A protein composition containing a dipeptide as a stabilizer

Abstract

The present invention relates to a pharmaceutical composition comprising a protein as an active ingredient, one or more dipeptides as stabilizers, and optionally an enzyme inhibitor, which is particularly useful for administration to the intestinal tract.

Description

Technical Field

[0001] The present invention relates to novel therapeutic compositions and their use. The composition has a protein, such as an antibody, as an active ingredient.

Background Art

[0002] Biopharmaceuticals, particularly proteins (including antibodies), are an increasingly important class of pharmaceuticals, and many therapeutic uses have been proposed and implemented. Monoclonal antibodies (mAbs) are the best-selling class of biotherapies. However, delivering such biopharmaceuticals to the desired target is a major challenge in the pharmaceutical industry and remains an unmet medical need for several diseases. Specifically, oral or rectal administration may be ineffective because the molecule is very unstable in the presence of gastric and intestinal fluids. Therefore, for this reason, intravenous or subcutaneous delivery remains the most feasible option for protein delivery. However, such delivery is usually not the most convenient method of drug administration to patients, and the problems include pain at the injection site, compliance, and frequent hospital visits for intravenous infusion. There may also be other significant drawbacks to intravenous or subcutaneous delivery, and for some applications, systemic high doses of mAbs have been reported to be associated with serious systemic side effects. High systemic exposure can also lead to a decrease in efficacy with repeated dosing due to the formation of anti-drug antibodies.

[0003] Currently, there are no commercially available oral biological therapies available for the treatment of chronic diseases such as diabetes or inflammatory bowel disease (IBD), and there is a clear unmet medical need for a safer, more effective, and patient-friendly treatment approach.

[0004] For some drugs, targeting drugs to the colon has been used as a means of achieving local or systemic therapy. For example, WO2007 / 122374 describes compositions having a delayed release coating that can be used to target the release of drugs from the core to the intestine, particularly the colon. The colon is susceptible to several medical conditions such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), constipation, diarrhea, infections, and cancer. IBD is a chronic and medically incurable disease associated with inflammation of the intestine. There are mainly two forms of IBD. Ulcerative colitis (UC) usually develops in the descending colon and rectum and may continue to spread to involve the entire colon (pancolitis). Crohn's disease (CD) is usually associated with the terminal part of the small intestine and the ascending colon. UC usually affects only the epithelial layer of the intestinal wall, while CD may affect all layers of the intestinal wall. Although several biological therapies are currently available for the treatment of IBD, due to the above stability problems, there is no biological therapy currently available as an oral treatment.

[0005] There remains an unmet need for methods of stabilizing proteins in the presence of luminal fluids found in the lower gastrointestinal tract, such as the small intestine and / or colon. Specifically, there remains a need for methods of sufficiently stabilizing proteins to deliver them to the ileum and / or colon, particularly by rectal or oral administration.

[0006] The inventors have found that dipeptides (e.g., diglycine and L-carnosine) or combinations thereof are effective in stabilizing proteins when in contact with body fluids found in the lower gastrointestinal tract, such as the small intestine and / or colon.

[0007] Surprisingly, the inventors have found that enhanced stability in GI fluid is seen when an enzyme inhibitor is included with a dipeptide in a composition having a protein as an active ingredient. In contrast, the inventors have found that when the enzyme inhibitor is used alone, it has little effect on the stability of the protein upon contact with body fluids found in the ileum and / or colon. SUMMARY OF THE INVENTION

[0008] Accordingly, the present invention provides a pharmaceutical composition comprising a protein as an active ingredient, one or more dipeptides, and optionally an enzyme inhibitor. The dipeptide and the enzyme inhibitor act as stabilizers.

[0009] In the composition of the present invention, the "active ingredient" is a protein. The protein can be an antibody or a fragment thereof.

[0010] The composition of the present invention can be in liquid, solid or semi-solid form, preferably in a form suitable for rectal administration or particularly for oral administration. The composition can be in a solid form suitable for oral administration, and the above composition has an enteric coating. Most preferably, it is in a solid or semi-solid form suitable for oral administration and is adapted for the selective release of the protein in the lower digestive tract, particularly the ileum and / or colon.

[0011] The composition can include a solid dosage form having a core and a coating for the core. The core can include a protein, a dipeptide, and optionally an enzyme inhibitor as active ingredients, and the coating can include a mixture of a digestible polysaccharide and a film-forming material having a solubility threshold of pH 6.0 or higher.

[0012] The composition can be an orally administrable pharmaceutical composition comprising a protein as an active ingredient, one or more dipeptides, and optionally an enzyme inhibitor.

[0013] The present invention also provides a solid dosage form for oral administration comprising a core containing a protein, one or more dipeptides, and optionally an enzyme inhibitor as active ingredients, and a delayed-release coating for the core.

[0014] The present invention also provides a rectally administrable pharmaceutical composition comprising a protein as an active ingredient, one or more dipeptides, and optionally an enzyme inhibitor.

[0015] The present invention also provides an enema preparation containing a protein as an active ingredient and optionally one or more dipeptides, and containing or not containing an enzyme inhibitor.

[0016] The present invention further provides a pharmaceutical composition according to the present invention for use in therapy. Furthermore, a pharmaceutical composition according to the present invention for use in the treatment or prevention of a disease or condition selected from inflammatory bowel disease, irritable bowel syndrome, constipation, diarrhea, infection or cancer is also provided. Preferably, the pharmaceutical composition is the pharmaceutical composition of the present invention.

