WO2018138292A1

WO2018138292A1 - A polypeptide having protease activity for use in treating otitis

Info

Publication number: WO2018138292A1
Application number: PCT/EP2018/052008
Authority: WO
Inventors: Kerstin BERGVALL; Mats Peter CLARSUND; Fredrik Lindberg
Original assignee: Enzymatica Ab
Priority date: 2017-01-26
Filing date: 2018-01-26
Publication date: 2018-08-02
Also published as: GB201701315D0

Abstract

The present invention provides polypeptides having protease activity for use in the treatment or prevention of otitis in a subject. For example, the polypeptide may be used to treat otitis caused by a microbial infection. In particular, the polypeptides are useful in the treatment or prevention of otitis externa. In one embodiment, the polypeptide is a trypsin enzyme from Atlantic cod, or a fragment or variant thereof.

Description

A POLYPEPTIDE HAVING PROTEASE ACTIVITY FOR USE IN TREATING OTITIS

Field of Invention The present invention relates to the use of polypeptides with protease activity for the treatment and prevention of otitis, in particular for otitis externa caused by microbial infections.

Background

Resistance to antibiotics is a rapidly growing problem throughout the world (18). There is a clear correlation between a high incidence of resistance and a high consumption of antibiotics. The problem is considerably exacerbated, furthermore, if antibiotics are available without prescription, as is the case in many countries. An additional significant factor is the use of antibiotics in animal husbandry. There are several examples of this leading to resistance to antibiotics that are significant for human medicine. It is now possible to envisage that the situation will arise in the near future in which it is not possible to treat bacterial infections from certain types of bacteria with any licensed antibiotic, due to antibiotic resistance.

Another "need and market driver" is that the FDA is banning several antiseptics. The FDA has announced that effective September 2017, it would prohibit the sale of "consumer antiseptic washes" containing triclosan or 18 other ingredients marketed as antimicrobials due to the FDA's findings of the lack of efficacy in these products (1 ). The FDA is also warning about the possibility that chlorhexidine can cause allergic reactions. Thus, there will be a large unmet market need for replacement products that do not contain triclosan and chlorhexidine. In parallel with the increasing problem of resistance, there is a conspicuous lack of development of new antibacterial pharmaceuticals. There are several reasons for this. For example, the lack of new, easily identifiable targets, which leads to major scientific and technical challenges and very high development costs.

Otitis (inflammation of the ear) is a common medical condition in both humans and in non- human mammals, for example dogs or cats. Otitis externa affects the external ear canal, from the tympanic membrane, and may also involve the ear pinna (2). Otitis may be caused by a microbial infection and so is commonly treated with antimicrobials such as antibiotics, optionally in combination with pain medication and glucocorticoids. However, some microbial infections are resistant to the anti-microbials typically administered, and present as treatment-resistant otitis.

For example, otitis caused by Pseudomonas bacteria can present as treatment resistant otitis, as Pseudomonas are resistant to many anti-microbials. In addition, they readily develop further resistance if treatment is ineffective as they have a large genome to express resistance genes and mutations, and are capable of plasmid, transposon and bacteriophage transfer. Once fluoroquinolone resistance is established other anti- Pseudomonas antimicrobials are indicated; these are often expensive, not licensed for animals and have to been given intravenously if used systemically (3).

Additionally, fungal infections may also cause otitis, and can also be resistant to treatment with anti-microbials.

For example, the fungal genus Malassezia comprises lipid-dependent and lipophilic yeast species that are part of the normal skin microbiota, including in the ear canal (2), but species of this genus, such as M. pachydermatis, can cause dermatitis and otitis in mammals.

As otitis can be caused by numerous different conditions, including microbial infections which may be resistant to treatment with conventional anti-microbials, there remains a need for improved therapies for treating otitis, in particular treatment-resistant otitis. With antibiotic resistance becoming more prevalent, this is a pressing issue.

Thus, the present invention seeks to provide new treatments for otitis. Summary of Invention

The first aspect of the invention provides a polypeptide having protease activity for use in the treatment or prevention of otitis in a mammal.

By "protease activity" we include any polypeptide which is capable of catalysing proteolysis in vivo, in the mammalian (e.g. human) body. Thus, any type of protease may be utilised in the invention, including but not limited to serine proteases (such as trypsins/chymotrypsins), threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.

By "otitis" we mean inflammation of the ear, typically associated with a localised infection of the outer, middle and/or inner ear. By "treatment" we include the alleviation, in part or in whole, of the symptoms of otitis, namely inflammation of the ear. Such treatment may include eradication, or slowing of population growth, of a microbial agent associated with the inflammation.

By "prevention" we include the reduction in risk of otitis developing in patients. However, it will be appreciated that such prevention may not be absolute, i.e. it may not prevent all such patients developing otitis. As such, the terms "prevention" and "prophylaxis" may be used interchangeably.

In one embodiment the otitis is external otitis (otitis externa). By external otitis, we mean that the otitis causes inflammation of the external ear canal. By external ear canal, we mean the portion of the ear canal distal to the tympanic membrane. The ear pinna may or may not be involved (2).

Otitis externa may be characterised by, for example, pain, itching, redness and swelling. Otitis externa may lead to a ruptured tympanum (ear drum) and otitis media. In animals, symptoms of otitis may include head shaking, ear scratching, odour, pain on manipulation of the ear, swelling, inflammation, exudate, ulceration, and erythema (redness) (2, 4).

Alternatively, the otitis to be treated may be of the middle ear (otitis media, also known as tympanitis) or the inner ear (otitis interna, also known as labyrinthitis, vestibular neuronitis or vestibular neuritis). In one embodiment the otitis is associated with a microbial infection.

In one embodiment the microbial infection is selected from the group consisting of bacterial infections, viral infections, fungal infections and yeast infections.

For example, the microbial infection may be a bacterial infection. The bacterial infection may be caused by Gram negative and/or Gram-positive bacteria.

In one embodiment the bacterial infection is caused by bacteria selected from the following genera: Pseudomonas (e.g. Pseudomonas aeruginosa), Staphylococcus (e.g. Staphylococcus intermedius, Staphylococcus epidermis, Staphylococcus aureus), Streptococcus (e.g. Streptococcus group D and Streptococcus group G), Corynebacterium, Entero- coccus, Proteus, Escherichia (e.g. Escherichia coir), Bacteroides, Peptostreptococcus, Haemophilus (e.g. Haemophilus influenza) and Clostridium.

In a preferred embodiment, the bacterial infection is caused by bacteria from the Pseudomonas genus, e.g. Pseudomonas aeruginosa.

In a further embodiment, the microbial infection may be a viral infection. Thus, the otitis may be associated with infection by rhinovirus, coronavirus, influenza virus, respiratory syncytial virus and the like. For example, subjects with the common cold frequently exhibit excess mucus production affecting the oropharyngeal cavity, which can lead to blockage of the Eustachian tubes resulting in otitis media. In the case of viral infection, it is believed that the protease-based therapies of the invention act to disarm/deactivate, rather than to destroy, the virus particles by cleavage of viral surface proteins.

In another embodiment, the microbial infection is a fungal infection.

In one embodiment the fungal infection is caused by fungi selected from the following genera: Malassezia (e.g. Malassezia pachydermatis), Candida (e.g. Candida albicans) and Aspergillus (e.g. Aspergillus niger). In a preferable embodiment the fungal infection is caused by fungi from the Malassezia genus, e.g. Malassezia pachydermatis. The otitis may be associated with multiple microbial infections, for example, both fungal and bacterial infections.

In one embodiment the microbial infection comprises formation of a biofilm.

Biofilms are heterogeneous, complex 3D matrices that comprise a population of microbial cells embedded in an extracellular matrix (ECM). They are not just a passive assembly of cells, but are structurally and dynamically complex biological systems that form local ecosystems. The microbial cells within a biofilm population appear to co-operate and take on special functions. By co-operating and forming a protective ECM, the biofilm provides the microorganisms with a protected mode of growth that allows them to colonise diverse environments. The biofilm mode of growth allows the bacteria to counteract the immune system of the host as well as antibiotics and similar bacteriostatic and bactericidal agents. The development of biofilm thus allows a population of bacteria to show resistance to antibiotics. Bacteria growing in biofilms are more difficult to defeat than their planktonic, i.e. free-living, counterparts (19, 20). Biofilm formation is an important step in the pathogenesis of many diseases.

