Compositions for oral delivery of biological agents - Patents.com

Abstract

The present invention relates to a method for stabilizing proteins and peptides in the lower gastrointestinal tract. A pharmaceutical composition is described that comprises as an active ingredient a protein or peptide; 5-aminolevulinic acid (5-ALA) or a pharma- ceutical acceptable salt thereof; and, optionally, a pH adjusting component and / or an enzyme inhibitor.

Description

[Technical field]

[0001] The present invention relates to novel compositions and their uses. [Background technology]

[0002] Biopharmaceuticals, particularly proteins including antibodies and peptides, are an increasingly important class of pharmaceuticals, with many therapeutic uses proposed and implemented. While the most convenient and patient-preferred method of drug administration is orally, nearly all biopharmaceuticals are currently delivered by injection, due to the instability of this class of molecules in the gastrointestinal (GI) tract and their inefficient crossing of GI membranes.

[0003] There is an unmet need for methods to sufficiently stabilize proteins used as biologics in the presence of luminal fluid found in the lower gastrointestinal tract, such as the small intestine and / or colon, so that they can be delivered either locally or systemically via the ileum and / or colon upon oral administration.

[0004] Furthermore, to optimize systemic delivery following oral administration, it is necessary to provide means to enhance uptake across the membranes of the GI tract and into the systemic circulation. Many such "penetration enhancing" compounds and methods have been described.

[0005] It has been shown that proteins can be protected from degradation in the GI tract by formulation in the presence of certain protective excipient molecules. In particular, it has been shown that antibodies can be protected from digestion in the colon lumen by co-formulation with enzyme inhibitor molecules such as aprotinin in combination with certain dipeptide molecules (WO 2020 / 229693). It has further been shown that peptides, particularly cyclic peptides and peptides containing non-naturally occurring amino acids (D-amino acids), can be stabilized in GI fluids, particularly colonic fluids, by formulation in the presence of certain amino acids (WO 2022 / 101322).

[0006] The present inventors have found that formulation of a protein or peptide with the non-proteinogenic amino acid 5-aminolevulinic acid (5-ALA) results in enhanced stability of the protein or peptide in fluids found in the lower gastrointestinal tract, such as the small intestine and / or colon. The addition of pH adjusting components to increase the pH of the mixture, and / or enzyme inhibitors, may serve to further enhance the stability of the protein or peptide.

[0007] The inventors have further shown that combining 5-ALA with a peptide or protein in a formulation results in enhanced penetration of the protein or peptide into and across the membranes of the GI tract, allowing for the potential for greater uptake of the protein or peptide into or across the GI tissues and into the systemic circulation. Summary of the Invention

[0008] Thus, the present invention provides pharmaceutical compositions comprising, consisting of, or consisting essentially of a protein or peptide as an active ingredient; 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof; and, optionally, a pH adjusting component and / or an enzyme inhibitor.

[0009] Preferably, when the active ingredient is a protein, the composition comprises, consists of, or consists essentially of the protein active ingredient, 5-ALA or a pharma- ceutically acceptable salt thereof; and a pH adjusting ingredient and / or an enzyme inhibitor. Preferably, the protein active ingredient is an antibody or a fragment thereof.

[0010] Preferably, when the active ingredient is a peptide, the composition comprises, consists of, or consists essentially of the peptide active ingredient, 5-ALA or a pharma- ceutically acceptable salt thereof; and an enzyme inhibitor. Preferably, the peptide active ingredient is a linear or cyclic peptide.

[0011] The protein may be an antibody or a fragment thereof. The peptide may be a linear or cyclic peptide.

[0012] The composition of the present invention can be in liquid, solid or semi-solid form, preferably in a form suitable for rectal or, in particular, oral administration.The composition can be in a solid form suitable for oral administration, said composition having an enteric coating.Most preferably, the composition is in a solid or semi-solid form suitable for oral administration and adapted for selective release of protein in the lower gastrointestinal tract, in particular in the ileum and / or colon.

[0013] The composition may include a solid dosage form having a core and a coating for the core, the core comprising a protein or peptide as an active ingredient, 5-ALA and optionally a pH adjusting ingredient and / or an enzyme inhibitor; and the coating comprising a mixture of a digestible polysaccharide and a film former having a solubility threshold at or above pH 6.0, i.e., dissolves at or above pH 6.0.

[0014] The composition may be an orally administrable pharmaceutical composition comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and optionally (c) a pH adjusting component and / or (d) an enzyme inhibitor.

[0015] The present invention also provides solid dosage forms for oral administration comprising a core comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and optionally (c) a pH adjusting component and / or (d) an enzyme inhibitor.

[0016] The present invention also provides a rectally administrable pharmaceutical composition comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and optionally (c) a pH adjusting component and / or (d) an enzyme inhibitor.

[0017] The present invention also provides an enema preparation comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and optionally (c) a pH adjusting component and / or (d) an enzyme inhibitor.

[0018] 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 (IBD); irritable bowel syndrome; constipation; diarrhea; infectious disease; or cancer is also provided.Preferably, the pharmaceutical composition is the pharmaceutical composition of the present invention.Preferably, the active ingredient is an antibody.

[0019] The present invention further provides a method for treating or preventing a disease or condition in a subject, comprising administering to the subject a pharmaceutical composition according to the present invention.Preferably, the disease or condition is selected from inflammatory bowel disease; irritable bowel syndrome; constipation; diarrhea; infectious disease; autoimmune disease or cancer.Preferably, the pharmaceutical composition is the pharmaceutical composition of the present invention.Preferably, the active ingredient is an antibody.

[0020] The present invention further provides a method for stabilizing a protein or peptide in the presence of intestinal fluids, comprising the step of delivering an active ingredient selected from a protein or peptide together with 5-ALA and, optionally, a pH modifier and / or an enzyme inhibitor.

[0021] The present invention also provides the use of 5-ALA, optionally with a pH adjusting component and / or an enzyme inhibitor, to stabilize a protein or peptide administered as a pharmacologic active ingredient and delivered to the lower gastrointestinal tract. Preferably, the pharmaceutical composition is the pharmaceutical composition of the present invention.

[0022] Release is followed by penetration of the drug into the GI tissues at or near the site of release in the GI lumen, allowing for localized therapy of diseases of the GI tract, as well as treatment of a wide range of diseases through penetration of the drug into the bloodstream.

[0023] The present invention also provides the use of 5-ALA, optionally with a pH adjusting component and / or an enzyme inhibitor, to improve the penetration of a protein or peptide into the tissue lining the GI tract when the protein or peptide is administered as a pharmacologic active ingredient of a pharmaceutical composition and delivered to the lower gastrointestinal tract. Preferably, the pharmaceutical composition is the pharmaceutical composition of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] As used herein, "lower gastrointestinal tract" refers to the gastrointestinal tract after the stomach. It 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. Preferably, the stabilizer (e.g., 5-ALA, pH adjusting component and / or enzyme inhibitor) reduces the degradation of protein or peptide in the ileum and / or colon.

