Oxidised papain-like protease

Abstract

The present invention relates generally to a particulate composition, and more particularly to a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof. The present disclosure also relates to processes of producing said compositions, as well as medical and non-medical uses thereof of oxidised papain-like proteases and particulate compositions comprising papain-like proteases.

Description

[0001] OXIDISED PAPAIN-LIKE PROTEASE

[0002] FIELD

[0003] [1] The present invention relates generally to a particulate composition, and more particularly to a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof. The present disclosure also relates to processes of producing said compositions, as well as medical and non-medical uses thereof of oxidised papain-like proteases and particulate compositions comprising papain-like proteases.

[0004] BACKGROUND

[0005] [2] Cysteine proteases are a large class of proteolytic enzymes that play important roles in a broad range of biological processes. These proteases are characterised by a relatively conserved catalytic dyad comprising a nucleophilic cysteine residue in close proximity to a histidine residue. Their mechanism of action is similar to that of other proteases; binding of the substrate causes activation of the nucleophilic cysteine residue, which then cleaves the substrate at a specific peptide residue.

[0006] [3] Papain-like proteases (also referred to as “PLPs”) are one of the most well-studied sub-families of cysteine proteases. This family comprises endo- and exopeptidases that share structural and functional properties with papain, a cysteine protease originally isolated from papaya. In animals, PLPs are also referred to as cysteine cathepsins. Depending on the organisms in which they are expressed, PLPs play different biological roles, ranging from cell differentiation to cellular senescence. Given the common origin of these proteases, their structures are relatively conserved. The mature form of PLPs is in general monomeric globular proteins composed of two halves known as the L- and R- domains, with the active site being situated in the cleft formed at the interface of these domains to which the peptide substrate binds. PLPs comprise a conserved catalytic dyad composed of a cysteine-histidine ion pair. Further residues are also required for proper positioning of the catalytic dyad in order to effect cleavage of the substrates.

[0007] [4] The biological importance of the PLP family is evidenced by the fact that a number of human PLPs represent important clinical drug targets. For example, various inhibitors of cathepsin K have been developed for the treatment of osteoporosis. In addition, the clinical benefit of PLPs themselves has also been previously recognised. In particular, PLPs have been shown to exhibit pleiotropic therapeutic effects, for example, anti-oedematous, anti- infective, anti-inflammatory, anticancer, antimetastatic, antithrombotic, and anti-fibrinolytic activities (see, e.g., Maurer, Cell. Mol. Life Sci. 58 (2001) 1234-1245). WO 2009 / 033536, for example, describes the treatment of a range of disorders using the PLP bromelain, including cancer and bacterial infection. Similarly, WO 2021 / 255625 describes the PLP ananain as demonstrating utility as an immunosuppressant and as a debriding agent. Both of these documents emphasise the need to prevent oxidation of the PLPs during their preparation for administration, in line with the general understanding in the field that oxidation of the PLP active cysteine residue would block its catalytic function.

[0008] [5] PLPs thus show clear promise as a therapy for a number of different conditions and are also widely taken as nutrition. Papain and bromelain, for example, are both widely used in nutritional supplements. Despite this promise, the biological mechanisms by which PLPs exert their therapeutic effects remains less clear, with further research being required to better understand the role PLPs play in the treatment of these conditions. There remains, therefore, an ongoing need in the art for further PLP compositions having use in therapy, including for the treatment of cancer and bacterial and viral infection.

[0009] [6] The present disclosure thus meets the foregoing needs with the various aspects and embodiments defined herein.

[0010] SUMMARY

[0011] [7] According to a first aspect, there is provided a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0012] [8] According to a second aspect, there is provided a pharmaceutical composition comprising a particulate composition according to the first aspect and one or more carriers, excipients, or diluents, wherein the pharmaceutical composition optionally further comprises a solvent.

[0013] [9] According to a third aspect, there is provided a process for preparing a composition according to the first aspect, the process comprising the steps of:

[0014] (ai) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less;

[0015] (aii) treating the particles with an oxidising agent; or

[0016] (bi) providing a papain-like protease or a fragment or a variant thereof; (bi i) treating the papain-like protease or the fragment or the variant thereof with an oxidising agent; and

[0017] (biii) preparing particles comprising the oxidised form of the papain-like protease or the fragment or the variant thereof, wherein the particles have a particle size of about 1000 nm or less.

[0018]

[0010] According to a fourth aspect, there is provided a composition obtained or obtainable by the process of the third aspect.

[0019]

[0011] According to a fifth aspect, there is provided an oxidised form of a papain-like protease or a fragment or a variant thereof, for use in therapy.

[0020]

[0012] According to a sixth aspect, there is provided an oxidised form of a papain-like protease or a fragment or a variant thereof for use in treating cancer.

[0021]

[0013] According to a seventh aspect, there is provided an oxidised form of a papain-like protease or a fragment or a variant thereof, for use in treating a bacterial or viral infection.

[0022]

[0014] According to an eighth aspect, there is provided a particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof for use in treating a bacterial or viral infection.

[0023]

[0015] According to a ninth aspect, there is provided a particulate composition according to the first aspect of the invention for use in therapy.

[0024]

[0016] According to a tenth aspect, there is provided a non-therapeutic use of a particulate composition according to the first aspect, wherein the use comprises applying the composition to a surface as a disinfectant.

[0025]

[0017] These aspects and embodiments are set out in the appended independent and dependent claims. It will be appreciated that features of the dependent claims may be combined with each other and with features of the independent claims in combinations other than those explicitly set out in the claims. Furthermore, the approaches described herein are not restricted to specific embodiments such as those set out below, but include and contemplate any combinations of features presented herein.

[0026]

[0018] The foregoing and other objects, features, and advantages of the present disclosure will appear more fully hereinafter from a consideration of the detailed description that follows along with the accompanying drawings. It is to be expressly understood, however, that the drawings are for illustrative purposes and are not to be construed as defining the limits of the disclosure.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

[0019] Figure 1A shows cytotoxicity IC50 data from Example 2 for the particulate composition of Comparative Example 1 following step D for the 5 cancer cell lines tested. The data is in the form of graphs plotting the % cytotoxicity achieved against the log concentration of the compositions (in mM) being tested.

[0029]

[0020] Figure 1B shows cytotoxicity IC50 data from Example 2 for the particulate composition of Example 1 following step B for the 5 cancer cell lines tested. The data is in the form of graphs plotting the % cytotoxicity achieved against the log concentration of the compositions (in mM) being tested.

[0030]

[0021] Figure 1C shows cytotoxicity IC50 data from Example 2 for the particulate composition of Example 1 following step C for the 5 cancer cell lines tested. The data is in the form of graphs plotting the % cytotoxicity achieved against the log concentration of the compositions (in mM) being tested.

[0031]

[0022] Figure 1D shows cytotoxicity IC50 data from Example 2 for the particulate composition of Example 1 following step D for the 5 cancer cell lines tested. The data is in the form of graphs plotting the % cytotoxicity achieved against the log concentration of the compositions (in mM) being tested.

[0032]

[0023] Figure 2 shows bacterial inhibition data from Example 3 in the form of a bar chart plotting bacterial inhibition (in mm diameter) achieved for the four bacterial strains tested for the oxidised bromelain compared with a control non-oxidised form.

[0033] DETAILED DESCRIPTION

[0034]

[0024] While various exemplary embodiments are described or suggested herein, other exemplary embodiments utilizing a variety of methods and materials similar or equivalent to those described or suggested herein are encompassed by the general inventive concepts. Those aspects and features of embodiments which are implemented conventionally may not be discussed or described in detail in the interests of brevity. It will thus be appreciated that aspects and features of apparatus and methods described herein which are not described in detail may be implemented in accordance with any conventional techniques for implementing such aspects and features. Such techniques are explained in the literature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any reference to prior art documents in this specification is not to be considered an admission that such prior art is widely known or forms part of the common general knowledge in the field. All publications mentioned in the specification are herein incorporated by reference.

[0035]

[0025] As used in this specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0036]

[0026] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” are used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0037]

[0027] The ranges provided herein provide exemplary amounts of each of the components. Each of these ranges may be taken alone or combined with one or more other component ranges.

[0038]

[0028] As used herein, the term “at least” includes the end value of the range that is specified.

[0039]

[0029] As used herein, wt% means “weight percentage” as the basis for calculating a percentage. Unless indicated otherwise, all % values are calculated on a weight basis, and are provided with reference to the total weight of the product in which the substance is present.

[0040]

[0030] As used herein, “substantially free” means no more than trace amounts, i.e. the amount of the substance(s) concerned is negligible. In various embodiments, “substantially free” means no more than 1000 ppm, preferably no more than 100 ppm, more preferably no more than 10 ppm, even more preferably no more than 1 ppm of the substance(s) concerned.

[0041]

[0031] In all aspects of the present disclosure, the disclosure includes, where appropriate, all enantiomers and tautomers of the compounds disclosed herein. A person skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and / or tautomers may be isolated / prepared by methods known in the art. Some of the compounds disclosed herein may exist as stereoisomers and / or geometric isomers - e.g. they may possess one or more asymmetric and / or geometric centres and so may exist in two or more stereoisomeric and / or geometric forms. The disclosure also includes, where appropriate, the corresponding isozymes or isoenzymes of the papain-like proteases disclosed herein.

[0032] The present disclosure contemplates the use of all the individual stereoisomers and geometric isomers of those compounds, and mixtures thereof. The terms used in the claims encompass these forms. The disclosure also includes, where appropriate, the corresponding isozymes or isoenzymes of the papain-like proteases disclosed herein.

[0042]

[0033] As used in this specification, the words “comprises”, “comprising”, and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean “including, but not limited to”. The terms “comprises”, “comprising”, and similar words also include the term “consisting of’.

[0043]

[0034] The general inventive concept of the invention is centred on the Applicant’s unexpected finding that oxidised PLPs resulted in improved efficacy in a number of different therapeutic applications compared with the native non-oxidised form. In particular, it was identified that oxidised PLPs exhibited improved cytotoxicity against a number of different cancer cell lines, compared with the non-oxidised form. Oxidised forms of the PLP were also identified as having an improved ability to inhibit the growth of bacteria compared with equivalent non-oxidised forms. It was further identified that oxidised forms of PLPs also exhibited effective virucidal activity. These advantages were surprising given the teachings in the prior art; for example, whilst WO 2009 / 033536 and WO 2021 / 255625 both describe the therapeutic utility of certain PLPs, both of these documents emphasise the need to prevent oxidation of the PLPs during production, in line with the general understanding in the field that oxidation of the PLP active cysteine residue would block its catalytic function.

[0044]

[0035] The inventive concept of the invention further encompasses the finding that PLPs had particular efficacy when administered in particulate form below a particular average particle size, specifically where the particles administered had an average particle size of about 1000 nm or less. The combination of particulate compositions comprising oxidised forms of PLPs were found to be particularly effective.

[0045]

[0036] For ease of reference, these and further features of the present disclosure are now discussed under appropriate section headings. However, the teachings under each section are not limited to the section in which they are found. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0046] PARTICULATE COMPOSITION

[0037] In a first aspect, a particulate composition is provided, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0047]

[0038] As used herein, the term “papain-like protease” (sometimes shortened to “PLP”) is used to refer to a cysteine protease in Clan CA as classified under the MEROPS classification system (see Clan CA contains the families of cysteine peptidases known to have a common origin, and thus which are structurally similar to, papain. Cysteine proteases in Clan CA also share the cysteine / histidine catalytic dyad of papain and have other similarities in the structure of the catalytic site. Further information on the classification of cysteine proteases into Clan CA and other clans may be found in Barrett and Rawlings, Perspectives in Drug Discovery and Design, Vol. 6, pp. 1-11 , which is incorporated herein by reference. The Clan CA of the MEROPS classification includes the cysteine peptidase family C1 , as well as other families containing papain-like proteases.

[0048]

[0039] As used herein, a papain-like protease that belongs to the “cysteine peptidase family C1” or “cysteine peptidase family C1A” is used to refer to a cysteine protease in the family C1 or C1 A as classified under the MEROPS classification system referred to above.

[0049]

[0040] As used herein, a “variant” of a papain-like protease is used to refer to a protease whose amino acid sequence has been modified in such a manner that the variant protease in question retains at least one or all of its endogenous functions, in particular its proteolytic activity. A variant protease can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the parent protease.

