WO2019097251A1 - Nouvelles compositions - Google Patents

Nouvelles compositions Download PDF

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Publication number
WO2019097251A1
WO2019097251A1 PCT/GB2018/053332 GB2018053332W WO2019097251A1 WO 2019097251 A1 WO2019097251 A1 WO 2019097251A1 GB 2018053332 W GB2018053332 W GB 2018053332W WO 2019097251 A1 WO2019097251 A1 WO 2019097251A1
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WO
WIPO (PCT)
Prior art keywords
carnitine
composition
antibody
salt
acyl
Prior art date
Application number
PCT/GB2018/053332
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English (en)
Inventor
Vipul YADAV
Abdul Waseh Basit
Original Assignee
Intract Pharma Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB1719085.1A external-priority patent/GB201719085D0/en
Priority claimed from GBGB1807971.5A external-priority patent/GB201807971D0/en
Priority claimed from GBGB1816543.1A external-priority patent/GB201816543D0/en
Application filed by Intract Pharma Limited filed Critical Intract Pharma Limited
Publication of WO2019097251A1 publication Critical patent/WO2019097251A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • This invention relates to novel compositions and their use.
  • Antibodies and antibody-derived molecules are an important class of pharmaceuticals, demonstrating high specificity for particular targets. Many therapeutic uses have been proposed and implemented, and monoclonal antibodies (mAbs) are the highest selling class of biotherapeutics.
  • mAbs monoclonal antibodies
  • delivery of the antibody to the desired target continues to be a major challenge in pharmaceutical industry as well as an unmet medical need for several diseases.
  • oral or rectal administration is generally not feasible for antibodies, because the antibody is highly unstable in the presence of gastric and intestinal fluids, and for this reason, intravenous or subcutaneous delivery remains the most feasible option for delivery of antibodies.
  • Such delivery is not, usually, however, the most convenient method of administration of a drug to a patient, and issues include pain at the site of injection, poor patient compliance, and frequent hospital visits for intravenous infusions.
  • WO 2007/122374 describes compositions having a delayed release coating which can be used to target release of a drug from a core to the intestine, particularly the colon.
  • the colon is susceptible to a number of disease states, including inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), constipation, diarrhoea, infection and carcinoma.
  • IBD is a chronic, medically incurable condition involving inflammation of the gut.
  • IBD ulcerative colitis
  • CD Crohn’s disease
  • PEGylation and glycosylation are established techniques in which the antibody is modified in order to increase the circulation time of the antibody in the bloodstream.
  • modification of the antibody clearly has disadvantages, and even for modified antibodies, the route of administration remains predominantly intravenous or subcutaneous.
  • the invention provides a pharmaceutical composition which comprises an antibody together with one or more excipients selected from (a) carnitine, an acyl carnitine, or a salt of carnitine or an acyl carnitine; (b) a salt or an ester of a bile acid; and (c) an alkylsaccharide; said composition being in solid or semi-solid form adapted for release of the active ingredient in the ileum and/or the colon.
  • excipients selected from (a) carnitine, an acyl carnitine, or a salt of carnitine or an acyl carnitine; (b) a salt or an ester of a bile acid; and (c) an alkylsaccharide; said composition being in solid or semi-solid form adapted for release of the active ingredient in the ileum and/or the colon.
  • the composition of the invention is in solid or semi-solid form, preferably in a form suitable for rectal or, especially, oral administration. Most preferably it is in a solid or semi-solid form suitable for oral administration, and adapted for selective release of the antibody in the ileum and/or the colon, especially the colon.
  • the excipients (a), (b) and (c) are well-known as enhancers of penetration (“penetration enhancers”), and have been proposed for use in a number of pharmaceutical formulations. However, they have not been proposed for use together with antibodies. Further, nothing in the prior art known to the inventors suggests that they might have any effect on the stabilising of any active ingredient administered as a pharmaceutical composition, and particularly an antibody so administered.