Biofilms can consist of mono or poly-bacterial populations adhering to virtually any biological or non-biological surface. In such multicellular populations, cells adhere to each other. A majority of bacterial species, as well as archaea, protozoa, fungi and algae, have the ability to adhere to surfaces and to each other and form biofilm structures. Formation of biofilms typically begins with the attachment of free-floating microorganisms to a surface. When the expressions of numerous genes are changed, a planktonic cell undergoes a phenotypic shift and switches from the free living mode to the biofilm mode of growth. The first colonists adhere to the surface initially through weak, reversible adhesion, which may become stronger by production of cell adhesion structures such as pili. Once colonization has begun, the biofilm grows through a combination of cell division and appearance and binding of new bacteria. The first colonists facilitate the arrival of other cells by providing more diverse adhesion sites and by beginning to build the matrix that holds the biofilm together.

By "biofilm" we include a cluster or population of microorganisms, which are adhered to each other within the extracellular matrix on a biological surface (e.g. skin, mucosa, etc).

For example, the infection may be associated with formation of a biofilm of Pseudomonas aeruginosa or Staphylococcus aureus. The protease-based therapies of the present invention are effective against biofilms, which can be difficult to treat with conventional antibiotics (see WO 2017/017027 to Enzymatica AB, the disclosures of which are incorporated herein by reference). In one embodiment, the otitis is associated with an allergic or dermatologic condition. Allergic disease is a major cause of otitis, for example allergic contact dermatitis. Examples of dermatologic conditions which may be associated with otitis include psoriasis, atopic dermatitis and seborrhoeic dermatitis. Otitis may also be caused by, for example, parasites, foreign bodies in the ear, keratinization disorders or autoimmune disease.

Excessive moisture in the ear canal is another contributory factor for otitis. Swimming and water exposure is a risk factor which increases the likelihood of otitis developing, as do the use of devices which obstruct the ear canal, such as hearing aids, earphones or swimming caps. Sweating, e.g. from heavy exercise, may also increase the risk of developing otitis.

Trauma from cleaning or scratching of the ear canal is a further risk factor for developing otitis. Additionally, radiation therapy can increase the risk of developing otitis, by causing ischemic ear canal changes, and changing cerumen production and epithelial migration (22).

In a further embodiment, the otitis is treatment resistant otitis. By "treatment resistant" we mean that the otitis has not been resolved by treatment with conventional therapies such as antibiotics.

The otitis may be either acute or chronic, and either unilateral or bilateral. In one embodiment, the mammal is human.

In an alternative embodiment, the mammal is a non-human mammal. Thus, the polypeptides of the invention may be used in a veterinary setting, for example in the treatment of otitis in domestic and/or farm animals (including dogs, cats, rabbits, horses, cattle, pigs, sheep and the like). In a preferred embodiment, the mammal is a dog. In one embodiment, the polypeptide having protease activity is selected from the group consisting of serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases. In one embodiment, the polypeptide having protease activity is a serine protease. By "serine protease" we include both naturally occurring and non-naturally occurring catalytic polypeptides capable of cleaving peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site of the polypeptide (as defined in accordance with EC Number 3.4.21 ). The serine protease may have chymotrypsin-like protease activity (i.e. trypsins, chymotrypsins and elastases) or subtilisin-like protease activity.

Thus, in one embodiment the protease is a trypsin or chymotrypsin, or a component of a mixture thereof. Thus, the polypeptides of the invention may exhibit trypsin activity. By "trypsin activity" we mean that the polypeptide exhibits a peptidase activity of a trypsin enzyme (EC 3,4,21 ,4) or of a related peptidase (such as chymotrypsin enzymes, EC 3,4,21 ,1 ). For example, the protease may be a naturally-occurring trypsin, of either eukaryotic or prokaryotic origin, or a mutated version of such a trypsin.

In one embodiment of the invention, the polypeptide having protease activity is cold- adapted, i.e. the polypeptide is psychrophilic. By "cold-adapted" we mean the polypeptide is derived from an organism from a cold environment, and is hence adapted to function at low temperatures. For example, the polypeptide having protease activity may exhibit protease activity for longer periods of time at 15°C than at higher temperatures, such as 25°C or 37°C (see Stefansson et a/., 2010, Comparative Biochem. Physiol: Part B - Biochem. & Mot. Biol., 155(2): 186-194, the disclosures of which are incorporated by reference) (21 ). The polypeptides of the invention may be naturally occurring or non-naturally occurring.

In one embodiment, the polypeptide having protease activity comprises or consists of the amino acid sequence of a naturally-occurring protease. For example, the polypeptide having protease activity may consist of the amino acid sequence of a naturally-occurring trypsin, of either eukaryotic or prokaryotic origin. In one embodiment, the polypeptide is a marine serine protease. The marine serine protease may be obtainable from, for example, cod, pollock, salmon or krill. Other possible sources of marine proteases include catfish, haddock, hoki, hake, redfish, roughies, tilapia, whiting and Chilean seabass. Specifically included are cold-adapted trypsins, such as a trypsin from Atlantic cod (Gadus morhua), Atlantic and Pacific salmon (e.g. Salmo salar and species of Oncorhynchus) and Alaskan Pollock (Theragra chalcogramma). For example, the polypeptide having serine protease activity may comprise or consist of the amino acid of SEQ ID NO:1 , as listed below. In a preferred embodiment, the marine serine protease is obtainable from Atlantic cod.

Naturally-occurring serine proteases may be purified from a source organism (e.g. Atlantic cod) or may be expressed recombinantly. Thus, it will be appreciated by persons skilled in the art that such naturally-occurring serine protease polypeptides of the invention must be provided in a form different to that in which they are found in nature. For example, the polypeptide of the invention may consist of the amino acid sequence of a naturally-occurring eukaryotic trypsin but lack the glycosylation moieties present on the protein as it is expressed in nature.

In a preferred embodiment, the marine serine protease is a trypsin, for example trypsin I, trypsin X, trypsin Y or trypsin ZT (for example, see below).

Three major isozymes of trypsin were originally characterised from Atlantic cod, designated Trypsin I, II and III (see Asgeirsson et ai, 1989, Eur. J. Biochem. 180:85-94, the disclosures of which are incorporated herein by reference). For example, trypsin I from Atlantic cod is defined in GenBank Accession No. ACO90397 (see Stefansson etal., 2010, Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 155 (2), 186-194, the disclosures of which are incorporated herein by reference). Subsequently, the trypsins produced by Atlantic cod have been further characterised and a number of distinct isoforms have now been characterised, including trypsin I, trypsin ZT, trypsin X and trypsin Y (see below).

In addition, Atlantic cod expresses two major isozymes of chymotrypsin, designated Chymotrypsin A and B (see Asgeirsson & Bjarnason, 1991 , Comp. Biochem. Physiol. B 998:327-335, the disclosures of which are incorporated herein by reference). For example, see GenBank Accession No. CAA55242.1. In one embodiment, the polypeptide having protease activity comprises or consists of an amino acid sequence of trypsin I from Atlantic cod (Gadus morhua), i.e. SEQ ID NO: 1 or SEQ ID NO: 2 IVGGYECTKHSQAHQVSLNSGYHFCGGSLVSKD VVSAAHCYKSRIEVRLGEHHIRVNEG TEQYISSSSVIRHPNYSSYNINNDIMLIKLSKPATLNQYVQPVALPTECAADGTMCTVSG WGNTMSSVADGDKLQCLSLPILSHADCANSYPGMITQSMFCAGYLEGGKDSCQGDSGGPV VCNGVLQGVVSWGYGCAERDHPGVYAKVCVLSGWVRDTMANY

[SEQ ID NO: 1]

IVGGYECTKHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHCYKSVLRVRLGEHHIRVNEG TEQYISSSSVIRHPNYSSYNINNDIMLI LTKPATLNQYVHAVALPTECAADAT CTVSG WGNTMSSVADGDKLQCLSLPILSHADCANSYPGMITQSMFCAGYLEGGKDSCQGDSGGPV VCNGVLQGVVSWGYGCAERDHPGVYAKVCVLSGWVRDTMANY

[SEQ ID NO: 2] or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) of SEQ ID NO: 1 or 2, which retains the trypsin activity of said amino acid sequences.

Further details of trypsin I can be found in (see GuQmundsdottir et a/., 1993, Eur J Biochem. 217(3):1091-7 and Stefansson et al., 2010, Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 155 (2), 186-194, (the disclosures of which are incorporated herein by reference).

Alternatively, the polypeptide having protease activity may comprise or consist of an amino acid sequence of a trypsin ZT isoform from Atlantic cod [Gadus morhua), e.g. SEQ ID NOs: 3 to 7 (see WO 2017/017012 to Enzymatica AB, the disclosures of which are incorporated herein by reference).