[0050]

[0041] In various embodiments, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the variant PLP retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. In preferred embodiments, a “variant” within the meaning of the present disclosure may have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0042] In various embodiments, conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0051]

[0043] As would be readily understood by the person skilled in the art, the effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool e.g. SIFT (Vaser, R., et al., 2016. Nature protocols, 11 (1), pp.1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76(1), pp.7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13(1), pp.1-12), REVEL (loannidis, N.M., et al., 2016. The American Journal of Human Genetics, 99(4), pp.877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24(8), pp.2125-2137), and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids research, 39(17), pp.el 18-e118) or based on clinical data e.g. ClinVar (Landrum, M.J., et al., 2016. Nucleic acids research, 44(D1), pp.D862-D868). All of these references being incorporated by reference. Suitable additions, deletions, substitutions, modifications, replacements and / or variations may be considered tolerated, benign, and / or likely benign.

[0052]

[0044] As above, in preferred embodiments, the variant of the papain-like protease retains proteolytic activity. The proteolytic activity of the variant may be determined by any suitable assay, and expressed as any suitable unit. The proteolytic activity of the variant may be determined by any suitable assay, and expressed as any suitable unit. In preferred embodiments, the proteolytic activity of the variant may be determined by measuring the rate at which it catalyses the cleavage of a model substrate, such as pGlu-Phe-Leu p-nitroanilide. In other preferred embodiments, the proteolytic activity may be expressed in terms of gelatin digesting units (GDU) per gram. One GDU corresponds to the amount of variant that will liberate, after 20 minutes digestion at 45°C, 1 mg of amino nitrogen from gelatin at pH 4.5.

[0053]

[0045] In various embodiments, the variant of the papain-like protease is a variant comprising an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the parent papain-like protease. In preferred embodiments, the variant of the papain-like protease is a variant comprising an amino acid sequence which is at least 90% identical to the amino acid sequence of the parent papain-like protease. In particularly preferred embodiments, the variant of the papain-like protease is a variant comprising an amino acid sequence which is at least 95% identical to the amino acid sequence of the parent papain-like protease. Most preferably, the variant of the papain-like protease is a variant comprising an amino acid sequence which is at least 98% identical to the amino acid sequence of the parent papain-like protease.

[0054]

[0046] As used herein, a “fragment” of a papain-like protease is used to refer to a variant sequence that represents a portion of the full-length papain-like protease. The portion may be of any size that retains at least one or all of its endogenous functions, in particular its proteolytic activity. In preferred embodiments, the fragment of the papain-like protease retains the proteolytic activity. The proteolytic activity of the fragment may be determined by any suitable assay, and expressed as any suitable unit. In preferred embodiments, the proteolytic activity of the fragment may be determined by measuring the rate at which it catalyses the cleavage of a model substrate, such as pGlu-Phe-Leu p-nitroanilide. In other preferred embodiments, the proteolytic activity may be expressed in terms of gelatin digesting units (GDU) per gram. One GDU corresponds to the amount of fragment that will liberate, after 20 minutes digestion at 45°C, 1 mg of amino nitrogen from gelatin at pH 4.5.

[0055]

[0047] The fragment of the papain-like protease may be both "naturally" derived, i.e. by permitting auto-digestion of protease, or artificially derived, e.g. by cleaving the protease with another protease.

[0056]

[0048] Whilst the present disclosure encompasses variants and fragments of the oxidised form of papain-like protease, in preferred embodiments the particulate composition comprises an oxidised form of the papain-like protease - i.e. the full-length and non-variant form of the papain-like protease.

[0057]

[0049] In various embodiments, the papain-like protease is naturally occurring. As used herein, the term “naturally occurring” in connection with a “papain-like protease” is used to refer to a papain-like protease that is naturally expressed by a living organism, such as a plant or an animal. Papain-like proteases that are naturally occurring are typically well-tolerated in humans and generally exhibit low toxicity.

[0050] In various embodiments, the papain-like protease is plant-derived. As used herein, the term “plant-derived” in connection with a papain-like protease is used to refer to a papain-like protease that is naturally expressed by a plant. Plant-derived papain-like proteases are in general well-tolerated and non-toxic in animals. For example, papain (the archetypal papainlike protease) is found naturally in the fruit of the papaya plant and has a long history of safe use in humans. Similarly, the papain-like protease bromelain is found naturally in both the stem and fruit of the pineapple plant and has a similar history of safe use in humans. Additionally, plant-derived proteases may in general be advantageously and relatively cheaply obtained by direct extraction from plant material, resulting in a more sustainable manufacturing process with lower energy requirements that does not require complicated synthetic production.

[0058]

[0051] Any suitable plant source may be used to obtain the plant-derived papain-like protease. The plant-derived papain-like protease may be expressed in a number of different plant species. Non-limiting examples of suitable plant families are the Caricaceae family (comprising for example, the papaya plant, Carica papaya) the Moraceae family (comprising for example, the fig plant species Ficus carica), the Anacardiaceae family (comprising for example, the mango plant species Mangifera indica), the Musaceae family (comprising for example, the banana plant species Musa acuminata and Musa balbisiana), the Brassicaceae family (comprising for example, the broccoli plant species Brassica oleracea var. italica), the Actinidiaceae family (comprising for example, the kiwifruit plant species Actinidia deliciosa), and the Bromeliaceae family (comprising for example, the pineapple plant species Ananas comosus).

[0059]

[0052] Thus, in preferred embodiments, the plant-derived papain-like protease may be derived from Carica papaya, Ficus carica, Mangifera indica, Musa acuminata, Musa balbisiana, Brassica oleracea var. italica, Actinidia deliciosa, or Ananas comosus. In particularly preferred embodiments, the papain-like protease is a pineapple-derived papainlike protease, that is, it is a papain-like protease naturally occurring in the plant species Ananas comosus.

[0060]

[0053] The plant-derived papain-like protease may be obtained by any suitable means known in the art. The papain-like protease may, for instance, be obtained by physical extraction from the plant material itself. Bromelain may, for example, be obtained by first pressing pineapple to obtain juice, following by centrifugation, ultrafiltration, and lyophilisation. Alternatively, the plant-derived papain-like protease may obtained by recombinant expression, as is described, for example, in WO 2009 / 033536 which is incorporated herein by reference for said process.

[0054] It will be appreciated that a naturally occurring papain-like protease may alternatively be animal-derived, that is, a papain-like protease that is naturally expressed in an animal. In other embodiments, the papain-like protease is therefore animal-derived. Preferred, but nonlimiting examples of such animal-derived papain-like proteases are cathepsins.

[0061]

[0055] In preferred embodiments, the papain-like protease is a fruit-derived papain-like protease. As used herein, the term “fruit-derived” in connection with a papain-like protease is used to refer to a papain-like protease that is naturally expressed by a plant in its fruit. Any suitable fruit may be used to derive the papain-like protease.

[0062]

[0056] In particularly preferred embodiments, the papain-like protease is a “pineapple- derived” papain-like protease, which herein refers to a papain-like protease that naturally occurs in the plant species Ananas comosus, a member of the Bromeliaceae plant family. Papain-like proteases naturally expressed in Ananas comosus are particularly well-studied, with their therapeutic utility established.

[0063]

[0057] In various embodiments, the papain-like protease is thus derived from a plant of the Bromeliaceae plant family, with the pineapple plant (Ananas comosus) of this family preferred as the plant source. Crude pineapple stem extract contains at least four distinct cysteine proteinases, with stem bromelain (EC 3.4.22.32; SEQ ID NO: 1), fruit bromelain (EC 3.4.22.33; SEQ ID NO: 2), ananain (EC 3.4.22.31 ; SEQ ID NO: 3), and comosain being most prominent. Each of these four cysteine proteases are papain-like proteases of the C1 peptidase family. That is, each of these proteases are closely related to one another and exhibit similar structural and functional properties. In preferred embodiments, the pineapple-derived papain-like protease is accordingly selected from stem bromelain, fruit bromelain, ananain, comosain, and mixtures thereof.

[0064]

[0058] In particularly preferred embodiments, the papain-like protease is bromelain. As used herein, the term “bromelain” is used to refer to either stem bromelain, fruit bromelain, or mixtures thereof. Sources of bromelain are known in the art (see e.g. Arshad, Z.I.M., et al., 2014. Applied microbiology and biotechnology, 98(17), pp.7283-7297). For example, bromelain may be obtained by physical extraction and purification from a crude pineapple source. Alternatively, bromelain may be obtained by recombinant expression, as is described, for example, in WO 2009 / 033536.

[0065]

[0059] As noted herein, the pineapple plant (Ananas comosus) is preferred as the plant source for the papain-like protease of the present disclosure. Pineapple is a well-known source of bromelain, which is found in various parts of the pineapple plant, including the fruit and stem. In the present disclosure, bromelain is a preferred example of a papain-like protease due to its wide availability at a commercial scale. Moreover, as discussed above and shown in the examples below, the therapeutic benefits of bromelain are well-established. However, it will be appreciated that the advantageous properties of particulate compositions comprising bromelain described herein would be applicable to other papain-like proteases, which are structurally similar to bromelain and share similar substrate specificity. In this respect, bromelain is used as an exemplary papain-like protease to demonstrate the general inventive concept on which the present disclosure is based. The present disclosure should not be construed as being limited to bromelain.

[0066]

[0060] In various embodiments, the papain-like protease belongs to the cysteine peptidase family C1. This family of cysteine proteases encompasses papain-like proteases that are closely homologous to one another. In particular, the papain-like proteases of the cysteine peptidase family C1 share a closely conserved catalytic active site, with similar substrate specificity. The cysteine peptidase family C1 comprises each of the dominant cysteine proteases present in crude pineapple stem extract, stem bromelain, fruit bromelain, ananain, and comosain.

[0067]

[0061] In preferred embodiments, the papain-like protease belongs to the cysteine peptidase subfamily C1 A. The cysteine peptidase family C1 A includes a subset of the cysteine peptidase family C1 that are even more closely conserved in structure, which includes stem bromelain, fruit bromelain, ananain, and comosain. The dominant specificity subsite in the peptidases of subfamily C1 A is provided by the S2 substrate binding pocket, shared by the proteases in this family. This subsite displays a preference for occupation by a bulky hydrophobic side chain (see Lecaille et al., Eur. J. Biochem., 268, 2733-2741 (2001)).

[0068]

[0062] In preferred embodiments, the papain-like protease may be a naturally occurring, plant-derived papain-like protease. In further preferred embodiments, the papain-like protease may be a naturally occurring, fruit-derived papain-like protease.

[0069]

[0063] In particularly preferred embodiments, the papain-like protease may be a plant-derived papain-like protease that belongs to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1A. In further particularly preferred embodiments, the papain-like protease may be a fruit-derived papain-like protease that belongs to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1A. In further particularly preferred embodiments, the papain-like protease may be a pineapple-derived papain-like protease that belongs to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1 A.

[0064] In especially preferred embodiments, the papain-like protease is derived from a member of the Bromeliaceae plant family that belongs to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1A.

[0070]

[0065] In various embodiments, the papain-like protease is bromelain. In such embodiments, the bromelain may comprise: a) stem bromelain or a fragment or a variant thereof; b) fruit bromelain or a fragment of a variant thereof; or c) a mixture of (a) and (b).

[0071]

[0066] In preferred embodiments, the bromelain may consist essentially of: a) stem bromelain or a fragment or a variant thereof; b) fruit bromelain or a fragment of a variant thereof; or c) a mixture of (a) and (b).

[0072]

[0067] In particularly preferred embodiments, the bromelain may consist of: a) stem bromelain or a fragment or a variant thereof; b) fruit bromelain or a fragment of a variant thereof; or c) a mixture of (a) and (b).

[0073]

[0068] The terms “fragment” and “variant” are defined above. In various embodiments, the variant of stem bromelain or fruit bromelain is therefore taken to include a polypeptide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to the subject sequence of SEQ ID NO: 1 (stem bromelain) or SEQ ID NO: 2 (fruit bromelain). Preferably, the variant of stem bromelain or fruit bromelain is therefore taken to include a polypeptide sequence which may be at least at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence of SEQ ID 1 (stem bromelain) or SEQ ID 2 (fruit bromelain). A “fragment” of stem bromelain or fruit bromelain as used herein refers to a variant sequence that is a portion of the full-length polypeptide in accordance with the definition of “fragment” above.

