concentrations of MTl-I peptide, irrelevant control peptide, MMP-2 and -9 inhibitor peptide or 10 μM of broad- spectrum MMP-inhibitor (Marimastat) at 37 ⁰ C. Also MT2-MMP (= MMP- 15), MT3- MMP (= MMP- 16), MT4-MMP (= MMP- 17), MMP-10, MMP-7, MMP-I l, MMP-I, MMP-3, MMP-8, MMP-9, MMP-12, MMP-13 and MMP-20 are tested under the same conditions. Thereafter, 52 μM β-casein or 0.1 mg of laminin-5 γ2-chain were added and incubation was continued for 2 hours at room temperature after which the reaction was stopped by boiling in Laemmli-buffer. β-Caseino lysis was analyzed by separating proteins on an 11% SDS-polyacrylamide gel stained with 0.1% Serva blue R and disappearance of the 21- kDaβ -casein band was regarded as MMP-activity. The gels were destained with 20% methanol, 10% acetic acid and photographed. The effects of peptides on proteolytic cleavage of the laminin-5 γ2-chain by MTl-MMP were analyzed by Western immunoblotting. Samples were separated on an 8% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane and incubated with Laminin-5 γ2-chain specific polyclonal antibody [dilution 1 : 1000] over night. Membranes were then incubated with alkaline phosphatase conjugated goat anti-rabbit IgG antibody. Immunoblots were developed in a solution of nitroblue tetrazolium and 5-bromo-4-chloro-3 indolyl phosphate.

AK-417 MTl-MMP Fluorecent Assay Kit for Drug Discovery (BIOMOL)

Human recombinant MTl-MMP enzyme (catalytic domain) and peptides were diluted in kit-assay buffer. The assay was performed in black flat bottom 96-well plate. MTl-MMP (12mU/ml) was preincubated (37 ⁰ C, 60min) with and without the peptides (43μM-430μM) or kit-NNGH control inhibitor (1.3μM) to allow inhibitor-enzyme interaction. The reaction was started by adding OmniMMP™ fluorescent substrate 4μM (Mca-PLGL-Dpa- AR-NH ₂ )

and then the substrate cleavage (0-15min) was continuously measured using Tecan Spectrafluor Plus densitometer using 340 nm filter for excitation and 394 nm filter for emission. Fluorescence data was plotted versus time for each sample and the range of initial time points during which the reaction was linear was selected for reaction velocity analysis (U/min).

Pepspot-assay

Binding of MTl-MMP to MTl-I peptide and its' variants was analyzed using pepspot membrane (Haartman institute, PeproLab). In the pepspot-membrane peptides are synthesised on top of nitrocellulose membrane. Here the effect of each amino acid of the MTl-I peptide was analyzed by replacement or alanine scan mutagenesis.

Pepspot membrane was thawed from -20 ⁰ C to RT and rinsed in methanol to solubilize the peptides then washed three times 10 min with TBS. The membrane was blocked by overnight incubation with 3% skim milk and 5% sucrose in TTBS and then washed again for lOmin with TTBS. Recombinant MMP- 14 enzyme containing a his-tag sequence (Invitek) was diluted 2μg/ml in TTBS and then incubated on top of the membrane for 2 h in RT. After elution of unbound MMP-14 by washing 3xlθmin with TTBS the formed peptide-MMP complexes were detected by incubating rabbit anti-his-tag-probes diluted 1 : 100 in TTBS (Santacruz) on top of the membrane for 1 h, unbound probes again washed of with TTBS (3xlθmin), the membrane incubated according to an ECL protocol using anti-rabbit ECL probes (Amersham Pharmacia Biotech). Briefly, ECL-antirabbit antibody was diluted 1 : 1000 in TTBS and incubated on top of the membrane for 2 h in RT, unbound antibodies washed of (4xl0min TTBS) and formed antibody complexes detected using kit- detection solutions and high performance X-ray films (based on chemiluminescence).