SEQ ID NO: 3 is the consensus sequence of the ZT- isoforms, ZT-1 to ZT-4, presented below. IX1GGX2X3CEPX4SRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDL RVFEGTEQLVKTNTIFWHEX5YDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGM LCSVSG GNMAMGEEVNLPTRLQCLDVPIVEX6VX7CX8AX9YPG ISPRMX10CX11GX12M DGGRDX13CNGDSGSPLVCEGVLTGLVSWGX14GCAX15PNX16PGVYVKVYEX17LSWIQTT LDANP

[SEQ ID NO: 3] wherein

Xi is selected from I and V;

X2 is selected from Q and H;

X3 is selected from D and E;

X₄ is selected from R and N;

X₅ is L;

Χβ is selected from T and P;

X₇ is selected from D and A;

Xs is selected from E and Q;

Xg is selected from A and S;

X10 is selected from V and M;

X11 is selected from A and V;

X12 is selected from Y and F;

Xi3 is selected from A and V;

Xi4 is selected from Q and R;

X15 is selected from L and E;

X16 is selected from Y and S; and

Xi7 is selected from Y and F.

Atlantic cod trypsin ZT-1 isoform:

IVGGHECEPNSRPF ASLNYGYHFCGGVLINDQWVLSVAHC YNPYYMQVMLGEHDLRVF EGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSV SGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGMISPRMMCVGFMDGGRDVCNGDS GSPLVCEGVLTGLVSWGRGCAEPNSPGVYVKVYEFLSWIQTTLDANP

[SEQ ID NO: 4] Atlantic cod trypsin ZT-2 isoform:

IVGGHECEPNSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVF EGTEQLVKTNTIF HELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSV SGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGYMDGGRDACNGDS GSPLVCEGVLTGLVSWGQGCALPNYPGVYVKVYEYLSWIQTTLDANP

[SEQ ID NO: 5]

Atlantic cod trypsin ZT-3 isoform:

IIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQVMLGEHDLRVF EGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSV SGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGMISPRMMCVGFMDGGRDVCNGDS GSPLVCEGVLTGLVSWGRGCAEPNSPGVYVKVYEFLSWIQTTLDANP

[SEQ ID NO: 6] Atlantic cod trypsin ZT-4 isoform:

IIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHC YNPYYMQVMLGEHDLRVF EGTEQLVKTNTIFWHELYDYQTLDYDMMMI LYHPVEVTQSVAPISLPTGPPDGGMLCSV SGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGY DGGRDACNGDS GSPLVCEGVLTGLVSWGQGCALPNYPGVYVKVYEYLSWIQTTLDANP

[SEQ ID NO: 7]

It will be appreciated by persons skilled in the art that the polypeptide may be present as a mixture of one or more of the above trypsin ZT isoforms, optionally in combination with trypsins I, X and/or Y.

Alternatively, the polypeptide having protease activity may comprise or consist of an amino acid sequence of trypsin X from Atlantic cod, e.g. SEQ ID NOs: 8 to 1 1 (see Stefansson et al., 2017, Biochim Biophys Acta. 1865(1 ):11-19, the disclosures of which are incorporated herein by reference). Atlantic cod trypsin X:

IVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHCYKSVLRVRLGEHHIRVNEG TEQFISSSSVIRHPNYSSYNIDNDIMLIKLTEPATLNQYVHAVALPTECAADATMCTVSG WGNTMSSVDDGDKLQCLNLPILSHADCANSYPGMITQSMFCAGYLEGGKDSCQGDSGGPV VCNGVLQGVVSWGYGCAERDNPGVYAKVCVLSGWVRDTMASY

[SEQ ID NO: 8]

Atlantic cod trypsin X-1 :

IVGGYECTRHSQAHQVSLNSGYHFCGGSLVS DWVVSAAHCYKSRIEVRLGEHHIRVNEG TEQFISSSSVIRHPNYSSYNIDNDIMLIKLSEPATLNQYVQPVALPTECAADGTMCTVSG GNTMSSVDDGDKLQCLNLPILSHADCANSYPGMITQSMFCAGYLEGGKDSCQGDSGGPV VCNGVLQGVVSWGYGCAERDNPGVYAKVCVLSGWVRDT ASY

[SEQ ID NO: 9] Atlantic cod trypsin X-2:

IVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHCYKSRIEVRLGEHHIRVNEG TEQFISSSSVIRHPNYSSYNIDNDIMLIKLSKPATLNQYVQTVALPTECAADGTMCTVSG GNTMSSVDDGDKLQCLNLPILSHADCSNSYPGMITQSMFCAGYLEGGKDSCQGDSGGPV VCNGVLQGVVSWGYGCAERDNPGVYAKVCVLSGWVRDTMASY

[SEQ ID NO: 10]

Atlantic cod trypsin X-3:

IVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKD VVSAAHCYKSRIEVRLGEHHIRVNEG TEQFISSSSVIRHPNYSSYNIDNDIMLIKLSEPATLNQYVQTVALPTECAADGTMCTVSG GNTMSSVDDGDKLQCLNLPILSHADCSNSYPGMITQSMFCAGYLEGG DSCQGDSGGPV VCNGVLQGVVSWGYGCAERDNPGVYAKVCVLSG VRDTMASY

[SEQ ID NO: 11]

Alternatively, the polypeptide having protease activity may comprise or consist of an amino acid sequence of trypsin Y from Atlantic cod, e.g. SEQ ID NO: 12 (see Palsdottir & Gudmundsdottir, 2008, Food Chem. 111(2):408-14, the disclosures of which are incorporated herein by reference).

Atlantic cod trypsin Y:

I IGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSVAHCWYNPYYMQV LGEHDLRVF EGTEQLVKTNTIF HEQYDYQTLDYDMMMIKLYHPVEVTQSVAPISLPTGPPDGGMLCSV SGWGNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPGMISPRMVCAGYMDGGRDACNGDS GSPLVCEGVLTGLVS GQGCALPNYPGVYVKVYEYLSWIQTTLDANP

[SEQ ID NO: 12]

Thus, in exemplary embodiments, the polypeptide having protease activity comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 1 to 12. Such a polypeptide may be purified from Atlantic cod, for example as described in Asgeirsson et a/., 1989, Eur. J. Biochem. 180:85-94 (the disclosures of which are incorporated herein by reference).

Suitable exemplary polypeptides of the invention, and methods for their production, are also described in European Patent No. 1 202 743 B (the disclosures of which are incorporated herein by reference) (7).

Like many proteases, trypsin I from Atlantic cod is produced as an inactive precursor, or zymogen, comprising a propeptide (or "activation") sequence that is cleaved off to generate the mature, active trypsin. The initial expression product for trypsin also comprises a signal sequence, which is removed following expression.

A zymogen sequence for trypsin I from Atlantic cod, including the signal sequence, is shown below as SEQ ID NO: 13 (and corresponds to Uniprot database accession no. P16049-1):

10 20 30 40 50

MKSLIFVLLL GAVFAEEDKL VGGYECTKHS QAHQVSLNSG YHFCGGSLVS

60 70 80 90 100

KD VVSAAHC YKSVLRVRLG EHHIRVNEGT EQYISSSSVI RHPNYSSYNI

110 120 130 140 150

NNDIMLIKLT KPATLNQYVH AVALPTECAA DATMCTVSGW GNTMSSVADG

160 170 180 190 200

DKLQCLSLPI LSHADCANSY PGMITQSMFC AGYLEGGKDS CQGDSGGPVV

210 220 230 240

CNGVLQGWS WGYGCAERDH PGVYAKVCVL SGWVRDTMAN Y

[SEQ ID NO: 13] wherein:

Signal peptide = amino acids 1 to 13 (underlined)

Propeptide = amino acids 14 to 19 (bold italics)

Mature trypsin = amino acids 20 to 241

The zymogen sequence for the variant trypsin I from Atlantic cod corresponding to SEQ ID NO: 2, including the signal sequence, is shown below as SEQ ID NO: 14 (and corresponds to Uniprot database accession no. P16049-1 ):

10 20 30 40 50

MKSLIFVLLL GAVFAEEPKI VGGYECTKHS QAHQVSLNSG YHFCGGSLVS

60 70 80 90 100

KDWVVSAAHC YKSVLRVRLG EHHIRVNEGT EQYISSSSVI RHPNYSSYNI

110 120 130 140 150

NNDIMLIKLT KPATLNQYVH AVALPTECAA DATMCTVSGW GNTMSSVADG

160 170 180 190 200

DKLQCLSLPI LSHADCANSY PGMITQSMFC AGYLEGGKDS CQGDSGGPVV

210 220 230 240

CNGVLQGWS WGYGCAERDH PGVYAKVCVL SGWVRDTMAN Y

[SEQ ID NO: 14] wherein:

Signal peptide = amino acids 1 to 13 (underlined)

Propeptide = amino acids 14 to 19 (bold italics)

Mature trypsin = amino acids 20 to 241

The zymogen sequence for the variant trypsin X corresponding to SEQ ID NO: 8, including the signal sequence, is shown below as SED ID NO: 15 (and corresponds to Genbank Accession No. Q91041.2).