[0017] The present invention further provides a method of treating or preventing a disease or condition in a subject, comprising administering a pharmaceutical composition according to the present invention to the subject. Preferably, the disease or condition is selected from inflammatory bowel disease, irritable bowel syndrome, constipation, diarrhea, infection, autoimmune disease or cancer.

[0018] The present invention further provides a method of stabilizing a protein in the presence of intestinal fluid, comprising contacting the protein with one or more dipeptides and optionally an enzyme inhibitor.

[0019] The present invention also provides the use of one or more dipeptides and optionally an enzyme inhibitor for the stabilization of a protein administered as a pharmaceutical composition and delivered to the lower gastrointestinal tract. Preferably, the pharmaceutical composition is the pharmaceutical composition of the present invention.

[0020] Following release, penetration of the drug into the GI tissue at or near the site of release in the GI lumen can occur, enabling local treatment of GI tract diseases and, through penetration of the drug into the bloodstream, treatment of a wide range of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021]

Figure 1

Figure 2

DETAILED DESCRIPTION OF THE INVENTION

[0022] As used herein, "lower digestive tract" refers to the gastrointestinal tract after the stomach. This includes the small intestine and the large intestine. The small intestine is composed of the duodenum, jejunum, and ileum, while the large intestine is also known as the colon. One or more dipeptides are used as stabilizers to reduce the degradation of proteins in the ileum and / or colon.

[0023] Protein therapeutic agent The active ingredient can be any protein that advantageously achieves a therapeutic effect by being administered via the lower digestive tract, preferably the ileum and / or colon, particularly the colon.

[0024] Classes of proteins for use in the present invention include, for example, the antibodies, enzymes, cytokines, chemokines, receptors, blood factors, hormones, toxins, transcription proteins, non-immunoglobulin binding protein scaffolds, and multimeric proteins described below. Fusion proteins containing the above proteins can also be used. Generally, "protein" as used herein contains more than 50 amino acids.

[0025] Antibody The active ingredient can be one or more antibodies.

[0026] As used herein, "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target antigen, such as a cytokine, protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, bacterium, virus, or combination thereof, via at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antigenic determinants of antibodies, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Preferably, the antibody is a monoclonal antibody. The antibody can comprise any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are based on the identity of the heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. Preferably, the antibody is an IgG antibody, more preferably IgG1 or IgG4. The term "antibody" is also intended to include conjugates of antibodies, e.g., conjugates with polyethylene glycol (PEG).

[0027] Furthermore, except where the context requires otherwise, the term "antibody" should be understood to encompass complete antibodies and antibody fragments containing the antigen-binding regions of complete antibodies. Antibody fragments include, for example, single domain antibodies (e.g., V HMultispecific antibodies formed from H domain antibodies), monovalent or divalent Fabs, Fab', F(ab')2, scFvs, Fcs, bispecific antibodies, diabodies, minibodies, or antibody fragments, such as scFv fragments or diabodies, and optionally minibodies composed of different arrangements of Fc fragments or CH domains, such as scFv-Fc, scFv-Fc-scFv, Fab-scFv, (Fab'ScFv)2, sc diabodies, sc diabody-Fc, sc diabody-CH3, scFv-CH3 and scFv-CH2-CH3 fusion proteins. Antibody fragments can be generated by enzymatic cleavage of a complete antibody, or by synthetic means, such as recombinant DNA technology, phage display or yeast display technology, or using transgenic mice, or by liquid or solid phase peptide synthesis. Antibody fragments can also form part of a fusion protein if their antigen-binding ability is retained.

[0028] Antibodies suitable for use in the present invention include adalimumab, infliximab, ruplizumab, certolizumab pegol, golimumab, natalizumab, vedolizumab, tildrakizumab, ustekinumab and combinations thereof.

[0029] When an antibody is used in the composition according to the present invention, it can be any antibody that advantageously achieves a therapeutic effect by administration via the colon. Particular antibodies of particular interest in the context of the present invention include existing commercially available antibodies for the treatment of IBD (e.g., adalimumab, infliximab, certolizumab pegol, golimumab, natalizumab, vedolizumab, tildrakizumab, ustekinumab) as well as additional antibodies (agonists or antagonists) under development for the treatment of IBD that target pathways and molecules involved in the etiology of IBD (e.g., CD40). Targeting the colon further offers the possibility of improving the treatment of colorectal cancer by targeting and localizing to tumors with antibodies for cancer treatment or different possible formats as described above.

[0030] Furthermore, the stabilization of biomolecules in the gastrointestinal (GI) tract and penetration into GI tissues can further provide the possibility for therapeutic agents to permeate into the systemic circulation through GI tissues, and can provide opportunities to target a much wider range of diseases.

[0031] Stabilizer Dipeptides and enzyme inhibitors are used as stabilizers.

[0032] The stabilizer helps to maintain intact proteins in the lower digestive tract. Methods for evaluating the stability of proteins in gastrointestinal fluids are described in the examples. One or more dipeptides and optionally enzyme inhibitors maintain more than 5% of the proteins present intact after 4 hours in gastrointestinal fluids. Preferably, more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or more of the proteins are maintained intact.

[0033] Dipeptide The compositions of the present invention contain at least one, preferably at least two dipeptides. Preferably, the composition contains glycine and / or L-carnosine.