[0025] Proteins or peptides The protein or peptide may be any protein or peptide having a beneficial therapeutic effect on a human or animal, which therapeutic effect is advantageously achieved by administration via the lower gastrointestinal tract, preferably to the ileum and / or colon, particularly the colon.

[0026] Classes of proteins for use in the present invention include, for example, antibodies, enzymes, cytokines, chemokines, receptors, blood factors, peptide hormones, toxins, transcription proteins, non-immunoglobulin binding protein scaffolds, and multimeric proteins, as described below.

[0027] antibody As used herein, "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target antigen, such as a cytokine, a protein, a polypeptide, a peptide, a carbohydrate, a polynucleotide, a lipid, a bacterium or a virus, or a combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term "antibody" encompasses polyclonal antibodies, monoclonal antibodies, multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigenic determinant portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, so long as the antibody exhibits the desired biological activity. Preferably, the antibody is a monoclonal antibody. Antibodies may include any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), 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 shapes.Preferably, the antibody is an IgG antibody, more preferably IgG1 or IgG4.The term "antibody" is also intended to include conjugates of the antibody, such as conjugates with polyethylene glycol, PEG.

[0028] Furthermore, unless the context requires otherwise, the term "antibody" should be understood to encompass complete antibodies and antibody fragments that contain the antigen-binding region of a complete antibody. Antibody fragments include, for example, single domain antibodies (e.g., V HH domain antibodies), monovalent or bivalent Fab, Fab', F(ab')2, scFv, Fc, bispecific antibodies, diabodies, minibodies or multispecific antibodies formed from antibody fragments, such as various permutations of scFv fragments or diabodies, and optionally minibodies composed of Fc fragments or CH domains, such as scFv-Fc, scFv-Fc-scFv, Fab-scFv, (Fab'ScFv)2, sc diabody, sc diabody-Fc, sc diabody-CH3, scFv-CH3, and scFv-CH2-CH3 fusion proteins. Antibody fragments can be produced by enzymatic cleavage of intact antibodies, or by the use of recombinant DNA techniques, phage display or yeast display technologies or transgenic mice, or by synthetic means such as solution or solid phase peptide synthesis.

[0029] When an antibody is used in the composition according to the invention, it may be any antibody whose therapeutic effect is advantageously realized by administration via the colon.Specific antibodies of particular interest in the context of the present invention include existing commercially available IBD therapeutic antibodies, such as adalimumab, infliximab, cetrolizumab pegol, golimumab, natalizumab, vedolizumab, tildrakizumab, ustekinumab, and additional antibodies in the development of IBD therapeutics that target pathways and molecules (agonists or antagonists) involved in the pathogenesis of IBD, such as CD40.Targeting to the colon further offers the possibility of improving the treatment of colorectal cancer by targeting and localizing anti-cancer therapeutic antibodies to tumors, or various possible forms of targeting and localization as described above.

[0030] peptide A peptide is a chain of amino acid monomers linked together by amide bonds. A peptide may be composed of two or more amino acid monomers. Preferably, a peptide comprises less than 50 amino acid monomers, preferably 2-50 amino acid monomers, more preferably 2-10 amino acid monomers, 5-20 amino acid monomers, 15-40 amino acid monomers or 18-30 amino acid monomers. Preferably, a peptide has a molecular weight of up to 5 kDa, more preferably 1-2.5 kDa. Suitable peptides include linear peptides, branched peptides and cyclic peptides with disulfide bridges. A cyclic peptide may comprise 1-5 disulfide bridges, preferably 1-3 disulfide bridges. Preferably, the peptide is a cyclic peptide.

[0031] Peptides are generally made from naturally occurring L-amino acids. Mutant peptides may contain one or more D- and / or non-natural amino acids, such as those with modified side groups. Preferably, mutant peptides have one, two, three, four or five D-amino acids. Preferably, in mutant peptides, L-amino acids are replaced by equivalent D-amino acids or equivalent amino acids with modified side groups. Furthermore, mutant forms of peptides can be linear or cyclic versions of normal cyclic or linear peptides, respectively.

[0032] One preferred peptide is oxytocin and its variants. Oxytocin has the amino acid sequence Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ ID NO: 2) and typically contains sulfide bridges between cysteine ​​residues.

[0033] Stabilizers 5-Aminolevulinic acid As used herein, "5-ALA" refers to the naturally occurring amino acid 5-aminolevulinic acid, its precursors, derivatives or pharma- ceutically acceptable salts. The compound 5-aminolevulinic acid, described below, has previously been utilized as a precursor of the photosensitizer PpIX to perform photodynamic diagnosis and therapy to identify and kill tumor cells. 5-ALA is converted to PpIX and heme in mitochondria by iron insertion catalyzed by iron-adding enzyme. HO-1 further catalyzes the conversion of heme to carbon monoxide (CO), biliverdin and bilirubin. 5-ALA (CAS No. 106-60-5) has the following general formula:

[0034] [ka]

[0035] A "pharmaceutically acceptable salt" is one that is safe for use in mammals and retains the desired biological activity. Suitable salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Preferably, the salt is a hydrochloride salt.

[0036] As used herein, "5-ALA" also encompasses precursors of 5-aminolevulinic acid. A "precursor" is a compound that is metabolized to 5-aminolevulinic acid after administration in the intestinal tract, for example, through the action of bacteria found in the gastrointestinal tract, particularly the lower gastrointestinal tract.

[0037] As used herein, "5-ALA" also includes derivatives of 5-aminolevulinic acid and their pharma- ceutically acceptable salts. Derivatives include esters, in which the -OH group of 5-aminolevulinic acid is replaced with -OR, where R is an alkyl group having 8 or less carbon atoms. Preferably, the alkyl group has 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. The alkyl group is preferably unbranched. Preferably, the alkyl group is methyl or hexyl. Suitable derivative esters include methyl aminolevulinic acid and hexyl aminolevulinic acid, and preferred salts include methyl aminolevulinate hydrochloride and hexyl aminolevulinate hydrochloride.

[0038] 5-ALA, optionally in combination with a pH modifier and / or an enzyme inhibitor, acts as a stabilizer that helps maintain intact proteins or peptides in the lower gastrointestinal tract. Methods for assessing the stability of proteins or peptides in gastrointestinal fluids are described in the Examples.

[0039] 5-ALA, optionally in combination with a pH modifier, and / or an enzyme inhibitor, causes at least 5% of the peptide or protein to remain intact in the gastrointestinal fluids after one hour, preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or more of the peptide or protein to remain intact.

