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WO2018193129A1 - Inhibiteurs de la signalisation bmp2 et bmp4 destinés à être utilisés dans le traitement de l'endobrachyoesophage - Google Patents

Inhibiteurs de la signalisation bmp2 et bmp4 destinés à être utilisés dans le traitement de l'endobrachyoesophage Download PDF

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Publication number
WO2018193129A1
WO2018193129A1 PCT/EP2018/060251 EP2018060251W WO2018193129A1 WO 2018193129 A1 WO2018193129 A1 WO 2018193129A1 EP 2018060251 W EP2018060251 W EP 2018060251W WO 2018193129 A1 WO2018193129 A1 WO 2018193129A1
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bmp4
bmp2
inhibitor
amino acid
antibody
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PCT/EP2018/060251
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English (en)
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Kausilia Krishnawatie KRISHNADATH
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Academisch Medisch Centrum
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary

Definitions

  • the invention is related to the field of compounds for the use in the preventive treatment of esophageal adenocarcinoma. More specifically, it relates to inhibitors of BMP2 and BMP4. The invention further relates to pharmaceutical compositions for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.
  • Barrett's esophagus is a condition in which the normal multi-layered squamous epithelium is substituted by a (specialized) columnar epithelium (i.e. intestinal or other columnar type of metaplasia). This process is assumed to be the result of longstanding gastro-esophageal reflux disease and is most prevalent in middle aged, Caucasian males.
  • the specialized intestinal type of columnar metaplasia in particular, confers a significantly increased risk for the development of esophageal adenocarcinoma (EAC).
  • EAC esophageal adenocarcinoma
  • Treatment of Barrett's oesophagus with no malignant features aim on reducing inflammation and includes treatment with compounds to relieve reflux or anti-reflux surgery.
  • malignant degeneration treatment is by endoscopic ablative therapies or surgically removing the affected part of the esophagus. The remains therefore a need in the art for an alternative treatment.
  • the present invention is based on the surprising finding that following treatment with an BMP2/4 inhibitor, implantations in mice of Barrett tissue biopsies from a patient suffering from Barrett's esophagus, the typical phenotypical histological symptoms disappeared.
  • the cells formed a multi-layered epithelium that expressed the squamous marker p63 and CK5 (Figure 3K-L), indicating the regeneration of normal multi-layered squamous epithelium of the esophagus and thus the disappearance of Barrett's esophagus.
  • the epithelial layer of the treated organoids also lack the presence of columnar and goblet cells (Figure 3J). This shows the effectivity of a BMP2/4 inhibitor in a treatment of Barrett's esophagus and is therefore effective in the prevention of esophageal adenocarcinoma.
  • the invention provides a kit of parts comprising an inhibitor of BMP2 and an inhibitor of BMP4 or an inhibitor of BMP2 and BMP4 for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.
  • said kit of parts comprises an inhibitor of BMP2 and a n inhibitor of BMP4 or an inhibitor of BMP2 and BMP4 is selected from the group consisting of: chordin, noggin, follistatin, BMP3, Inhibin, an anti BMP2 and an anti BMP4 antibody, an antibody binding to BMP2 and BMP4.
  • said inhibitor of BMP2 and BMP4 is an isolated, synthetic or recombinant antibody, which binds within residues 24-31, 57-68, 70-72, 89, 91, 101, 103, 104 and 106 of SEQ ID NO:l.
  • the isolated, synthetic or recombinant antibody according to the invention binds to: a. at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28, Asn29,Asp30,Trp31, b. at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61,
  • the isolated, synthetic or recombinant antibody according to the invention binds to Asp30, Trp31, Leu66 and LyslOl.
  • said isolated, synthetic or recombinant antibody is a single chain antibody.
  • said single chain antibody comprises: a. a heavy chain CD 1 consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence not differing more than 1 amino acid thereof, b. a heavy chain CD 2 consisting of the amino acid sequence of SEQ ID NO:6, or a sequence not differing more than 1 amino acid thereof, and c. a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 7 or a sequence not differing more than 1 amino acid thereof.
  • the isolated, synthetic or recombinant antibody according to the invention comprises the amino acid sequence of SEQ I D NO: 12 or a sequence at least 70%, more preferably 71, 72, 73, 74, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical thereto.