  • the present invention further provides a method of stabilising an antibody in the presence of intestinal fluid, which comprises delivering the antibody in a pharmaceutical composition to the intestine together with an excipient selected from (a), (b) and (c) above. Further, the invention provides the use of an excipient selected from (a), (b) and (c) above for the stabilisation of an antibody which has been administered as a pharmaceutical composition and delivered to the intestine, especially to the ileum and/or the colon. It is an advantage of the present invention that not only do said excipients stabilise the antibody, they also continue to act as penetration enhancers, facilitating
  • compositions which, in a preferred embodiment, is capable of delivering an antibody to the ileum and/or the colon, following oral or rectal administration, and in which penetration of the antibody into the intestinal tissue can occur without significant degradation of the antibody.
  • antibody means an immunoglobulin molecule that recognizes and specifically binds to a target antigen, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, bacteria or virus, or combination thereof through at least one antigen recognition site within the variable region of the immunoglobulin molecule.
  • a target antigen such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, bacteria or virus, or 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 comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity.
  • An antibody can include any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy- chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations.
  • the antibody is IgGl or IgG4.
  • the term“antibody” is also intended to include conjugates of the antibody, for example conjugates with polyethylene glycol, PEG.
  • antibody should be understood to encompass complete antibodies and antibody fragments comprising an antigen binding region of the complete antibody.
  • Antibody fragments may for example be single domain antibodies (e.g. V H H domain antibodies), monovalent or divalent Fab, Fab', F(ab')2, scFv, Fc, bispecific antibodies, diabodies, minibodies or multispecific antibodies formed from antibody fragments, for example minibodies composed of different permutations of scFv fragments or diabodies, and optionally Fc fragments or CH domains, such as scFv-Fc, scFv-Fc-scFv, Fab-scFv, (Fab'ScFv)2, scDiabodies, scDiabody-Fc, scDiabody-CH3, scFv- CH3, and scFv-CH2-CH3 fusion proteins.
  • An antibody fragment can be produced by enzymatic cleavage of a complete antibody,
  • an antibody may be any one whose therapeutic effect is advantageously realised by administration via the colon.
  • Specific antibodies of particular interest in the context of the present invention include existing commercial IBD therapeutic antibodies such as adalimumab, infliximab, cetrolizumab pegol, golimumab, natalizumab, vedolizumab, ustekinumab, and additional antibodies in development for IBD treatment which target pathways and molecules (agonists or antagonists) implicated in pathogenesis of IBD, such as, for example CD40.
  • Targeting to the colon additionally affords the possibility to improve treatment of colorectal cancer by targeting and localization of anti-cancer therapeutic antibodies, or of different possible formats as mentioned above, to the tumour.
  • GI gastrointestinal
  • stabilisation of biomolecules in the gastrointestinal (GI) tract and penetration into GI tissues additionally may offer the potential for transmission of therapeutics through GI tissue and into the systemic circulation, thus affording the opportunity to target a much larger range of diseases.
  • compositions and any of these may be used in the compositions of the invention.
  • compositions of the invention may comprise carnitine, or a salt thereof.
  • compositions of the invention may comprise an acyl carnitine, or a salt thereof.
  • compositions of the invention may comprise carnitine and an acyl carnitine, or a salt thereof.
  • compositions of the invention may comprise L- or D-forms of carnitine or an acyl carnitine.
  • compositions of the invention comprise the L-form of carnitine, herein referred to as L-camitine, or the L-form of an acyl carnitine, herein referred to as an acyl-L-carnitine.
  • acyl carnitine has the general formula:
  • R represents an alkyl or alkenyl group having from 1 to 20 carbon atoms, for example at least 2, at least 8, at least 10 or at least 12 carbon atoms. R may for example have from 6 to 16 carbon atoms.
  • Specific acyl carnitines of interest include, acetyl carnitine, lauroyl carnitine, myristoyl carnitine, stearoyl carnitine, propionyl carnitine,
  • palmitoylcamitine valeryl carnitine, hexanoyl carnitine and octanoyl carnitine, especially hexanoyl carnitine, octanoyl carnitine and lauroyl carnitine.
  • Most preferred acyl carnitines are valeryl carnitine, hexanoyl carnitine, octanoyl carnitine, lauroyl carnitine, myristoyl carnitine and stearoyl carnitine.
  • Salts of carnitine or an acyl carnitine which are suitable for use in the present invention are those wherein a counterion is pharmaceutically acceptable.
  • Suitable salts include those formed with organic or inorganic acids or bases.