[0040] pH adjusting ingredient The pH adjusting component is one that changes the pH of gastric fluid compared to gastric fluid containing administered 5-ALA. The pH adjusting component neutralizes the effect of 5-ALA so that the pH of gastrointestinal fluid does not change when the composition is administered. Preferably, the pH adjusting component increases the pH so that the gastric fluid has a pH of about 6.0 after addition of the composition. The pH adjusting component is preferably a base. Suitable pH adjusting components include arginine, sodium hydroxide, ammonia, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, and trolamine. Preferably, the pH adjusting component is arginine.

[0041] Enzyme inhibitors Preferably, the enzyme inhibitor is a protease inhibitor, such as aprotinin, ovomucoid type II-O (containing ovoinhibitor), Bowman-Birk inhibitor (BBI), or Kunitz-type trypsin inhibitor, for protection of proteins.

[0042] Preferably the enzyme inhibitor is aprotinin or an analogue thereof.

[0043] Aprotinin The pharmaceutical compositions of the present invention preferably include aprotinin, or a fragment or analogue of aprotinin. It will of course be understood that aprotinin, a fragment or analogue of aprotinin is not present in the compositions of the present invention as an active ingredient.

[0044] Preferably, the composition does not include: (a) carnitine, an acylcarnitine, or a salt of carnitine or an acylcarnitine; (b) a salt or ester of a bile acid; or (c) a combination of aprotinin or an analog of aprotinin with an alkyl saccharide.

[0045] Aprotinin is a protease inhibitor that is known to inhibit trypsin and other similar proteases. It is often also called bovine pancreatic trypsin inhibitor (BPTI). Aprotinin is a 58 amino acid protein formed after processing of a 100 amino acid polypeptide that contains a signal peptide, a propeptide domain, and a Kunitz-type domain. In its mature 58 amino acid form, aprotinin is commercially available and sold under the trade name Trasylol®, and it is indicated for prophylactic use to reduce blood loss during surgical procedures.

[0046] Aprotinin for use in the present invention may comprise the amino acid sequence RPDFCLEPPYTGPCKARMIRYFYNAKAGLCQPFVYGGCRAKRNNFKSSEDCMRTCGGA (SEQ ID NO: 1).

[0047] Aprotinin, fragments or aprotinin analogs 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 chemistry techniques such as solution or solid phase peptide synthesis), or it can be a naturally occurring protein from an animal source, such as, for example, of bovine origin.

[0048] Furthermore, unless the context requires otherwise, the term "aprotinin" should be understood to include aprotinin, aprotinin analogs and aprotinin fragments. Suitable aprotinin analogs and fragments for use in the present invention are those that, when combined with 5-ALA, enhance the stability of a protein or peptide in the presence of fluids found in the lower gastrointestinal tract, such as the ileum and / or colon. Methods for confirming the ability of aprotinin, aprotinin fragments or aprotinin analogs to enhance the stability of a protein or peptide in the presence of fluids found in the lower gastrointestinal tract, such as the ileum and / or colon, are described in the Examples below. As used herein, "enhanced" stability means that at least 15% of the peptide or protein remains intact in gastrointestinal fluid after 1 hour, preferably after 2 hours. Preferably, at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95% or more of the peptide or protein remains intact.

[0049] For example, an aprotinin fragment for use in the present invention may comprise a fragment of the sequence defined by SEQ ID NO: 1. For example, an aprotinin fragment may comprise at least 30, 40, 50, 51, 52, 53, 54, 55, 56 or 57 consecutive amino acids of the sequence defined by SEQ ID NO:1.

[0050] An analog of aprotinin for use in the present invention may include a protein that includes a sequence similar to the amino acid sequence defined in SEQ ID NO:1, which when combined with 5-ALA enhances the stability of the peptide or protein in the presence of fluids found in the lower gastrointestinal tract, such as the ileum and / or colon. For example, the aprotinin analog may have at least 70%, 80%, 90% or at least 95% similarity to the sequence defined in SEQ ID NO:1 or a fragment of the sequence defined by SEQ ID NO:1. Alternatively, the aprotinin analog may have at least 70%, 80%, 90% or at least 95% identity to the sequence defined in SEQ ID NO:1 or a fragment of the sequence defined by SEQ ID NO:1. For example, the aprotinin analog may include an amino acid sequence that is identical to 50, 51, 52, 53, 54, 55, 56 or 57 amino acids of the sequence defined in SEQ ID NO:1. The similar or identical amino acids may be contiguous or non-contiguous.

[0051] Programs 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. This program is capable of calculating the amino acid identity or similarity (identity plus conservation of amino acid types) for optimal alignment. Programs such as BLASTx align the longest stretches of similar sequences and assign a value to the matches. It is therefore possible to obtain a comparison in which several regions of similarity are found, each with a different score. Both types of analysis are envisaged in the present invention. The identity or similarity is preferably calculated over the entire length of SEQ ID NO:1.

[0052] Analogs of aprotinin may contain one or more amino acid substitutions, insertions and / or deletions.

[0053] Amino acid substitution means that an amino acid residue is replaced with a substitute amino acid residue at the same position. Amino acid substitution can be conservative, which means that the substituted amino acid has similar chemical properties as the original amino acid. Those skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids 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.

[0054] The inserted amino acid residues may be inserted at any position, and some or all of the inserted amino acid residues may be inserted immediately adjacent to one another, or none of the inserted amino acid residues may be inserted immediately adjacent to another inserted amino acid residue. For example, an aprotinin analog may include 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 in SEQ ID NO:1.

[0055] One, two or three amino acids may be deleted from the sequence of SEQ ID NO: 1. Each deletion may occur at any position in SEQ ID NO: 1.

[0056] The inserted amino acid and the replacement amino acid may be naturally occurring or non-naturally occurring amino acids, e.g., may contain non-naturally occurring side chains and / or may be linked together via non-natural peptide bonds. Such modified peptide ligands are known in the art. When more than one amino acid residue is replaced and / or inserted, the replaced / inserted amino acid residues may be the same as each other or different from each other. Each replacement amino acid may have a different side chain from the amino acid it replaces.

[0057] Analogs of aprotinin may contain one or more modified bases, and amino acid residues may be chemically modified. Examples of chemical modifications include those that correspond to post-translational modifications, such as phosphorylation, acetylation, and deamidation. Chemical modifications may not correspond to those present in vivo. For example, the N-terminus or C-terminus of aprotinin peptides may be modified to improve peptide stability, bioavailability, and / or affinity. Further examples of non-natural modifications include the incorporation of uncoded α-amino acids, photoreactive crosslinking amino acids, N-methylated amino acids, and β-amino acids, backbone reduction, retroinversion by using d-amino acids, N-terminal methylation and C-terminal amidation, and PEGylation.

[0058] Pharmaceutical preparations The pharmaceutical compositions according to the invention are preferably in solid or semi-solid form and preferably they are suitable for oral or rectal administration.

[0059] The composition may also be in the form of a lotion, cream, foam, emulsion or gel. Such formulations may be prepared by a number of known methods well established in the art.