[0034] A dipeptide is a molecule containing two amino acids linked by a peptide bond. The amino acids can be the naturally occurring version or a chemically modified version. Examples of dipeptides, as described below, are glycine or L-carnosine.

[0035] Diglycine Glycine is a dipeptide consisting of two glycine amino acids linked together via a peptide bond. Glycine has the following structure.

[0036]

Chemical formula

[0037] L-Carnosine L-carnosine (also known as beta-alanyl-L-histidine) is a dipeptide of beta-alanine and histidine and has the following structure.

[0038]

Chemical formula

[0039] Enzyme inhibitor Preferably, the composition contains an enzyme inhibitor as a further stabilizer.

[0040] Preferably, the enzyme inhibitor is a protease inhibitor for protein protection, for example, aprotinin, ovomucoid type II-O (including ovomucoid inhibitor), Bowman-Birk inhibitor (BBI) or Kunitz type trypsin inhibitor. Preferably, the enzyme inhibitor is aprotinin.

[0041] Preferably, the enzyme inhibitor is aprotinin, and the dipeptide is diglycine and / or L-carnosine. The composition may contain aprotinin, diglycine and L-carnosine as stabilizers.

[0042] Aprotinin The pharmaceutical composition of the present invention may contain aprotinin, an aprotinin analog or a fragment thereof. Of course, it will be understood that aprotinin, an aprotinin analog or a fragment thereof does not exist as an active ingredient in the composition of the present invention.

[0043] Aprotinin is a protease inhibitor and is known to inhibit trypsin and other similar proteases. It is also often called bovine pancreatic trypsin inhibitor (BPTI). Aprotinin is a 58 - amino - acid protein formed after the processing of a 100 - amino - acid polypeptide containing a signal peptide, a propeptide domain, and a knotted domain. The mature 58 - amino - acid form of aprotinin is commercially available and is also sold under the trade name Trasylol®, which has been shown to have prophylactic use for reducing blood loss during surgery.

[0044] Aprotinin for use in the present invention may comprise the amino acid sequence RPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA (SEQ ID NO: 1).

[0045] Aprotinin or a fragment thereof or an aprotinin analog or a fragment thereof for use in the present invention can be prepared recombinantly (e.g., in E. coli, mammalian cells, or insect cells), synthetically (e.g., using standard organic chemical techniques such as liquid - phase or solid - phase peptide synthesis), or it can be a natural protein derived from animals, e.g., bovine - derived.

[0046] Furthermore, unless the context requires otherwise, the term "aprotinin" should be understood to include aprotinin, aprotinin analogs, aprotinin fragments, and fragments of aprotinin analogs. Analogs and fragments suitable for use in the present invention enhance the stability of proteins in the presence of body fluids found in the lower gastrointestinal tract, such as the ileum and / or colon, when combined with one or more dipeptides. Methods for confirming the ability of aprotinin, aprotinin fragments, aprotinin analogs, or fragments thereof to enhance the stability of the active ingredient (protein) in the presence of body fluids found in the lower gastrointestinal tract, such as the ileum and / or colon, are described in the following examples. As used herein, "enhanced" stability means that more than 50% of the active ingredient present remains intact after 4 hours in gastrointestinal fluid. Preferably, more than 60%, 70%, 75%, 80%, 90%, 95% or more of the active ingredient remains intact.

[0047] For example, a fragment of aprotinin for use in the present invention may include a fragment of the sequence defined by SEQ ID NO: 1. For example, a fragment of aprotinin may include 30 or more, 40 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 or more, 55 or more, 56 or more, or 57 or more consecutive amino acids of the sequence defined by SEQ ID NO: 1.

[0048] An aprotinin analog for use in the present invention may comprise a protein comprising a sequence similar to the amino acid sequence defined in SEQ ID NO: 1, and when combined with one or more dipeptides, enhances the stability of the protein in the presence of body fluids found in the lower digestive tract, such as the ileum and / or colon. For example, the aprotinin analog may have a similarity of 70% or more, 80% or more, 90% or more, or 95% or more to the sequence defined in SEQ ID NO: 1 or a fragment of the sequence defined in SEQ ID NO: 1. Alternatively, the aprotinin analog may have an identity of 70% or more, 80% or more, 90% or more, or 95% or more to the sequence defined in SEQ ID NO: 1 or a fragment of the sequence defined in SEQ ID NO: 1. For example, the aprotinin analog may comprise an amino acid sequence that is identical to 50, 51, 52, 53, 54, 55, 56, or 57 amino acids of the sequence defined by SEQ ID NO: 1. The similar or identical amino acids may or may not be adjacent.

[0049] A program, such as the CLUSTAL program, can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces into either sequence as necessary. It is possible to calculate the identity or similarity of amino acids (conservation of amino acid type in addition to identity) for the optimal alignment. Programs such as BLASTx align the longest stretches of similar sequences and assign values to the fits. Thus, a comparison can be obtained when several similar regions are found, each having a different score. In the present invention, both types of analysis are contemplated. Identity or similarity is preferably calculated over the full length of SEQ ID NO: 1.

[0050] An aprotinin analog may contain one or more amino acid substitutions, insertions, and / or deletions.