  • suitable salts formed with acids according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids, or with organic sulfonic acids, such as (C1-C4) alkyl or aryl sulfonic acids which are unsubstituted or substituted, for example by halogen.
  • Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxalic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine.
  • Suitable salts formed with bases include those formed with base salts include ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D- glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl or di methyl-propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono , di or triethanolamine. Corresponding internal salts may furthermore be formed.
  • compositions and any of these may be used in the compositions of the invention.
  • Salts and esters of bile acids such as taurocholic, turodexycholic, deoxycholic, cholic, lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic, dehydrocholic and fusidic acids, and their salts and/or esters, are examples.
  • Suitable cations which may be present in salts include alkali metal cations, especially sodium, or ammonium or amino cations.
  • esters are those with alkylcarboxylic acids having from 1 to 20, especially having at least 8, preferably at least 10, for example from 6 to 16, carbon atoms in the alkyl group, as for example in lauroylcholine chloride.
  • a salt is used.
  • Most preferred is the use of sodium taurocholate.
  • the saccharide is a mono- or, especially, di-saccharide, for example a maltose or sucrose.
  • the alkyl saccharide may for example be an ether-linked alkyl saccharide.
  • the alkyl group suitably has from 1 to 20, especially having at least 8, preferably at least 10, carbon atoms in the alkyl group.
  • the alkyl group may for example be a lauryl, myristyl, stearyl or palmityl group.
  • Suitable alkylsaccharides include lauryl-P-D-maltoside (also known as dodecyl-P-D-maltoside), lauryl sucrose, myristyl sucrose, and palmityl sucrose.
  • compositions according to the invention may be in any suitable form, but as stated above, it is preferred that they are in solid or semi-solid form, and preferably they are suitable for oral or rectal administration. They may be prepared by any known method.
  • the antibody and the required excipient may be admixed together, optionally together with other excipients required in the dosage form.
  • compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, tablets, mini-tablets, or pellets, or as powders, granules or crystals.
  • the minimum diameter of each particle is typically at least l0 4 m, usually at least 5 x l0 4 m and, preferably at least l0 3 m.
  • the maximum diameter is usually no more than 30 mm, typically no more than 20 mm and, preferably, no more than 10 mm.
  • the particle has a diameter from about 0.2 mm to about 15 mm, preferably from about 1 mm to about 4 mm (e.g. for pellets or mini-tablets) or from about 6 mm to about 12 mm (e.g. for certain tablets or capsules).
  • the term "diameter" refers to the largest linear dimension through the particle.
  • compositions according to the invention may of course contain any further conventional excipients as required.
  • Excipients used in solid forms 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, gelatine, natural sugars such as glucose or beta-lactose, com sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate,
  • Disintegrators include without limitation starch, methylcellulose, agar, bentonite, xanthan gum and the like.
  • Fast dissolving diluents include mannitol, lactose, sucrose and/or cyclodextrins.
  • Lubricants, glidants, flavours, colouring agents and stabilizers may also be added for ease of fabrication and use.
  • Lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the antibody. Preferred examples of coatings are given below.
  • Capsules may have solid, semi-solid or non-solid contents.
  • Exemplary contents for capsules may include suspensions which can contain, for example, microcrystalline cellulose for imparting 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 above.
  • Formulations for rectal administration may be presented as a suppository with the usual carriers such as cocoa butter, synthetic glyceride esters or polyethylene glycol. Such carriers are typically solid at ordinary temperatures, but liquefy and/or dissolve in the rectal cavity to release the drug.
  • Preferred unit dosage formulations are those containing an effective dose, or an appropriate fraction thereof, of the active ingredient. Release from certain formulations may be sustained, if the composition contains suitable controlled-release excipients.
  • compositions according to the invention will typically comprise a therapeutically effective amount of the antibody which may be from 0.01 wt % to 99 wt %, based on the total weight of the composition.
  • the actual dosage would be determined by the skilled person using common general knowledge.
  • "low" dose formulations typically comprise no more than 20 wt % of the antibody, and preferably comprise from 1 wt % to 10 wt %, e.g. 5 wt %, of the antibody.
  • “High” dose formulations typically comprise at least 40 wt % of the antibody, and preferably from 45 wt % to about 85 wt %, e.g. 50 wt % or 80 wt %.
