AU2008100719A4 - Papaya resin for gluten digestion and uses thereof - Google Patents

Description

00 0 0 P/00/009A Section 29

AUSTRALIA

Patents Act 1990 INNOVATION PATENT SPECIFICATION Invention Title: Applicant: PAPAYA RESIN FOR GLUTEN DIGESTION AND USES

THEREOF

Glutagen Pty Ltd The invention is described in the following statement: 1 7019 00 PAPAYA RESIN FOR GLUTEN DIGESTION AND USES THEREOF la The present invention relates generally to compositions for the prophylaxis or therapy Sof conditions related to gluten intolerance, and uses thereof.

BACKGROUND

Gluten intolerance is found predominantly in areas where wheat is a major food source, Europe, North America and Australia). In these areas, the incidence of the disease is about 1 per 100 head of population (DA Van Heel et al, Gut 2006; 55:1037- 0 1046). The symptoms of this condition include abdominal pain, bloating and diarrhoea.

SIn severe and long term cases, such as in coeliac disease, there are inflammatory changes to the intestinal mucosa, resulting in malabsorption of nutrients, fatigue, chronic diarrhoea, weight loss, abdominal distension, anaemia, increased tendency to haemorrhage, as well as increased risk of gastrointestinal malignancies, such as lymphoma and carcinoma.

The pathogenesis of gluten intolerance (or coeliac disease, coeliac sprue) appears to have both genetic and environmental factors. Whilst genetic predisposition is a major factor (about 10% of first degree relatives are affected), the fact that monozygotic twins have a concordance rate of only about 75% suggests that environment also plays a part in the development of the disease.

Patients with gluten intolerance characteristically have T cells present in the intestinal mucosa which recognize certain sequences present in toxic gluten peptides. Evidence suggests that these T cells play a crucial role in the immunopathogenesis of the disease by recognising peptides containing specific sequences of amino acids associated with toxicity. For instance, the proliferation of gliadin-specific HLA-DQ2restricted T cell clones from the intestine of coeliac disease patients can be initiated in vitro by the addition of a water-soluble, partially digested form of gliadin to HLA-DQ2 carrying antigen-presenting cells.

A related disease associated with severe gluten intolerance is dermatitis herpetiformis, which presents as a chronic eruption characterized by clusters of intensely pruritic vesicles, papules, and urticaria-like lesions. Studies have shown that IgA deposits occur in almost all normal-appearing and perilesional skin. Asymptomatic glutensensitive enteropathy is found in 75 to 90% of patients and in some of their relatives.

W:F dWe%83265~83265_speci_v040808 doc 00 The onset of dermatitis herpetiformis is usually gradual, progressing to severe itching O and burning of the affected surface. Moreover, scratching often obscures the primary lesions with eczematization of nearby skin, leading to an erroneous diagnosis of eczema.

Gluten is a protein fraction found, for example, in cereal dough, which can be subdivided into glutenins and prolamins. Prolamins may also be subclassified as gliadins, secalins, hordeins, and avenins from wheat, rye, barley and oat, respectively.

Among gluten proteins with potential harmful effect to gluten intolerant patients are the storage proteins of wheat, species of which include Triticum aestivum; Triticum 00 Saethiopicum; Triticum baeoticum; Triticum militinae; Triticum monococcum; Triticum sinskajae; Triticum timopheevii; Triticum turgidum; Triticum urartu, Triticum vavilovii; Triticum zhukovskyi; etc. (see, for example, Colot, Genet Eng (NY) 12:225-41, 1990).

Gliadin is the 70% alcohol-soluble protein fraction of wheat gluten. Derived from wheat flour, gliadins can be classified into several groups according to their electrophoretic mobility, including a-type, I-type, y-type and w-type. Gliadins are typically rich in glutamine and proline, particularly in the N-terminal part. For example, the first 100 amino acids of a and y-gliadins contain about 35% and about 20% of glutamine and proline residues, respectively. Different gliadins are present in each subcultivar of wheat, with variations in the amino acid sequences within each type. Gliadins are typically characterized by a molecular mass of around 30-50 kDalton and their insolubility in neutral aqueous solutions. Examples of gliadin sequences include but are not limited to wheat a-gliadin sequences, for example as provided in Genbank, accession numbers AJ133612; AJ133611; AJ133610; AJ133609; AJ133608; AJ133607; AJ133606; AJ133605; AJ133604; AJ133603; AJ133602; D84341.1; U51307; U51306; U51304; U51303; U50984; and U08287. A sequence of wheat omega gliadin is set forth in Genbank accession number AF280605.