[0069] As discussed above, the particulate composition may in various embodiments comprise the full-length, non-variant form of the papain-like protease. Thus, in particularly preferred embodiments, the bromelain may comprise: a) stem bromelain; b) fruit bromelain; or c) a mixture of (a) and (b).

[0074]

[0070] In particularly preferred embodiments, the bromelain may consist essentially of: a) stem bromelain; b) fruit bromelain; or c) a mixture of (a) and (b).

[0075]

[0071] In particularly preferred embodiments, the bromelain may consist of: a) stem bromelain; b) fruit bromelain; or c) a mixture of (a) and (b).

[0076]

[0072] In a particularly preferred embodiment, the papain-like protease is stem bromelain or a fragment or a variant thereof. In an especially preferred embodiment, the papain-like protease is stem bromelain.

[0077]

[0073] The central inventive concept of the present disclosure is based on using an oxidised form of a papain-like protease or a fragment or a variant thereof.

[0078]

[0074] As used herein, the term “oxidised form” used in relation to a papain-like protease is used to refer to a form of a papain-like protease comprising at least one amino acid residue having an atom in a higher oxidation state than in the native form of the protein. For example, an oxidised form of a papain-like protease may refer to a papain-like protease where one of the cysteine residues (sulfur atom in the -2 oxidation state) has been oxidised to a cystine (sulfur atom in the -1 oxidation state) - i.e. a disulfide bond - or a cysteic acid (sulfur in the +4 oxidation state) that is not present in the native form of the papain-like protease. A further example would be where one or more of the native disulfide bonds present in the native papain-like protease have been further oxidised, e.g. to cysteic acid. It will be appreciated that amino acid residues other than cysteine also present in a papain-like protease may also be oxidised in the oxidised form. The oxidised form of the papain-like protease also encompasses forms where, for example, a methionine thioether group has been oxidised to a sulfoxide or sulfone, or where a tyrosine residue has been oxidised to a dityrosine group or a L-3,4- dihydroxyphenylalanine group.

[0079]

[0075] The oxidised form of the papain-like protease may be obtained by any suitable method known to the person skilled in the art. In various embodiments, the oxidised form of the papainlike protease may be obtained by a reaction of the papain-like protease or a fragment or a variant thereof with an oxidising agent. Any suitable oxidising agent may be used for this purpose. Non-limiting examples of such suitable oxidising agents are peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof. In this respect, the oxidising agent disclosed herein in relation to the process of the third aspect may be combined with the oxidised form embodiments for the particulate composition of the first aspect.

[0080]

[0076] In various embodiments, the oxidised form of the papain-like protease may be obtained by a reaction of the papain-like protease or a fragment or a variant thereof with an oxidising agent which is a peroxide. Peroxides are a class of oxidising agents characterised by two oxygen atoms being linked together by a single covalent bond. Non-limiting examples of suitable peroxide oxidising agents are hydrogen peroxide, benzoyl peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, acetyl peroxide, methyl ethyl ketone peroxide, di-tert- butyl peroxide, urea peroxide (carbamide peroxide), sodium peroxide, potassium peroxide, or mixtures thereof. A particularly suitable example includes hydrogen peroxide.

[0081]

[0077] Thus, in various embodiments, the oxidised form of the papain-like protease may be an oxidised form of a naturally occurring papain-like protease obtained by reaction of the naturally occurring papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0082]

[0078] In further preferred embodiments, the oxidised form of the papain-like protease may be an oxidised form of a plant-derived papain-like protease obtained by reaction of the plant- derived papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0083]

[0079] In further preferred embodiments, the oxidised form of the papain-like protease may be an oxidised form of a papain-like protease belonging to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1A, obtained by reaction of the papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0084]

[0080] In particularly preferred embodiments, the oxidised form of the papain-like protease may be an oxidised form of bromelain obtained by reaction of the bromelain with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0085]

[0081] In various embodiments, the oxidised form of the papain-like protease or the fragment or variant thereof comprises one or more oxidised thiol groups. In preferred embodiments, the one or more oxidised thiol groups may comprise at least one oxidised cysteine residue. The one or more oxidised thiol groups preferably comprise at least one group selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds. In preferred embodiments, the one or more oxidised thiol groups comprise at least one sulfonic acid group.

[0086]

[0082] The oxidised form of the papain-like protease may also comprise other oxidised groups. For example, the oxidised form of the papain-like protease may comprise one or more oxidised thioether groups, preferably wherein the one or more oxidised thioether groups comprise at least one oxidised methionine residue. The oxidised form of the papain-like protease may also comprise one or more oxidised tyrosine residues and / or one or more oxidised tryptophan residues.

[0087]

[0083] In various embodiments, the oxidised form of the papain-like protease may be an oxidised form of a naturally occurring papain-like protease obtained by reaction of the naturally occurring papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, wherein the oxidised form of the naturally occurring papain-like protease comprises one or more oxidised thiol groups preferably selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0088]

[0084] In various embodiments, the oxidised form of the papain-like protease may be an oxidised form of a plant-derived papain-like protease obtained by reaction of the plant-derived papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, wherein the oxidised form of the plant-derived papain-like protease comprises one or more oxidised thiol groups preferably selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0089]

[0085] In various embodiments, the oxidised form of the papain-like protease may be an oxidised form of a papain-like protease belonging to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1A, obtained by reaction of the papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, wherein the oxidised form of the papain-like protease comprises one or more oxidised thiol groups preferably selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0090]

[0086] In particularly preferred embodiments, the oxidised form of the papain-like protease may be an oxidised form of bromelain obtained by reaction of bromelain with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, wherein the oxidised form of bromelain comprises one or more oxidised thiol groups preferably selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0091]

[0087] In addition to the oxidised form, the general inventive concept of the present disclosure encompasses embodiments where the particles of the particulate composition have an average particle size of about 1000 nm or less. Nanoparticle-based drug delivery systems have in general been shown to overcome several challenges associated with conventional drug therapy, including poor solubility, poor permeability and high toxicity. In particular, studies have shown that nanoparticulate agents may exhibit higher cellular uptake than their nonnanoparticulate equivalents, thus providing more effective delivery of the therapeutic to the target cells. In cancer treatment in particular, it has been identified that nanoparticle-based delivery systems may exhibit enhanced penetration and retention effects at the tumour site, especially within the tumour microenvironment.

[0092]

[0088] As used herein, the term “average particle size” refers to the median particle size by volume of the measured particles, also referred to as the Dv50 value. The skilled person will appreciate that there exist many suitable methods of measuring Dv50 values in the art, such as dynamic light scattering, where Mie theory is used in both methods for calculation of the Dv50 value. In the present invention, the average particle size (Dv50 value) is thus measured by dynamic light scattering using Mie theory.

[0093]

[0089] The particles may otherwise be referenced herein as “nanoparticles”. As used herein, the term “nanoparticles” is used generally to refer to particles having an average particle size - i.e. a Dv50 value - of about 1000 nm or less. In preferred embodiments, the term “nanoparticles” refers to particles having an average particle size of about 100 nm or less.

[0094]

[0090] In various embodiments, the particles of the particulate composition have an average particle size of about 1 nm or more, such that the average particle size is from about 1 nm to about 1000 nm. In preferred embodiments, the particles have an average particle size of about 100 nm or less, such as an average particle size of about 1 nm to about 100 nm.

[0095]

[0091] In particularly preferred embodiments, the particles of the particulate composition comprise an oxidised form of a papain-like protease belonging to the cysteine peptidase family C1 , preferably the cysteine peptidase subfamily C1A, obtained by reaction of the papain-like protease with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, wherein the oxidised form of the papain-like protease comprises one or more oxidised thiol groups preferably selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds, wherein the particles have an average particle size of about 100 nm or less. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0092] In especially preferred embodiments, the particles of the particulate composition comprise an oxidised form of bromelain, obtained by reaction of bromelain with an oxidising agent, wherein the oxidising agent is selected from peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, wherein the oxidised form of bromelain comprises one or more oxidised thiol groups preferably selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds, wherein the particles have an average particle size of about 100 nm or less. Preferably, the oxidising agent is a peroxide, more preferably hydrogen peroxide.

[0096]

[0093] In various embodiments, the particulate composition may be in dry form, i.e. having a moisture content by weight of about 10 wt.% or less, 5 wt.% or less, 3 wt.% or less, 1 wt.% or less, or being substantially free of moisture.

[0097]

[0094] In other embodiments, the composition may further comprise a solvent in which the particles are suspended. The term “suspended” as used herein refers to particles that are not dissolved in the solvent, but are present as a solid distributed throughout the solvent medium.

[0098]

[0095] The solvent is not limited and may include any solvent used in the preparation of the particulate composition. Thus, the embodiments of the solvent disclosed herein in relation to the preparation of the particulate composition (the process of the third aspect) may be combined with the embodiments of the particulate composition of the first aspect. In such embodiments, the solvent may preferably be an aqueous solvent. As used herein, an “aqueous solvent” refers to a solvent where more than 50% of the solvent by volume is water. In preferred embodiments, an “aqueous solvent” may refer to a solvent where more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, and more preferably more than 99% of the solvent by volume is water. Most preferably, an “aqueous solvent” refers to a solvent which is water.

[0099]

[0096] It will also be appreciated that the particles of the particulate composition are not limited to comprising only the oxidised form of the papain-like protease or fragment or variant thereof, and may further comprise suitable additives. Non-limiting examples of such additives include bulking agents, anti-caking agents, emulsifiers and glazing agents. Such additives are known in the art and suitable materials would be readily identified by the person skilled in the art.

[0100] PROCESS

[0101]

[0097] A further aspect of the invention relates to a process for preparing a particulate composition as described above, the process comprising the steps of: (ai) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and

[0102] (aii) treating the particles with an oxidising agent; or

[0103] (bi) providing a papain-like protease or a fragment or a variant thereof;

[0104] (bii) treating the papain-like protease or the fragment or the variant thereof with an oxidising agent; and

[0105] (biii) preparing particles comprising the oxidised form of the papain-like protease or the fragment or the variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0106]

[0098] That is, the papain-like protease may be oxidised when in particulate form, or the papain-like protease may first be oxidised, and then prepared into particulate form. Both processes are within the scope of the present disclosure. In preferred embodiments, the papain-like protease may be oxidised when in particulate form - i.e. the process above preferably comprises steps (ai) and (aii).

[0107]

[0099] The oxidising agent used in the process may be as defined above for the particulate composition.

[0108]

[0100] In various embodiments, the particles are provided in step (ai) as a suspension in a solvent. Preferably, the solvent is an aqueous solvent as described above in relation to the particulate composition of the first aspect. More preferably, the aqueous solvent is water. It will be appreciated, however, that other solvents would also be suitable. For example, the particles could also be provided in an alcohol-based solvent such as ethanol in step (ai). As used herein, the term “suspension” refers to particles that are not dissolved in the solvent, but are present in solid form distributed throughout the solvent medium.

[0109]

[0101] In various embodiments, the process further comprises a step of treating the papainlike protease or fragment or variant thereof with a thiosulphate. This step may be carried out more than once and at different stages of the process. For example, the process may further comprise a step of treating the papain-like protease or fragment or variant thereof with a thiosulphate following step (ai) and before step (aii), following step (bi) and before step (bii), following step (aii), and / or following step (bii) and before step (biii). In preferred embodiments, the thiosulphate comprises sodium thiosulphate. Without being bound by theory, it is thought that the thiosulphate acts to prevent undesirable aggregation or autolysis of the papain-like protease both before and following the oxidation step.

[0110]

[0102] The oxidation reaction in steps (aii) and (bii) may be carried out under any conditions suitable for oxidation to occur. In particular, the pH, temperature, and duration of the oxidation reaction in steps (aii) and (bii) are not especially limited. In various embodiments, steps (aii) and (bii) may be carried out at a pH of from about 6 to about 8, preferably from about 7 to about 8. In various embodiments, steps (aii) and (bii) may be carried out at a temperature of from about 20 °C to about 60 °C, preferably from about 25 °C to about 55 °C, more preferably from about 30 °C to about 40 °C, yet more preferably from about 35 °C to about 40 °C, most preferably at about 37 °C. In various embodiments, steps (aii) and (bii) are carried out for a length of at least about 10 minutes, preferably from about 10 minutes to about 300 minutes.