[0060] For example, the protein or peptide and the required excipients may be mixed together, optionally with other excipients required in the dosage form.

[0061] Pharmaceutical compositions according to the invention suitable for oral administration may be in the form of any of tablets, capsules, minitablets, pellets, powders, granules, microparticles, nanoparticles or hydrogels.

[0062] The compositions of the present invention suitable for oral administration may be present as discrete units such as capsules, tablets, minitablets, or pellets, or as powders, granules, or crystals. In solid compositions, the smallest diameter of each particle is typically at least 10. -4 m, typically at least 5 × 10 -4 m, preferably at least 10-3 m. 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 (e.g., for pellets or minitablets) or about 6 mm to about 12 mm (e.g., for certain tablets or capsules). The term "diameter" refers to the longest linear dimension through the particle.

[0063] As well as the required 5-ALA or a pharma- ceutically acceptable salt thereof, and optional pH adjusting components and / or enzyme inhibitors, the composition according to the invention may of course contain any further conventional excipients, such as binders, extenders, disintegrants, diluents and lubricants, as necessary. 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, extenders, disintegrants, diluents and lubricants known in the art. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn syrup, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Fast-dissolving diluents include mannitol, lactose, sucrose and / or cyclodextrin. Lubricants, flow agents, 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.

[0064] Tablets may be produced by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricant, surfactant or dispersant. Molded tablets may be produced by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may optionally be coated or scored, and may be formulated to provide sustained, delayed or controlled release of the antibody. Examples of suitable coatings are given below.

[0065] Capsules may have solid, semi-solid or non-solid contents. A typical content for a capsule may include a suspension, which may include, for example, microcrystalline cellulose to provide bulk, alginic acid or sodium alginate as a suspending agent, and methylcellulose as a viscosity enhancer, as well as any of the solid or semi-solid forms described above.

[0066] Formulations for rectal administration may be presented as a suppository using conventional carriers such as cocoa butter, synthetic glyceride esters, or polyethylene glycols. Such carriers are typically solid at normal room temperature (up to 25° C.) but liquefy and / or melt in the rectal cavity to release the drug.

[0067] The composition may also take the form of an enema preparation, such as a liquid or foam enema administered rectally to the lower colon. The enema preparation typically contains the peptide or protein, along with 5-ALA, optionally in combination with a pH regulator, and / or an enzyme inhibitor, dissolved or dispersed in a suitable fluid carrier medium, such as deionized water and / or distilled water. The preparation may be thickened with one or more thickening agents. The preparation may also contain a buffer and may also contain an effective amount of a lubricant, such as a natural or synthetic fat or oil, such as a tris-fatty acid glycerate or lecithin. A non-toxic non-ionic surfactant may also be included as a wetting agent and dispersant. A buffer is preferably added to the liquid or foam enema to stabilize the pH. The pH of the enema preparation is preferably 3.5 to 7.5, especially 6.5 to 7.5.

[0068] The unit dose of the enema formulation can be administered from a pre-filled bag or syringe. In the case of a pressurized enema formulation, the carrier medium can also include an effective amount of a foaming agent, such as n-butane, propane or i-butane, or the foaming agent / propellant can be kept separate from the composition, such as in the bag-in-bag or bag-in-can system described in WO-A-9603115 (hereby incorporated by reference). The enema foam can also include a foaming agent and a foam stabilizer.

[0069] Liquid enemas typically have a volume of 50-200 cm 3 , preferably about 100 cm 3 The volume of a foam enema is typically 20-40 cm. 3 The suitable dosage of 5-ALA or its pharmaceutical salt for the enema administered is 1 mg / ml to 20 mg / ml, preferably 2 mg / ml to 10 mg / ml.

[0070] Preferred unit dosage formulations are those containing an effective amount, or an appropriate fraction thereof, of the active ingredient. Release from certain formulations may be sustained if the composition contains appropriate controlled release excipients. However, in preferred formulations, release is pulsatile.

[0071] The compositions according to the invention typically contain a therapeutically effective amount of protein or peptide, which may be 0.01 wt% to 99 wt%, based on the total weight of the composition. The actual dosage will be determined by the skilled artisan using common general knowledge. However, as an example, a "low" dose formulation typically contains 20 wt% or less of protein or peptide, preferably 1 wt% to 10 wt%, such as 5 wt% of protein or peptide. A "high" dose formulation typically contains at least 40 wt% of protein or peptide, preferably 45 wt% to about 85 wt%, such as 50 wt% or 80 wt%.

[0072] The protein or peptide active agent is typically present in an amount to provide a concentration in the lower gastrointestinal tract of 0.1-25 mg / ml. Each dosage form may contain 50-5000 mg of protein or peptide, typically 100-1000 mg; 250-750 mg or 300-500 mg.

[0073] The composition according to the present invention typically comprises an effective amount of stabilizer (i.e., the total amount of 5-ALA or a pharma- ceutically acceptable salt thereof, and pH regulator and / or enzyme inhibitor, if present). Typically, the formulation comprises 0.01 wt% to 99 wt% stabilizer, based on the total weight of the composition. The composition may preferably comprise 20 wt% or less of stabilizer, and preferably 1 wt% to 10 wt%, for example 5 wt% stabilizer. Alternatively, the composition may comprise at least 40 wt% of stabilizer, and preferably 45 wt% to about 85 wt%, for example 50 wt% or 80 wt%.

[0074] 5-ALA is typically present in an amount to provide a concentration in the lower gastrointestinal tract of 0.1-25 mg / ml. Each dosage form may contain 50-5000 mg of 5-ALA, typically 100-1000 mg; 250-750 mg or 300-500 mg.

[0075] When included in the formulation, enzyme inhibitors such as aprotinin are typically present in an amount to provide a concentration in the lower gastrointestinal tract of 0.1-25 mg / ml. Each dosage form may contain 50-5000 mg of aprotinin, typically 100-1000 mg; 250-750 mg or 300-500 mg.

[0076] When included in the formulation, a pH modifier such as arginine is typically present in an amount to provide a pH in the range of 5.5 to 7.5 in the lower gastrointestinal tract. Typically, the pH modifier is present in a concentration of 0.1 to 25 mg / ml in the lower gastrointestinal tract. Each dosage form may contain 50 to 5000 mg of aprotinin, typically 100 to 1000 mg; 250 to 750 mg or 300 to 500 mg.

[0077] Protein or peptide can be used as the only active ingredient in the composition according to the present invention, but it is also possible that protein or peptide is used in combination with one or more additional therapeutic agents, such as additional protein or peptide, or non-biological preparations.Therefore, the present invention also provides a composition according to the present invention that contains an additional therapeutic agent in addition to protein or peptide.If necessary, the composition according to the present invention can be administered together with the additional composition by simultaneous, sequential or separate administration.