[0051] An amino acid substitution means that an amino acid residue is replaced at the same position by a substituted amino acid residue. The amino acid substitution can be conservative, which means that the substituted amino acid has similar chemical properties to the original amino acid. A person skilled in the art understands which amino acids share similar chemical properties. For example, the following amino acid groups share similar chemical properties, such as size, charge, and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

[0052] The inserted amino acid residue can be inserted at any position, and some or all of the inserted amino acid residues can be inserted so that they are directly adjacent to each other, or the inserted amino acid residues can be inserted so that none of them are directly adjacent to another inserted amino acid residue. For example, aprotinin analogs can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acids at the N-terminus and / or C-terminus of the amino acid sequence defined by SEQ ID NO: 1.

[0053] One, two, or three amino acids can be deleted from the sequence of SEQ ID NO: 1. Each deletion can be carried out at any position of SEQ ID NO: 1.

[0054] The inserted and substituted amino acids can be naturally occurring amino acids or can be non-naturally occurring amino acids, for example, can contain non-natural side chains and / or can be linked together via non-natural peptide bonds. Such modified peptide ligands are known in the art. When two or more amino acid residues are substituted and / or inserted, the substituted / inserted amino acid residues may be the same as or different from each other. Each substituted amino acid can have a different side chain from the amino acid being substituted. Analogs of aprotinin can contain one or more modified bases where the amino acid residues can be chemically modified. Examples of chemical modifications include chemical modifications corresponding to post-translational modifications, for example, phosphorylation, acetylation, and deamidation. The chemical modifications may not correspond to chemical modifications that can exist in vivo. For example, the N- or C-terminus of a peptide can be modified to improve the stability, bioavailability, and / or affinity of the peptide. Further examples of non-natural modifications include incorporation of non-coded α-amino acids, photoreactive crosslinking amino acids, N-methylated amino acids, and β-amino acids; backbone reduction; retro-inversion by using d-amino acids; N-terminal methylation; and C-terminal amidation and pegylation.

[0055] Pharmaceutical preparation The pharmaceutical compositions according to the invention are preferably in liquid, solid or semi-solid form and are preferably suitable for oral or rectal administration.

[0056] The compositions can also be in the form of a lotion, cream, foam, emulsion or gel. Such formulations can be prepared by many known methods established in the art.

[0057] For example, the active ingredient (protein) and the necessary stabilizer can be mixed together, optionally with other excipients required for the dosage form.

[0058] The pharmaceutical composition of the present invention suitable for oral administration can be presented in any form of tablets, capsules, mini-tablets, pellets, powders, granules, microparticles, nanoparticles or hydrogels.

[0059] The composition of the present invention suitable for oral administration can be presented as separate units, such as capsules, tablets, mini-tablets or pellets, or as powders, granules or crystals. In the solid composition, the minimum diameter of each particle is typically 10 -4 m or more, usually 5×10 -4 m or more, preferably 10 -3 m or more. The maximum diameter is usually 30 mm or less, typically 20 mm or less, preferably 10 mm or less. In a preferred embodiment, the particles have a diameter of about 0.2 mm to about 15 mm, preferably about 1 mm to about 4 mm (for example, in the case of pellets or mini-tablets) or about 6 mm to about 12 mm (for example, in the case of certain tablets or capsules). The term "diameter" refers to the maximum linear dimension passing through the particle.

[0060] As with the necessary stabilizers, the compositions according to the invention may, of course, optionally contain any further conventional excipients, such as binders, fillers, disintegrants, diluents and lubricants, as required. Excipients used in solid form include, for example, microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate, calcium sulfate, sorbitol, glucose and / or lactose, and / or other excipients, binders, fillers, disintegrants, diluents and lubricants known in the art. Suitable binders include starch, gelatin, natural sugars (such as glucose or beta-lactose), corn sweeteners, natural and synthetic gums (such as acacia, tragacanth or sodium alginate), carboxymethyl cellulose, polyethylene glycol, waxes and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Fast-dissolving diluents include mannitol, lactose, sucrose and / or cyclodextrin. Lubricants, glidants, flavors, colorants and stabilizers may also be added to facilitate manufacture and use. Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride. The compositions according to the invention may, of course, further optionally contain any further conventional excipients, such as binders, fillers, disintegrants, diluents and lubricants, as required.

[0061] Tablets can be manufactured by compression or molding, optionally using one or more supplementary components. Compressed tablets can be prepared by mixing the active ingredient in free-flowing form, such as a powder or granules, optionally with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant, and compressing with a suitable machine. Molded tablets can be made by shaping a mixture of the powdered compound moistened with an inert liquid diluent with a suitable machine. Tablets can optionally be coated or scored and formulated to provide sustained, delayed or controlled release of the antibody. Preferred examples of coatings are given below.

[0062] The capsule can have solid, semi-solid or non-solid contents. Exemplary contents of the capsule can include, for example, microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a thickening agent, and a suspension that can include either of the above solid or semi-solid forms.

[0063] Formulations for rectal administration can be presented as suppositories containing conventional carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at normal room temperature (up to 25 °C), but liquefy and / or dissolve in the rectal cavity to release the drug.

[0064] The composition can also take the form of an enema preparation, liquid or foaming enema, which is rectally administered to the lower colon. Enema preparations typically contain a protein together with a suitable fluid carrier vehicle, such as deionized water and / or distilled water, and a stabilizer. The preparation can be thickened with one or more thickening agents. They can also contain a buffer solution, and can also contain an effective amount of a lubricant, such as a natural or synthetic lipid or oil, such as tris-fatty acid glyceric acid or lecithin. A non-toxic non-ionic surfactant can also be included as a wetting and dispersing agent. It is preferred to add a buffer solution to the liquid or foaming enema to stabilize the pH. The pH is preferably from 3.5 to 7.5, particularly from 6.5 to 7.5.