[0111]

[0103] In various embodiments, the process further comprises a step of treating the the papain-like protease or fragment or variant thereof with N-acetyl-L-cysteine. Without being bound by theory, it is thought that the N-acetyl-L-cysteine helps preserve the oxidised groups generated in the oxidation step, whilst also preventing aggregation and autolysis of the papain-like protease following the oxidation step.

[0112]

[0104] In various embodiments, the process comprises the steps of:

[0113] (ai) providing particles comprising a naturally occurring papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and

[0114] (aii) treating the particles with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; or

[0115] (bi) providing a naturally occurring papain-like protease or a fragment or a variant thereof;

[0116] (bii) treating the naturally occurring papain-like protease or the fragment or the variant thereof with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; and

[0117] (biii) preparing particles comprising the oxidised form of the naturally occurring papain-like protease or the fragment or the variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0118]

[0105] In various embodiments, the process comprises the steps of:

[0119] (ai) providing particles comprising a plant-derived papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and

[0120] (aii) treating the particles with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; or

[0121] (bi) providing a plant-derived papain-like protease or a fragment or a variant thereof;

[0122] (bii) treating the plant-derived papain-like protease or the fragment or the variant thereof with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; and

[0123] (biii) preparing particles comprising the oxidised form of the plant-derived papainlike protease or the fragment or the variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0124]

[0106] In particularly preferred embodiments, the process comprises the steps of: (ai) providing particles comprising a papain-like protease belonging to the cysteine peptidase family C1 or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and

[0125] (aii) treating the particles with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; or

[0126] (bi) providing a papain-like protease belonging to the cysteine peptidase family C1 or a fragment or a variant thereof;

[0127] (bii) treating the papain-like protease belonging to the cysteine peptidase family C1 or the fragment or the variant thereof with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; and

[0128] (biii) preparing particles comprising the oxidised form of the papain-like protease belonging to the cysteine peptidase family C1 or the fragment or the variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0129]

[0107] In especially preferred embodiments, the process comprises the steps of:

[0130] (ai) providing particles comprising bromelain or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and

[0131] (aii) treating the particles with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; or (bi) providing bromelain or a fragment or a variant thereof;

[0132] (bii) treating the bromelain or the fragment or the variant thereof with an oxidising agent selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide; and

[0133] (biii) preparing particles comprising the bromelain or the fragment or the variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0134]

[0108] In various embodiments, the process comprises the steps of:

[0135] (i) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have a particle size of about 1000 nm or less;

[0136] (i-a) treating the particles with a thiosulphate;

[0137] (ii) treating the particles with an oxidising agent;

[0138] (iii) treating the particles with a thiosulphate;

[0139] (iv) treating the particles with N-acetyl-L-cysteine; and

[0140] (v) optionally, adding an aqueous solvent; wherein the steps are carried out in the order (i)-(v).

[0141]

[0109] In various embodiments, the process comprises the steps of:

[0142] (i) providing particles comprising a naturally occurring papain-like protease or a fragment or a variant thereof, wherein the particles have a particle size of about 1000 nm or less;

[0143] (i-a) treating the particles with thiosulphate, preferably sodium thiosulphate;

[0144] (ii) treating the particles with an oxidising agent selected from selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide;

[0145] (iii) treating the particles with a thiosulphate, preferably sodium thiosulphate;

[0146] (iv) treating the particles with N-acetyl-L-cysteine; and

[0147] (v) optionally, adding an aqueous solvent; wherein the steps are carried out in the order (i)-(v).

[0148]

[0110] In various embodiments, the process comprises the steps of:

[0149] (i) providing particles comprising a plant-based papain-like protease or a fragment or a variant thereof, wherein the particles have a particle size of about 1000 nm or less;

[0150] (i-a) treating the particles with thiosulphate, preferably sodium thiosulphate;

[0151] (ii) treating the particles with an oxidising agent selected from selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide;

[0152] (iii) treating the particles with a thiosulphate, preferably sodium thiosulphate;

[0153] (iv) treating the particles with N-acetyl-L-cysteine; and

[0154] (v) optionally, adding an aqueous solvent; wherein the steps are carried out in the order (i)-(v).

[0155]

[0111] In various embodiments, the process comprises the steps of:

[0156] (i) providing particles comprising a papain-like protease belonging to the cysteine peptidase family C1 or a fragment or a variant thereof, wherein the particles have a particle size of about 1000 nm or less;

[0157] (i-a) treating the particles with thiosulphate, preferably sodium thiosulphate; (ii) treating the particles with an oxidising agent selected from selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide;

[0158] (iii) treating the particles with a thiosulphate, preferably sodium thiosulphate;

[0159] (iv) treating the particles with N-acetyl-L-cysteine; and

[0160] (v) optionally, adding an aqueous solvent; wherein the steps are carried out in the order (i)-(v).

[0161]

[0112] In various embodiments, the process comprises the steps of:

[0162] (i) providing particles comprising bromelain or a fragment or a variant thereof, wherein the particles have a particle size of about 1000 nm or less;

[0163] (i-a) treating the particles with thiosulphate, preferably sodium thiosulphate;

[0164] (ii) treating the particles with an oxidising agent selected from selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof, preferably wherein the oxidising agent is a peroxide, more preferably hydrogen peroxide;

[0165] (iii) treating the particles with a thiosulphate, preferably sodium thiosulphate;

[0166] (iv) treating the particles with N-acetyl-L-cysteine; and

[0167] (v) optionally, adding an aqueous solvent; wherein the steps are carried out in the order (i)-(v).

[0168]

[0113] The solvent is not limited and may include any solvent used in the preparation of the particulate composition.

[0169]

[0114] A further aspect of the present disclosure relates to a composition obtained or obtainable by the process described above. The embodiments disclosed herein in relation to the preparation of the particulate composition (the process of the third aspect) may accordingly be combined with the embodiments of the particulate composition of the first aspect.

[0170] THERAPEUTIC USES

[0171]

[0115] A further aspect of the present disclosure relates to particulate compositions and / or oxidised forms of papain-like proteases as described herein for use in medicine.

[0172]

[0116] In one aspect of the present disclosure, an oxidised form of a papain-like protease or a fragment or a variant thereof is provided, for use in therapy.

[0173]

[0117] In various embodiments, the oxidised form of a papain-like protease or a fragment or variant thereof is for use in treating a disorder selected from cancer, a bacterial infection, a helminth infection, or a viral infection. Preferably, the disorder is selected from cancer, a bacterial infection, or a viral infection.

[0174]

[0118] In various embodiments, the oxidised form of the papain-like protease or a fragment or a variant thereof is for use in treating cancer. Herein, the term “cancer” is to be understood as referring to an abnormal proliferation of human cells both solid tumours and liquid cancers. The type of cancer is not limited. In various embodiments, the cancer may be lung cancer, colon cancer, prostate cancer, leukaemia, pancreatic cancer, or a combination thereof.

[0175]

[0119] In other embodiments, the oxidised form of the papain-like protease or a fragment or a variant thereof, for use in treating a bacterial or viral infection.

[0176]

[0120] In various embodiments, the bacterial infection is caused by gram-negative bacteria, gram-positive bacteria, mycobacteria, or a combination thereof.

[0177]

[0121] As used herein, the term “bacterial infection" includes, but is not limited to, references to infections caused by organisms of the following classes and specific types:

[0178]