[0078] Unless the context requires otherwise, throughout this specification and claims, any reference to a pharmaceutical composition in solid or semi-solid form should be understood to include individual solid or semi-solid particles or unit forms that are solid or semi-solid throughout, as well as those that have a solid or semi-solid exterior and a non-solid, e.g., liquid or gel, interior. For example, a capsule may have a liquid or gel content.

[0079] Delivery to the lower gastrointestinal tract The compositions according to the invention are preferably adapted for delayed or selective release of proteins or peptides in the lower gastrointestinal tract, particularly in the ileum and / or colon, following rectal or, in particular, oral administration. This may be achieved by the use of specific coatings. The compositions of the invention may be delayed release oral (DRO) compositions. DRO compositions pass the stomach substantially unchanged and deliver the active ingredient to the lower gastrointestinal tract, typically the ileum and / or colon (i.e., the site of the diseased mucosa).

[0080] The composition according to the invention may have an enteric coating. The enteric coating protects the active ingredient in the composition from attack and degradation in the stomach, but dissolves and releases the contents of the dosage form in the intestine, usually due to a change in pH. Suitable enteric coatings are well known in the art. The optimal coating for any particular formulation will vary depending on the exact intended use, and the coating may be tailored to release the active ingredient in a particular region of the intestine or at a particular time after ingestion. Such formulations may contain one or more intermediate layers between the active ingredient and the outer enteric coating, if necessary. In this case, the composition of the invention is capable of releasing a portion of its contents in one particular region of the intestine and a further portion of its contents in a second region of the intestine, such as the colon. Preferably, the composition of the invention is in solid or semi-solid form, including an enteric coating adapted to release the protein or peptide in the colon. Useful enteric coatings are those that remain intact in the low pH environment of the stomach, but dissolve readily when the optimal pH for dissolution is reached. This may vary from pH 3 to 7.5, preferably 5 to 7, depending on the chemical composition of the coating. The thickness of the coating required depends on the solubility of the coating and the area intended to be treated. Typically, the coating is 25 to 200 μm, especially 75 to 150 μm.

[0081] The compositions of the present invention are adapted to release the active ingredient to the portion of the lower gastrointestinal tract where the disease is prevalent. Typically, the enteric coating should dissolve at the pH of the jejunum (about pH 5.5), the pH of the ileum (about pH 6) and / or the pH of the colon (pH 6-7), where the majority of the protein or peptide active ingredient is released at the desired site.

[0082] WO 2007 / 122374, the contents of which are incorporated herein by reference, describes compositions for selective release in the colon, which form a preferred embodiment of the present invention. Thus, the present invention further provides compositions comprising particles having a core and a coating for the core, the core comprising a protein or peptide together with 5-ALA and optionally other stabilizing agents such as pH modifiers and / or enzyme inhibitors, and the coating comprising a mixture of digestible polysaccharides and a film former having a solubility threshold at a pH of 6.0 or greater, preferably at a pH of 7 or greater.

[0083] The digestive polysaccharide is susceptible to attack by intestinal bacteria. Preferably the digestive polysaccharide is selected from the group consisting of starch, amylose, amylopectin, chitosan, chondroitin sulfate, cyclodextrin, dextran, pullulan, carrageenan, scleroglucan, chitin, curdulan and levan.

[0084] For example, the polysaccharide may be starch, amylose or amylopectin.

[0085] The film-forming material is an enteric material that has a pH solubility threshold below which it is insoluble and at or above which it is soluble. The pH of the surrounding medium triggers the dissolution of the second material. The normal pH of gastric fluid is usually in the range of 1-3, while the pH of intestinal fluid increases gradually from about 5.5 in the duodenum to about 7-8 in the colon. Thus, the second material, when used in the composition of the present invention, has a pH solubility threshold of 6.0 or higher, in particular 7 or higher.

[0086] The film former is typically selected from acrylate polymers, cellulose polymers, or polyvinyl polymers. Examples of suitable cellulose polymers include cellulose acetate phthalate ("CAP"); cellulose acetate trimellitate ("CAT"); and hydroxypropyl methylcellulose acetate succinate. Examples of suitable polyvinyl polymers include polyvinyl acetate phthalate ("PVAP"). The film former is preferably a polymer selected from (meth)acrylic acid and (meth)acrylic acid C. l-4 Copolymers of alkyl esters, for example copolymers of methacrylic acid and methacrylic acid methyl ester. Such polymers include those available under the tradenames Eudragit L, Eudragit S and Eudragit FS. The use of Eudragit S as a film former is particularly preferred.

[0087] In such compositions, multiple unit dosage forms containing particles with a diameter of less than 3 mm are preferred. The "core" is usually a single solid body. The core may consist of 5-ALA and a protein or peptide together with optionally a pH regulator acting as a stabilizer, and / or an enzyme inhibitor. More commonly, however, the core comprises a mixture of the protein or peptide and the above-mentioned 5-ALA, optionally with a pH regulator, and / or an enzyme inhibitor, and optionally one or more additional excipients. The core may, for example, comprise a filler or diluent material, for example a cellulosic material such as lactose or microcrystalline cellulose; a binder, for example polyvinylpyrrolidone (PVP); a disintegrant, for example croscarmellose sodium; and / or a lubricant, for example magnesium stearate. The core may be a compressed granule containing at least some of these materials.

[0088] Release from such compositions is delayed until the lower gastrointestinal tract, particularly the ileum and / or colon. Such compositions have application in multiphasic release compositions comprising at least two multiple particles, e.g. coated pellets, in the same dosage form, e.g. capsule, one multiple particle being differentiated from the other multiple particle by a coating. The coating may differ from the multiple in terms of coating thickness or composition, e.g. ratio and / or identity of components. Multiphasic release formulations are particularly suitable for patients with Crohn's disease, which affects various areas along the intestine, including the ileum and / or colon.

[0089] Medical Use The present invention provides a pharmaceutical composition according to the present invention for use in therapy.The present invention also provides a method for treating or preventing a disease or condition in a subject, particularly a human subject, which comprises administering to the subject a pharmaceutical composition comprising a protein or peptide as an active ingredient; 5-aminolevulinic acid (5-ALA) or a pharma-ceutically acceptable salt thereof; and optionally a pH adjusting component and / or an enzyme inhibitor, via the lower gastrointestinal tract, particularly the ileum and / or colon.Preferably, the composition is adapted for administration via oral or rectal route.The present invention finds utility in treating diseases of the lower gastrointestinal tract, particularly the ileum and / or colon, but it also has application as an entry point for protein or peptide into the systemic circulation by absorption from the lower gastrointestinal tract, particularly the ileum and / or colon, and thus finds utility in treating a wide range of diseases and conditions.

[0090] The following non-limiting examples illustrate the invention.