  • the antibody may be used as the sole active ingredient in a composition according to the invention, it is also possible for the antibody to be used in combination with one or more further therapeutic agents.
  • the invention also provides a composition according to the invention containing a further therapeutic agent in addition to the antibody. If desired, the composition according to the invention may be administered together with a further composition, by simultaneous, sequential or separate administration.
  • 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 which are solid or semi-solid throughout, as well as those having a solid or semi-solid exterior and a non-solid, for example liquid or gel, interior.
  • a capsule may have liquid or gel contents.
  • compositions according to the invention may have an enteric coating.
  • Enteric coatings which protect the active ingredients in a composition from attack and degradation in the stomach, and permit release within the intestines, are well known.
  • the optimal coating for any particular formulation depends on the exact intended use, and coatings may be tailored to release the active ingredient in a particular region of the intestines, or at a particular time following ingestion.
  • the composition of the present invention is in a solid or semi-solid form which comprises an enteric coating.
  • Such a formulation may if desired contain one or more intermediate layers between the active ingredient and the outer enteric coating. In this case, it is possible for a composition of the invention to release a portion of its contents at one particular region of the intestines, and a further portion of its contents at a lower region of the intestines. Delivery to the ileum and/or colon
  • formulations which provide release only within a specific part of the GI tract, particularly the ileum or, especially, the colon.
  • WO 2007/122374 (the contents of which are incorporated herein by reference) describes such formulations, and these form one preferred embodiment of the invention.
  • the invention further provides a composition
  • a composition comprising a particle with a core and a coating for the core, the core comprising an antibody together with one or more excipients selected from (a) carnitine, an acyl carnitine, or a salt of carnitine or an acyl carnitine ; (b) a bile acid or a salt or an ester thereof; and (c) an alkylsaccharide;
  • the coating comprising a mixture of a first material which is susceptible to attack by intestinal bacteria and a second material which has a solubility threshold at pH 5 or above, preferably pH 7 or above, wherein the first material comprises a polysaccharide selected from the group consisting of starch; amylose; amylopectin; chitosan; chondroitin sulfate; cyclodextrin;
  • dextran dextran; pullulan; carrageenan; scleroglucan; chitin; curdulan and levan.
  • the polysaccharide is selected from the group consisting of starch; amylose; amylopectin; chitosan; chondroitin sulfate; cyclodextrin; dextran; and carrageenan.
  • the polysaccharide may be starch, amylose or amylopectin.
  • the second material is an enteric material which has a pH threshold which is the pH below which it is insoluble and at or above which it is soluble.
  • the pH of the surrounding medium triggers dissolution of the second material.
  • the normal pH of gastric juice is usually in the range of 1 to 3, while the pH of intestinal juice gradually increases from about 5.5 in the duodenum to about 7 to 8 in the colon.
  • the second material preferably has a pH threshold of 6.5 or greater, especially 7 or greater.
  • the second material is typically a film-forming polymeric material such as an acrylate polymer, a cellulose polymer or a polyvinyl-based polymer.
  • suitable cellulose polymers include cellulose acetate phthalate ("CAP”); cellulose acetate trimellitate (“CAT”); and hydropropylmethylcellulose acetate succinate.
  • suitable polyvinyl-based polymers include polyvinyl acetate phthalate (“PVAP”).
  • the second material is preferably a co-polymer of a (meth)acrylic acid and a (meth)acrylic acid CM alkyl ester, for instance, a copolymer of methacrylic acid and methacrylic acid methyl ester.
  • Such polymers include those available under the Trade Marks Eudragit L, Eudragit S and Eudragit FS. The use of Eudragit S as the second material is particularly preferred.
  • the core is usually a single solid body.
  • the core may consist of the antibody together with one or more of excipients (a), (b) and (c). More usually, however, the core will comprise a mixture of the antibody and said excipient together with one or more additional excipients.
  • the core may for example include a filler or diluent material, e.g. lactose or cellulose material such as microcrystalline cellulose; a binder, e.g.
  • the core may be a compressed granulate comprising at least some of these materials.
  • compositions have application in a multi-phasic release composition comprising at least two pluralities of particles, e.g. coated pellets, in the same dosage form, e.g. a capsule, in which the particles of one plurality are differentiated from the particles of the or each other plurality by the coating.