[0065] The unit dose of the enema preparation can be administered from a pre-filled bag or syringe. In the case of a pressurized enema preparation, the carrier vehicle can also contain an effective amount of a foaming agent, such as n-butane, propane or i-butane, or the foaming agent / propellant can be separately held from the composition in a bag-in-bag or bag-in-can system as described in, for example, WO-A-9603115 (incorporated herein by reference). The enema foam can also contain a swelling agent and a foaming stabilizer.

[0066] The volume of the liquid enema is typically 50 - 200 cm 3 , preferably about 100 cm 3 . The volume of the foaming enema is typically 20 - 40 cm 3 . The appropriate dosage of the stabilizer, particularly aprotinin, in the enema to be administered is 1 mg / mL - 20 mg / mL, preferably 2 mg / mL - 10 mg / mL. The dipeptide is usually present in an amount to result in a concentration of 0.1 - 100 mM, typically 25 - 80 mM, or 50 - 75 mM.

[0067] A preferred unit dosage formulation is a formulation containing an effective dose of the active ingredient or an appropriate fraction thereof. If the composition contains a suitable sustained-release excipient, the release from a particular formulation can also be sustained. However, in a preferred formulation, the release is pulsatile.

[0068] The composition according to the invention typically contains a therapeutically effective amount of the active ingredient (protein) which can be 0.01 wt% - 99 wt% based on the total weight of the composition. The actual dosage is determined by a person skilled in the art using general common knowledge. However, by way of example, a "low" dosage formulation typically contains 20 wt% or less of the active ingredient, preferably 1 wt% - 10 wt% of the active ingredient, for example 5 wt% of the active ingredient. A "high" dosage formulation typically contains 40 wt% or more, preferably 45 wt% - about 85 wt%, for example about 50 wt% or 80 wt% of the active ingredient.

[0069] The composition according to the invention typically contains an effective amount of a stabilizer. Usually, the formulation contains 0.01 wt% - 99 wt% of the stabilizer based on the total weight of the composition. The composition may preferably contain 20 wt% or less of the stabilizer, preferably 1 wt% - 10 wt% of the stabilizer, for example 5 wt% of the stabilizer. Alternatively, the composition may contain 40 wt% or more, preferably 45 wt% - about 85 wt%, for example about 50 wt% or 80 wt% of the stabilizer.

[0070] Dipeptides are usually present in the lower gastrointestinal tract in an amount to achieve a concentration of 0.1 to 100 mM, typically 25 to 80 mM, or 50 to 75 mM. Each dosage form may contain 50 to 5000 mg, typically 100 to 1000 mg, 250 to 750 mg or 300 to 500 mg of dipeptide.

[0071] Aprotinin is usually present in the lower gastrointestinal tract in an amount to achieve a concentration of 0.1 to 20 mg / mL. Each dosage form may contain 50 to 5000 mg, typically 1000 to 1000 mg, 250 to 750 mg or 300 to 500 mg of aprotinin.

[0072] The protein can be used as the sole active ingredient in the compositions according to the invention, but it is also possible for the protein to be used in combination with one or more further therapeutic agents. Accordingly, the invention also provides compositions according to the invention which contain, in addition to the protein, a further therapeutic agent. If desired, the compositions according to the invention can be administered together with, simultaneously with, sequentially or separately from, further compositions.

[0073] Unless the context requires otherwise, throughout this specification and the claims, references to pharmaceutical compositions in solid or semi-solid form are to be understood to include discrete solid or semi-solid particles or unit forms (which are solid or semi-solid throughout, and those having an exterior which is solid or semi-solid and an interior which is non-solid, such as liquid or gel). For example, a capsule can have a liquid or gel content.

[0074] Delivery to the lower digestive tract The composition according to the invention is adapted for the delayed or selective release of the active ingredient in the lower digestive tract, particularly the ileum and / or especially the colon, following rectal administration or particularly oral administration. This can be achieved by using a specific coating. The composition of the invention can be a delayed release oral (DRO) composition. The DRO composition passes through the stomach substantially unchanged and delivers the active ingredient to the lower digestive tract, typically the ileum and / or colon (i.e., the site of diseased mucosa).

[0075] The composition according to the invention can have an enteric coating. The enteric coating protects the active ingredient in the composition from attack and degradation in the stomach but usually dissolves in the intestine due to a change in pH and releases the contents of the dosage form. Suitable enteric coatings are well known in the art. The coating optimal for a particular formulation depends on the exact purpose of use and can be adjusted to release the active ingredient in a particular region of the intestine or at a particular time after ingestion. Such formulations can optionally include one or more intermediate layers between the active ingredient and the outer enteric coating. In this case, the composition of the invention can release a part of its contents in a particular region of the intestine and release a further part of its contents in a second region of the intestine, for example, the colon. Preferably, the composition of the invention is in solid or semi-solid form comprising an enteric coating adapted to release the protein in the colon. A useful enteric coating is one that maintains its integrity in the low pH environment of the stomach but dissolves as soon as the pH optimal for dissolution is reached. This can vary depending on the chemical composition of the coating, from pH 3 to 7.5, preferably 5 to 7. The required thickness of the coating depends on the solubility of the coating and the site of the target being treated. Usually, the coating is 25 to 200 μm, particularly 75 to 150 μm.