It has been discovered that in gluten intolerant individuals, enzymes normally present in the small bowel that are necessary for the digestion of gluten are missing. Peptide fragments produced by incomplete digestion of grain protein are toxic to such individuals; the most toxic peptides being those derived from a-gliadin, or a similar protein called A-gliadin.

Serine-containing peptides, (containing PSQQ and possibly also QQQP motifs, as found in residues 11-19 of A-gliadin), appear to have a cytotoxic effect. Tyrosine- W:VF es2653265PspeeU~vO4O8O8 do 00 containing peptides, (containing QQPY and/or QPYP motifs, as found in residues 0 86 of A-gliadin), are associated with immunological activity through T-cell mediation (Ni and hence, toxicity.

Experiments indicate that active serine-containing peptides like 11-19 and active Styrosine-containing peptides like 75-86 are incompletely digested by mucosal enzymes in patients suffering from coeliac disease. The residual peptide sequences, such as 11-18 and 77-84, are still toxic, which suggests that the aetiology of coeliac disease is connected to defective mucosal digestion and that the pathogenesis of the disease results from the action of the undigested peptides on the mucosa. This may ultimately 0 be due to deficiency in a single enzyme in coeliac disease patients, but at least two Sdifferent types of peptide residues build up and cause damage to mucosal tissue.

(Ni At present, there is no effective therapy for treating the effects of gluten intolerance other than to impose a gluten-free diet on the patient. However, due to the number of food products containing either cereals comprising gluten, or gluten per se, this approach constitutes a severe restriction to the food choices available to a patient.

Moreover, although gluten withdrawal has improved the prognosis of gluten intolerant patients, some people still die of the disease, presumably from lymphoreticular disease (especially intestinal lymphoma), particularly in those people who present with severe gluten intolerance at the outset. It appears that gluten withdrawal diminishes the risk of developing lymphoreticular disease, whilst apparent clinical remission is often associated with histologic relapse that is detected only by review biopsies or by an increased IgA class anti-endomysial antibody (EMA) titre.

In view of the serious and widespread nature of gluten intolerance, improved methods of treating, preventing or ameliorating the effects of this condition are needed.

Accordingly, it is an aspect of the present invention to overcome, or at least partly alleviate, some of the aforementioned problems of the art by providing improved compositions for preventing or treating conditions arising from gluten intolerance and methods for their use.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

W. FIesr8326D9B3265speci vO4OO dmo c 00

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O SUMMARY OF THE INVENTION

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SIn one aspect of the present invention, there is provided a composition for the prophylaxis or treatment of a condition related to gluten intolerance, the composition Sincluding an extract of papaya resin, or a functional analogue thereof.

In some embodiments of the present invention, the extract of papaya resin is derived from papaya fruit, papaya leaves, papaya root, papaya latex, or any combination thereof.

00 SIn some embodiments of the present invention, the extract of papaya resin is derived from species of papaya classified in the genera selected from the group consisting of Carica, Cylicomorpha, Jacaratia, Jarilla, Horovitzia, Vasconcella, and a cultivar thereof.

In some embodiments of the present invention, the papaya resin is a papaya oleoresin.

In some embodiments, the composition of the present invention, as herein described, is formulated as an enterically coated tablet or capsule.

In another aspect of the present invention, there is provided a method for the prophylaxis or treatment of a condition related to gluten intolerance, the method including administering to a subject in need thereof a composition according to the present invention, as herein described.

DETAILED DESCRIPTION OF THE INVENTION Compositions In one aspect of the present invention, there is provided an oral composition for the prophylaxis or treatment of a condition related to gluten intolerance, the composition including an extract of papaya resin, or a functional analogue thereof.

It has been found by the present inventors that papaya resin (also referred to herein as a papaya resin extract) is capable of inactivating toxic oligopeptides that are produced following gluten ingestion to produce non-toxic peptides, thereby offering an improved W:\F es\3265l326590_espec_040O08.doc 00 method of preventing or at least partly alleviating their toxic effect in gluten intolerant Sindividuals. Without being bound by theory, it is expected that the effect is dosedependent of the concentration of enzyme and the amount of ingested gluten.

The papaya is a fast-growing tree-like herbaceous plant that produces fruit (also called papaya but sometimes referred to as mambo, tree melon, fruta bomba, lechosa or pawpaw. In Australia, red and pink-fleshed cultivars are often known as 'papaya' to distinguish them from the yellow-fleshed fruits, known as 'paw paw', but both of these common names refer to the same plant species. Irrespective of its flesh colour, C. papaya is generally known as 'papaya' in other countries. In some areas, an 0 unrelated plant, Asiminia triloba (Annonaceae), native to North America, is also called paw paw. Until recently, the Caricaceae species of papaya (Carica papaya was thought to comprise 31 species in three genera (namely Carica, Jacaratia and Jarilla) from tropical America and one genus, Cylicomorpha, from equatorial Africa (Nakasone Paull 1998; Tropical fruits. CAB International, Wallingford). However, a more recent taxonomic revision proposed that some species formerly assigned to Carica were more appropriately classified in the genus Vasconcella (Badillo 2002; Carica L. vs.