MKSLIFVLLLGAVFAEED IVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHC YKSVLRVRLGEHHIRVNEGTEQFISSSSVIRHPNYSSYNIDNDIMLIKLTEPATLNQYVH AVALPTECAADATMCTVSGWGNTMSSVDDGDKLQCLNLPILSHADCANSYPGMITQS FC AGYLEGGKDSCQGDSGGPWCNGVLQGVVSWGYGCAERDNPGVYAKVCVLSGWVRDTMAS

Y

[SEQ ID NO: 15]

(wherein the signal sequence and propeptide are underlined and in bold italics, respectively).

The zymogen sequence for the variant trypsin X-1 corresponding to SEQ ID NO: 9, including the signal sequence, is shown below as SED ID NO: 16 (and corresponds to Genbank Accession No. AOX15769.1)

MKSLIFVLLLGAVFAEEDKIVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHC YKSRIEVRLGEHHIRVNEGTEQFISSSSVIRHPNYSSYNIDNDI LIKLSEPATLNQYVQ PVALPTECAADGTMCTVSGWGNTMSSVDDGDKLQCLNLPILSHADCANSYPGMITQSMFC AGYLEGGKDSCQGDSGGPVVCNGVLQGVVS GYGCAERDNPGVYAKVCVLSGWVRDTMAS

Y

[SEQ ID NO: 16]

(wherein the signal sequence and propeptide are underlined and in bold italics, respectively). The zymogen sequence for the variant trypsin X-2 corresponding to SEQ ID NO: 10, including the signal sequence, is shown below as SED ID NO: 17 (and corresponds to Genbank Accession No. AOX15770.1 ) MKSLI FVLLLGAVFAEEDJCIVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHC YKSRIEVRLGEHHIRVNEGTEQFI SSSSVIRHPNYS SYNI DNDIML IKLSKPATLNQYVQ TVALPTECAADGTMCTVSGWGNTMSSVDDGDKLQCLNLPILSHADCSNSYPGMITQS FC AGYLEGGKDSCQGDSGGPVVCNGVLQGVVSWGYGCAERDNPGVYAKVCVLSGWVRDTMAS

Y

[SEQ ID NO: 17]

(wherein the signal sequence and propeptide are underlined and in bold italics, respectively). The zymogen sequence for the variant trypsin X-3 corresponding to SEQ ID NO: 11 , including the signal sequence, is shown below as SED ID NO: 18 (and corresponds to Genbank Accession No. AOX15771.1 )

MKSLI FVLLLGAVFAEED IVGGYECTRHSQAHQVSLNSGYHFCGGSLVSKDWVVSAAHC YKSRIEVRLGEHHIRVNEGTEQFI SSS SVIRHPNYSSYNI DNDIMLIKLSEPATLNQYVQ TVALPTECAADGTMCTVSG GNTMSSVDDGDKLQCLNLP ILSHADCSNSYPGMITQS FC AGYLEGGKDSCQGDSGGPVVCNGVLQGVVS GYGCAERDNPGVYAKVCVLSGWVRDTMAS

Y

[SEQ ID NO: 18]

The zymogen sequence for the variant trypsin Y corresponding to SEQ ID NO: 12, including the signal sequence, is shown below as SED ID NO: 19 (and corresponds to Genbank Accession No. CAD30563.1 ) MIGLALLMLLGAAAAVP2¾EDGRIIGGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSV AHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHEQYDYQTLDYDMMMIKLYHPVEV TQSVAPISLPTGPPDGGMLCSVSGWGNMA GEEVNLPTRLQCLDVPIVETVDCEAAYPGM ISPRMVCAGYMDGGRDACNGDSGSPLVCEGVLTGLVSWGQGCALPNYPGVYVKVYEYLSW IQTTLDANP

[SEQ ID NO: 19]

The trypsin ZT isoforms represented by SEQ ID NOs: 3 to 7 represent the active variants of these trypsins, i.e. variants that have been activated by cleavage of the N terminus of the trypsins. These trypsins are proteins expressed in the pyloric caeca / pancreas (pancreatic tissue in fish) with a number of amino acids on the N terminal end that are important for secretion out of the cells and for keeping the enzyme inactive.

For example, the full-length trypsin ZT isoforms are also disclosed herein as:

Uncleaved Atlantic cod trypsin ZT-1 isoform:

MIGLALL LLGAAAAAVPRDVGKIVGGHECEPNSRPFMASLNYGYHFCGGVLINDQ VLS VAHCWYNPYY QVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYDMMMIKLYHPVE VTQSVAPISLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPG MISPRMMCVGFMDGGRDVCNGDSGSPLVCEGVLTGLVSWGRGCAEPNSPGVYVKVYEFLS IQTTLDANP

[SEQ ID NO: 20]

Uncleaved Atlantic cod trypsin ZT-2 isoform:

MIGLALLMLLGAAAAAVPRDVGKIVGGHECEPNSRPFMASLNYGYHFCGGVLINDQWVLS VAHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNTIFWHELYDYQTLDYD MMIKLYHPVE VTQSVAPISLPTGPPDGGMLCSVSG GNMAMGEEVNLPTRLQCLDVPIVETVDCEAAYPG MISPRMVCAGYMDGGRDACNGDSGSPLVCEGVLTGLVSWGQGCALPNYPGVYVKVYEYLS WIQTTLDANP

[SEQ ID NO: 21] Uncleaved Atlantic cod trypsin ZT-3 isoform:

MI GLALLMLLGAAAAVPREDGRI I GGQDCE PRSRPFMASLNYGYHFCGGVLINDQWVLSV AHCWYNPYYMQVMLGEHDLRVFEGTEQLVKTNT I FWHELYDYQTLDYDMMMIKLYHPVEV TQSVAPI SLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVPIVEPVACQASYPGM I S PRM CVGFMDGGRDVCNGDSGS PLVCEGVLTGLVSWGRGCAE PNS PGVYVKVYE FLSW IQTTLDAN P

[SEQ ID NO: 22]

Uncleaved Atlantic cod trypsin ZT-4 isoform:

MIGLALLMLLGAAAAVPREDGRI I GGQDCEPRSRPFMASLNYGYHFCGGVLINDQWVLSV AHC YNPYYMQVMLGEHDLRVFEGTEQLV TNT I FWHELYDYQTLDYDMMMIKLYHPVEV TQSVAP I SLPTGPPDGGMLCSVSGWGNMAMGEEVNLPTRLQCLDVP IVETVDCEAAYPGM I S PRMVCAGYMDGGRDACNGDSGS PLVCEGVLTGLVSWGQGCAL PNYPGVYVKVYEYLSW I QTTLDANP

[SEQ ID NO: 23]

The term 'amino acid' as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the 'D' form (as compared to the natural V form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as 'alanine' or 'Ala' or TV, the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.

In accordance with convention, the amino acid sequences disclosed herein are provided in the N-terminus to C-terminus direction.

In one embodiment, the polypeptides of the invention comprise or consist of L-amino acids. Persons of skill in the art will appreciate that the polypeptide having protease activity may comprise or consist of a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) of one of the above amino acid sequences, e.g. SEQ ID NOs: 1 to 12, provided that said fragment, variant, derivative or fusion retains (at least in part) the trypsin activity of said amino acid sequences.

Trypsin activity may be determined using methods well known in the art. For example, trypsin assay kits are commercially available from Abeam, Cambridge, UK (see Cat No. ab102531 ) and other suppliers. In one embodiment, trypsin activity is measured using Cbz-Gly-Pro-Arg-p-nitroanilide (Cbz-GPR-pNA) as a substrate (see EP 1 ,202,743 B and Stefansson et al., 2010, Comp Biochem Physiol B Biochem Mol Biol. 155(2): 186-94, the disclosures of which are incorporated herein by reference).

Typically, the protease polypeptide has a specific activity of at least 1 U/mg of polypeptide, for example at least 10 U/mg, at least 50 U/mg, at least 100 U/mg, at least 200 U/mg or at least 500 U/mg. 'U' as used herein means an enzyme unit (one U is the amount of enzyme that catalyzes the conversion of 1 micro-mole of substrate per minute).

In one embodiment the polypeptide comprises or consists of a fragment of the amino acid sequence according to SEQ ID NO: 1 , wherein the fragment exhibits protease activity.