  • the invention further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a kit of parts comprising an inhibitor of BMP2 and an inhibitor of BMP4 or an inhibitor of BMP2 and BMP4 as defined above and a pharmaceutically acceptable carrier or diluent for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.
  • Figure 1 shows the generation of in vivo BE organoids.
  • Figure 2 shows that in vivo organoid recapitulates the original patient biopsy.
  • Figure 3 shows tissue after periodic treatment with C8C8, an antiBMP2 and BMP antibody, show expression of the squamous markers p63 and CK5.
  • Figure 4 A-E show the Inhibition of BMP2/4 drives the development of squamous epithelium in a conditional Noggin knockout model.
  • Figure 4(A) shows that Rosa26-cre mice were crossed with loxp [noggin] loxp mice and injected with tamoxifen for 3 days (Img, i.p.). Noggin-/- mice were sacrificed after 4, 8, 12, 16 or 20 weeks.
  • Figure 4 (B) shows a H&E staining of multilayered glands (MLGs) developed at the squamo-columnar junction (SCJ) in noggin-/- mice upon tamoxifen injection (3 days, Img i.p.) at different time points.
  • Figure 4 (C) shows that Rosa26-cre mice were crossed with loxp [noggin] loxp mice and injected with tamoxifen for 3 days (Img, i.p.). Noggin-/- mice were injected with tamoxifen for 3 days (Img, i.p.) and treated with saline or VHH BMP2/4 antibodies for 8 weeks. All mice were treated with proton pump inhibitors (PPIs) during the experiment.
  • MLGs multilayered glands
  • SCJ squamo-columnar junction
  • Figure 4 (D) shows SCJ in wild type or noggin-/- mice after treatment with saline or VHH BMP2/4 antibodies.
  • the SCJ with affected areas (dotted line) was stained for columnar marker K19 and squamous markers p63, K14 and K5.
  • Figure 4 shows MLGs at the SCJ in noggin-/- mice treated with saline or VHH BMP2/4 antibodies. Affected areas were IHC stained for BMP4, downstream target pSMADl,5,8, squamous markers K5 and K14 (blue) and K7 (brown, 3 rd panel)) and ki67 (brown, 4 th panel).
  • Barrett's esophagus is a serious complication of gastroesophageal reflux disease (GE D).
  • GE D gastroesophageal reflux disease
  • normal tissue lining the esophagus is replaced by tissue that resembles the lining of the intestine.
  • Barrett's esophagus does not have any specific symptoms, although patients with Barrett's esophagus may have symptoms related to GERD. It does, though, increase the risk of developing esophageal adenocarcinoma, which is a serious, potentially fatal cancer of the esophagus.
  • Diagnosis of Barrett's oesophagus may be done using endoscopy, histology, and/or using biomarkers, for instance as described in US 20120009597 Al.
  • BMP2 is used to refer to mature bone morphogenic protein 2, preferably of human origin.
  • the nucleotide sequence of human pro-BMP2 is publicly available by reference to GenBank Accession No. NM_001200.
  • BMP4 is used to refer to human mature bone morphogenic protein 4.
  • the nucleotide sequence of human pro-BMP4 is publicly available by reference to GenBank Accession No. NM_130851.
  • the term “inhibitor” of BMP2 or 4 refers to any compound which interferes with, blocks or decreases the signalling of said molecule. Said compound may be a small molecule or antibody.
  • BMP4 signalling refers to the ability of BMP4 to activate the canonical (the phosphorylation of SMAD 1/5/8) Assays to test BMP4 signalling are described for instance in Shaifur Rahman et al., "TGF- ⁇ / ⁇ signaling and other molecular events: regulation of osteoblastogenesis and bone formation” in Bone Research 3, Article number: 15005 (2015).
  • BMP2 signalling refers to the ability of BMP2 to activate the canonical (the phosphorylation of SMAD 1/5/8).
  • C4C4 refers to single chain antibody which binds to the hydrophobic groove of the wrist of BMP4.
  • C4C4 refers to a dimer of said C4 single chain antibody.
  • C8 refers to an antibody according to the invention binds within residues 24-31, 57-68,70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (as depicted in SEQ ID NO:l of
  • This region represents a "hydrophobic pocket" within the wrist epitope of BMP4.