[0091] 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. The model was set up using an anaerobic workstation (Electrotek 500TG™ workstation, Electrotek, West Yorkshire, UK) that maintained 37°C and 70% relative air humidity. The fecal material was transferred to the anaerobic workstation and diluted with freshly prepared basal medium to obtain 25% w / w colonic fluid by homogenization. The basal medium provides nutrients and growth factors to the microbiota, allowing survival for up to 24 hours. The homogenized bacterial medium was sieved through an open mesh fabric (SefarNitex™, pore size 350 μm) to remove any heterogeneous fibrous material. The pH was maintained at approximately 7.0 to mimic the human colonic luminal pH.

[0092] Rat cecal slurry The rat cecal slurry was based on a combination of cecal and proximal colon fluids collected from healthy Wistar rats weighing 250 g. The fluids were collected immediately after the rats were sacrificed and transferred to an anaerobic workstation (Electrotek 500TG™ Workstation, Electrotek, West Yorkshire, UK) that maintained 37°C and 70% relative air humidity. The fluids were diluted with freshly prepared basal medium to obtain 50% w / w cecal fluid by homogenization. The homogenized mixture was sieved through an open mesh fabric. The pH of the fluid was measured to be 5.5.

[0093] Antibody incubation studies The antibody stock solution (infliximab) was prepared at a concentration of 2 mg / ml in PBS. 5-ALA was prepared at concentrations of 10 mg / ml, 20 mg / ml or 40 mg / ml. Methyl-ALA, hexyl-ALA, sodium sarcaprozate, (SNAC) and sodium chenodeoxycholate were prepared at a concentration of 20 mg / ml. Basic L-arginine was prepared at 11 mg / ml, 22 mg / ml or 44 mg / ml and the stabilizer aprotinin was prepared at a concentration of 2 mg / ml. Stock solutions containing specific infliximab combinations (Examples 1-5) were added in a 1:1 dilution to either 25% w / w human colonic fluid (human colon model) or 50% w / w rat cecal slurry to give incubation concentrations of 1 mg / ml infliximab, 5 mg / ml, 10 mg / ml or 20 mg / m 5-ALA, 5.5 mg / ml, 11 mg / ml or 22 mg / ml L-arginine, 10 mg / ml methyl-ALA, 10 mg / ml hexyl-ALA, 10 mg / ml sodium sarcaprozate, 10 mg / ml sodium chenodeoxycholate, and 12.5% ​​w / w human colonic fluid or 25% w / w rat cecal slurry. Samples were removed at the appropriate time points and added to a protease inhibitor cocktail (Sigma-Aldrich, P2714) in a 1:3 ratio. Samples were centrifuged at 9.6 g for 10 min and the supernatant was analyzed by size-exclusion-HPLC (SE-HPLC).

[0094] Peptide incubation studies Peptide stock solutions (oxytocin) were prepared at a concentration of 2 mg / ml in PBS. Concentrations of 10 mg / ml and 40 mg / ml of 5-ALA, 40 mg / ml methyl-ALA, 40 mg / ml hexyl-ALA, and 2 mg / ml aprotinin were prepared. Stock solutions containing specific oxytocin combinations were added to 50% rat cecal slurry to obtain incubation concentrations of 1 mg / ml peptide, 5 mg / ml or 20 mg / ml 5-ALA, 20 mg / ml methyl-ALA, 20 mg / ml hexyl-ALA, 1 mg / ml aprotinin, and 25% w / w rat cecal slurry (Examples 6 and 7). Samples were removed at appropriate time points and added to 0.1 N HCl solution in a ratio of 1:3. Samples were centrifuged at 9.6 g for 10 minutes and the supernatant was analyzed by reverse phase-HPLC (RP-HPLC).

[0095] Ussing chamber system A NaviCyte Vertical Ussing System (Harvard Apparatus, Cambridge, UK) was used to measure transport across epithelial membranes, which are polar structures that hold an apical (mucosal) and basolateral (serosal) side. The chambers are fabricated from solid acrylic and support the tissue membrane so that each side of the membrane is isolated and faces different chambers representing the luminal (apical) and blood (basolateral) compartments. The working system consists of a unit that fits up to six vertical chambers, a gas manifold for carbogen purging (95% O2, 5% CO2) and a heater block to maintain the temperature of the chambers at 37 °C during the experiment using a circulating water bath. The chambers are two-part assemblies held together by high spring tension retaining rings to ensure leak-free operation during the experiment.

[0096] An EVOM™ volt-ohmmeter (World Precision Instruments, Hertfordshire, UK) and Ag / AgCl electrodes (Harvard Apparatus, Cambridge, UK) were used to measure the transepithelial electrical resistance (TEER) of tissue samples. TEER monitors the presence of functional tight junctions, which serve a barrier function and limit the paracellular permeation of water and solutes. 200 Ω / cm 2 A TEER value of 0.01 was set as the lower limit to ensure tissue viability and tight junction integrity.

[0097] For tissue permeability studies, freshly excised small intestine (jejunum) and proximal colon tissues of healthy Wistar rats were harvested and transferred to an ice-cold solution of Krebs-Ringer bicarbonate solution (KBr) at pH 7.4. The tissues were cut open transversely, washed with KBr solution to remove luminal contents, and then mounted in Ussing chambers. The mucosal surface of the colonic tissue faced the apical chamber, and the endothelial surface of the tissue faced the basolateral chamber. The exposed tissue area on each side of the chamber was 0.29 cm. 2 and tissue fixation area was 4×8 mm. The volume of KBr in the apical and basolateral chambers was 5 ml and pH was maintained at 7.4. Tissues were incubated with KBr for 15 min before addition of drugs. Antibodies and peptides; infliximab and oxytocin were tested in specific combinations (examples 8-14) with and without ALA, methyl-ALA, hexyl-ALA, L-arginine, sodium caprate, sodium chenodeoxycholate, SNAC, labrasol, Na2EDTA and N-acetylcysteine ​​(NAC). Drug concentrations tested during permeation experiments were 0.2 mg / ml and 1 mg / ml for antibody and peptide studies, respectively. Antibody / peptide permeation in small intestinal and colonic tissues was tested in a minimum of two rats for 2 h. Tissues without drugs were incubated in parallel for the same time and served as negative controls. The chamber was purged with carbogen and maintained at 37° C. with a water jacket during incubation. TEER was continuously monitored during the experiment to ensure tissue viability and integrity. 200 Ω / cm2 Tissues with TEER values ​​below were not used in the experiments.

[0098] SE-HPLC Infliximab sample analysis (stability study) was performed using a size-exclusion-high performance liquid chromatography (SE-HPLC) system (Agilent Technologies, 1260Infinity II Series™) equipped with a pump (Model G7111A), autosampler (Model G7129A) and a diode array UV detector (Model G7114A). A 300×8.0 mm YMC-Pack Diol-200 S-5 μm, 20 nm (YMC Co., Ltd., Kyoto, Japan) size-exclusion (SE) chromatography column was used for sample separation using phosphate buffered saline (pH 7.3) prepared with sterile HPLC grade water as the mobile phase for elution at a flow rate of 0.5 ml / min. The analysis was performed at room temperature and the UV detection wavelength was set at 214 nm. Each sample was run for 40 min to allow complete elution of sample proteins and reduce run-over. The retention time for infliximab was 15 min.