[0122] Gram-positive cocci, such as Staphylococci (e.g. Staph, aureus, Staph, epidermidis, Staph, saprophyticus, Staph, auricularis, Staph, capitis capitis, Staph, c. ureolyticus, Staph, caprae, Staph, cohnii cohnii, Staph, c. urealyticus, Staph, equorum, Staph, gallinarum, Staph, haemolyticus, Staph, hominis hominis, Staph, h. novobiosepticius, Staph, hyicus, Staph, intermedius, Staph, lugdunensis, Staph, pasteuri, Staph, saccharolyticus, Staph, schleiferi schleiferi, Staph, s. coagulans, Staph, sciuri, Staph, simulans, Staph, warneri and Staph. xylosus); Streptococci (e.g. beta-haemolytic, pyogenic streptococci (such as Strept. agalactiae, Strept. canis, Strept. dysgalactiae dysgalactiae, Strept. dysgalactiae equisimilis, Strept. equi equi, Strept. equi zooepidemicus, Strept. iniae, Strept. porcinus and Strept. pyogenes), microaerophilic, pyogenic streptococci (Streptococcus “milleri”, such as Strept. anginosus, Strept. constellatus constellatus, Strept. constellatus pharyngidis and Strept. intermedius), oral streptococci of the “mitis” (alpha-haemolytic - Streptococcus “viridans”, such as Strept. mitis, Strept. oralis, Strept. sanguinis, Strept. cristatus, Strept. gordonii and Strept. parasanguinis), “salivarius” (non-haemolytic, such as Strept. salivarius and Strept. vestibularis) and “mutans” (tooth-surface streptococci, such as Strept. criceti, Strept. mutans, Strept. ratti and Strept. sobrinus) groups, Strept. acidominimus, Strept. bovis, Strept. faecalis, Strept. equinus, Strept. pneumoniae and Strept. suis, or Streptococci alternatively classified as Group A, B, C, D, E, G, L, P, U or V Streptococcus); Enterococci (e.g. Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus and Enterococcus solitarius)- Micrococci (e.g. M. aloeverae, M. antarcticus, M. cohnii, M. endophyticus, M. flavus, M. luteus, M. lylae, M. terreus, and M. yunnanensis): Bacillaceae, such as Bacillus anthracis, Bacillus subtilis, Bacillus thuringiensis, Bacillus stearothermophilus and Bacillus cereus: Gramnegative cocci, such as Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria cinerea, Neisseria elongata, Neisseria flavescens, Neisseria lactamica, Neisseria mucosa, Neisseria sicca, Neisseria subflava and Neisseria weaver , Enterobacteriaceae, such as Escherichia coll, Enterobacter (e.g. Enterobacter aerogenes, Enterobacter agglomerans and Enterobacter cloacae), Citrobacter (such as Citrob. freundii and Citrob. divernis), Hafnia (e.g. Hafnia alvei), Erwinia (e.g. Erwinia persicinus), Morganella (e.g. Morganella morganii), Salmonella (Salmonella enterica and Salmonella typhi), Shigella (e.g. Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei), Klebsiella (e.g. Klebs, pneumoniae, Klebs, oxytoca, Klebs, ornitholytica, Klebs, planticola, Klebs, ozaenae, Klebs, terrigena, Klebs, granulomatis (Calymmatobacterium granulomatis) and Klebs, rhinoscleromatis), Proteus (e.g. Pr. mirabilis, Pr. rettgeri and Pr. vulgaris), Providencia (e.g. Providencia alcalifaciens, Providencia rettgeri and Providencia stuartii), Serratia (e.g. Serratia marcescens and Serratia liquifaciens), and Yersinia (e.g. Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis)- Helicobacter (e.g. Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae)- Acinetobacter (e.g. A. baumanii, A. calcoaceticus, A. haemolyticus, A. johnsonii, A. junii, A. Iwoffi and A. radioresistens); Pseudomonas (e.g. Ps. aeruginosa, Ps. maltophilia (Stenotrophomonas maltophilia), Ps. alcaligenes, Ps. chlororaphis, Ps. fluorescens, Ps. luteola. Ps. mendocina, Ps. monteilii, Ps. oryzihabitans, Ps. pertocinogena, Ps. pseudalcaligenes, Ps. putida and Ps. stutzeri): Bacteriodes fragilis- Peptococcus (e.g. Peptococcus niger): Peptostreptococcus; Clostridium (e.g. C. perfringens, C. difficile, C. botulinum, C. tetani, C. absonum, C. argentinense, C. baratii, C. bifermentans, C. beijerinckii, C. butyricum, C. cadaveris, C. carnis, C. celatum, C. clostridioforme, C. cochlearium, C. cocleatum, C. fallax, C. ghonii, C. g / yco / icum, C. haemo / yticum, C. hastiforme, C. histolyticum, C. indolis, C. innocuum, C. irregulare, C. leptum, C. limosum, C. ma / enominatum, C. novyi, C. oroticum, C. paraputrificum, C. piliforme, C. putrefasciens, C. ramosum, C. septicum, C. sordelii, C. sphenoides, C. sporogenes, C. subterminale, C. symbiosum and C. tertium); Brevundimonas (e.g. B. abyssalis, B. alba, B. albigilva, B. aurantiaca, B. aveniformis, B. bacteroides, B. balnearis, B. basaltis, B. bullata, B. canariensis, B. denitrificans, B. diminuta, B. faecalis, B. fluminis, B. goettingensis, B. halotolerans, B. humi, B. intermedia, B. kwangchunensis, B. lenta, B. lutea, B. mediterranea, B. mongoliensis, B. naejangsanensis, B. nasdae, B. poindexterae, B. pondensis, B. staleyi, B. subvibrioides, B. terrae, B. vancanneytii, B. variabilis, B. vesicularis, and B. viscosa); Mycoplasma (e.g. M. pneumoniae, M. hominis, M. genitalium and M. urealyticum); Mycobacteria (e.g. Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium leprae, Mycobacterium smegmitis, Mycobacterium africanum, Mycobacterium alvei, Mycobacterium asiaticum, Mycobacterium aurum, Mycobacterium bohemicum, Mycobacterium bovis, Mycobacterium branded, Mycobacterium brumae, Mycobacterium celatum, Mycobacterium chubense, Mycobacterium confluentis, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium flavescens, Mycobacterium gadium, Mycobacterium gastri, Mycobacterium genavense, Mycobacterium gordonae, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium hassicum, Mycobacterium intracellulare, Mycobacterium interjectum, Mycobacterium heidelberense, Mycobacterium lentiflavum, Mycobacterium malmoense, Mycobacterium microgenicum, Mycobacterium microti, Mycobacterium mucogenicum, Mycobacterium neoaurum, Mycobacterium nonchromogenicum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium shimoidei, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium thermoresistabile, Mycobacterium triplex, Mycobacterium triviale, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium vaccae, Mycobacterium wolinskyi and Mycobacterium xenopi); Haemophilus (e.g. Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parain fluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus); Actinobacillus (e.g. Actinobacillus actinomycetemcomitans, Actinobacillus equuli, Actinobacillus hominis, Actinobacillus lignieresii, Actinobacillus suis and Actinobacillus ureae); Actinomyces (e.g. Actinomyces israelii); Brucella (e.g. Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis); Campylobacter (e.g. Campylobacter jejuni, Campylobacter coll, Campylobacter lari and Campylobacter fetus); Listeria monocytogenes; Vibrio (e.g. Vibrio cholerae and Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio fluvialis, Vibrio furnissii, Vibrio hollisae, Vibrio metschnikovii, Vibrio mimicus and Vibrio vulnificus)-, Erysipelothrix rhusopathiae: Corynebacteriaceae (e.g. Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium urealyticum)- Spirochaetaceae, such as Borrelia (e.g. Borrelia recurrentis, Borrelia burgdorferi, Borrelia afzelii, Borrelia andersonii, Borrelia bissettii, Borrelia garinii, Borrelia japonica, Borrelia lusitaniae, Borrelia tanukii, Borrelia turdi, Borrelia valaisiana, Borrelia caucasica, Borrelia crocidurae, Borrelia duttoni, Borrelia graingeri, Borrelia hermsii, Borrelia hispanica, Borrelia latyschewii, Borrelia mazzottii, Borrelia parked, Borrelia persica, Borrelia turicatae and Borrelia venezuelensis) and Treponema (Treponema pallidum ssp. pallidum, Treponema pallidum ssp. endemicum, Treponema pallidum ssp. pertenue and Treponema carateum)-, Pasteurella (e.g. Pasteurella aerogenes, Pasteurella bettyae, Pasteurella canis, Pasteurella dagmatis, Pasteurella gallinarum, Pasteurella haemolytica, Pasteurella multocida multocida, Pasteurella multocida gallicida, Pasteurella multocida septica, Pasteurella pneumotropica and Pasteurella stomatis)- Bordetella (e.g. Bordetella bronchiseptica, Bordetella hinzii, Bordetella holmseii, Bordetella parapertussis, Bordetella pertussis and Bordetella trematum)- Nocardiaceae, such as Nocardia (e.g. Nocardia asteroides and Nocardia brasiliensis)- Rickettsia (e.g. Ricksettsii or Coxiella burnetii)- Legionella (e.g. Legionalla anisa, Legionalla birminghamensis, Legionalla bozemanii, Legionalla cincinnatiensis, Legionalla dumoffii, Legionalla feeleii, Legionalla gormanii, Legionalla hackeliae, Legionalla israelensis, Legionalla jordanis, Legionalla lansingensis, Legionalla longbeachae, Legionalla maceachernii, Legionalla micdadei, Legionalla oakridgensis, Legionalla pneumophila, Legionalla sainthelensi, Legionalla tucsonensis and Legionalla wadsworthii)- Moraxella catarrhalis- Cyclospora cayetanensis; Entamoeba histolytica; Giardia lamblia; Trichomonas vaginalis; Toxoplasma gondii; Stenotrophomonas maltophilia- Burkholderia stenotrophomonas; Burkholderia cepacia- Burkholderia mallei and Burkholderia pseudomallei-, Francisella tularensis: Gardnerella (e.g. Gardneralla vaginalis and Gardneralla mobiluncus)- Streptobacillus moniliformis- Flavobacteriaceae, such as Capnocytophaga (e.g. Capnocytophaga canimorsus, Capnocytophaga cynodegmi, Capnocytophaga gingivalis, Capnocytophaga granulosa, Capnocytophaga haemolytica, Capnocytophaga ochracea and Capnocytophaga sputigena)- Bartonella (Bartonella bacilliformis, Bartonella clarridgeiae, Bartonella elizabethae, Bartonella henselae, Bartonella quintana and Bartonella vinsonii arupensis)- Leptospira (e.g. Leptospira biflexa, Leptospira borgpetersenii, Leptospira inadai, Leptospira interrogans, Leptospira kirschneri, Leptospira noguchii, Leptospira santarosai and Leptospira weilii)- Spirillium (e.g. Spirillum minus)- Baceteroides (e.g. Bacteroides caccae, Bacteroides capillosus, Bacteroides coagulans, Bacteroides distasonis, Bacteroides eggerthii, Bacteroides forsythus, Bacteroides fragilis, Bacteroides merdae, Bacteroides ovatus, Bacteroides putredinis, Bacteroides pyogenes, Bacteroides splanchinicus, Bacteroides stercoris, Bacteroides tectus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides ureolyticus and Bacteroides vulgatus)- Prevotella (e.g. Prevotella bivia, Prevotella buccae, Prevotella corporis, Prevotella dentalis (Mitsuokella dentalis), Prevotella denticola, Prevotella disiens, Prevotella enoeca, Prevotella heparinolytica, Prevotella intermedia, Prevotella loeschii, Prevotella melaninogenica, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulora, Prevotella tannerae, Prevotella venoralis and Prevotella zoogleoformans) Porphyromonas (e.g. Porphyromonas asaccharolytica, Porphyromonas cangingivalis, Porphyromonas canoris, Porphyromonas cansulci, Porphyromonas catoniae, Porphyromonas circumdentaria, Porphyromonas crevioricanis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas levii and Porphyromonas macacae): Fusobacterium (e.g. F. gonadiaformans, F. mortiferum, F. naviforme, F. necrogenes, F. necrophorum necrophorum, F. necrophorum fundiliforme, F. nucleatum nucleatum, F. nucleatum fusiforme, F. nucleatum polymorphum, F. nucleatum vincentii, F. periodonticum, F. russii, F. ulcerans and F. varium) Chlamydia (e.g. Chlamydia trachomatis)’ Cryptosporidium (e.g. C. parvum, C. hominis, C. cam's, C. felis, C. meleagridis and C. muris): Chlamydophila (e.g. Chlamydophila abortus (Chlamydia psittaci), Chlamydophila pneumoniae (Chlamydia pneumoniae) and Chlamydophila psittaci (Chlamydia psittaci))’ Leuconostoc (e.g. Leuconostoc citreum, Leuconostoc cremoris, Leuconostoc dextranicum, Leuconostoc lactis, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides)’ Gemella (e.g. Gemella bergeri, Gemella haemolysans, Gemella morbillorum and Gemella sanguinis)’ Aeromonas (e.g. Aeromonas hydrophila, Aeromonas caviae and Aeromonas veronii biovar sobria)’ and Ureaplasma (e.g. Ureaplasma parvum and Ureaplasma urealyticum).

[0179]

[0123] In various embodiments, the bacterial infection is caused by gram-negative bacteria selected from Enterobacteriaceae and Brevundimonas. In preferred embodiments, the bacterial infection may, for example be caused by Escherichia coli or Brevundimonas diminuta.

[0180]

[0124] In other embodiments, the bacterial infection is caused by gram-positive bacteria selected from Staphylococcus, Micrococcus, and Bacillaceae. In preferred embodiments, the bacterial infection is caused by Staphylococcus aureus, Micrococcus luteus, and Bacillus megaterium.

[0181]

[0125] In other embodiments, the bacterial infection is caused by mycobacteria. In preferred embodiments, the bacterial infection is caused by Mycobacterium smegmitis.

[0182]

[0126] In various embodiments, the viral infection is caused by an enveloped virus. An “enveloped virus” herein refers to a virus that has a lipid bilayer membrane (or “envelope”) surrounding the protein capsid on the outer part of the virus. Conversely, a “non-enveloped virus” does not comprise such a lipid bilayer membrane structure.

[0183]

[0127] Non-limiting examples of enveloped viruses are the DNA viruses: Herpesviridae, Poxviridae Hepadnaviridae, and Asfarviridae; and the RNA viruses: Flaviviridae, Alphaviridae, Togaviridae, Coronaviruses, Hepatitis D, Orthomyxoviruses, Paramyxoviridae, Rhabdoviruses, Bunyaviruses, and Filoviruses.

[0184]

[0128] In preferred embodiments enveloped virus is preferably a Coronavirus. As used herein, “coronavirus” is used to refer to an RNA virus of the Coronaviridae family. This family also includes the subfamilies Letovirinae and Orthocoronavirinae. Preferably, the Coronavirus is SARS-CoV-2.

[0185]

[0129] In other embodiments, the viral infection is caused by a non-enveloped virus. Nonlimiting examples of non-enveloped viruses are the DNA viruses: Adenoviruses, Parvoviruses, Polyomaviruses, and Anelloviruses; and the RNA viruses: Caliciviruses, Picornaviruses, Reoviruses, Astroviruses, and Hepeviridae.

[0186]

[0130] In various embodiments, the viral infection is caused by a non-enveloped virus, preferably a Picornavirus, more preferably hepatitis A virus.

[0187]

[0131] In various embodiments, the oxidised form of the papain-like protease is for use in treating a helminth infection. In various embodiments, the helminth infection may be caused by a gastrointestinal parasite, preferably a helminth of the phylum Nematoda. In various embodiments, the helminth infection may be caused by a helminth selected from the group consisting of Anaplocephala, Ancylostoma, Anecator, Ascaris, Capillaria, Cooperia, Dipylidium, Dirofilaria, Echinococcus, Enterobius, Fasciola, Haemonchus, Oesophagostumum, Ostertagia, Toxocara, Strongyloides, Toxascaris, Trichinella, Trichuris, and Trichostrongylus. Non-limiting examples of particular helminth infections that may be treated are those caused by the following species: Ancylostoma duodenale, Ancylostoma ceylanicum, Acylostoma braziliensis, Ancylostoma spp., Ascaris lubricoides, Ascaris spp., Brugia malayi, Brugia timori, Bunostomum spp., Chabertia spp., Clonorchis spp., Cooperia spp., Dicrocoelium spp, Dictyocaulus filaria, Diphyllobothrium latum, Dracunculus medinensis, Echinococcus granulosus, Echinococcus multilocularis, Enterobius vermicularis, Faciola spp., Haemonchus spp., Heterakis spp., Hymenolepis nana, Hyostrongulus spp., Loa Loa, Nematodirus spp., Oesophagostomum spp., Opisthorchis spp., Onchocerca volvulus, Ostertagia spp., Paragonimus spp., Schistosomen spp., Strongyloides fuelleborni, Strongyloides stercoralis, Stronyloides spp., Taenia saginata, Taenia solium, Trichinella spiralis, Trichinella nativa, Trichinella britovi, Trichinella nelson!, Trichinella pseudopsiralis, Trichostrongulus spp., Trichuris trichuria, and Wuchereria bancrofti.