Vasconcella St. Hil. (Caricaceae) con la Rehabilitacion de este Ultimo. Ernstia 10: 74- 79). Accordingly, the family's classification has been revised to comprise Cylicomorpha and five South and Central American genera (Carica, Jacaratia, Jarilla, Horovitzia and Vasconcella) (Badillo 1971; Monografia de la familie Caricaceae.

Associacion de Profesores, Universidad Central de Venezuela, Maracay, Venezuela), with Carica papaya the only species within the genus Carica (Badillo 2002). Papaya is now grown in most tropical countries and many sub-tropical regions of the world.

Cultivars of papaya include, but are not limited to, Kamiya, Mexican Red, Mexican Yellow, Solo, Sunrise (Sunrise Solo), Sunset (Sunset Solo), Vista Solo, and Waimanalo (Waimanalo Solo, X-77).

Thus, in some embodiments of the present invention, the extract of papaya resin is derived from a species of papaya classified in the genera selected from the group consisting of Carica, Cylicomorpha, Jacaratia, Jarilla, Horovitzia, Vasconcella, or a cultivar thereof.

Papaya resin may be extracted or isolated from any part of the papaya plant (including, but not limited to, the fruit, leaves, root, latex, or any combination thereof) in accordance with conventional methods known to the skilled addressee.

W ,iWe3265 3265s ecvO4080oc 00 Methods useful for the extraction or isolation of papaya resin from a papaya plant (or 0 a part thereof) include, but are not limited to, precipitation with inorganic salts, solid- (Ni liquid extraction, liquid-liquid extraction, solid-phase extraction, membrane filtration, ;ultrafiltration, dialysis, electrophoresis, solvent concentration, centrifugation, ultracentrifugation, liquid or gas phase chromatography (including size exclusion Schromatography, affinity chromatography, etc) with or without high pressure, lyophilisation, evaporation, precipitation with various "carriers" antibodies), and any combination thereof. The skilled addressee would also understand how to use such options, in a sequential fashion, in order to enrich each successive fraction for papaya resin, if necessary, by following its biological activity throughout the purification 0 process. The activity of the papaya resin can be measured using a variety of methods known to the skilled addressee.

(Ni Solid-liquid extraction includes, but is not limited to, the use of various solvents, vortex shakers, ultrasounds and other means to enhance extraction, as well as recovery by filtration, centrifugation and related methods as described in the art (see, Cannell RJP, Natural Products Isolation, Humana Press, 1998). Examples of solvents that may be used include, but are not limited to, hydrocarbon solvents, chlorinated solvents, organic esters, organic ethers, alcohols, water, and combinations thereof.

Liquid-liquid extraction includes, but is not limited to, the use of solvents known in the art such as hydrocarbon solvents, chlorinated solvents, organic esters, organic ethers, alcohols, water, various aqueous solutions, and combinations thereof. The liquid-liquid extraction can be facilitated manually, or it can be automated (completely or in part), and the solvent can be removed and/or concentrated by standard techniques in the art.

Membrane, reverse osmosis and ultrafiltration include, but are not limited to, the use of various types of membranes known in the art, as well as the use of pressure, vacuum, centrifugal force, and/or other means that can be utilised in membrane and ultrafiltration processes.

Dialysis typically includes the use of membranes having a molecular weight cut-off that is selective for the removal of various constituents from the natural source so as to increase the relative purity of papaya resin in a sample. The present invention also encompassed the recovery of purified and/or fractionated extracts from either the dialysate or the retentate by various means known in the art including, but not limited to, lyophilization and crystallization.

W J.IeC\83265g'.3265D SPeaO40808 OC 00 oO 0 O Chromatography includes, but is not limited to, the use of regular column (Ni chromatography, flash chromatography, high performance liquid chromatography (HPLC), medium pressure liquid chromatography (MPLC), supercritical fluid chromatography (SFC), countercurrent chromatography (CCC), moving bed chromatography, simulated moving bed chromatography, expanded bed chromatography, and planar chromatography. Examples of sorbents that may be used in chromatography include, but are not limited to, silica gel, alumina, fluorisil, cellulose and modified cellulose, various modified silica gels, ion-exchange resins, size exclusion 10 gels, chemically modified gels, and other sorbents known to those skilled in the art. The 0 0present invention also includes the use of two or more salt gradients to effect the fractionation and/or partial purification of papaya resin by chromatographic methods.