[0099] RP-HPLC Peptide sample analysis (stability and permeability studies) was performed using a reversed-phase liquid chromatography (RP-HPLC) system (Agilent Technologies, 1260Infinity II Series™) equipped with a pump (model G7111A), an autosampler (G7129A) and a diode array UV detector (model G7114A). A 150 x 4.6 mm Luna 5 μm C18 100 Å (Phenomenex, Torrance, CA) column was used with a gradient system as follows:

[0100] [Table 1]

[0101] The injection volume of each sample was 20 μl, the flow rate of the mobile phase was 1 ml / min, and peptides were detected at 214 nm. Each sample was run for 23 min.

[0102] ELISA Infliximab sample analysis (permeability study) was performed using an indirect ELISA platform assay. 96-well plates were coated with 1ug / ml TNF-α. 5% BSA in PBS-T was used to block the plates to avoid non-specific binding. Drug samples were incubated at 37°C for 1 hour. Fc-specific secondary antibody was added to specifically bind to infliximab. TMB substrate was added to the wells and allowed to incubate in the dark to promote a color change depending on the level of antibody binding before addition of stop solution. Between each step, the plate was washed with 0.1% Tween 20 in PBS. Plates were read at 650 nm wavelength using a plate reader (Synergy LX multimode reader, Biotek).

[0103] Example 1. Stability of the monoclonal antibody, infliximab, in the presence of 5-ALA. The stability of the drug / antibody infliximab in human colonic fluid was measured with or without 5-ALA at various concentrations. Each experiment was performed in duplicate / triplicate. Remaining antibody at various time points was measured by SE-HPLC. The results are shown in the table below.

[0104] [Table 2]

[0105] The results are shown in the table above and indicate that 5-ALA confers antibody stability. However, increasing concentrations of 5-ALA result in a decrease in the pH of the drug solution. 5-ALA does not stabilize the antibody in a dose-dependent manner.

[0106] Example 2. Stability of the monoclonal antibody, infliximab, in the presence of 5-ALA and the base L-arginine. The stability of the drug infliximab in human colonic fluid was measured with or without 5-ALA and L-arginine at various concentrations. Each experiment was performed in duplicate / triplicate. The remaining antibody at various time points was measured by SE-HPLC.

[0107] [Table 3]

[0108] The results are shown in the table above and demonstrate that increasing concentrations of 5-ALA in the presence of the pH adjusting component L-arginine helps act as a stabilizer and increases the amount of antibodies present in the colonic fluid.

[0109] Example 3. Stability of the monoclonal antibody, infliximab, in the presence of 5-ALA and aprotinin. The stability of the drug infliximab in human colonic fluid was measured with or without 5-ALA and aprotinin at various pHs. Each experiment was performed in duplicate / triplicate. The remaining antibody at various time points was measured by SE-HPLC.

[0110] [Table 4]

[0111] The results are shown in the table above and show that 5-ALA helps act as a stabilizer and increases the amount of antibody present in the colonic fluid to a greater extent than does the addition of the enzyme inhibitor aprotinin. pH 6.5 is ideal for antibody stabilization, as increasing the pH to 7.8 reduces stability.

[0112] Example 4. Stability of the monoclonal antibody, infliximab, in the presence of 5-ALA, lipophilic derivatives of 5-ALA and aprotinin. The stability of the drug infliximab in human colonic fluid was measured with or without 5-ALA, methyl-ALA and hexyl-ALA and aprotinin. Each experiment was performed in duplicate / triplicate. The remaining antibody at various time points was measured by SE-HPLC.

[0113] [Table 5]

[0114] The results are shown in the table above and indicate that 5-ALA helps act as a stabilizer and increases the amount of antibody present in the colonic fluid. Lipophilic derivatives of 5-ALA; methyl-ALA and hexyl-ALA were tested and shown to greatly stabilize the antibody to a greater extent with the addition of aprotinin.

[0115] Example 5. Stability of the monoclonal antibody, infliximab, in the presence of the penetration enhancers, lipophilic derivatives of 5-ALA and aprotinin. The stability of the drug infliximab in rat cecal slurry was measured with or without SNAC, sodium chenodeoxycholate, hexyl-ALA and aprotinin. Each experiment was performed in duplicate / triplicate. The remaining antibody at various time points was measured by SE-HPLC.

[0116] [Table 6]

[0117] The results are shown in the table above and indicate that aprotinin alone provided antibody stabilization benefits, the penetration enhancers, SNAC and sodium chenodeoxycholate alone provided no stabilization, and hexyl-ALA both alone and in combination with aprotinin provided good antibody stabilization.

[0118] Example 6. Stability of the peptide drug oxytocin in the presence of 5-ALA and aprotinin. The stability of the drug oxytocin in rat cecal slurry was measured with or without 5-ALA and the enzyme inhibitor aprotinin. Each experiment was performed in duplicate / triplicate. The remaining oxytocin at various time points was measured by RP-HPLC.

[0119] [Table 7]

[0120] The results are shown in the table above and indicate that 5-ALA acts as a stabilizer, increasing the amount of peptide present in the cecal slurry, and that the addition of aprotinin slightly improves the stability of oxytocin.

[0121] Example 7. Stability of the peptide oxytocin in the presence of 5-ALA, lipophilic derivatives of 5-ALA and aprotinin. The stability of the drug oxytocin in rat cecal slurry was measured with or without methyl-ALA, hexyl-ALA and the enzyme inhibitor aprotinin. Each experiment was performed in duplicate / triplicate. The remaining oxytocin at various time points was measured by RP-HPLC.

[0122] [Table 8]

[0123] The results are shown in the table above and show that methyl-ALA and hexyl-ALA provide some peptide stabilization (but not as much as 5-ALA - see Example 6) and that in the cecal slurry the addition of aprotinin slightly improves stability. 5-ALA naturally lowers the pH of the oxytocin solution, which helps to stabilize the peptide, but its lipophilic derivative does not lower the pH as much, which therefore results in a poorer stability profile.

[0124] Example 8. Penetration of the antibody, infliximab, through GI tissues in the presence of 5-ALA and the base L-arginine. The permeability of the drug infliximab across rat small intestinal (jejunal) and colonic tissues was measured with or without 5-ALA and the base L-arginine. Antibodies in the tissues at 2 hours were measured by ELISA.

[0125] [Table 9]

[0126] The results are shown in the table above and demonstrate that 5-ALA, with or without the base L-arginine, did not result in improved penetration or tissue uptake of infliximab in either small intestinal or colonic tissue.