[0188]

[0132] In any of the above embodiments, the oxidised form of the papain-like protease or the fragment or the variant thereof may be as defined as above for the particulate composition of the first aspect.

[0189]

[0133] Another aspect of the present disclosure relates to a method of treating a disorder as described above comprising administering an oxidised form of a papain-like protease or a fragment or a variant thereof as described herein to a subject.

[0190]

[0134] Another aspect of the present disclosure relates to a method of treating a disorder as described above in a subject. The method according to this aspect is effected by administering to a subject in need thereof a therapeutically effective amount of the oxidised papain-like protease or a fragment or variant thereof, as described hereinabove, either per se, or, more preferably, as a part of a pharmaceutical composition, mixed with, for example, a pharmaceutically acceptable carrier, as is detailed hereinafter. Preferably, the subject is a mammal, more preferably a human.

[0191]

[0135] The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0192]

[0136] Herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease or disorder, substantially ameliorating clinical symptoms of a disease or disorder or substantially preventing the appearance of clinical symptoms of a disease or disorder.

[0193]

[0137] The term “therapeutically effective amount” refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disease or disorder being treated.

[0194]

[0138] Another aspect of the present disclosure relates to the use of an oxidised form of a papain-like protease or a fragment or a variant thereof as described above for the manufacture of a medicament for treating a disorder as described above.

[0195]

[0139] In a further aspect of the disclosure, a particulate composition is provided, wherein the particles comprise a papain-like protease or a fragment or a variant thereof for use in treating a bacterial or viral infection, wherein the particles have an average particle size of about 1000 nm or less. The particulate composition and the bacterial or viral infection may be as defined above.

[0196]

[0140] In a further aspect of the disclosure, a particulate composition is provided, wherein the particles comprise a papain-like protease or a fragment or a variant thereof for use in treating a helminth infection, wherein the particles have an average particle size of about 1000 nm or less. The particulate composition and the helminth infection may be as defined above.

[0197]

[0141] Another aspect of the disclosure relates to a method of treating a bacterial or viral infection as described above comprising administering a particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof, to a subject, wherein the particles have an average particle size of about 1000 nm or less. The particulate composition and the bacterial or viral infection may be as defined above.

[0198]

[0142] Another aspect of the disclosure relates to a method of treating a helminth infection as described above comprising administering a particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof, to a subject, wherein the particles have an average particle size of about 1000 nm or less. The particulate composition and the helminth infection may be as defined above.

[0199]

[0143] Another aspect of the disclosure relates to the use of a particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof, for the manufacture of a medicament for treating a bacterial or viral infection, wherein the particles have an average particle size of about 1000 nm or less. The particulate composition and the bacterial or viral infection may be as defined above.

[0200]

[0144] Another aspect of the disclosure relates to the use of a particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof, for the manufacture of a medicament for treating a helminth infection, wherein the particles have an average particle size of about 1000 nm or less. The particulate composition and the helminth infection may be as defined above.

[0201]

[0145] In another aspect of the disclosure, there is provided a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, for use in therapy.

[0202]

[0146] In various embodiments, the particulate composition is for use in treating cancer. Preferably, the cancer is selected from the group consisting of lung cancer, colon cancer, prostate cancer, leukaemia, pancreatic cancer, or a combination thereof.

[0147] In further preferred embodiments, the particulate composition is for use in treating a bacterial or viral infection. The bacterial or viral infection may be as defined above.

[0203]

[0148] In various embodiments, the oxidised form of the papain-like protease is for use in treating a helminth infection. The particulate composition and the helminth infection may be as defined above.

[0204]

[0149] Another aspect of the invention relates to a method of treating cancer comprising administering a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, to a subject. The particulate composition and the cancer may be as defined above.

[0205]

[0150] Another aspect of the invention relates to a method of treating a bacterial or viral infection, comprising administering a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, to a subject. The particulate composition and the bacterial or viral infection may be as defined above.

[0206]

[0151] Another aspect of the invention relates to a method of treating a helminth infection, comprising administering a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, to a subject. The particulate composition and the helminth infection may be as defined above.

[0207]

[0152] Another aspect of the invention relates to the use of a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, for the manufacture of a medicament for treating cancer. The particulate composition and the cancer may be as defined above.

[0208]

[0153] Another aspect of the invention relates to the use of a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, for the manufacture of a medicament for treating a bacterial or viral infection. The particulate composition and the bacterial or viral infection may be as defined above.

[0209]

[0154] Another aspect of the invention relates to the use of a particulate composition, wherein the particles comprise an oxidised form of a papain-like protease or a fragment or a variant thereof, and wherein the particles have an average particle size of about 1000 nm or less, for the manufacture of a medicament for treating a helminth infection. The particulate composition and the helminth infection may be as defined above.

[0210]

[0155] In the above aspects, the oxidised form of the papain-like protease, the cancer, and the bacterial or viral infection, may be as defined above.

[0211]

[0156] Another aspect of the invention relates to a non-therapeutic use of the particulate composition described herein, wherein the use comprises applying the composition to a surface as a disinfectant. As used herein, the term “disinfectant” refers to a composition that acts to inactivate or kill microorganisms, such as bacteria or viruses. The surface may be any inert surface in need of treatment.

[0212] PHARMACEUTICAL COMPOSITIONS

[0213]

[0157] In a further aspect of disclosure, a pharmaceutical composition is provided, the pharmaceutical composition comprising a particulate composition according to the first aspect as described above, and further comprising one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may optionally further comprise a solvent. In preferred embodiments, the solvent is an aqueous solvent as defined above.

[0214]

[0158] The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2ndEdition, (1994), Edited by A Wade and PJ Weller. The carrier, or, if more than one be present, each of the carriers, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0215]

[0159] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), buffer(s), flavouring agent(s), surface active agent(s), thickener(s), preservative(s) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Suspending agents may be also used.

[0216]

[0160] Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g., by inhalation. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the particulate composition with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

[0217]

[0161] Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of the papain-like protease or fragment or variant thereof. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the particulate composition in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surfaceactive agent or dispersing agent. Moulded tablets may be made by moulding the particulate composition with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling the particulate composition, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein the particulate composition together with any accessory ingredient(s) is sealed in a rice paper envelope. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water liquid emulsion. Formulations for oral administration include controlled release dosage forms, e.g., tablets wherein the particulate composition is formulated in an appropriate release - controlling matrix, or is coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.

[0218]

[0162] Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of the particulate composition with the softened or melted carrier(s) followed by chilling and shaping in moulds. Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the particulate composition in aqueous or oleaginous vehicles.

[0219]

[0163] Injectable preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, the particulate composition may be constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

[0220]

[0164] Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, chelating agents, inert gases and the like.

[0221]

[0165] Formulations suitable for topical formulation may be provided for example as gels, creams or ointments. Such preparations may be applied e.g. to a wound or ulcer either directly spread upon the surface of the wound or ulcer or carried on a suitable support such as a bandage, gauze, mesh or the like which may be applied to and over the area to be treated.

[0222]

[0166] Liquid or powder formulations may also be provided which can be sprayed or sprinkled directly onto the site to be treated, e.g. a wound or ulcer. Alternatively, a carrier such as a bandage, gauze, mesh or the like can be sprayed or sprinkle with the formulation and then applied to the site to be treated.

[0167] According to a further aspect, there is provided a process for the preparation of a pharmaceutical composition as described above, the process comprising bringing the particulate composition into association with the carrier, for example by admixture.

[0223]

[0168] In general, the formulations are prepared by uniformly and intimately bringing into association the particulate composition with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. The disclosure extends to methods for preparing a pharmaceutical composition comprising bringing the particulate composition described herein into conjunction or association with a pharmaceutically acceptable carrier or vehicle.

[0224]

[0169] The pharmaceutical composition may in preferred embodiments be in the form of a nutritional supplement. The nutritional supplement may be provided as a powder or liquid suitable for adding by the consumer to a food or beverage. For example, in some embodiments, the nutritional supplement can be administered to an individual in the form of a powder, for instance to be used by mixing into a beverage, or by stirring into a semi-solid food such as a pudding, topping, sauce, puree, cooked cereal, or salad dressing, for instance, or by otherwise adding to a food.

[0225]

[0170] The nutritional supplement may contain further ingredients in addition to those discussed above, such as one or a combination of other vitamins, minerals, fiber and other dietary supplements (e.g., protein, amino acids, choline, lecithin, omega-3 fatty acids). Selection of one or several of these ingredients is a matter of formulation, design, consumer preference and end-user. The amounts of these ingredients added to the supplements are readily known to the skilled person. Further vitamins and minerals that can be added include, but are not limited to, calcium phosphate or acetate, tribasic; potassium phosphate, dibasic; magnesium sulfate or oxide; salt (sodium chloride); potassium chloride or acetate; ascorbic acid; ferric orthophosphate; niacinamide; zinc sulfate or oxide; calcium pantothenate; copper gluconate; riboflavin; beta-carotene; pyridoxine hydrochloride; thiamine mononitrate; folic acid; biotin; chromium chloride or picolonate; potassium iodide; sodium selenate; sodium molybdate; phylloquinone; vitamin D3; cyanocobalamin; sodium selenite; copper sulfate; vitamin A; vitamin C; inositol; and potassium iodide.

[0226] ADMINISTRATION

[0227]

[0171] The pharmaceutical compositions of the present disclosure may be adapted for rectal, nasal, intrabronchial, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intraarterial and intradermal), intratumoural, intraperitoneal or intrathecal administration. Preferably the formulation is an orally administered formulation. The formulations may conveniently be presented in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose. By way of example, the formulations may be in the form of tablets and sustained release capsules, and may be prepared by any method well known in the art of pharmacy.

[0228]

[0172] Formulations for oral administration in the present invention may be presented as: discrete units such as capsules, vials, gellules, drops, cachets, pills or tablets each containing a predetermined amount of the particulate composition; as a powder or granules; as a solution, emulsion or a suspension of the particulate composition in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; or as a bolus etc.

[0229]

[0173] For compositions for oral administration (e.g. tablets and capsules), the term “acceptable carrier” includes vehicles such as common excipients e.g. binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose and starch; fillers and carriers, for example corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silica. Flavouring agents such as peppermint, oil of Wintergreen, cherry flavouring and the like can also be used. It may be desirable to add a colouring agent to make the dosage form readily identifiable. Tablets may also be coated by methods well known in the art.

[0230]

[0174] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the particulate composition in a free flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may be optionally be coated or scored and may be formulated so as to provide slow or controlled release of the particulate composition.

[0231]

[0175] Other formulations suitable for oral administration include lozenges comprising the particulate composition in a flavoured base, usually sucrose and acacia ortragacanth; pastilles comprising the particulate composition in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the particulate composition in a suitable liquid carrier.

[0176] Other forms of administration comprise solutions or emulsions which may be injected intravenously, intratumourally, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions.

[0232]

[0177] The pharmaceutical compositions may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.

[0233]

[0178] The pharmaceutical compositions are administered in an amount to be effective for the intended application and the subject to be treated. To this end, the dosage of the composition and other constituents may vary depending on age, weight, and condition of the subject. In general, the active agent is preferably administered at a concentration that will afford effective results without causing any harmful or deleterious side effects, and may be administered either as a single unit dose, or if desired in convenient subunits administered at suitable times throughout the day. The pharmaceutical composition may be administered multiple times a day, for example, from two to five times adding up for the necessary amount for one day, once a day, or continuously for a necessary term.

[0234]

[0179] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

[0235] EXAMPLES

[0236] Example 1 - Preparation of particulate composition comprising oxidised papain-like protease

[0237]

[0180] An exemplary particulate composition according to the present disclosure was prepared by the following process.

[0238] Step A - 500 g of commercially obtained bromelain powder was pulverised in a planetary ball mill to produce a nanoparticulated composition. A 2M suspension of the bromelain was prepared by adding 90 g of the bromelain to 200 ml distilled water and centrifuging to disperse and mix the suspension and prevent aggregation. The supernatant, containing the particulate composition, was then transferred to a separate container.

[0239] Step B - To the supernatant from step A was added 3 ml of 0.5M sodium thiosulphate. Step C - 5 ml of 0.5M Tris buffer pH 7.4 was then added, followed by 4 ml of 0.05% hydrogen peroxide solution. The resulting mixture was incubated at 37 °C for a sufficient period of time to allow the oxidation of bromelain to proceed, typically between 10 and 300 minutes.