[0076] The composition of the present invention is adapted for the release of the active ingredient into the portion of the lower digestive tract where the disease is spreading. Usually, an enteric coating needs to dissolve at the pH of the jejunum (about pH 5.5), ileum (about pH 6) and / or colon (pH 6 - 7) so that most of the therapeutic molecules are released at the desired site. WO2007 / 122374 (the content of which is incorporated herein by reference) describes compositions for selective release in the colon, and these form a preferred embodiment of the present invention. Accordingly, the present invention further provides a composition comprising particles having a core and a coating for the core, wherein the core comprises an active ingredient, one or more stabilizers, and optionally an enzyme inhibitor, and the coating comprises a mixture of a digestible polysaccharide and a film-forming material having a dissolution threshold of pH 6.0 or higher, preferably pH 7.0 or higher.

[0077] The digestible polysaccharide is sensitive to attack by intestinal bacteria. Preferably, the digestible polysaccharide is selected from the group consisting of starch, amylose, amylopectin, chitosan, chondroitin sulfate, cyclodextrin, dextran, pullulan, carrageenan, scleroglucan, chitin, curdlan and levan.

[0078] For example, the polysaccharide can be starch, amylose or amylopectin.

[0079] The film-forming material is an enteric material having a pH threshold at which it is insoluble below that and soluble above that. The pH of the surrounding medium causes the dissolution of the second material. The normal pH of gastric juice is usually 1 - 3, while the pH of intestinal juice gradually increases from about 5.5 in the duodenum to about 7 - 8 in the colon. Accordingly, the second material has a pH threshold of 6.0 or higher, particularly 7 or higher, when used in the composition of the present invention.

[0080] The film-forming material is typically selected from acrylate polymers, cellulose polymers, or polyvinyl-based polymers. Examples of suitable cellulose polymers include cellulose acetate phthalate ("CAP"), cellulose acetate trimellitate ("CAT"), and hydroxypropyl methylcellulose phthalate acetate succinate. Examples of suitable polyvinyl-based polymers include polyvinyl acetate phthalate ("PVAP"). The film-forming material is preferably a copolymer of (meth)acrylic acid and a C 1-4 alkyl ester of (meth)acrylic acid, for example, a copolymer of methacrylic acid and methyl methacrylate. Such polymers include those available under the trademarks Eudragit L, Eudragit S, and Eudragit FS. It is particularly preferred to use Eudragit S as the film-forming material.

[0081] In such compositions, a multi-unit dosage form containing particles having a diameter of less than 3 mm is preferred. The "core" is usually a single solid. The core can consist of an active ingredient (protein), one or more dipeptides, and optionally an enzyme inhibitor. However, more generally, the core contains a mixture of an active ingredient and a stabilizer, and optionally one or more additional excipients. The core can contain, for example, a filler or diluent material, such as lactose or a cellulose material, such as microcrystalline cellulose; a binder, such as polyvinylpyrrolidone (PVP); a disintegrant, such as croscarmellose sodium; and / or a lubricant, such as magnesium stearate. The core can be a compressed granule containing at least some of these materials.

[0082] Release from such compositions is delayed until the lower gastrointestinal tract, particularly the ileum and / or colon. Such compositions have use in multi-phasic release compositions comprising at least two pluralities of particles (e.g., coated pellets in the same dosage form (e.g., capsule), where one plurality of particles is a coated pellet that is distinguished therein by the coating from another plurality of particles or pluralities of particles from each other). The coatings may differ from one plurality of coatings to the next with respect to the thickness or composition of the coating (e.g., component ratio and / or identity of components). The multi-phasic release formulations are particularly suitable for patients with Crohn's disease that affects different regions along the intestine including the ileum and / or colon.

[0083] Medical application The present invention provides a pharmaceutical composition according to the present invention for use in therapy. The present invention also provides a method of treating or preventing a disease or condition in a subject, particularly a human subject, the method comprising administering to the subject a pharmaceutical composition via the lower gastrointestinal tract, particularly the ileum and / or colon, wherein the pharmaceutical composition comprises a protein as an active ingredient, one or more dipeptides, and optionally an enzyme inhibitor. Preferably, the composition is adapted for administration via the oral or rectal route. The present invention has found utility in the treatment of diseases of the lower gastrointestinal tract, particularly the ileum and / or colon, but it also has use as an entry point for the entry of proteins into the systemic circulation by absorption from the lower gastrointestinal tract, particularly the ileum and / or colon, and thus has found utility in the treatment of a wide range of diseases and conditions. It may, for example, find utility in the treatment or prevention of autoimmune diseases.

[0084] The present invention finds particular utility in the treatment or prevention (including maintenance of remission or prevention of recurrence) of diseases or conditions of the ileum and / or colon, particularly the colon, such as inflammatory bowel diseases (including ulcerative colitis and Crohn's disease), IBS, constipation, diarrhea, infections or cancer. Accordingly, the present invention further provides the use of one or more dipeptides and optionally enzyme inhibitors in the manufacture of a medicament comprising a protein for the treatment and / or prevention of one or more of these conditions. The treatment and / or prevention of IBD is of particular importance.

Examples

[0085] The following examples illustrate the present invention and refer to the following figures.