RESULTS

MTl-I peptide inhibits MTl-MMP activity. MTl-I peptide inhibits proteolytic activity of MMP-14 in Quantizyme™ fluorescent assay (Fig. 8). Anti-MMP effect of the MTl-I - peptide was also studied by incubating peptide, recombinant enzymes and Ln-5 γ2-chain or β-casein as a substrate. MTl-I peptide inhibited MTl-MMP mediated proteolysis of the Ln-5 γ2-chain at 100 μM (Fig.2a) and β-casein at 50 μM (Fig.2b). The non-relevant CTRL-peptide in 500μM did not have any effect on MTl-MMP activity (Fig.2b). MTl-I peptide did not inhibit MMP-2 mediated proteolysis of β-casein (Fig.2c) and neither that of

MMP-I, -3, -7, -8, -9, -10, -11, -12,-13, -15, -17, -20 (Fig. 10). In comparison, the antigelatinolytic CTT-peptide inhibited β-casein proteolysis by MMP-2 as expected (Fig.2c).

MTl-I peptide inhibits cancer cell migration and invasion. Antimigratory and - invasive potential of MTl-I peptide was studied in vitro using Transwell- and Matrigel assays. MTl-I peptide at 100 μM inhibited HT 1080 fibrosarcoma cell migration on fϊbronectin (Fig.l A) and the inhibition was comparable to that of the MMP-2 and -9 specific CTT-peptide. Migration of C8161 melanoma cell on both fibronectin (Fig.l B) and Ln-5 (Fig.l C) was also inhibited on that same concentration. MTl-I peptide at 100 μg/ml effectively reduced Matrigel-invasion of C8161 melanoma (Fig.l D) and HSC-3 human tongue carcinoma cells (Fig. 1 E) while having no effect on HSC-3 cell migration (not shown). MTl-I peptide inhibited migration of both MTl-MMP transfected (Fig.4A) as well as vector (Fig.4 B) transfected SCC25 oral carcinoma cells on Ln-5. MTl-I peptide inhibited MTl-MMP transfected SCC25 oral carcinoma cell migration over Ln-5 dose- dependently. The Ctrl-peptide at 500 μM did not show any effect (Fig.5). MTl-I peptide up to concentrations of 500 μM did not affect C8161 melanoma cell adhesion (Fig. 3 A), cell proliferation (Fig. 3 B), or cell viability (not shown). Similar results were found for the CTT and C-peptide. To further analyze possible effects of MTl-I peptide to MDA-MB- 435 breast cancer cell line (Koivunen et al. 1999) we studied cell migration and invasion. MTl-I peptide had no effect on MDA-MB-435 migration while these cells had almost none invasive potential in the Matrigel invasion assay (not shown).

Mice bearing HSC-3 xenograft tumours survive longer if treated with MTl-I peptide.

Anticancer potential of the MTl-I peptide in vivo was studied using HSC-3 human tongue squamous cell carcinoma xenograft model on athymic nude mice. The HSC-3 tumour xenografts grew significantly slower in the MTl-I peptide treated group as compared to the irrelevant controls (Fig.6.); in 2 mice out of 10 the tumour volume even reduced thus the effect being permanent. Also the survival of the mice in the MTl-I peptide treated group was longer because tumours of the MTl-I peptide treated mice reached euthanasia threshold slower (Fig.7.). At the end of the experiment 2 mice were still alive in the MTl-I peptide treated group.

Cysteines are important for MMP binding. Binding of MTl-MMP to MTl-I peptide and its variants was analyzed using pepspot membrane where individual amino acids were

replaced by alanin scan mutagenesis. The most dramatic effect on MMP-binding was detected when both of the cysteines were replaced. Also changing any amino acid between the cysteines reduced MMP-binding; the serine being most effective. Exocyclin aminoacids had less effect on MMP-binding (Fig. 9).

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