  • An advantage of antibodies which bind to this region is that these antibodies have a very high affinity for BMP4 and BMP2 and are capable of efficiently inhibiting BMP4 and BMP2 signalling.
  • C8C8 refers to a dimer of said C8 antibody.
  • esophageal cancer refers to cancer that starts in the esophagus, including but not limited to squamous cell carcinoma and adenocarcinoma.
  • Embodiments The invention is based on the finding that inhibition of both BMP2 and BMP4 signalling effectively restores the normal tissue lining the esophagus, and is therefore effective in the preventive treatment of esophageal adenocarcinoma.
  • the invention therefore provides a kit of parts comprising an inhibitor of BMP2 and an inhibitor of BMP4.
  • a compound which inhibits BMP2 and BMP4 signalling is also suitable for use in the treatment of Barrett's Esophagus or for the prevention of esophageal adenocarcinoma.
  • any combination of compounds which inhibit BMP2 and BMP4 signalling may be used in the treatment of the invention.
  • any compound which inhibits BMP2 signalling and another compound which inhibits BMP4 signalling may be used.
  • Such compounds may be provided simultaneously or sequentially in a treatment.
  • an inhibitor which simultaneously inhibits of BMP2 and BMP4 signalling is provided for use in the treatment of the invention.
  • Preferred compounds include, but are not limited to chordin, noggin, follistatin, BMP3, Inhibin (see Ezra Wiater and Wylie Vale, March 7, 2003, The Journal of Biological Chemistry, 278, 7934-7941).
  • Antibodies capable of inhibiting BMP2 signalling and/or BMP4 signalling are known in the art and described for instance in WO2016/042050.
  • said epitope is located in the wrist within residues 10-17, 24-31, 45-72, 89, 91, 101, 103, 104, and 106 of BMP4.
  • an antibody according to the invention binds within residues 10-17, 45-56, and 69 of BMP4.
  • This epitope contains a hydrophobic groove, which the inventors believe is important for BMP4 specific binding.
  • An advantage of the antibodies according to this embodiment is that said antibodies have a low affinity for other members of the BMP family and are highly effective in specifically inhibiting BMP4 signaling.
  • said antibody does not substantially bind to BMP2, BMP5, BMP6 or BMP7.
  • a further advantage thereof is that said antibody does not inhibit BMP2 mediated signaling, thereby diminishing or even avoiding adverse side effects when used in vivo. More preferably, said antibody specifically binds to at least one residue selected from the group consisting of LyslO, Asnll, Lysl2, Asnl3, Cysl4, Argl5, Argl6, and Hisl7, at least one residue selected from the group consisting of Gly45, Asp46, Cys47, Pro48, Phe49, Pro50, Leu51, Ala52, Asp53, His54, Leu55 and Asn56, and to Ser69 of BMP4. Preferably, said antibody binds to more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 preferably 16 residues thereof. In a highly preferred
  • said antibody specifically binds to at least Lysl2, Argl5, Asp46, and Pro50 of BMP4.
  • an antibody according to the invention is a single chain antibody.
  • said antibody comprises a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 3 or a sequence not differing more than 2 amino acid thereof, a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO:4, or a sequence not differing more than 1 amino acid thereof, and preferably further a heavy chain CDRl consisting of the amino acid sequence of SEQ ID NO: 2 or a sequence not differing more than 1 amino acid thereof.
  • said antibody comprises the amino acid sequence of SEQ ID NO: 11.
  • an antibody according to the invention binds within residues 24-31, 57- 68,70-72, 89, 91, 101, 103, 104 and 106 of BMP4 (of SEQ ID NO:l). This region represents a
  • hydrophobic pocket within the wrist epitope of BMP4.
  • An advantage of antibodies which bind to this region is that these antibodies have a very high affinity for BMP4 and BMP2 and are also capable of efficiently inhibiting BMP4 and BMP2 signaling.
  • said antibody specifically binds to at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28,
  • Asn29,Asp30,Trp31 at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61, Ile62, Val63, Gln64, Thr65, Leu66, Val67, and Asn68; at least one residue selected from the group consisting of Val70, Asn71 and Ser72; at least one residue selected from the group consisting of Tyrl03 and Glnl04; and Met89, Tyr91, LyslOl, and to Metl06 of BMP4.
  • said antibody binds to more than 9, 10, 11, 12, 13 preferably 14 residues thereof.