Thus, where the polypeptide comprises an amino acid sequence according to any one of SEQ ID NOs: 1 to 12, it may comprise additional amino acids at its N- and/or C- terminus beyond those of SEQ ID NOs: 1 to 12. Likewise, where the polypeptide comprises a fragment, variant or derivative of an amino acid sequence according to SEQ ID NOs: 1 to 12, it may comprise additional amino acids at its N- and/or C- terminus.

Alternatively, the polypeptide having protease activity may correspond to a fragment of such a wildtype trypsin, such as SEQ ID NOs: 1 to 12, provided that said fragment retains (at least in part) the trypsin activity of the naturally occurring trypsin protein from which it is derived. Thus, the polypeptide may comprise or consist of at least 10 contiguous amino acids of SEQ ID NOs: 1 to 12, e.g. at least 15,16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 contiguous amino acids any one of SEQ ID NOs: 1 to 12. For example, the fragment may comprise or consist of amino acid residues 61 to 77 of any one of SEQ ID NOs:1 to 12. Alternatively, or in addition, the fragment may comprise or consist of amino acid residues 225 to 241 of any one of SEQ ID NOs: 1 to 12. It will be appreciated by persons skilled in the art that the polypeptide of the invention may alternatively comprise or consist of a variant of the amino acid sequence according to any one of SEQ ID NOs: 1 to 12 (or fragments thereof). Such a variant may be a non-naturally occurring variant. By 'variants' of the polypeptide we include insertions, deletions and substitutions, either conservative or non-conservative. In particular, we include variants of the polypeptide where such changes retain, at least in part, the trypsin activity of the said polypeptide.

Such variants may be made using the methods of protein engineering and site-directed mutagenesis well known in the art using the recombinant polynucleotides (see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2000, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference) (8).

In one embodiment, the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to any one of SEQ ID NOs: 1 to 12, or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (as described in Thompson et al., 994, Nuc. Acid Res. 22:4673-4680, which is incorporated herein by reference) (9).

The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1 , window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

Scoring matrix: BLOSUM. Alternatively, the BESTFIT program may be used to determine local sequence alignments.

In one embodiment, the polypeptide having protease activity is a variant of SEQ ID NO:1 or 2 comprising one or more mutated amino acids selected from the group consisting of amino acid positions:

E21 , H25, H29, V47, K49, D50, L63, H71 , H72, R74, N76, T79, Y82, S85, S87, S89, N98, I99, V121 , M135, V138, M145, V148, D150, K154, L160, M175, S179, A183, L185, V212, Y217, P225, A229, V233, L234, V238, N240, Y241 and/or M242. or a fragment thereof which exhibits protease activity (wherein the amino acid sequence and numbering is according to Protein Data Bank [PDB] entry '2EEK!', with the initial isoleucine of SEQ ID NO: 1 or 2 being numbered as position 1-16).

Thus, the polypeptide having protease activity may be a variant of SEQ ID NO:1 or 2 comprising one or more amino acids mutations selected from the group consisting of:

E21T, H25Y, H29(Y/N), V47I, K49E, D50Q, L63I, H71 D, H72N, R74(K/E), N76(T/L), T79(S/N), Y82F, S85A, S87(K/R), S89R, N98T, I99L, V121 I, M135Q, V138I, M145(T/LA /E/K), V148G, D150S, K154(T/V), L160(l/A), M175(K/Q), S179N, A183V, L185G, V212I, Y217(D/H/S), P225Y, A229V, V233N, L234Y, V238I, N240S, Y241 N and/or M242I; or a fragment thereof which exhibits protease activity. In one embodiment, the polypeptide having protease activity may comprise or consist of the amino acid sequence of SEQ ID NO:1 or 2 with one of the following defined mutations in Table 1 (or combinations thereof). Table 1

Sequences of exemplary trypsin polypeptides

Polypeptide name Mutations relative to

SEQ ID NO:1 or 2*

EZA-030 M135Q

EZA-031 M145K, V148G

EZA-032 M175Q

EZA-033 L63I, S85A

EZA-034 L160I

EZA-035 V138I, L160A, A183V

EZA-036 V121 I

EZA-037 V47I, V238I, M242I

EZA-038 V238I

EZA-039 L234Y

Likewise, the polypeptide having protease activity may comprise or consist of the amino acid of SEQ ID NO:1 or 2 with one of the following defined mutations (or combinations thereof):

(a) H25N, N76T

(b) H25N, H29Y

(c) H25N, M135Q

(d) H29Y, T79N, M135Q

(e) I99L, V121 I, L160I, Y217H

(f) V121 I, L160I

(g) H72N, R74E, S87K

(h) H25N, M135Q, Y217H

(i) T79N, V121 I, V212I

(j) H29Y, N76T, I99L, M135Q

(k) K49E, D50Q, N76L, Y82F, S179N, V233N

(I) M145K, V148G, N76L, Y82F, S179N, V233N

(m) H25N, N76T, S87K, K154T

(n) H25Q

(o) H25D

(P) H25S

(q) K24E, H25N

(r) Y97N (S) N100D

(t) A120S, A122S

(u) M135E

(v) V204Q, A122S

(W) T79D

(X) R74D

(y) K49E

(z) K49S.D50Q

(aa) D50Q

(bb) Q178D

(cc) S87R

In one preferred embodiment, the polypeptide having protease activity is a variant of the amino acid sequence of SEQ ID NO:1 or 2 which does not comprise histidine at position 25.

For example, the polypeptide having protease activity may comprise or consist of the amino acid sequence of SEQ ID NO:3 (comprising an H25N mutation; see box in sequence below):

16

IVGGYECTK§SQAHQVSLNSGYHFCGGSLVSKDWVVSAAHCYKSVLRVRLGEHHIRVNEG 79

TEQYISSSSVIRHPNYSSYNINNDIMLIKLTKPATLNQYVHAVALPTECAADAMCTVSG 141

WGNTMSSVADGDKLQCLSLPILSHADCANSYPG ITQS FCAGYLEGGKDSCQGDSGGPV 200

VCNGVLQGVVSWGYGCAERDHPGVYAKVCVLSGWVRDTMANY

[SEQ ID NO: 24] In an alternative preferred embodiment, the polypeptide having protease activity is a variant of the amino acid sequence of SEQ ID NO:1 or 2 which does not comprise lysine at position 160. For example, the polypeptide having protease activity may comprise or consist of the amino acid sequence of SEQ ID NO: 4 (comprising an L160I mutation; see box in sequence below): 16

IVGGYECTKHSQAHQVSLNSGYHFCGGSLVSKDWWSAAHCYKSVLRVRLGEHHIRVNEG 79

TEQYISSSSVIRHPNYSSYNINNDIMLIKLTKPATLNQYVHAVALPTECAADAMCTVSG

141

WGNTMSSVADGDKLQCLSQJPILSHADCANSYPGMITQS FCAGYLEGGKDSCQGDSGGPV 200

VCNGVLQGVVSWGYGCAERDHPGVYAKVCVLSGWVRDT ANY

[SEQ ID NO: 25]

It will be appreciated by persons skilled in the art that the above identified mutations (defined by reference to the amino acid sequence of trypsin I of Atlantic cod, SEQ ID NO:1 or 2) could also be made in trypsins from other species. For example, the specific mutations highlighted in SEQ ID NOS: 3 and 4 (H25N and L160I, respectively) could be made in the trypsin from Alaskan Pollock (for example see GenBank: BAH70476.3, wherein the amino acid sequence of the active trypsin commences at position I20, such that H25 corresponds to H29 in BAH70476.3, etc).

In a further embodiment of the first aspect of the invention, the polypeptide comprises or consists of a fusion protein. By 'fusion' of a polypeptide we include an amino acid sequence corresponding to a polypeptide having protease activity (such as SEQ ID NOS: 1 to 12 or a fragment or variant thereof) fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said polypeptide are also included in the scope of the invention. The fusion may comprise a further portion which confers a desirable feature on the said polypeptide of the invention; for example, the portion may be useful in augmenting or prolonging the therapeutic effect. For example, in one embodiment the fusion comprises human serum albumin or a similar protein.

Alternatively, the fused portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.

In a further embodiment of the first aspect of the invention, the polypeptide, or fragment, variant, fusion or derivative thereof, comprises or consists of one or more amino acids that are modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, i-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications.

It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. Thus, by 'polypeptide' we include peptidomimetic compounds which have an anti-inflammatory activity of the polypeptide of any of SEQ ID NOS: 1 to 12. The term 'peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent. For example, the polypeptides of the invention include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference (10). This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the polypeptide of the invention may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH2NH)- bond in place of the conventional amide linkage.