  • the coatings may differ from one plurality to the next in terms of coating thickness or composition, e.g. the ratio and/or identity of
  • Multi-phasic release formulations would be particularly suitable for suffers of Crohn's disease affecting different regions along the intestine, including the ileum and/or colon.
  • the present invention provides a pharmaceutical composition according to the invention for use in therapy. It also provides a method of treating or preventing a disease or condition in a subject, especially a human subject, which comprises administering to the subject a pharmaceutical composition according to the invention in which the antibody in the composition is medically indicated for treatment or prevention of said disease or condition.
  • the compositions are adapted for administration via the oral or rectal route, and although in one embodiment the invention finds utility in the treatment of diseases of the intestine and particularly in the treatment of diseases of the ileum and/or the colon, it also has application as a portal for entry of the antibody into the systemic circulation by absorption from the intestine, and particularly the ileum and/or the colon, and hence finds 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.
  • the invention finds particular utility in the treatment or prevention, including maintenance of remission or prevention of relapse, of a disease or condition of the ileum and/or the colon, especially the colon, for example inflammatory bowel disease (including ulcerative colitis and Crohn’s disease), constipation, diarrhoea, infection, or cancer, and the invention therefore further provides the use of at least one excipient (a), (b) and/or (c) in a method of
  • manufacture of a medicament comprising an antibody for the treatment and/or prevention of one or more of these conditions.
  • the treatment and/or prevention of IBD is of particular importance.
  • a rat colonic model based on a mixed fecal inoculum was used to mimic the luminal environment of the rat large intestine.
  • An anaerobic workstation (Electrotek 500TGTM workstation, Electrotek, West Yorkshire, ETC) maintained at 37 °C and 70% relative air humidity was used to set up the model.
  • Three healthy male wistar rats were sacrificed and the fecal contents were collected.
  • the fecal material was transferred in the anaerobic workstation and diluted with freshly prepared basal medium to obtain 20% w/w slurry by
  • the basal media provides nutrients and growth factors to the microbiota allowing viability for upto 24 hours.
  • the homogenized bacterial media was sieved through an open mesh fabric (SefarNitexTM, pore size 350 pm) to remove any nonhomogeneous fibrous material.
  • the pH was maintained at approximately 6.5 to mimic the colonic luminal pH of the rat.
  • a human colonic model based on a mixed fecal inoculum was used to mimic the luminal environment of the human large intestine.
  • An anaerobic workstation (Electrotek 500TGTM workstation, Electrotek, West Yorkshire, ETC) maintained at 37°C and 70% relative air humidity was used to set up the model.
  • the fecal material was transferred in the anaerobic workstation and diluted with freshly prepared basal medium to obtain 20% w/w slurry by homogenization.
  • the basal media provides nutrients and growth factors to the microbiota allowing viability for up to 24 hours.
  • the homogenized bacterial media was sieved through an open mesh fabric (SefarNitexTM, pore size 350 pm) to remove any nonhomogeneous fibrous material.
  • the pH was maintained at approximately 7 to mimic the colonic luminal pH of the human.
  • Antibody stock solution (infliximab, anti-CD40, vedolizumab) was prepared in PBS at 2 mg/ml and added to 20% human or rat faecal slurry to obtain an incubation concentration of 1 mg/ml and 10% w/w faecal slurry. Samples were withdrawn at appropriate time points and added to a protease inhibitor cocktail (Sigma, P2714) in a ratio of 1 :3. The samples were centrifuged at 9.6 g for 10 mins and the supernatant was analysed by size exclusion-HPLC (SE-HPLC).
  • SE-HPLC size exclusion-HPLC
  • HPLC high performance liquid chromatography
  • the chambers are made of solid acrylic and supports the tissue membrane in such a way that each side of the membrane is isolated and faces a different chamber representing the apical and basolateral chamber.
  • the working system consists of a unit to fit a maximum of six vertical chambers, a gas manifold for carbogen purging (95% 0 2 , 5% CO2) and a heater block to maintain the temperature of the chambers at 37°C during the experiments with the use of a circulating water bath.
  • the chambers are two-piece assemblies held together by a high spring-tension retaining ring to ensure leak-free operation during the experiments.