[0086] Figure 1 shows the levels of antibody in tissue after administration via enteric-coated capsules with or without a stabilizer, or intravenous administration.

[0087] Figure 2 shows the plasma levels of antibody after administration via enteric-coated capsules with or without a stabilizer, or intravenous administration.

[0088] Materials and methods Human colon model A human colon model based on a mixed fecal inoculum was used to mimic the luminal environment of the human large intestine. For the model setup, an anaerobic workstation (Electrotek 500TG™ workstation, Electrotek, West Yorkshire, UK) maintained at 37 °C and 70% relative humidity was used. Fecal material was transferred to the anaerobic workstation and diluted with freshly prepared basal medium to obtain a 20% w / w slurry by homogenization. The basal medium provides nutrients and growth factors for the microbiota and enables survival for up to 24 hours. The homogenized bacterial medium was sieved through an open mesh fabric (SefarNitexTM, pore size 350 μm) to remove all non-uniform fibrous material. The pH was maintained at approximately 7 to mimic the human colonic luminal pH.

[0089] Antibody incubation test Prepare a 2 mg / mL antibody stock solution (vedolizumab, rupatadine, infliximab, ustekinumab, infliximab Fab fragment, or a combination of infliximab and ustekinumab) in PBS and add it to 20% human or rat fecal slurry to obtain fecal slurries with incubation concentrations of 1 mg / mL and 10% w / w. Samples were collected at appropriate time points and added to a protease inhibitor cocktail (Sigma-Aldrich, P2714) at a ratio of 1:3. The samples were centrifuged at 9.6 g for 10 minutes, and the supernatant was analyzed by size exclusion HPLC (SE-HPLC).

[0090] SE-HPLC Sample analysis was performed using a high-performance liquid chromatography (HPLC) system (Agilent Technologies, 1260 Infinity II Series (trademark)) equipped with a pump (model G1311C), an autosampler (model G1329B), and a diode array UV detector (model G1314B). For sample separation, a 600×7.8-mm Biosep (trademark) 5μm SEC-s3000 290Å (Phenomenex, Torrance, CA) size exclusion (SE) chromatography column was used, and phosphate-buffered saline (pH 7.3) prepared with sterile HPLC-grade water was used as the mobile phase for elution at a flow rate of 1 mL / min. The analysis was performed at room temperature, and the UV detection wavelength was set at 280 nm. Each sample was run for 40 minutes to allow complete elution of the sample protein and reduce overrun. The retention times of the IgG1 antibody, F(ab’)2, and Fab / Fc fragments were 17 minutes, 18.2 minutes, and 20.3 minutes, respectively.

[0091] In vivo administration of infliximab capsules with or without stabilizer to Wistar rats The freeze-dried antibody (infliximab) was formulated at a volume of 0.7 mg / kg into rat capsules (5 mm × 2.6 mm) co-formulated with mannitol or a stabilizer (a combination of aprotinin, L-carnosine, and diglycine). The capsules were coated with an enteric coating consisting of a mixture of a pH-sensitive polymer and an enterobacteria-digestible polysaccharide.

[0092] Healthy male Wistar rats (Charles River Laboratories) weighing 350 - 400 g were housed in groups of 4 per cage and acclimated for at least 5 days before entering the study. After an overnight fast, the rats were dosed with the capsules and fasted from dosing until sacrifice. The animals had free access to water throughout the study. The rats were sacrificed 7 hours after administration, and the entire GI tract, plasma, and feces were collected, and the antibody levels were analyzed by ELISA.

[0093] To compare the tissue and plasma concentrations of the antibody, a parallel treatment group regarding IV injection of the same dose of the antibody was also tested in rats.

[0094] ELISA Each well of a 96-well plate was coated with 100 μL of 1 μg / mL antigen (recombinant human TNF-α protein) diluted with PBS and incubated overnight at 4°C. The wells were washed 3 times with 200 μL / well of wash buffer and blocked for 1 hour at 37°C. After subsequent washing, the test samples were added and incubated for 1 hour at 37°C. After 3 washing cycles, the secondary antibody solution was added (goat anti-human IgG Fc secondary antibody HRP-conjugated) and incubated for 45 minutes at 37°C. After the washing step, the wells were incubated with 150 μL / well of K-BLUE substrate and incubated in the dark at room temperature for 10 minutes. After 10 minutes, 50 μL of RED STOP solution was added to each well, and the plate was read at 650 nm.

[0095] Example 1 Stability of monoclonal antibodies in a human colon model in the presence of L-carnosine, diglycine (Gly-Gly) and optionally aprotinin As described in the Methods section, colonic stability was evaluated using a human colon model by the amount of intact antibody (vedolizumab, rupatadine, infliximab or ustekinumab) remaining at each time point evaluated by SE-HPLC. Experiments were conducted in the absence of dipeptide, aprotinin or excipient and then in the presence of 50 mM or 100 mM L-carnosine. Experiments were also conducted in the presence of L-carnosine and 0.5 mg / mL aprotinin (Sigma-Aldrich).

[0096] The results are shown in the following table.

[0097]

Table 1

[0098] The results indicate that infliximab, vedolizumab and rupatadine were degraded by the human colonic microbiota in the absence of the tested stabilizer molecules. The antibody + aprotinin (0.5 mg / mL) control was observed to result in less antibody degradation. A significant stabilizing effect on the antibody was obtained in the presence of 100 mM L-carnosine and 100 mM diglycine. This stabilizing effect of L-carnosine was significantly enhanced by including 0.1 and 0.5 mg / mL aprotinin in the test samples.