In a further alternative, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond. It will be appreciated that the polypeptide may conveniently be blocked at its N- or C- terminus so as to help reduce susceptibility to exoproteolytic digestion.

A variety of uncoded or modified amino acids such as D-amino acids and N-methyl amino acids have also been used to modify polypeptides. In addition, a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber ef al., 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thorsett er a/., 1983, Biochem. Biophys. Res. Comm. 111 :166, which are incorporated herein by reference (1 1 , 12). In one preferred embodiment, however, the polypeptide of the invention comprises one or more amino acids modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

It will be appreciated by persons skilled in the art that the polypeptides of the invention may be of any suitable length. Preferably, the polypeptides are between 10 and 30 amino acids in length, for example between 10 and 20, 12 and 18, 12 and 16, or 15 and 20 amino acids in length. Alternatively, the polypeptide may be between 150 and 250 amino acids in length, for example between 200 and 250, 210 and 240, 220 and 230, or 220 and 225 amino acids in length.

In one embodiment, the polypeptide is linear.

In a further embodiment, the polypeptide is a recombinant polypeptide.

The polypeptides of the invention, as well as nucleic acid molecules, vectors and host cells for producing the same, may be made using methods well known in the art (for example, see Green & Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference) (8).

Recombinant methods for producing polypeptides having protease activity, such as trypsins, are disclosed in WO 2015/150799 to Enzymatica, the disclosures of which are incorporated by reference.

Alternatively, the polypeptides of the invention may be synthesised by known means, such as liquid phase and solid phase synthesis (for example, t-Boc solid-phase peptide synthesis and BOP-SPPS).

It will be appreciated by persons skilled in the art that the present invention also includes pharmaceutically acceptable acid or base addition salts of the above described polypeptides. The acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e. salts containing pharmacologically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesu!phonate, ethanesulphonate, benzenesulphonate, p-toluenesulphonate and pamoate [i.e. 1 ,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.

Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the polypeptides. The chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds. Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.

It will be further appreciated that the polypeptides of the invention may be lyophilised for storage and reconstituted in a suitable carrier prior to use. Any suitable lyophilisation method (e.g. spray drying, cake drying) and/or reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate. Preferably, the lyophilised (freeze dried) polypeptide loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (prior to lyophilisation) when rehydrated.

The polypeptides of the invention are typically provided in the form of a therapeutic composition, in which the polypeptide is formulated together with a pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient. Additional compounds may be included in the compositions, including, chelating agents such as EDTA, citrate, EGTA or glutathione. The antimicrobial/therapeutic compositions may be prepared in a manner known in the art that is sufficiently storage stable and suitable for administration to humans and animals. The therapeutic compositions may be lyophilised, e.g., through freeze drying, spray drying, spray cooling, or through use of particle formation from supercritical particle formation.

It will be appreciated by persons skilled in the art that the polypeptides of the invention may also be added to cosmetic formulations, in order to impart a therapeutic and/or prophylactic benefit to a cosmetic product (such as eardrops, wash compositions and the like). The terms 'pharmaceutical composition' and 'medicament' as used herein are to be construed accordingly.

By "pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the trypsin activity of the polypeptide of the invention. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference) (13, 14).

The term "buffer" is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the peptide in the therapeutic preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term "adjuvant" is intended to mean any compound added to the formulation to increase the biological effect of the polypeptide of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as polyvinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

In one embodiment, the polypeptide may be provided together with a stabiliser, such as calcium chloride. The polypeptides of the invention may be formulated into any type of therapeutic composition known in the art to be suitable for the delivery of polypeptide agents.

In one embodiment, the polypeptides may simply be dissolved in water, saline, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers.

In a preferred embodiment, the invention provides a protease polypeptide as described above in an osmotically active solution. For example, the polypeptide may be formulated in glycerol or glycerine. Without wishing to be bound by theory, it is believed that hyperosmotic solutions (such as cod trypsin in glycerol) impart a drying effect in the vicinity of the ear canal, which can create a localised environment less suitable for microbial growth and/or can reduce swelling by drying out an oedema. In parallel, the protease component of the treatment is able to break down dead tissue in the ear canal and disarm or destroy any infecting microbial agents present (e.g. by dissolving a bacterial biofilm and/or disarming virus particles).

An osmotically active solution may also facilitate movement of fluid from within microbial cells to the extracellular milieu. This, in turn, is believed to facilitate the therapeutic effect of the polypeptides of the invention by creating a thin, active barrier that inhibits (at least, in part) the uptake of microbial cells such as bacteria and viruses by the host epithelial cells, e.g. of the ear canal.

In a further embodiment, the therapeutic compositions of the invention may be in the form of a liposome, in which the polypeptide is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example US 4,235,871 , the disclosures of which are incorporated herein by reference (15).

The therapeutic compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. Preparations of such microspheres can be found in US 5,851 ,451 and in EP 0 213 303, the disclosures of which are incorporated herein by reference (16, 17).

In a further embodiment, the therapeutic compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the peptide. The polymers may also comprise gelatin or collagen.

It will be appreciated that the therapeutic compositions of the invention may include ions and a defined pH for potentiation of action of the polypeptides. Additionally, the compositions may be subjected to conventional therapeutic operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc. In one preferred embodiment, the therapeutic composition comprises the polypeptide in a Tris or phosphate buffer, together with one or more of EDTA, xylitol, sorbitol, propylene glycol and glycerol.

In one embodiment the polypeptide is for administration in combination with glycerol and a buffer. The therapeutic compositions according to the invention may be administered via any suitable route known to those skilled in the art. Thus, possible routes of administration include oral, buccal, parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, parenteral, vaginal and rectal. Also administration from implants is possible.

In one preferred embodiment, the therapeutic compositions are administered topically, in a form suitable for delivery to the ear canal. For example, the polypeptide may be formulated as a spray, gel, cream or liquid or conventional liquid for administration.

In an alternative embodiment, the therapeutic compositions are administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. They are conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The therapeutic compositions will be administered to a patient in a pharmaceutically effective dose. A 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, efc, as is well known in the art. The administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a prolonged period.

In one embodiment, the formulation comprises the protease polypeptide at a concentration of between 0.01 and 100 U/g of the formulation, e.g. between 1 and 10 U/g of the formulation. For example, the formulation (e.g. mouthwash, gel, ointment, etc.) may comprise at least 0.1 U/g, at least 0.5 U/g, at least 1 U/g, at least 5 U/g, at least 10 U/g, or at least 50 U/g of the protease polypeptide in the formulation. Thus, the formulation (e.g. mouthwash, gel, ointment, efc.) may comprise no more than 50 U/g, no more than 20 U/g, no more than 10 U/g, no more than 5 U/g, no more than 1 U/g, or no more than 0.1 U/g of the protease polypeptide in the formulation.

Thus, the therapeutic formulation may comprise an amount of a polypeptide, or fragment, variant, fusion or derivative thereof, sufficient to disarm, kill or slow the growth of microorganisms, such as viruses, bacteria and yeasts, within the ear.

Where the polypeptide is formulated for administration to the ear canal (for example as a spray or gel), the therapeutic composition may comprise the polypeptide dissolved in water and glycerol. Exemplary formulations have been marketed as Coidzyme® (by Enzymatica AB, Lund, Sweden) and Penzim ® (by Zymetech ehf, Reykjavik, Iceland). Further suitable spray and gel formulations have been marketed as Kalvatin® (by Enzymatica AB, Lund, Sweden). A particularly preferred therapeutic composition of the invention is described in Example A below.

In one embodiment the polypeptide can be provided in a delivery device, for example in a spray container, which may be configured for ease of delivery to the ear.

In one embodiment the polypeptide is for use in combination with one or more additional active agents. For example, the additional active agents may be selected from the group consisting of antimicrobial agents (including antibiotics, antiviral agents and anti-fungal agents), antiinflammatory agents (including steroids and non-steroidal anti-inflammatory agents) and antiseptic agents. In one embodiment the active agents are one or more antimicrobial agents, for example antibiotics selected from the group consisting of penicillins, cephalosporins, fluoroquinolones, aminoglycosides, monobactams, carbapenems and macrolides.

For example, the antibiotics may be selected from the group consisting of amikacin, amoxicillin, ampicillin, azithromycin, carbenicillin, carbapenems, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, cephalosporins, chloramphenicol, ciprofloxacin, clindamycin, dalacin, dalfopristin, daptomycin, doxycycline, enrofloxacin, ertapenem, erythromycin, fluoroquinolones, gentamicin, marbofloxacin, meropenem, metronidazole, minocycline, moxifloxacin, nafcillin, ofloxacin, oxacillin, penicillin, quinupristin, rifampin, silver sulfadiazine, sulfamethoxazole, teicoplanin, tetracycline, tobramycin, trimethoprim, vancomycin, bacitracin and polymyxin B, or a mixture thereof.