  • TEER trans- epithelial electrical resistance
  • the freshly excised colon of male Wistar rats was collected and transferred to an ice-cold solution of Krebs-Bicarbonate Ringer solution (KBr) of pH 7.4.
  • KBr Krebs-Bicarbonate Ringer solution
  • the tissue was cut open transversally and was washed with KBr solution to remove the luminal contents and was then mounted in the Ussing chambers.
  • the mucosal surface of the colon tissue was facing the apical chamber, and the endothelial surface of the tissue was facing the basolateral chamber.
  • the exposed tissue area on each side of the chamber was 0.29 cm 2 and the tissue mounting region was 4 x 8 mm ( Figure 3.6).
  • the volume of KBr in apical and basolateral chamber was 5 ml and the pH was maintained at 7.4.
  • the tissue was allowed to incubate with KBr for 20 minutes before addition of the drug. Antibody concentrations tested during the penetration experiments was 0.5 mg/ml. The penetration of drugs into the tissue was tested for 3 hours and in a minimum of 3 rats. The tissue without drug was incubated in parallel for the same time which acted as the negative control. The chambers were purged with carbogen and kept at 37°C by water jackets during incubation. The TEER was continuously monitored during the experiment to confirm the viability and integrity of the tissue. Tissues with TEER value below 200 were not used for the experiments.
  • the tissue section exposed to the drug was precisely cut at the end of the experiment and immediately transferred to a cryostat (Leica CM3050, Leica Microsystems, Milton Keynes, UK) at -30°C. The tissue was allowed to freeze for 15-20 minutes. After the tissues were frozen, thin sections of the tissue (10 pm) were sliced and mounted on adherent microscope slides (SuperFrost ® Plus, VWR International, Leuven, Belgium). Up to 10 sections from the entire length of the tissue exposed to the drug were sliced. Similarly, sections were cut from the tissue not exposed to drug that served as negative controls.
  • the slides were kept at room temperature for 15 minutes before the initiation of the staining procedure.
  • the tissue sections were fixed in 4% paraformaldehyde (Sigma- Aldrich, UK) for 10 minutes followed by incubation with 0.1% Triton X-100 (Sigma- Aldrich, UK) surfactant for 5 minutes to open up the tight junctions.
  • the sections were then incubated with 1% bovine serum albumin (BSA) (Sigma- Aldrich, UK) for 30 minutes to avoid any protein- protein non-specific binding. Washing steps were included at each stage using PBS pH 7.4.
  • BSA bovine serum albumin
  • the sections were then stained with secondary antibody, 10pg/ml (Red) (anti-human IgG from goat, Alexa Flour ® 633, Molecular Probes, UK) for 1 hour. This was followed by staining with CellMaskTM green plasma membrane stain (Green) (Molecular Probes, UK)
  • Hard SetTM mounting medium with DAPI Blue
  • DAPI Blue
  • the slides were stored at 2-8°C in the dark until analysis by confocal laser-scanning microscopy (LSM 710, Zeiss, Cambridge, UK).
  • LSM 710 Zeiss, Cambridge, UK
  • Zen 2012 imaging software Carl Zeiss Ltd., Cambridge, United Kingdom
  • an anti-TNF a antibody detection ELISA kit (Alpha Diagnostic International, Texas, USA) was used.
  • the kit comprised of TNF-a coated 96 well plate with 12 removal strips of 8 microwells each, an anti-human Fc domain specific IgG labelled with HRP, wash solution, dilution buffer and TMB substrate.
  • HRP horseradish Phosphosine
  • the extraction buffer supernatant obtained following tissue homogenization and extraction of infliximab was diluted using dilution buffer.
  • 100 m ⁇ of the diluted test sample, positive control (infliximab in PBS), negative control (homogenate of colon tissue without drug) or calibration curve samples (1000, 500, 100, 50, 25, 10 ng/ml infliximab) were added into the wells of the 96 well plate in triplicates and incubated at 50 rpm shaking for 1 hr at room temperature. Post incubation, the wells were washed with wash buffer 4 times for 5 mins each.
  • Anti-human IgG-HRP conjugated (lOOx) solution was diluted to lx using dilution buffer and 100 m ⁇ of the solution was added to each well. The plate was incubated for
  • TMB substrate 100 m ⁇ was added and incubated in the dark for 15 mins. The wells began to turn blue due to the reaction between HRP and TMB.