(Ni When water or an aqueous phase is used, it may contain varying amounts of inorganic or organic salts, and/or the pH may be adjusted to different values with an acid or a base such that fractionation and/or purification is enhanced.

The process of extracting papaya resin from a papaya plant (or part thereof) may also include further concentrating the extracted resin, for example, by fractionation and/or purification of papaya resin from any extraneous material using techniques known to those skilled in the art including, but not limited to, rotary evaporation, distillation (normal and reduced pressure), centrifugal vacuum evaporation (speed-vac), lyophilization and combinations thereof.

In some embodiments, the composition of the present invention includes papaya resin in an amount at least about one or a few percent by weight of the total weight of the composition, for example, at least about five percent by weight of the total weight of the composition. In some embodiments, the papaya resin constitutes at least about twenty percent by weight of the total weight of the composition of the present invention. In some embodiments, the papaya resin constitutes at least about fifty percent by weight of the total weight of the composition of the present invention. In some embodiments, the papaya resin constitutes at least about eighty percent by weight of the total weight of the composition of the present invention. In some embodiments, the papaya resin constitutes at least about ninety percent or at least about ninety-five percent or more by weight of the total weight of the composition of the present invention.

W: Fites 6326591832659_speci_v040808 Ooc 00 In some embodiments of the present invention, the papaya resin is a papaya oleo- 0 resin. As used herein, the term "papaya oleo-resin" typically means a mixture that 0 includes papaya resin and an essential oil which exudes from the papaya plant (or a ;part or parts thereof). Papaya oleoresin may be isolated extracted) from a papaya plant, or a part or parts thereof, by any means known to the skilled addressee. For Sexample, papaya oleoresin may be prepared by the method described by Dietmar et al.

(2002, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 132(4):721-728).

Essential oils are typically oils derived from a plant (or a part or parts thereof, including, 00 but not limited to, fruit, leaves, stems, flowers, bark, roots and twigs) and usually carry Sthe odor and/or flavour of the plant (or a part or parts thereof). Essential oils are typically a complex mixture of odorous, volatile or extractable organic compounds, which are synthesized by many types of plants. Typically, the principle constituents of essential oils are aromatic compounds. Essential oils may be extracted from plant material by any methods known to the skilled addressee, including, but not limited to, steam distillation, cold pressing, CO 2 extraction, or extraction with organic solvents. In some embodiments, the essential oil is extracted from the plant (or a part of parts thereof) by the same process for the extraction of the papaya resin. Alternatively, or in addition, the essential oil may be extracted from the plant (or a part of parts thereof) by a process known to the skilled addressee that is separate or distinct from the process for the extraction of the papaya resin.

Assay for Biological Activity The composition of the present invention can be tested for its potential efficacy in the prophylaxis or treatment of a condition related to gluten intolerance by testing its ability to reduce the toxicity of toxic gluten peptides. This can be achieved by any means known to those skilled in the art, including any one or more of the procedures described herein. It will be understood by the skilled addressee that methods of extracting papaya resin from a papaya plant (or a part thereof), or from any other suitable source, will be such that at least some biological activity of the papaya resin is retained so as to provide for the prophylaxis or treatment of a condition related to gluten intolerance. One skilled in the art would appreciate that there are numerous methods and techniques for measuring qualitatively and/or quantitatively the ability of papaya resin to reduce the toxicity of toxic gluten peptides, either in vitro or in vivo.

W:.File8326583259seciv040808oOOdc 00 For instance, the biological activity of papaya resin may be identified by its ability to

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Sinactivate toxic gluten oligopeptides, where the substrate may include, but is not limited Sto, a gliadin, hordein, secalin or avenin protein. Toxic gliadin oligopeptides include Speptides derived during normal human digestion of gliadins and related storage proteins, as herein described, from dietary cereals wheat, rye, barley, and the like). Such oligopeptides are believed to act as antigens for T cells in patients suffering from a condition arising from gluten intolerance, such as Celiac Sprue. For binding to Class II MHC proteins, immunogenic peptides are usually from about 8 to 20 amino acids in length, more usually from about 10 to 18 amino acids. Such peptides may include QQPY or related tyrosine-containing motifs. Determination of whether an 0 oligopeptide is immunogenic for a particular patient is readily determined by standard T Scell activation and other assays known to those of skill in the art. The ability of papaya resin, or a functional analogue thereof, as herein described, to inactivate a toxic gluten peptide can be determined by any methods known in the art, for example, by using a Rat Liver Lysosome (RLL) assay, as described, for example, in WO 2003/100051 and Cornell and Townley (1974; Gut, 15(11):862-869), the entire contents of which are incorporated herein by reference.