In one embodiment, the additional antibiotics may be for topical or oral administration. In one embodiment, the invention provides an implantable medical device which is impregnated, coated or otherwise treated with a polypeptide as described herein. The implantable medical device may be a device suitable for use within the ear, for example a grommet for insertion into the tympanic membrane. A second, related aspect of the invention provides a polypeptide as defined above in the preparation of a medicament for the treatment or prevention of otitis in a subject. Exemplary embodiments of the second aspect of the invention are described above in relation to the first aspect of the invention.

For example, in one embodiment of the second aspect of the invention the polypeptide is a trypsin or chymotrypsin.

In a further embodiment, the polypeptide comprises or consists of an amino acid sequence of any of SEQ ID NOs: 1 to 12 or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the trypsin activity of said amino acid sequence.

For example, the polypeptide may consist of an amino acid sequence of any of SEQ ID NOs: 1 to 12. In one embodiment the otitis is external otitis (otitis externa).

In one embodiment the otitis is associated with a microbial infection.

A third, related aspect of the invention provides a method for the treatment or prevention of otitis in a subject, the method comprising administering to the subject a therapeutically- effective amount of a polypeptide having protease activity, as defined above in relation to the first aspect of the invention.

Exemplary embodiments of the third aspect of the invention are described above in relation to the first aspect of the invention.

For example, in one embodiment the polypeptide is a trypsin or chymotrypsin.

In one embodiment, the polypeptide comprises or consists of an amino acid sequence of any of SEQ ID NOs: 1 to 12 or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the trypsin activity of said amino acid sequence.

For example, the polypeptide may consist of an amino acid sequence of any of SEQ ID NOs: 1 to 12.

In one embodiment, the otitis is external otitis (otitis externa). In one embodiment, the otitis is associated with a microbial infection.

Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, in one embodiment the invention provides a variant of SEQ ID NO: 1 for use in the treatment of otitis externa caused by a fungal infection.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the above description. It should be understood, however, that the above description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and/or rearrangements.

Preferred, non-limiting examples which embody certain aspects of the invention will now be described. EXAMPLES

Example A: Exemplary therapeutic formulation An exemplary stock solution of a polypeptide of the invention, trypsin I from Atlantic cod (SEQ ID NO:1 or 2), may be formulated as shown in Table 2:

Table 2

Exemplary formulation

Water is used as solvent and the pH is adjusted to 7.5.

Optionally, the formulation may comprise a bitter agent to deter oral consumption, such as 20 ppm of Denatonium benzoate.

Example B: Case study I - Pseudomonas otitis

Subject A German Shepard dog, male, 6 years old, diagnosed with chronic, treatment resistant Pseudomonas otitis, with rupture of tympanic membranes.

The dog had had otitis for the last 10 months. The dog was recruited to evaluate Kalvatin dermal gel as a "last option prior to euthanasia" treatment.

Treatment

Following ear flush under general anesthesia, Kalvatin dermal gel applied to the affected area of the ears.

Kalvatin dermal gel was applied in the ear canals twice daily for 14 days.

Result

The treatment resistant Pseudomonas otitis infection was cleared at the 14 days recheck at the clinic.

Treatment has been maintained without relapse for 14 months (twice weekly application of Kalvatin in the ears).

The subject remains in remission.

Example C: Case study II - Malassezia otitis

Subject A female welsh springer spaniel.

The dog was diagnosed with chronic, treatment resistant, purulent Malassezia otitis, with which it had suffered for several months. Treatment

Following ear flush under general anesthesia, Kalvatin dermal gel applied to the affected area of the ears. Kalvatin dermal gel was applied in the ear canals twice daily for 14 days. Result

The infection cleared upon commencement of treatment with Kalvatin.

Treatment has been maintained without relapse for over 6 months, June-Dec 2016 (twice weekly application of Kalvatin in the ears).

The subject remains in remission.

Example D: Case study III - External otitis (swimmer's ear)

Case report on two human patients with external otitis (Swimmer's ear). Patient 1. Sixty-year-old male physician. Healthy, no skin disorders. Served as Swedish Navy Diving physician during the 1980s and developed a recurrent external otitis, which has continued to appear once or twice a year in spite of a regular life with only occasional scuba diving. At least once presented with a massive external otitis requiring acute ENT specialist care with removal of debris from the ear canal, insertion of a wick impregnated with alcohol, and subsequent treatment with corticosteroid and broad-spectrum antibiotic solution. The patient has since treated annual relapses with antibiotic-steroid ear drops through early symptom intervention. In Nov 2017, the patient again experienced an external otitis that was neglected due to travelling and thus not treated while symptoms were still moderate. Patient developed painful swelling of the ear canal, otorrhea and unilateral hearing loss due to extensive edema, and presented with a 1-2 mm broad and inflamed skin crack at the orifice of the ear canal. Being a physician and familiar with the mode of action for ColdZyme® Throat Spray (Enzymatica AB, Lund, Sweden) - with a drying effect from the hyperosmotic action of glycerol, a debris degrading effect of the trypsin, and a biofilm-degrading and micro-organism deactivating effect of the trypsin - the patient conducted an experiment on himself wherein ColdZyme was sprayed in the ear canal twice before going to bed. Next morning, hearing loss and most of the pain had disappeared as a sign of de-swelling. Patient continued to applicate ColdZyme® twice more during the next 12 hours and the following day the external otitis was completely healed, without any signs or symptoms. At follow-up 8 weeks later, patient was still without problems.

Patient 2. Sixty-year-old female dentist. Experienced decades of recurrent episodes of external otitis with otorrhea and moderate pain. Patient kept the disorder under control with intermittent early intervention with acetic acid-based ear spray (Bulow's solution, Otinova®, Amellnova, Sweden). The patient presented for the first time with swollen ear canal, and severe pain when gently pushing and moving the ear canal. She sprayed ColdZyme® Throat Spray in the ear when going to bed. Next morning, after one treatment the evening before, most of the pain was gone. The patient treated herself once more and 12 hours later she was symptom free. At follow-up 4 weeks later, no relapse had occurred. REFERENCES

(1 ) U.S. Food and Drug Administration. (2 September 2016). FDA issues final rule on safety and effectiveness of antibacterial soaps [online] Available at: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm517478.htm

[Accessed 8 September 2016],

(2) Moriello, K. (n.d.). Overview of Otitis Externa [online] Available at: http://www.merckvetmanual.com/eye-and-ear/otitis-externa/overview-of-otitis-externa [Accessed 18 January 2017]

(3) Nuttall, T. (2016) "Successful management of otitis externa." In Practice 38.Suppl 2 (2016): 17-21.

(4) Hillier, A (2005). Treatment of Pseudomonas otitis in the dog [online] Available at: http://veterinarymedicine.dvm360.com/treatment-pseudomonas-otitis-dog-sponsored- pfizer [Accessed 18 January 2017]

(5) Asgeirsson B, Fox JW, Bjarnason JB.(1989) Purification and characterization of trypsin from the poikilotherm Gadus morhua. Eur J Biochem. 1989;180:85-94.

(6) Asgeirsson & Bjarnason (1991 ) Structural and kinetic properties of chymotrypsin from Atlantic cod (Gadus morhua). Comparison with bovine chymotrypsin. Comp. Biochem. Physiol. B 998:327-335 (7) EP1202743 B1 , Bjarnason

(8) Green & Sambrook (2012) Molecular Cloning: a Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press (9) Thompson et al. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nuc. Acid Res. 22:4673-4680

(10) Meziere et al. (1997) In vivo T helper cell response to retro-inverso peptidomimetics. J. Immunol. 159, 3230-3237 (11 ) Veber ei al. (1978) Conformationally restricted bicyclic analogs of somatostatin Proc. Natl. Acad. Sci. USA 75:2636

(12) Thorsett ei al. (1983) Dipeptide mimics. Conformationally restricted inhibitors of angiotensin-converting enzyme Biochem. Biophys. Res. Comm. 1 11 :166

(13) Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) (14) Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000)

(15) US 4,235,871 , Papahadjopoulos & Szoka (16) US 5,851 ,451 , Takeda Chemical lndustries, Ltd.

(17) EP0213303 B1 , Ekman & Lindahl

(18) Spellberg, Brad, John G. Bartlett, and David N. Gilbert. The future of antibiotics and resistance. New England Journal of Medicine 368.4 (2013): 299-302.