[0240] Step D - Following this incubation, 3 ml of 0.5M sodium thiosulphate was added. 2 ml of 0.1 M N-acetyl-L-cysteine was then added followed by mixing. Finally, 2 ml of water was added to provide the final formulation.

[0241] Comparative Example 1 - Preparation of particulate composition comprising oxidised papain-like protease

[0242]

[0181] For comparative purposes, a crude particulate composition comprising an oxidised form of the papain-like protease was also prepared. This composition was prepared according to the process comprising steps A-D as for Example 1 , except that no pulverisation step with the planetary ball mill was performed. The other steps were performed as for Example 1 .

[0243] Example 2 - Cancer cell line cytotoxicity

[0244]

[0182] The particulate compositions of Example 1 and Comparative Example 1 were assessed for their cytotoxic potential in 5 different cancer cell lines: HPAC pancreatic adenocarcinoma; 22Rv1 prostate carcinoma; A549 lung carcinoma; HCT116 colorectal carcinoma; and K562 chronic myeloid leukaemia. In addition to testing the final products of Example 1 (i.e. after step D), the cytotoxicity of the particulate compositions following step B (i.e. prior to oxidation), and step C (i.e. immediately after oxidation) was also tested. The cytotoxicity assays were carried out as follows.

[0245]

[0183] The following cell densities of the 5 cell lines were seeded in 22.5 pl growth medium and the plate incubated overnight at 37 °C and 5% CO2.

[0246] Table 1

[0247]

[0184] After 24 hours of incubation, the cells were treated with the test compounds in the concentration range from 200 mM to 0.0001 mM. The plate was then incubated for 72 hours at 37 °C and 5% CO2The assay was terminated by the addition of 25 pl of CellTitre-Glo reagent, followed by reading the plate for luminescence, allowing determination of the relative number of viable cells in each well of the plate. The cytotoxicity IC50 values of Examples 1 and Comparative Example 1 were thus calculated, as shown in Figures 1A-D and Table 2 below.

[0248] Table 2

[0249]

[0185] As can be seen from Table 2 above, following the oxidation step C, a significant reduction in the cytotoxicity IC50 of the particulate compositions was observed across all cell lines tested, indicating that the oxidised form of the papain-like protease according to the present disclosure exhibited significantly greater cytotoxic activity than the non-oxidised form. As discussed above, this result was unexpected given the conventional understanding in the art to avoid oxidation of papain-like proteases during production. A comparison of the final products following step D for both Example 1 and Comparative Example 1 also showed an unexpected improvement in cytotoxicity across all cell lines tested when the composition of the oxidised papain-like protease comprising nanoscale particles was used as opposed to the commercially available coarse powder.

[0250] Example 3 - Antimicrobial activity

[0251]

[0186] The final particulate compositions of Example 1 and Comparative Example 1 (i.e. those following step D) were further assessed for their antimicrobial activity against three different bacterial strains: Escherichia coli, S. aureus, and M. smegmatis. Each microbial strain was procured from the ATCC and stored at -70 ± 10 °C prior to use. Table 3 below provides a summary of the strains used:

[0252] Table 3

[0253] Microbial strain revival and usage

[0187] Test organisms were revived using suitable media and sub-cultured prior to commencement of each experiment. All cultures used were no more than 5 passages when subcultures from original ATCC reference culture. Revival: Working vials of test organisms were removed from storage and allowed to thaw. Microbial suspension aliquot was transferred onto MHA plates, streaked across the surface of medium and incubated at 35±2°C for 20-21 hours aerobically except M. smegmatis was inoculated onto SCDA plates incubated at 35±2°C for 69-70 hours aerobically. Subculture: Culture from revival plate was used for sub-culturing on MHA plates and incubated at 35±2°C for ~20 hours aerobically. M. smegmatis was subcultured on SCDA and CAMHB and was incubated at 35±2°C for 71-72 hours aerobically.

[0254] Minimum inhibitory concentration determination

[0255]

[0188] A bacterial inoculum suspension was prepared from freshly grown culture on MHA / SCDA plates in sterile CAMHB. Turbidity of bacterial culture was adjusted spectrophotometrically at 625 nm. Adjusted cultures were further diluted (example 1 :100 times) using sterile CAMHB such that after inoculation each well finally contains ~5 x 105 cfu / mL (range 2-8 x 105 cfu / mL).

[0256]

[0189] Counts and purity check: The inoculum suspensions were subjected to purity and count check. For purity check, an aliquot of the inoculum suspension was sub-cultured onto SCDA / MHA plate for simultaneous incubation. Colony counts of final adjusted inoculum suspension were estimated by ten-fold serial log dilutions and plating each dilution on agar plate. After incubation, the plates were observed for growth and microbial counts determined.

[0257]

[0190] The particulate compositions were tested at a final concentration range of 200-0.2 mM (200 mM, 100 mM, 0 mM, 25 mM, 12.5 mM, 6.25 mM, 3.12 mM, 1.56 mM, 0.8 mM, 0.4 mM and 0.2 mM).

[0258]

[0191] To each of the wells of the microtiter tray containing serial dilutions of the compounds, 50 pL of the above diluted inoculum suspension was inoculated to obtain a final inoculum density of ~5 x 105 cfu / mL (2-8 x 105 cfu / mL). For M. smegmatis, 100 pL of the diluted organism was added to 100 pL of serial dilutions of the compounds. Microtiter plates were incubated at 35± 2 °C in an ambient air incubator for 16-20 hours except M. smegmatis. M. smegmatis was incubated at 35 ± 2°C for 3 days aerobically. After incubation, plates were observed for presence or absence of growth. Organism control, organism control with vehicle, broth control, vehicle control and compound control were set up.

[0259]

[0192] The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism in broth dilution susceptibility test. After the incubation period, growth of organism in the wells was thus detected by unaided eye facilitated by a viewing device. The amount of growth in the drugcontaining wells was compared with the amount of growth in the organism-control wells. The lowest concentration of an antimicrobial agent that completely inhibits growth of the microorganism as detected by the unaided eye was taken as MIC. Additionally, MIC plates were read spectrophotometrically at 625 nm, and optical density was recorded using a plate reader.

[0260]

[0193] The MIC results are shown below in Table 4:

[0261] Table 4

[0262]

[0194] As shown in the table above, the particulate composition of Example 1 showed measurable antimicrobial activities against each of the bacterial strains tested. In addition, the nanoparticulate composition of Example 1 also exhibited unexpectedly improved activity compared with the compositions of Comparative Example 1 .

[0263] Example 4 - Further antimicrobial activity assay

[0264]

[0195] As a further investigation of the effect of oxidising a papain-like protease on antimicrobial activity, an additional bromelain composition was prepared according to the following process.

[0265]

[0196] Commercially prepared bromelain (Bromelain 500mg; Solgar, UK) was used. The following method was used prior to each assay to obtain the aqueous bromelain test samples: six 500mg bromelain tablets were crushed in a mortar and pestle and mixed with 10ml distilled water. The 0.013M solution was centrifuged and the liquid supernatant was removed from the sediment and used as the test sample in the assays described below.

[0266]

[0197] To prepared the oxidised bromelain samples, 30% hydrogen peroxide (H2O2), the oxidising agent, was prepared to fourth-order serial dilutions (ie. 1 :10, 1 :100, 1 :1000, 1 :10,000). Approximately 2ml of each H2O2 dilution was correspondingly added to 2ml of the above pre-prepared bromelain dilutions. On mixing further with 2ml Tris / HCL buffer the dilutions together with controls were incubated at 37°C for a sufficient length of time for oxidation to proceed, typically between 10 and 300 minutes.

[0267]

[0198] Following incubation, the oxidised sample and non-oxidised controls were plated out aseptically against selected bacteria. Each dilution and control was tested for antibacterial activity and involved aseptically pipetting 20pl of diluent onto a filter disk per pre-prepared species-specific bacterial lawn.

[0268]

[0199] The level of antibacterial activity was assessed by measuring the diameter surrounding the plated samples in which bacterial growth was not observed. The assays were carried out for four different bacterial strains: Eschericia coli, Brevundimonas diminuta, Micrococcus luteus, and Bacillus megaterium. The results are shown in Figure 2.

[0269]

[0200] As seen in Figure 2, in each of the tested bacterial strains, the oxidised form of the papain-like protease tested exhibited improved antibacterial activity compared with the nonoxidised control.

[0270] Example 5 - Virucidal activity assay

[0271]

[0201] The virucidal activity of the particulate composition of the disclosure was further investigated. Specifically, the composition of Example 1 following oxidation as described in step C was tested for its virucidal activity in human coronavirus, strain 229E, ATCC VR-7401 P4; and Hepatitis A virus, strain HM 175 / 18f, ATCC VR-1402 / P6.

[0272]

[0202] The test method was carried out to the standard method EN 14476:2013+A2:2019, “Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of virucidal activity in the medical area - Test method and requirements (Phase 2, step 1)”.

[0273]

[0203] In particular, the particulate composition was tested for its virucidal activity at contact times of 5 minutes (± 10 seconds), 30 minutes (± 10 seconds), and 60 minutes (± 10 seconds). Following this contact time, the average log reduction in infectious virus particles was determined. The results are shown in Table 5 below. Table 5

[0274]

[0204] As shown in the table above, the particulate composition of the disclosure showed measurable virucidal activity against both virus strains tested.

[0275]

[0205] The various embodiments described herein are presented only to assist in understanding and teaching the claimed features. These embodiments are provided as a representative sample of embodiments only, and are not exhaustive and / or exclusive. It is to be understood that advantages, embodiments, examples, functions, features, structures, and / or other aspects described herein are not to be considered limitations on the scope of the invention as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope of the claimed invention. Various embodiments of the invention may suitably comprise, consist of, or consist essentially of, appropriate combinations of the disclosed elements, components, features, parts, steps, means, etc., other than those specifically described herein. In addition, this disclosure may include other inventions not presently claimed, but which may be claimed in future.

[0276] EMBODIMENTS

[0277]

[0206] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered clauses.

[0278] 1. A particulate composition, wherein the particles comprise an oxidised form of a papainlike protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0279] 2. A composition according to clause 1 , wherein the papain-like protease is naturally occurring.

[0280] 3. A composition according to any preceding clause, wherein the papain-like protease is a plant-derived papain-like protease.

[0281] 4. A composition according to any preceding clause, wherein the papain-like protease is a fruit-derived papain-like protease.

[0282] 5. A composition according to any preceding clause, wherein the papain-like protease is derived from a member of the Bromeliaceae plant family.

[0283] 6. A composition according to any preceding clause, wherein the papain-like protease is a pineapple-derived papain-like protease. 7. A composition according to any preceding clause, wherein the papain-like protease belongs to the cysteine peptidase family C1 .

[0284] 8. A composition according to any preceding clause, wherein the papain-like protease belongs to the cysteine peptidase subfamily C1 A.

[0285] 9. A composition according to any preceding clause, wherein the papain-like protease is bromelain.

[0286] 10. A composition according to clause 9, wherein the bromelain comprises: a) stem bromelain or a fragment or a variant thereof; b) fruit bromelain or a fragment of a variant thereof; or c) a mixture of (a) and (b).

[0287] 11. A composition according to clause 9, wherein papain-like protease is stem bromelain.

[0288] 12. A composition according to any preceding clause, wherein the oxidised form of the papain-like protease or the fragment or the variant thereof is obtained by reaction of a papainlike protease or a fragment or a variant thereof with an oxidising agent.

[0289] 13. A composition according to clause 12, wherein the oxidising agent is selected from the group consisting of peroxides, persulphates, peroxynitrites, nitric oxide, halogens, hypochlorites, hypothiocyanites, halamines, diamides, iodosobenzoates, iodoacetamide, nitrobenzoic acids, alkylating agents, ozone, and mixtures thereof.

[0290] 14. A composition according to clause 12 or clause 13, wherein the oxidising agent is a peroxide.

[0291] 15. A composition according to clause 13 or clause 14, wherein the peroxide is hydrogen peroxide, benzoyl peroxide, tert-butyl hydroperoxide, cumene hydro peroxide, acetyl peroxide, methyl ethyl ketone peroxide, di-tert-butyl peroxide, urea peroxide (carbamide peroxide), sodium peroxide, potassium peroxide, or mixtures thereof.

[0292] 16. A composition according to any of clauses 13 to 15, wherein the peroxide comprises hydrogen peroxide.