In some embodiments, the biological activity of papaya resin is determined by testing its ability to inactivate toxic peptides such as those containing PSQQ, QQQP and QQPY motifs, and the A-gliadin wheat peptides QNPSQQQPQ (residues 11-19), RPQQPYPQPQPQ (residues 7.5-86), LGQQQPFPPQQPY (residues 31-43), PQPQPFPSQQPY (residues 44-55) and LGQGSFRPSQQN (residues 206-217).

The ability of papaya resin to modify a substrate can be determined, for example, by measuring the ability of an enzyme to increase the concentration of free NH 2 -termini in a reaction mixture containing 1 mg/ml substrate and 10 mg/ml of papaya resin, incubated at 37 0 C for 1 hour. The biological activity of papaya resin will typically be evidenced by an increase in the concentration of free amino termini under such conditions, usually by at least about 10%, more usually by at least about 25%, and preferably by at least about 50%. The biological activity of papaya resin may also be evidenced by a reduction in the toxicity of oligopeptides greater than about 1000 Da in a 0.1 ml of 50mg/ml substrate after a 2 hour incubation with 0.2 ml of 10mg/ml of the resin by at least about 2-fold, usually by at least about 5-fold, and preferably by at least about 10-fold. The toxicity of such oligopeptides can be determined by methods known in the art, for example, by using a Rat Liver Lysosome (RLL) assay, as described, for W files32659%832659 5peiov w.8O8 ooc 00 example, in WO 2003/100051 and Cornell and Townley (1973; Clin.Chim. Acta 49:

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0 181-188), the entire contents of which are incorporated herein by reference.

In some embodiments of the present invention, papaya resin will display detoxifying activity (as measured, by an RLL assay) of at least about 20%, optionally of at Sleast about 50% or optionally of at least about 90% protection to the lysosomes.

Other Compositions In some embodiments, the compositions of the present invention may include papaya I 10 resin in combination with other enzymes capable of inactivating toxic gluten 00 oligopeptides, including, but not limited to caricain and bromelain (or an analogue or Sbiologically active fragment thereof) and an intestinal extract, such as is described in WO 2003/100051, the entire contents of which is incorporated herein by reference.

Bromelain is a plant protease which is typically isolated from pineapple (Ananas comosus). It has an optimum pH range of 5 to 8 depending upon the substrate with broad specificity for peptide bonds. Bromelain may be purified from sources such as pineapple using any standard technique known in the art, including, but not limited to, the method of purification described by Yamada F. et al. ("Purification and characterization of a proteinase from pineapple fruit, fruit bromelain FA2"; J Biochem (Tokyo). 1976; 79(6):1223-34). Isolated and purified bromelain is also available commercially.

The intestinal extract, as herein described, may be derived from any portion of a gastrointestinal tract, including, but not limited to, the duodenum. The intestinal extract may be derived from any species, as long as it displays an ability to detoxify toxic gliadin peptides alone, or in a synergistic capacity with papaya resin, or a functional analogue thereof. In a certain embodiment of the present invention, the intestinal extract in derived from porcine intestine. Other animal forms of these proteins may be used, or modified forms may be isolated from other commercially available sources.

The compositions of the present invention may also include other enzymes, such as (but not limited to) fungal proteases from Aspergillus spp. such as Aspergillus oryzae Byun et al. (2001) J. Agric. Food Chem. 49, 2061-2063) and Lactobacilli spp. such as Lactobacillus helveticus Vesanto et al., (1995) Microbiol. 141, 3067-3075), and Lactococcus lactis (Mayo et al., (1991) Appl. Environ. Microbiol. 57, 38-44).

W:Fi deOs832650832650 speciv040808.0oc 00 As used herein and with reference to caricain and bromelain, the term "biologically active fragment' typically refers to a fragment that retains its ability to detoxify gluten peptides, in vitro or in vivo.

Fragments of interest include, but are not limited to, fragments of at least about Scontiguous amino acids, more usually at least about 50 contiguous amino acids, and may comprise 100 or more amino acids, up to the complete protein, and may extend further to include additional sequences. In each case, the key criterion is whether the fragment retains the ability to modify the toxic oligopeptides that contribute to a condition related to gluten intolerance.

00 SAs used herein and with reference to papaya resin, the term "functional analogue" typically denotes a material that has at least part of the biological activity of naturallyoccurring papaya resin enzymes extracted from the plant (or a part thereof).