[0127] Example 9. Permeation of the antibody, infliximab, through GI tissues in the presence of penetration enhancers. The permeability of the drug infliximab across rat small intestine (jejunum) was measured with or without sodium caprate, SNAC, sodium chenodeoxycholate, labrasol and Na2EDTA. Antibodies in tissues at 2 hours were measured by ELISA.

[0128] [Table 10]

[0129] The results are shown in the table above and indicate that there was some increase in tissue levels of infliximab with all penetration enhancers except sodium chenodeoxycholate.

[0130] Example 10. Penetration of the antibody, infliximab, through GI tissues in the presence of lipophilic derivatives of 5-ALA. The permeability of infliximab across the rat small intestine (jejunum) was measured with or without hexyl-ALA. Antibodies in tissues at 2 hours were measured by ELISA.

[0131] [Table 11]

[0132] The results are shown in the table above and indicate that there was some increase in tissue levels of infliximab with hexyl-ALA.

[0133] Example 11. Permeation of the antibody, infliximab, through GI tissues in the presence of a lipophilic derivative of 5-ALA, a penetration enhancer and N-acetylcysteine. The permeability of the drug infliximab across rat colonic tissue was measured with or without sodium caprate, SNAC, sodium chenodeoxycholate and hexyl-ALA. N-acetylcysteine ​​(NAC) was applied to the tissue prior to the addition of antibody to stimulate tissue damage. Antibody in the tissue at 2 hours was measured by ELISA.

[0134] [Table 12]

[0135] The results are shown in the table above and demonstrate that using NAC to mimic diseased tissue conditions was able to result in increased tissue levels of antibodies via sodium caprate, SNAC, sodium chenodeoxycholate and hexyl-ALA.

[0136] Example 12. Permeation of oxytocin through GI tissues in the presence of 5-ALA. The permeability of the drug oxytocin across rat small intestinal (jejunum) and colonic tissues was measured with or without 5-ALA. The peptides in the tissues at 2 hours were measured by RP-HPLC.

[0137] [Table 13]

[0138] The results are shown in the table above and demonstrate that 5-ALA acts as a permeability enhancer and increases the amount of oxytocin present in both small intestinal and colonic tissue.

[0139] Example 13. Permeation of oxytocin through GI tissues in the presence of 5-ALA and lipophilic derivatives of 5-ALA. The permeability of oxytocin across the rat small intestine (jejunum) was measured with or without 5-ALA, methyl-ALA and hexyl-ALA. The peptides in the tissues at 2 hours were measured by RP-HPLC.

[0140] [Table 14]

[0141] The results are shown in the table above and indicate that 5-ALA acts as a permeability enhancer and increases the amount of oxytocin present in the small intestinal tissue. The lipophilic derivatives, methyl-ALA and hexyl-ALA, also show enhanced permeation of oxytocin, but to a lesser extent compared to 5-ALA.

[0142] Example 14. Permeation of oxytocin through GI tissues in the presence of penetration enhancers and lipophilic derivatives of 5-ALA. The permeability of oxytocin across the rat small intestine (jejunum) was measured with or without sodium caprate, SNAC, labrasol, methyl-ALA and hexyl-ALA. Peptides in tissues at 2 hours were measured by RP-HPLC.

[0143] [Table 15]

[0144] The results are shown in the table above and demonstrate that the penetration enhancers and lipophilic derivatives of 5-ALA act as permeability enhancers and increase the amount of oxytocin present in the small intestinal tissue. Sodium caprate and SNAC showed the greatest increase in tissue levels of oxytocin.

Claims

1. A pharmaceutical composition comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and optionally (c) a pH adjusting component and / or (d) an enzyme inhibitor.

2. The pharmaceutical composition of claim 1 , wherein the active ingredient is a protein.

3. The pharmaceutical composition of claim 2 , wherein the active ingredient is an antibody.

4. The pharmaceutical composition of claim 1 , wherein the active ingredient is a peptide.

5. 5. The pharmaceutical composition of claim 1, wherein the enzyme inhibitor is aprotinin.

6. 6. A pharmaceutical composition according to any one of claims 1 to 5 in liquid, solid or semi-solid form suitable for oral or rectal administration.

7. 7. A pharmaceutical composition according to any one of claims 1 to 6 in a solid form suitable for oral administration having an enteric coating.

8. 8. A pharmaceutical composition according to any one of claims 1 to 7 adapted for selective release of the peptide or protein in the lower gastrointestinal tract.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, comprising a solid dosage form having a core and a coating for said core, said core comprising said active ingredient, said 5-aminolevulinic acid and optionally said base or enzyme inhibitor, and said coating comprising a mixture of a digestible polysaccharide and a film former having a solubility threshold at a pH of 6.0 or greater.

10. 10. The pharmaceutical composition of claim 9, wherein the digestible polysaccharide is selected from the group consisting of starch; amylose; amylopectin; chitosan; chondroitin sulfate; cyclodextrin; dextran; pullulan; carrageenan; scleroglucan; chitin; curdulan and levan.

11. The pharmaceutical composition according to claims 9 and 10, wherein the film-forming material is an acrylate polymer, a cellulose polymer or a polyvinyl-based polymer.

12. 12. The pharmaceutical composition of claim 11, wherein the film-forming material is selected from cellulose acetate phthalate; cellulose acetate trimellitate; hydroxypropyl methylcellulose acetate succinate; and polyvinyl acetate phthalate.

13. An orally administrable pharmaceutical composition comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof.

14. A solid dosage form for oral administration comprising a core comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and (c) a pH adjusting component and / or (d) an enzyme inhibitor.

15. A rectally administrable pharmaceutical composition comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and (c) a pH adjusting component and / or (d) an enzyme inhibitor.

16. An enema preparation comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and (c) a pH adjusting component and / or (d) an enzyme inhibitor.

17. 1. A method of treating or preventing a disease or condition in a subject, comprising administering to the subject via the ileum and / or colon a pharmaceutical composition comprising (a) a protein or peptide as an active ingredient and (b) 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof, and (c) a pH adjusting component and / or (d) an enzyme inhibitor.

18. 18. The method of claim 16 or claim 17, wherein the disease or condition is inflammatory bowel disease; irritable bowel syndrome; constipation; diarrhea; infectious disease; autoimmune disease or cancer.

19. 14. A composition according to any one of claims 1 to 13 for use in therapy.

20. A method for stabilizing a protein or peptide in the presence of intestinal fluids comprising contacting the protein or peptide with 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof.

21. Use of 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof for stabilizing an active ingredient, including a protein or peptide, administered as a pharmaceutical composition and delivered to the lower gastrointestinal tract.

22. Use of 5-aminolevulinic acid (5-ALA) or a pharma- ceutically acceptable salt thereof to increase colonic permeability resulting in increased uptake of a protein or peptide administered as a pharmaceutical composition and delivered to the lower gastrointestinal tract.

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