[0099] Example 2 Stability of combinations of monoclonal antibodies infliximab and ustekinumab in a human colon model in the presence of aprotinin, L-carnosine and diglycine (Gly-Gly) As described in the Methods section, colonic stability was evaluated using a human colon model by the amount of intact antibody (infliximab, ustekinumab, or a combination of infliximab and ustekinumab) remaining at each time point evaluated by SE-HPLC. Experiments were conducted in the absence of dipeptide, aprotinin or excipient and then in the presence of L-carnosine, diglycine and aprotinin (Sigma-Aldrich).

[0100] The results are shown in the following table.

[0101]

Table 2

[0102] Example 3 In vivo PK of antibodies in Wistar rats after oral administration in capsules After oral capsule administration with or without a stabilizer (a combination of aprotinin, L-carnosine, and diglycine), and after IV injection at a dose of 0.7 mg / kg, the intestinal tissue concentration of the antibody (infliximab) was measured.

[0103] The results are shown in Figures 1 and 2.

[0104] As a result, it was shown that the antibody concentration in the intestinal tissue (ileum colon and colon) after formulating a stabilizer in an enteric-coated capsule was higher compared to the case of formulating the enteric-coated capsule without a stabilizer and the case of IV injection of the same dose of the antibody. Plasma exposure of the antibody was not observed after capsule administration (with and without a stabilizer), but IV injection administration showed a high concentration of the antibody floating in the systemic circulation.

Claims

1. A pharmaceutical composition comprising an antibody or a fragment thereof as an active ingredient, one or more dipeptides selected from carnosine and diglycine, and aprotinin which is an enzyme inhibitor and has 90% or more identity to SEQ ID NO:

1.

2. The pharmaceutical composition according to claim 1, which stabilizes the antibody in the presence of intestinal fluid by reducing the degradation of the antibody in the ileum and / or colon.

3. The pharmaceutical composition according to claim 1 or 2, which is in a liquid, solid or semi-solid form suitable for oral or rectal administration.

4. The pharmaceutical composition according to claim 3, which is in a solid form suitable for oral administration and has an enteric coating, preferably an enteric coating adapted for the selective release of the antibody or its fragment in the lower digestive tract.

5. The pharmaceutical composition includes a solid dosage form having a core and a coating for the core, the core includes the active ingredient, a first stabilizer, and optionally at least one second stabilizer, and the coating includes a mixture of a digestible polysaccharide and a film-forming material having a dissolution threshold of pH 6.0 or higher. The pharmaceutical composition according to any one of claims 1 to 4.

6. The pharmaceutical composition according to claim 5, wherein the digestible polysaccharide is selected from the group consisting of starch, amylose, amylopectin, chitosan, chondroitin sulfate, cyclodextrin, dextran, pullulan, carrageenan, scleroglucan, chitin, curdlan and levan.

7. The pharmaceutical composition according to claim 5 or 6, wherein the film-forming material is an acrylate polymer, a cellulose polymer or a polyvinyl-based polymer, preferably cellulose acetate phthalate, cellulose acetate trimellitate, hydroxypropyl methylcellulose acetate succinate or polyvinyl acetate phthalate.

8. An orally administrable pharmaceutical composition comprising an antibody or a fragment thereof as an active ingredient, one or more dipeptides selected from carnosine and diglycine, and aprotinin which is an enzyme inhibitor and has 90% or more identity to SEQ ID NO:

1.

9. A core comprising an antibody or a fragment thereof as an active ingredient, one or more dipeptides selected from carnosine and diglycine, and aprotinin which is an enzyme inhibitor and has 90% or more identity to SEQ ID NO: 1 A delayed release coating for the core and a solid dosage form for oral administration.

10. A rectally administrable pharmaceutical composition comprising an antibody or a fragment thereof as an active ingredient, one or more dipeptides selected from carnosine and diglycine, and aprotinin which is an enzyme inhibitor and has 90% or more identity to SEQ ID NO:

1.

11. An enema preparation comprising an antibody or a fragment thereof as an active ingredient, one or more dipeptides selected from carnosine and diglycine, and aprotinin which is an enzyme inhibitor and has 90% or more identity to SEQ ID NO:

1.

12. The pharmaceutical composition is a pharmaceutical composition for treating or preventing a disease or condition in a subject, The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition is administered to the subject via the ileum and / or colon.

13. The pharmaceutical composition according to claim 12, wherein the disease or condition is inflammatory bowel disease, irritable bowel syndrome, constipation, diarrhea, infectious disease, autoimmune disease or cancer.

14. The composition according to any one of claims 1 to 11 for use in therapy.

15. A method for stabilizing an antibody or a fragment thereof in the presence of intestinal fluid, the method comprising contacting the antibody or a fragment thereof with one or more dipeptides selected from carnosine and diglycine and aprotinin which is an enzyme inhibitor and has at least 90% identity to SEQ ID NO:

1.

16. Use of one or more dipeptides selected from carnosine and diglycine and aprotinin which is an enzyme inhibitor and has at least 90% identity to SEQ ID NO: 1 for stabilizing an antibody or a fragment thereof administered as a pharmaceutical composition and delivered to the lower gastrointestinal tract.

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