Analogues of papaya resin enzymes may be prepared by in vitro synthesis using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers CS936X Peptide Synthesizer, CSBio Company, Inc.). Using such synthesizers, a skilled person can readily substitute for the naturally occurring amino acids one or more unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups can be introduced into the protein during synthesis that allow for linking to other molecules or to a surface.

For example, cysteines can be used to make thioethers, histidines can be used for linking to a metal ion complex, carboxyl groups can be used for forming amides or esters, amino groups can be used for forming amides, and the like.

As used herein and with reference to caricain and/or bromelain, the term "analogue" typically denotes a peptidase that has an amino acid sequence that is substantially identical to the amino acid sequence of the respective naturally occurring enzyme and has at least part of the biological activity of naturally-occurring caricain and/or bromelain.

The term "substantially identical', as used in regards to an analogue, typically denotes a substitution or addition of one or more amino acids such that the resulting analogue has at least some of the biological activity of the naturally occurring enzyme.

Analogues may be naturally occurring, such as an allelic variant or an mRNA splice W:Fidesf32659382659_5Deci_040808 dOC 00 variant, or they may be constructed using synthetic or recombinant techniques available to one skilled in the art.

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SIn some embodiments of the present invention, modifications may be made to the papaya resin (or an analogue thereof) to modulate enhance) its biological activity Sand/or bioavailability, including, but not limited to, chemical derivatization of proteins acetylation or carboxylation), glycosylation those made by modifying the glycosylation patterns of a protein during its synthesis and processing or in further processing steps), as well as sequences that have phosphorylated amino acid residues 10 phosphotyrosine, phosphoserine, or phosphothreonine).

00 SAlso useful in the practice of the present invention is papaya resin or an analogue thereof that has been modified using molecular biological techniques and/or chemistry so as to improve its resistance to proteolytic degradation and/or to acidic conditions such as those found in the stomach, and to optimize solubility properties or to render them more suitable as a therapeutic agent.

Pharmaceutical Compositions The compositions according to the present invention, as hereinbefore described, may be in the form of a pharmaceutical composition, in which the composition further includes a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.

Pharmaceutical compositions of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.

As used herein, the phrase "pharmaceutically acceptable carriel" includes, but is not limited to, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Typically, the route of administration is oral ingestion).

Generally, the pharmaceutical composition is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion W.F iles>83265983265_ speci v00808 DOC 00 medium containing, for example, water, ethanol, polyol (for example, glycerol, Spropylene glycol, or liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion or by the use of surfactants. Prevention of the action of microorganisms can Sbe achieved by incorporation of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugar, sodium chloride or polyalcohols such as mannitol, or sorbitol, in the composition.

SOral compositions generally comprise an inert diluent or an edible carrier. For the Spurpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or modified corn starch; a lubricant such as magnesium stearate or other stearates; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.

In some embodiments, the pharmaceutical composition of the present invention is prepared with carriers that will protect the composition against rapid elimination from the body, such as a controlled release formulation, including microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.

It is advantageous to formulate oral compositions in dosage unit form for ease of administration and uniformity of dosage. "Dosage unit form", as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The toxicity and therapeutic efficacy of such compounds can be determined by standard W: \Fles%32659 32659 sespvv0o80 oc 00 pharmaceutical procedures including in vitro assays, cell cultures or experimental 0 animals, for determining the LD 5 0 (the dose lethal to 50% of the population) and N the ED50 (the dose therapeutically effective in 50% of the population) depending on the ;compound studied. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio ED 50

/LD

50 Compounds which Sexhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.

oO 0The data obtained from the in vitro studies, cell culture assays and animal studies can Sbe used in formulating a range of dosages for use in humans. The dosage lies preferably within a range of circulating concentrations that include the ED 5 0 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The therapeutically effective dose of an enzyme can be estimated initially from in vitro assays. Such information can be used to more accurately determine useful doses in humans. In some embodiments of the present invention, the effective dose of papaya resin (or a functional analogue thereof) is at least about 0.1 mg per kg body weight taken with each meal for adults and typically half that dose for children.

Depending on the patient and condition being treated and on the administration route, the compositions of the present invention may be administered in dosages of 0.1 mg to mg/kg body weight per day. For instance, a typical daily dose of papaya resin, or a functional analogue thereof, in patients will be in at least about 100 mg/adult, more usually at least about 1000 mg; and preferably not more than about 5000 mg. Dosages will be appropriately adjusted for pediatric formulation. In children, the effective daily dose may be lower, for example at least about 0.1 mg, or 0.5 mg. In combination therapy involving, for example, papaya resin and intestinal extract, a comparable dose of the composition may be given; however, the ratio will be influenced by the relative stability of the composition toward gastric inactivation.

Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific activity of the papaya resin (or analogue thereof), the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given composition can be readily determined by those of skill in the art by a variety of means, W:Wees 59326559s285 peo_4OO08doc 00 such as measuring the biological activity of a given composition required to overcome the symptoms of gluten intolerance.

(Ni ;The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including the activity of the specific Scompound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, the degree of expression or activity to be modulated, sensitivity to gluten, previous treatments and other diseases present.

0 The pharmaceutical compositions can be included in a container, pack, or dispenser Stogether with instructions for administration.

(Ni For oral preparations, the compositions according to the present invention can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose variants, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

In some embodiments of the present invention, the oral formulations comprise enteric coatings, so that the active agent is delivered to the intestinal tract. Enteric formulations are often used to protect an active ingredient from the strongly acid contents of the stomach. Such formulations can be created by coating a solid dosage form with a film of a polymer that is insoluble in acid environments, and soluble in basic environments. Exemplary films are cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, methacrylate copolymers, cellulose acetate phthalate and acrylic coating systems such as Acryl-Ease (Colorcon).

Other enteric formulations comprise engineered polymer microspheres made of biologically erodable polymers, which display strong adhesive interactions with gastrointestinal mucus and cellular linings and can traverse both the mucosal absorptive epithelium and the follicle-associated epithelium covering the lymphoid tissue of Peyer's patches. The polymers maintain contact with intestinal epithelium for W:I de \83265 832650 speo_v04080B.€1oc 00 extended periods of time and actually penetrate it, through and between cells (see, for 0 example, Mathiowitz et al. (1997) Nature 386 (6623): 410-414. Drug delivery systems can also utilize a core of superporous hydrogels (SPH) and SPH composite (SPHC), as described by Dorkoosh et al. (2001) J Control Release 71 (3):307-18).

SMethods of Prophvlaxis or Treatment In another aspect of the present invention, there is provided a method for the prophylaxis or treatment of a condition related to gluten intolerance, the method including administering to a subject in need thereof a composition according to the 0 present invention, as herein described.

The method of the present invention can be used for prophylaxis or safeguarding, as well as for therapeutic purposes. Accordingly, as used herein, the term "treatment" and the like refers to any diminution in the severity of a pre-existing disease, condition or symptom of gluten intolerance, particularly as measured by the severity of symptoms such as, but not limited to, fatigue, chronic diarrhoea, and malabsorption of nutrients, weight loss, abdominal distension and anaemia. As used herein, the term "prophylaxis" and the like refer to the prevention of a condition related to gluten intolerance. Prevention of the condition may be complete no evidence of a symptom of gluten intolerance) or partial a diminution in the severity of any one or more of the symptoms of gluten intolerance). Other indices of gluten intolerance include, but are not limited to, the presence of antibodies specific for glutens, the presence of antibodies specific for tissue transglutaminase, the presence of proinflammatory T cells and cytokines, damage to the villous structure of the small intestine as evidenced by histological or other examination, enhanced intestinal permeability, and the like.

Subjects that can benefit from the methods of the present invention may be of any age and include adults and children. Children in particular benefit from prophylactic treatment, as prevention of early exposure to toxic gluten peptides can prevent initial development of the disease. Children suitable for prophylaxis can be identified by genetic testing for predisposition, for example, by HLA typing, by family history, by T cell assay, or by other means known to the skilled addressee.

In some embodiments of the present invention, the method of prophylaxis or treatment further includes administering to the subject in need thereof an effective dose of W: e 3265M832659_3pe_.040808 Ooc 18 00 caricain or bromelain, or biologically active fragments, analogues or variants thereof,

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Sor an intestinal extract, as herein described. Caricain, bromelain and the intestinal extract may be administered together with papaya resin (or an analogue thereof) or as Sseparate dosages, as required.

SThe methods according to the present invention may also be performed in combination with other modalities, including, but not limited to, administering to a subject in need thereof, an inhibitor of tissue transglutaminase, an anti-inflammatory agent, an antiulcer agent, a mast cell-stabilizing agents, and/or and an-allergy agent. Examples of such agents include HMG-CoA reductase inhibitors with anti-inflammatory properties 00 such as compactin, lovastatin, simvastatin, pravastatin and atorvastatin; anti-allergic Shistamine H1 receptor antagonists such as acrivastine, cetirizine, desloratadine, ebastine, fexofenadine, levocetirizine, loratadine and mizolastine; leukotriene receptor antagonists such as montelukast and zafirlukast; COX2 inhibitors such as celecoxib and rofecoxib; p38 MAP kinase inhibitors such as BIRB-796; and mast cell stabilizing agents such as sodium chromoglycate (chromolyn), pemirolast, proxicromil, repirinast, doxantrazole, amlexanox nedocromil and probicromil.