  • said antibody specifically binds to Asp30, Trp31, Leu66 and LyslOl.
  • said antibody is a single chain antibody.
  • said single chain antibody is capable of binding to a "hydrophobic pocket" region within the wrist epitope of BMP4 as described above.
  • an antibody according to the invention comprises a heavy chain CD 3 consisting of the amino acid sequence of SEQ ID NO: 7 or a sequence not differing more than 1 amino acid thereof. Without wishing to be bound by theory, the inventors believe that this CDR3 is important for the binding interaction with the hydrophobic pocket of the wrist of BMP4.
  • said antibody further comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 5 or a sequence not differing more than 1 amino acid thereof, and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:6, or a sequence not differing more than 1 amino acid thereof.
  • said antibody comprises the amino acid sequence of SEQ ID NO: 12.
  • said antibody binds specifically within residues 34, 35, 39, 86-88, 90, 97, 98, 100, 102 and 109 of BMP4.
  • This region represents the so called "knuckle" epitope of BMP4.
  • Antibodies specifically binding to residues in this region also have a high affinity for BMP4, but in addition also a high affinity for BMP2 and slightly less for BMP5, and BMP6.
  • said antibody binds specifically binding to Ala34, Gln39, Ser88, Leu90 and LeulOO.
  • said antibody is a single chain antibody.
  • an antibody capable of binding to said knuckle as described above comprises a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 8 or a sequence not differing more than 1 amino acid thereof and a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO:9, or a sequence not differing more than 1 amino acid thereof.
  • Said antibody preferably further comprises a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 10 or a sequence not differing more than 1 amino acid thereof.
  • said antibody comprises the amino acid sequence of SEQ ID NO: 13.
  • BMP4 inhibitors are known in the art and described for instance in Calpe et al. , MABS 2016, VOL. 8, NO. 4, 678-688.
  • BMP2 inhibitors are known in the art and described for instance in Khattab et al. Bone.
  • antibodies described on p. 20-22 which bind to the hydrophobic groove of the wrist of BMP4 and inhibit BMP4 signalling.
  • antibodies which bind to the hydrophobic pocket of the wrist of BMP4 are used. These antibodies inhibit the signalling of both BMP2 and BMP4.
  • Such antibodies are disclosed in WO2016/042050 on p. 22-24.
  • said antibody specifically binds to at least one residue selected from the group consisting of Ser24, Asp25, Val26, Gly27, Trp28, Asn29,Asp30,Trp31; at least one residue selected from the group consisting of Ser57, Thr58, Asn59, His60, Ala61, Ile62, Val63, Gln64, Thr65, Leu66, Val67, and Asn68; at least one residue selected from the group consisting of Val70, Asn71 and Ser72; at least one residue selected from the group consisting of Tyrl03 and Glnl04; and Met89, Tyr91, LyslOl, and to Metl06 of BMP4.
  • said antibody binds to more than 9, 10, 11, 12, 13 preferably 14 residues thereof. In a highly preferred
  • said antibody specifically binds to Asp30, Trp31, Leu66 and LyslOl.
  • said antibody is a single chain antibody.
  • an antibody according to the invention comprises a heavy chain CD 3 consisting of the amino acid sequence of SEQ ID NO: 7 or a sequence not differing more than 1 amino acid thereof. Without wishing to be bound by theory, the inventors believe that this CDR3 is important for the binding interaction with the hydrophobic pocket of the wrist of BMP4.
  • said an inhibitor of BMP2 and BMP4 comprises a hetero or homo multimeric molecule with increased antigen affinity for the antigens and/or an increased inhibitory effect on BMP signalling.
  • the invention therefore provides a multimeric antibody comprising at least one, more preferably at least two antibodies which bind to the hydrophobic pocket of the wrist of BMP4 as described above.
  • the present invention provides a composition, e.g., a pharmaceutical composition, containing an inhibitor of BMP2 and an inhibitor of BMP4 or an inhibitor of BMP2 and BMP4, formulated together with a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising antibodies binding to BMP4 or antigen binding portions thereof are well known in the art and are described in more detail for example in WO2008030611.
  • Such compositions may include one or a combination of (e.g., two or more different) antibodies or a multimeric antibody.
  • an antibody or multimeric antibody as defined herein is used.
  • Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. combined with other agents.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e. antibody, or antigen binding fragment thereof or multimeric antibody of the invention
  • the active compound may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.
  • the pharmaceutical compounds of the invention may include one or more pharmaceutically acceptable salts.
  • a "pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g., Berge et al, J. Pharm. ScL 66:1- 19 (1977)). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like
  • nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl- substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as ⁇ , ⁇ '-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • a pharmaceutical composition of the invention also may include a pharmaceutically acceptable anti- oxidant.
  • pharmaceutically acceptable antioxidants include: (1) water soluble
  • antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.
  • EDTA ethylenediamine tetraacetic acid
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.
  • dispersions are prepared by
  • a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredient, typically from about 0.1 per cent to about 70 per cent, most typically from about 1 per cent to about 30 per cent of active ingredient in combination with a pharmaceutically acceptable carrier.
  • IL2 receptor gamma chain knockout NSG
  • mice NOD-scid interleukin-2 receptor gamma chain knockout mice
  • AMC Animal Research Institute of the Academic Medical Center of Amsterdam
  • All mice were maintained in specific pathogen-free conditions, given autoclaved standard pellet feed and water, and housed in ventilated racks in the Animal Research Institute of the AMC. All animal experiments were approved by the Animal Experimental Committee (DEC) of the AMC.
  • DEC Animal Experimental Committee
  • Human tissue biopsies were obtained from Barrett Esophagus (BE) patients during the routine surveillance program at the Gastroenterology and Hepatology Department in the AMC. All patients were on long-term proton pump inhibition therapy and after a confirmed diagnosis by endoscopy and histology. The collection and use of human tissue for this project was in accordance with the legislation in the Netherlands and approved by the medical ethical committee in the AMC. After obtaining informed consent, fresh samples were collected endoscopically from BE patients. Samples were placed in chilled phosphate-buffered saline (PBS) without MgCI 2 and CaCI 2 and supplemented with 200 U/ml of penicillin and 200 ⁇ g/ml of streptomycin, and kept on ice.
  • PBS chilled phosphate-buffered saline
  • Biopsies were divided into approximately l-2mm 3 -sized pieces discarding any necrotic areas and blood clots. Representative samples were fixed in 10% buffered formalin for histological evaluation and the remaining pieces for implantation were placed in Matrigel (354234, Corning) with or without anti-BMP2/4 llama-derived antibody (C8C8; 500 ⁇ g/ml) or anti-BMP4 llama-derived antibody (C4C4; 500 ⁇ g/ml) (Calpe et al., 2015) and kept on ice until implantation. All biopsies were implanted within 2h of collection.
  • mice were anesthetized via an intraperitoneal injection (ip) of ketamine (lOOmg/ml) and xylazine (20mg/ml) solution. After weighing, mice are injected with 10 ⁇ of the anaesthetic solution per gram of body weight. The dorsum of the mouse was shaved and prepared with a 2 % (v/v) chlorhexidine gluconate/70 % (v/v) isopropyl alcohol solution. Under aseptic conditions, a 15-mm midline incision was made immediately caudal to the dorsal hump at the level of the renal angle. Using blunt dissection, a skin flap was raised and the skin retracted laterally in order to expose the implantation site.
  • a superficial stay suture was placed in the dorsal musculature immediately caudal to the lowest rib using a 4/0 braided absorbable suture. After tenting the muscle fibres, an intramuscular pocket was created using a combination of sharp and blunt dissection until it was just large enough to accommodate the BE piece. The BE biopsy coated in matrigel was then placed in the intramuscular (IM) pocket prior to suture closing. One or two separate transplantation sites were used per mouse. The skin was closed using 3/0 braided absorbable suture. Implants were cultured for a period of three months in order to form the in vivo organoid structures. During this period of time, mice received ip treatment three time per week of C4C4 or C8C8 (treatment groups) or saline (control group). Mice were closely monitored for any sign of discomfort and/or stress throughout the experiment.
  • mice were culled using C0 2 inhalation after three months. Immediately following culling, mice were shaved and their skin prepared. After identifying the non-absorbable marking suture, the muscle was incised around the site of xenograft leaving a 2mm wide margin. Following this, the muscle was retracted medially and any obvious lump or cyst carefully harvested. Samples were fixed in 10% buffered formalin to be assessed both histologically and immunohistochemically.