(19) Pozo & Patel, 2007. The challenge of treating biofilm-associated bacterial infections. Clin. Pharmacol. Ther. 82:204-9 (20) Stewart & Costerton, 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358:135-8

(21 ) Stefansson ei al., 2010. Characterization of cold-adapted Atlantic cod (Gadus morhua) trypsin I— Kinetic parameters, autolysis and thermal stability. Comparative Biochem. Physiol: Part B - Biochem. & Mol. Biol., 155(2): 186-194

(22) Goguen et al. 2014 External otitis: Pathogenesis, clinical features, and diagnosis [online] Available at: http://www.uptodate.com/contents/external-otitis-pathogenesis- clinical-features-and-diagnosis

Claims

1. A polypeptide having protease activity for use in the treatment or prevention of otitis in a mammal.

2. A polypeptide for use according to Claim 1 , wherein the otitis is external otitis (otitis externa).

3. A polypeptide for use according to Claim 1 or 2, wherein the otitis is associated with a microbial infection.

4. A polypeptide for use according to Claim 3, wherein the microbial infection is selected from the group consisting of bacterial infections, viral infections, fungal infections and yeast infections.

5. A polypeptide for use according to Claim 4, wherein the microbial infection is a bacterial infection.

6. A polypeptide for use according to Claim 5, wherein the bacterial infection is caused by bacteria selected from the following genera: Pseudomonas, Staphylococcus,

Streptococcus, Corynebacterium, Enterococcus, Proteus, Escherichia, Bacteroides, Peptostreptococcus, Haemophilus and Clostridium.

7. A polypeptide for use according to Claim 4, wherein the microbial infection is a fungal infection.

8. A polypeptide for use according to Claim 7, wherein the fungal infection is caused by fungi selected from the following genera: Malassezia, Candida and Aspergillus.

9. A polypeptide for use according to any one of Claims 3 to 8, wherein the microbial infection comprises formation of a biofilm.

10. A polypeptide for use according to Claim 4, wherein the microbial infection is a viral infection.

11. A polypeptide for use according to Claim 10, wherein the viral infection is caused by virus selected from the following genera: rhinovirus, coronavirus, influenza virus and respiratory syncytial virus.

12. A polypeptide for use according to any one of the preceding claims, wherein the otitis is associated with an allergic or dermatologic condition (such as eczema).

13. A polypeptide for use according to any one of the preceding claims, wherein the otitis is treatment-resistant otitis.

14. A polypeptide for use according to any one of the preceding claims, wherein the otitis is acute.

15. A polypeptide for use according to any one of the preceding claims, wherein the otitis is chronic.

16. A polypeptide for use according to any one of the preceding claims, wherein the otitis is unilateral.

17. A polypeptide for use according to any one of the preceding claims, wherein the otitis is bilateral.

18. A polypeptide for use according to any one of the preceding claims, wherein the mammal is human.

19. A polypeptide for use according to any one of Claims 1 to 17, wherein the mammal is a non-human mammal, for example a dog.

20. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide having protease activity is selected from the group consisting of serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases and metalloproteases.

21. A polypeptide for use according to Claim 20, wherein the protease is a serine protease.

22. A polypeptide for use according to Claim 20 or 21 , wherein the protease is a trypsin or chymotrypsin, or a component of a mixture thereof.

23. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide having protease activity is cold-adapted.

24. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is naturally occurring.

25. A polypeptide for use according to Claim 24, wherein the polypeptide is a marine serine protease.

26. A polypeptide for use according to Claim 25, wherein the marine serine protease is obtainable from cod, pollock, salmon or krill.

27. A polypeptide for use according to Claim 26, wherein the marine serine protease is obtainable from Atlantic cod.

28. A polypeptide for use according to any one of Claims 25 to 27, wherein the marine serine protease is a trypsin.

29. A polypeptide for use according to any one of the preceding claims wherein the polypeptide is trypsin I, trypsin X or trypsin ZT from Atlantic cod.

30. A polypeptide for use according to any one of the preceding claims wherein the activity of the trypsin ranges from 1 U/mg to 1 U/g of the polypeptide, for example between 50 U/mg and 500 U/mg of the polypeptide.

31. A polypeptide for use according to any one of Claims 1 to 23 wherein the polypeptide is non-naturally occurring.

32. A polypeptide for use according to any one of the preceding claims wherein the polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 12, or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the protease activity of said amino acid sequence.

33. A polypeptide for use according to Claim 32 wherein the polypeptide comprises or consists of an amino acid sequence selected from any one of SEQ ID NOs: 1 to 12.

34. A polypeptide for use according to any one of Claims 1 to 32 wherein the polypeptide comprises or consists of a fragment of the amino acid sequence according to

SEQ ID NOs: 1 to 12.

35. A polypeptide for use according to Claim 34 wherein the fragment comprises or consists of at least 15 contiguous amino acid of any one of SEQ ID NOs: 1 to 12, for example at least 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230 or 240 contiguous amino acids of any one of SEQ ID NOs: 1 to 12.

36. A polypeptide for use according to any one of Claims 1 to 32 wherein the polypeptide comprises or consists of a variant of the amino acid sequence according to any one of SEQ ID NOs: 1 to 12.

37. A polypeptide for use according to Claim 36 wherein the variant is a non-naturally occurring variant.

38. A polypeptide for use according to Claim 36 or 37 wherein the variant has an amino acid sequence which has at least 50% identity with the amino acid sequence according to any one of SEQ ID NOs: 1 to 12, or a fragment thereof, for example at least 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% or at least 99% identity.

39. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide comprises or consists of a fusion protein.

40. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide, or fragment, variant, fusion or derivative thereof, comprises one or more amino acids that are modified or derivatised.

41. A polypeptide for use according to Claim 40, wherein the one or more amino acids are modified or derivatised by PEGylation, amidation, esterification, acylation, acetylation and/or alkylation.

42. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is between 150 and 250 amino acids in length, for example between 200 and 250, 210 and 240, 220 and 230, or 220 and 225 amino acids in length.

43. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is linear.

44. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is a recombinant polypeptide.

45. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is provided as an osmotically active solution.

46. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is administered in combination with glycerol and/or a buffer.

47. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is provided in a form suitable for delivery to the ear canal.

48. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is provided in a spray, gel, cream or liquid.

49. A polypeptide for use according to any one of the preceding claims, wherein the polypeptide is for use in combination with one or more additional active agents.

50. A polypeptide for use according to Claim 49, wherein the additional active agents are selected from the group consisting of antimicrobial agents (including antibiotics, antiviral agents and anti-fungal agents), anti-inflammatory agents (including steroids and non-steroidal anti-inflammatory agents) and antiseptic agents.

51. A polypeptide for use according Claim 50, wherein the one or more antimicrobial agents are antibiotics selected from the group consisting of penicillins, cephalosporins, fluoroquinolones, aminoglycosides, monobactams, carbapenems and macrolides.

52. Use of a polypeptide having protease activity in the preparation of a medicament for the treatment or prevention of otitis in a subject, wherein the polypeptide is as defined in any one of Claims 1 to 44 above.

53. The use according to Claim 52, wherein the polypeptide is a trypsin or chymotrypsin.

54. The use according to Claim 52 or 53, wherein the polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 12 or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the trypsin activity of said amino acid sequence.

55. The use according to Claim 54, wherein the polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 12.

56. The use according to any one of Claims 52 to 55, wherein the otitis is external otitis (otitis externa).

57. The use according to any one of Claims 52 to 56, wherein the otitis is associated with a microbial infection.

58. A method for treatment or prevention of otitis in a subject, comprising administering to the subject a therapeutically-effective amount of a polypeptide having protease activity, wherein the polypeptide is as defined in any one of Claims 1 to 44 above.

59. The method according to Claim 58, wherein the polypeptide is a trypsin or chymotrypsin.

60. The method according to Claim 58 or 59, wherein the polypeptide comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 12 or a fragment, variant, derivative or fusion thereof (or a fusion of said fragment, variant or derivative) which retains the trypsin activity of said amino acid sequence.

61. The method according to Claim 60, wherein the polypeptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 12.

62. The method according to any one of Claims 58 to 61 , wherein the otitis is external otitis (otitis externa).

63. The method according to any one of Claims 58 to 62, wherein the otitis is associated with a microbial infection.

64. A polypeptide for use in the treatment or prevention of otitis in a subject as described herein with reference to the description.

65. Use of a polypeptide in the preparation of a medicament for the treatment or prevention of otitis in a subject as described herein with reference to the description.

66. A method for the treatment or prevention of otitis in a subject with reference to the description.