Various methods for administration may be employed, preferably using oral administration, for example with meals. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.

The initial dose can be larger, followed by smaller maintenance doses. The dose can be administered as infrequently as weekly or biweekly, or more often fractionated into smaller doses and administered daily, with meals, semi-weekly, or otherwise as needed to maintain an effective dosage level.

The therapeutic effect can be measured in terms of clinical outcome or can be determined by immunological or biochemical tests. Suppression of the deleterious Tcell activity can be measured by enumeration of reactive Thl cells, by measuring the release of cytokines at the sites of lesions, or using other assays for the presence of autoimmune T cells known in the art. Alternatively, one can look for a reduction in severity of the symptoms of the disease.

W:\F esB326561832659_ slci v040808.0oc 00 Preparation of Food Products 0 In another aspect of the present invention, there is provided a method of preparing an Sarticle of food derived from a gluten-containing material, the method including treating the article of food or the gluten-containing material with papaya resin, or a functional Sanalogue thereof, as herein described, so as to reduce the amount of toxic glutenderived oligopeptides present in the article of food.

Finally it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

00 SCompositions for the prophylaxis or treatment of conditions related to gluten intolerance according to certain embodiments of the present invention will now be described in the following examples. It should be understood, however, that the following examples are illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLES

Example 1: Detoxifying activity of papaya oleoresin Papaya oleoresin extract was prepared as a mix of proteolytic enzymes isolated from papaya fruit. In two Rat Liver Lysosome (RLL) assays, papaya oleoresin extract offered good protection of approximately 92%-94% at a concentration of about using the normal amount of gliadin digest which is 5 mg for the assay.

The Rat Liver Lysosome (RLL) assay, as described, for example, in Cornell and Townley (1973; Clinica Chimica Acta 49:181-188) and Cornell and Townley (1974; Gut, 15(11):862-869), is based on the fact that a peptic-tryptic-pancreatinic digest of wheat gliadin disrupts rat liver lysosomes, causing a reduction in optical density (at 400nm) of a suspension of these organelles. This is evidence of a cytotoxic reaction by the peptides present in the digest. However, if the enzyme extract is pre-incubated with the toxic gliadin digest for two hours, the change in optical density is much less after incubation with the lysosomes. By comparing a control (no toxic digest), a toxic sample (lysosomes incubated with a toxic digest of gliadin) and an extract treated sample (toxic digest of gliadin pre-incubated with enzyme extract prior to addition of lysosomes), the extent of protection can be determined. A protection index can be calculated from: W:\F les\32659832659_ peci_vO0408e.OC 00

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O P.I. reduction toxic sample reduction extract treated sample x 100 reduction toxic sample The detoxifying effect of papaya oleoresin was compared against a pig intestinal enzyme extract (as described, for example, in WO 2003/100051), crude caricain and a combination of crude caricain and the pig intestinal extract. Crude caricain was prepared by dissolving papaya latex extract in water, adjusting the concentration of ammonium sulphate to 60%, collecting the resulting precipitate by filtration, dialysing it and freeze-drying. This material was further enriched by chromatography on CM Sephadex using phosphate buffers with elution of the crude caricain with 0.7 M sodium 0 0 chloride, followed by dialysis and freeze-drying. The enzyme preparations were Sassayed using the RLL assay at a concentration of 6mg/ml.

Pig intestinal Crude Caricain Combination Papaya oleoextract (6mg/ml) (6mg/ml) (6mg/ml) resin extract Protection Index 61 71 92 94 W T-des 3265gW32659_speci~vG4080B~dOC

Claims (5)

1. A composition for the prophylaxis or treatment of a condition related to gluten intolerance, the composition including an extract of papaya resin, or a functional Sanalogue thereof.

2. The composition according to claim 1, wherein the extract of papaya resin is S 10 derived from a species of papaya classified in the genera selected from the group 0 consisting of Carica, Cylicomorpha, Jacaratia, Jarilla, Horovitzia, Vasconcella, or Sa cultivar thereof.

3. The composition according to claim 1 or 2, wherein the papaya resin is a papaya oleoresin.

4. The composition according to any one of claims 1 to 3, formulated as an enterically coated tablet or capsule.

5. A method for the prophylaxis or treatment of a condition related to gluten intolerance, the method including administering to a subject in need thereof a composition according to any one of claims 1 to 4. W:\Flles32G98B32SspeO4O~88,dac