  • Sections of formalin-fixed paraffin-embedded tissues from all of the original patient-derived biopsies and the in vivo organoids were stained with hematoxylin and eosin (H&E) and Alcian Blue.
  • H&E hematoxylin and eosin
  • Alcian Blue Alcian Blue.
  • IHC hematoxylin and eosin
  • the in vivo organoid structures were stained for the squamous markers CK5 (1:200) (ab52635; Abeam) and p63 (1:100) (sc-8431; Santa Cruz Biotechnology, Inc.).
  • the in vivo organoids show the same degree of differentiation (Figure 3A-F) and expression of molecular markers of intestinal differentiation (CK8, CDX2 and villin) as the original biopsy.
  • C8C8 organoids demonstrate a tendency to form a multi-layered epithelium that expressed the squamous marker p63 and CK5 ( Figure 3K-L).
  • the epithelial layer of the treated organoids also lack the presence of goblet cells ( Figure 3J).
  • treatment with C4C4 were only BMP4 is inhibit, the same differentiation doesn't occur and the in vivo organoids resemble the control biopsy and lacking the expression of the squamous markers p63 and CK5.
  • Example 2 A Inhibition of BMPs by Noggin drives the development of squamous epithelium in an ablation model.
  • mice During the healing process we treated the mice with either saline (control group) or Noggin (treatment group). At day 14 we were able to investigate the re-population of the ablation area. In the control group we observed that, after ablation, the stomach healed normally being able to recognize the columnar part, the SCJ and the neo-squamous cells. Like in the animals treated with BMP2/4, treatment with Noggin resulted in re-epithelization with squamous cells observed at the ablation proximal to the original SCJ, where normally columnar cells reside. The presence of neo-squamous epithelium was validated by immunohistochemistry
  • Rosa26-cre mice were crossed with loxp-noggin-loxp mice (fig4A).
  • immunohistochemistry IHC for noggin was negative confirming the deletion of noggin.
  • mice development columnar metaplasia that resembles the human Barrett's esophagus at the SCJ.
  • the metaplastic epithelium seem to arise from multi-lineage glands (MLGs) (Fig4B).
  • MLGs multi-lineage glands
  • the MLGs consist of an outer layer staining positive for squamous markers, including K5, K14 and p63 (Fig4).
  • the inner layer stains positive for columnar markers including K19, K8, PAS and Alcian Blue (Fig4D).

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Abstract

L'invention concerne un kit de pièces comprenant un inhibiteur de BMP2 et un inhibiteur de BMP4 ou un inhibiteur de BMP2 et BMP4 pour une utilisation dans le traitement de l'endobrachyœsophage ou pour la prévention de l'adénocarcinome œsophagien. L'invention concerne en outre une composition pharmaceutique comprenant un kit de pièces comprenant un inhibiteur de BMP2 et un inhibiteur de BMP4 ou un inhibiteur de BMP2 et BMP4 tels que définis dans la description et un véhicule ou un diluant pharmaceutiquement acceptable pour une utilisation dans le traitement de l'endobrachyœsophage ou pour la prévention d'un adénocarcinome œsophagien.
PCT/EP2018/060251 2017-04-22 2018-04-20 Inhibiteurs de la signalisation bmp2 et bmp4 destinés à être utilisés dans le traitement de l'endobrachyoesophage WO2018193129A1 (fr)

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* Cited by examiner, † Cited by third party
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WO2023142801A1 (fr) * 2022-01-29 2023-08-03 中国医学科学院阜外医院 Utilisation de l'omentine-1 dans l'inhibition d'une protéine morphogénétique osseuse
WO2023237431A1 (fr) 2022-06-07 2023-12-14 Esocap Ag Système d'administration de médicament comprenant un agent efficace dans le traitement ou la prévention d'une maladie oesophagienne pour l'application à des membranes muqueuses oesophagiennes

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023142801A1 (fr) * 2022-01-29 2023-08-03 中国医学科学院阜外医院 Utilisation de l'omentine-1 dans l'inhibition d'une protéine morphogénétique osseuse
WO2023237431A1 (fr) 2022-06-07 2023-12-14 Esocap Ag Système d'administration de médicament comprenant un agent efficace dans le traitement ou la prévention d'une maladie oesophagienne pour l'application à des membranes muqueuses oesophagiennes

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