  • the enzyme- substrate reaction was stopped by addition of 100 m ⁇ of stop solution (1% sulfuric acid).
  • optical density (O.D) readings of the calibration curve samples were plotted against log concentration (3, 2.699, 2, 1.699, 1.398, 1) in a 4-parameter logistic regression curve fit (4-PL) using software GraphPad Prism 7 (GraphPad Software Inc., San Diego, USA).
  • the equation obtained from the calibration curve is as follows:
  • Example 3 Tissue uptake of infliximab in rat ascending colon tissue and translocation of infliximab across the tissue into the basolateral compartment.
  • the amount of intact IgG remaining in the tissue was detected by ELISA post
  • Colon stability was assessed using the Human Colon Model with the amount of intact antibody remaining at each time point assessed by SE-HPLC as described in the Methods section.
  • the experiment was carried out in the absence of any excipient, and then in the presence of 50 mM L-carnitine, valeryl-L-carnitine, hexanoyl-L-camitine, octanoyl-L- camitine, lauroyl-L-camitine, myristoyl-L-carnitine or stearoyl-L-carnitine.

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  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
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  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une composition pharmaceutique qui comprend un anticorps conjointement avec un ou plusieurs excipients choisis parmi (a) la carnitine, une acylcarnitine, ou un sel de carnitine ou d'une acylcarnitine; (b) un sel ou un ester d'un acide biliaire; et (c) un alkylsaccharide; ladite composition étant sous une forme solide ou semi-solide conçue pour libérer le principe actif dans l'iléon et/ou le côlon. L'invention concerne également des méthodes de stabilisation d'un anticorps en présence de fluide intestinal et des méthodes de prévention d'une maladie ou d'une pathologie chez un sujet.
PCT/GB2018/053332 2017-11-17 2018-11-16 Nouvelles compositions WO2019097251A1 (fr)

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GB1719085.1 2017-11-17
GBGB1719085.1A GB201719085D0 (en) 2017-11-17 2017-11-17 Novel Compositions
GB1807971.5 2018-05-16
GBGB1807971.5A GB201807971D0 (en) 2018-05-16 2018-05-16 Novel compositions
GBGB1816543.1A GB201816543D0 (en) 2018-10-10 2018-10-10 Novel compounds
GB1816543.1 2018-10-10

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780028A (en) * 1993-09-20 1998-07-14 Anadis Ltd. Method of obtaining immunoglobulins from colostrum and their use in pharmaceutical composition
WO2017173068A1 (fr) * 2016-03-30 2017-10-05 University Of Maryland, Baltimore Système microparticulaire pour l'administration de médicaments au niveau du côlon
WO2018071655A1 (fr) * 2016-10-12 2018-04-19 Curirx Inc. Formulations destinées à l'administration entérique d'agents thérapeutiques

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5780028A (en) * 1993-09-20 1998-07-14 Anadis Ltd. Method of obtaining immunoglobulins from colostrum and their use in pharmaceutical composition
WO2017173068A1 (fr) * 2016-03-30 2017-10-05 University Of Maryland, Baltimore Système microparticulaire pour l'administration de médicaments au niveau du côlon
WO2018071655A1 (fr) * 2016-10-12 2018-04-19 Curirx Inc. Formulations destinées à l'administration entérique d'agents thérapeutiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CAON THIAGO ET AL: "Enhancing the Buccal Mucosal Delivery of Peptide and Protein Therapeutics", PHARMACEUTICAL RESEARCH, SPRINGER NEW YORK LLC, US, vol. 32, no. 1, 29 August 2014 (2014-08-29), pages 1 - 21, XP035420365, ISSN: 0724-8741, [retrieved on 20140829], DOI: 10.1007/S11095-014-1485-1 *
SHAJI JESSY ET AL: "Protein and Peptide drug delivery: oral approaches", INDIAN JOURNAL OF PHARMACEUTICAL SCIENCESI,, vol. 70, no. 3, 1 May 2008 (2008-05-01), pages 269 - 277, XP009174425, ISSN: 1998-3743, DOI: 10.4103/0250-474X.42967 *

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