[0293] 17. A composition according to any preceding clause, wherein the oxidised form of the papain-like protease or the fragment or the variant thereof comprises one or more oxidised thiol groups. 18. A composition according to clause 17, wherein the one or more oxidised thiol residues comprise at least one oxidised cysteine residue.

[0294] 19. A composition according to clause 17 or clause 18, wherein the one or more oxidised thiol groups comprise at least one residue selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds.

[0295] 20. A composition according to any preceding clause, wherein the particles are nanoparticles.

[0296] 21. A composition according to any preceding clause, wherein the particles have an average particle size of about 100 nm or less.

[0297] 22. A composition according to any preceding clause, having a moisture content by weight of about 10 wt.% or less, 5 wt.% or less, 3 wt.% or less, 1 wt.% or less, or being substantially free of moisture.

[0298] 23. A composition according to any preceding clause, wherein the particulate composition comprises a solvent in which the particles are suspended.

[0299] 24. A composition according to clause 23, wherein the solvent is an aqueous solvent, preferably wherein the aqueous solvent is water.

[0300] 25. A pharmaceutical composition comprising a particulate composition according to any preceding clause and one or more carriers, excipients, or diluents, wherein the pharmaceutical composition optionally further comprises a solvent.

[0301] 26. A pharmaceutical composition according to clause 25, wherein the solvent is an aqueous solvent, preferably wherein the aqueous solvent is water.

[0302] 27. A process for preparing a particulate composition according to any preceding clause, the process comprising the steps of:

[0303] (ai) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and

[0304] (aii) treating the particles with an oxidising agent; or

[0305] (bi) providing a papain-like protease or a fragment or a variant thereof; (bii) treating the papain-like protease or the fragment or the variant thereof with an oxidising agent; and

[0306] (biii) preparing particles comprising the oxidised form of the papain-like protease or the fragment or the variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

[0307] 28. A process according to clause 27, wherein the oxidising agent is as defined in any of clauses 13 to 16.

[0308] 29. A process according to clause 27 or clause 28, wherein the particles are provided in step (ai) as a suspension in a solvent.

[0309] 30. A process according to clause 29, wherein the solvent is an aqueous solvent, preferably wherein the aqueous solvent is water.

[0310] 31 . A process according to any of clauses 27 to 30, further comprising a step of treating the particles with a thiosulphate, preferably wherein the thiosulphate is sodium thiosulphate.

[0311] 32. A process according to any of clauses 27 to 31 , wherein step (aii) and step (bii) are carried out at a pH of from about 6 to about 8, preferably from about 7 to about 8.

[0312] 33. A process according to any of clauses 27 to 32, wherein step (aii) and step (bii) are carried out at a temperature of from about 20 °C to about 60 °C, preferably from about 25 °C to about 55 °C, more preferably from about 30 °C to about 40 °C, yet more preferably from about 35 °C to about 40 °C, most preferably at about 37 °C.

[0313] 34. A process according to any of clauses 27 to 33, wherein step (aii) and step (bii) are carried out for a length of at least about 10 minutes, preferably from about 10 minutes to about 300 minutes.

[0314] 35. A process according to any of clauses 27 to 34, further comprising a step of treating the particles with N-acetyl-L-cysteine.

[0315] 36. A process according any of clauses 27 to 35, comprising the steps of:

[0316] (i) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less;

[0317] (i-a) treating the particles with a thiosulphate; (ii) treating the particles with an oxidising agent;

[0318] (iii) treating the particles with a thiosulphate;

[0319] (iv) treating the particles with N-acetyl-L-cysteine; and

[0320] (v) optionally, adding a solvent; wherein the steps are carried out in the order (i)-(v).

[0321] 37. A composition obtained or obtainable by the process according to any of clauses 27 to 36.

[0322] 38. An oxidised form of a papain-like protease or a fragment or a variant thereof, for use in therapy.

[0323] 39. An oxidised form of a papain-like protease or a fragment or a variant thereof, for use in treating cancer.

[0324] 40. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 39, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, prostate cancer, leukaemia, and pancreatic cancer.

[0325] 41 . An oxidised form of a papain-like protease or a fragment or a variant thereof, for use in treating a bacterial or viral infection.

[0326] 42. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 41 , wherein the bacterial infection is caused by gram-negative bacteria, gram-positive bacteria, or mycobacteria.

[0327] 43. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 42, wherein the bacterial infection is caused by gram-negative bacteria selected from Enterobacteriaceae and Brevundimonas, preferably Escherichia coli or Brevundimonas diminuta.

[0328] 44. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 42, wherein the bacterial infection is caused by gram-positive bacteria selected from Staphylococcus, Micrococcus, and Bacillaceae, preferably Staphylococcus aureus, Micrococcus luteus, and Bacillus megaterium. 45. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 42, wherein the bacterial infection is caused by gram-positive bacteria selected from Mycobacteriaceae, preferably Mycobacterium.

[0329] 46. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 41 , wherein the viral infection is caused by an enveloped virus, preferably a coronavirus.

[0330] 47. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to clause 41 , wherein the viral infection is caused by a non-enveloped virus, preferably a hepatitis A virus.

[0331] 48. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to any of clauses 38 to 48, wherein the oxidised form of the papain-like protease or the fragment or the variant thereof is defined as in any of clauses 2 to 26.

[0332] 49. A particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof, for use in treating a bacterial or viral infection, wherein the particles have an average particle size of about 1000 nm or less.

[0333] 50. A composition for use according to clause 49, wherein the bacterial or viral infection is as defined in any of clauses 42 to 47.

[0334] 51 . A composition for use according to clause 49 or clause 50, wherein the composition is as defined as in any of clauses 1 to 26.

[0335] 52. A particulate composition according to any of clauses 1 to 26 or 37, for use in therapy.

[0336] 53. A composition according to any of clauses 1 to 26 or 37, for use in treating cancer.

[0337] 54. A composition for use according to clause 53, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, prostate cancer, leukaemia, and pancreatic cancer.

[0338] 55. A composition according to any of any of clauses 1 to 26 or 37, for use in treating a bacterial or viral infection.

[0339] 56. A composition for use according to clause 55, wherein the bacterial or viral infection is as defined in any of clauses 42 to 47.

[0340] 57. A non-therapeutic use of a composition according to any of clauses 1 to 26 or 37, wherein the use comprises applying the composition to a surface as a disinfectant. AMINO ACID SEQUENCES

[0341] Stem bromelain sequence (SEQ ID NO: 1):

[0342] AVPQSIDWRDYGAVTSVKNQNPCGACWAFAAIATVESIYKIKKGILEPLSEQQVLDCAKG YGCKGGWEFRAFEFIISNKGVASGAIYPYKAAKGTCKTDGVPNSAYITGYARVPRNNESS MMYAVSKQPITVAVDANANFQYYKSGVFNGPCGTSLNHAVTAIGYGQDSIIYPKKWGAKW GEAGYIRMARDVSSSSGICGIAIDPLYPTLEE

[0343] Fruit bromelain sequence (SEQ ID NO: 2):

[0344] MASKVQLVFLFLFLCAMWASPSAASRDEPNDPMMKRFEEWMAEYGRVYKDDDEKMRRF QIFKNNVKHIETFNSRNENSYTLGINQFTDMTKSEFVAQYTGVSLPLNIEREPWSFDDVNI SAVPQSIDWRDYGAVNEVKNQNPCGSCWSFAAIATVEGIYKIKTGYLVSLSEQEVLDCAVS YGCKGGWVNKAYDFIISNNGVTTEENYPYLAYQGTCNANSFPNSAYITGYSYVRRNDERS MMYAVSNQPIAALIDASENFQYYNGGVFSGPCGTSLNHAITIIGYGQDSSGTKYWIVRNSW GSSWGEGGYVRMARGVSSSSGVCGIAMAPLFPTLQSGANAEVIKMVSET

[0345] Ananain sequence (SEQ ID NO: 3):

[0346] MTSKVQLVFLFLFLCVMWASPSAASCDEPSDPMMKQFEEWMAEYGRVYKDNDEKMLRFQ IFKNNVNHIETFNNRNGNSYTLGINQFTDMTNNEFVAQYTGLSLPLNIKREPWSFDDVDISS VPQSIDWRDSGAVTSVKNQGRCGSCWAFASIATVESIYKIKRGNLVSLSEQQVLDCAVSYG CKGGWINKAYSFIISNKGVASAAIYPYKAAKGTCKTNGVPNSAYITRYTYVQRNNERNMMY AVSNQPIAAALDASGNFQHYKRGVFTGPCGTRLNHAIVIIGYGQDSSGKKFWIVRNSWGAG WGEGGYIRLARDVSSSFGLCGIAMDPLYPTLQSGPSVEVI

[0347] Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A particulate composition, wherein the particles comprise an oxidised form of a papainlike protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less.

2. A composition according to claim 1 , wherein the papain-like protease belongs to the cysteine peptidase family C1 .

3. A composition according to claim 1 or claim 2, wherein the papain-like protease is a plant-derived papain-like protease.

4. A composition according to any preceding claim, wherein the papain-like protease is bromelain.

5. A composition according to any preceding claim, wherein the papain-like protease is stem bromelain.

6. A composition according to any preceding claim, wherein the oxidised form of the papain-like protease or the fragment or the variant thereof is obtained by reaction of a papainlike protease or a fragment or a variant thereof with an oxidising agent.

7. A composition according to claim 6, wherein the oxidising agent is a peroxide.

8. A composition according to claim 7, wherein the peroxide comprises hydrogen peroxide and preferably is hydrogen peroxide.

9. A composition according to any preceding claim, wherein the oxidised form of the papain-like protease or the fragment or the variant thereof comprises one or more oxidised thiol groups.

10. A composition according to claim 9, wherein the one or more oxidised thiol groups comprise at least one residue selected from the group consisting of sulfenic acid, sulfinic acid, sulfonic acid, and disulfide bonds.

11. A composition according to any preceding claim, wherein the particles have an average particle size of about 100 nm or less.

12. A pharmaceutical composition comprising a particulate composition according to any preceding claim and one or more carriers, excipients, or diluents, wherein the pharmaceutical composition optionally further comprises a solvent.

13. A process for preparing a composition according to any preceding claim, the process comprising the steps of:(ai) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of about 1000 nm or less; and(aii) treating the particles with an oxidising agent; or(bi) providing a papain-like protease or a fragment or a variant thereof;(bii) treating the papain-like protease or the fragment or the variant thereof with an oxidising agent; and(biii) preparing particles comprising the oxidised form of the papain-like protease or the fragment or the variant thereof, wherein the particles have an average particle size of 1000 nm or less.

14. A process according to claim 13, wherein the oxidising agent is as defined in claim 7 or claim 8.

15. A process according to claim 13 or claim 14, comprising the steps of:(i) providing particles comprising a papain-like protease or a fragment or a variant thereof, wherein the particles have an average particle size of 1000 nm or less;(i-a) treating the particles with a thiosulphate;(ii) treating the particles with an oxidising agent;(iii) treating the particles with a thiosulphate;(iv) treating the particles with N-acetyl-L-cysteine; and(v) optionally, adding a solvent; wherein the steps are carried out in the order (i)-(v).

16. A composition obtained or obtainable by the process according to any of claims 13 to 15.

17. An oxidised form of a papain-like protease or a fragment or a variant thereof, for use in therapy.

18. An oxidised form of a papain-like protease or a fragment or a variant thereof, for use in treating cancer.

19. An oxidised form of a papain-like protease or a fragment or a variant thereof, for use in treating a bacterial or viral infection.

20. An oxidised form of a papain-like protease or a fragment or a variant thereof for use according to any of claims 17 to 19, wherein the oxidised form of the papain-like protease or the fragment or the variant thereof is defined as in any of claims 2 to 11 .

21. A particulate composition, wherein the particles comprise a papain-like protease or a fragment or a variant thereof for use in treating a bacterial or viral infection, wherein the particles have an average particle size of about 1000 nm or less.

22. A composition for use according to claim 21 , wherein the composition is as defined as in any of claims 1 to 11.

23. A composition according to any of claims 1 to 11 or 16 for use in therapy.

24. A composition according to any of claims 1 to 11 or 16 for use in treating cancer, a bacterial infection, or a viral infection.

25. A non-therapeutic use of a composition according to any of claims 1 to 11 or 16, wherein the use comprises applying the composition to a surface as a disinfectant.

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