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WO1998006751A1 - Antagonistes des recepteurs de mcp-3, rantes et mip-1alpha - Google Patents

Antagonistes des recepteurs de mcp-3, rantes et mip-1alpha Download PDF

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Publication number
WO1998006751A1
WO1998006751A1 PCT/US1997/014485 US9714485W WO9806751A1 WO 1998006751 A1 WO1998006751 A1 WO 1998006751A1 US 9714485 W US9714485 W US 9714485W WO 9806751 A1 WO9806751 A1 WO 9806751A1
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Prior art keywords
mcp
rantes
analog
mlp
native
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Application number
PCT/US1997/014485
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English (en)
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WO1998006751A9 (fr
Inventor
Ian Clark-Lewis
Jiang-Hong Gong
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Research Corporation Technologies, Inc.
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Publication of WO1998006751A1 publication Critical patent/WO1998006751A1/fr
Publication of WO1998006751A9 publication Critical patent/WO1998006751A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to NH 2 - terminally truncated analogs of Monocyte Chemoattractant Protein - 3 (MCP-3), RANTES and Macrophage Inflammatory Protein- l (MlP-l ⁇ ) and compositions containing same and methods of employing said compositions for treating inflammatory diseases as well as autoimmune disorders.
  • MCP-3 Monocyte Chemoattractant Protein - 3
  • MlP-l ⁇ Macrophage Inflammatory Protein- l
  • RANTES Normal T-Cell Expressed and Secreted
  • MCP-3 Monocyte Chemoattractant Protein- 3
  • MlP-l ⁇ Macrophage Inflammatory Protein- l ⁇
  • chemokine reflects the ability of these mediators to induce directed migration of several types of leukocytes, including monocytes, lymphocytes, basophils and eosinophils into sites of inflammation.
  • RANTES, MCP-3 and MlP-l ⁇ are collectively known as "CC” chemokines because the first two cysteines in each molecule are adjacent.
  • the CC chemokines contain disulfide bridges.
  • the CC 1 chemokines have been implicated in a number of allergic and chronic inflammatory diseases such as arthritis and various lung diseases. In such conditions, early monocytic infiltration of monocytes, 5 T- lymphocytes and other leukocytes may be a key event in the progression of the disease.
  • chemokine receptor CCKR-1 which binds MlP-l ⁇ , RANTES and MCP-3
  • CCKR- 2 which binds MCP-3 and MCP-1
  • CCKR-3 which binds to
  • RANTES comprises a 68 amino acid polypeptide
  • MCP-3 comprises
  • MlP-l ⁇ comprises a 70 amino acid polypeptide.
  • the present inventors have not only discovered the antagonistic effects of truncated RANTES, MCP-3 and MlP-l ⁇ , but also have discovered that the analogs are useful for treating inflammatory diseases and autoimmune disorders, e.g., rheumatoid arthritis.
  • the inventors have also discovered that the analogs competitively bind and cross-bind to several different receptors. Inflammation involves the infiltration of multiple cell types that occurs through the interaction of different chemokines with distinct functional receptors.
  • the inventors have further discovered that blocking the infiltration of multiple effector cells is highly effective in breaking the inflammation and autoimmune cycle.
  • the present invention is directed towards
  • the present invention is also directed to inhibiting the biological activities of three native, mammalian chemokines MCP-3, RANTES and MlP-l ⁇ .
  • the present invention is further directed to treating inflammatory diseases and autoimmune disorders such as rheumatoid arthritis and multiple sclerosis, for example.
  • the present invention is also directed to pharmaceutical compositions comprising NH 2 - terminally truncated che okine analogs .
  • One aspect of the present invention is directed to an analog of mammalian MCP-3 lacking NH 2 - terminal amino acids corresponding to amino acid residues 1-6, 1-7, 1-8, 1-9 or 1-10, of MCP-3, having substantial homology to the native MCP-3 molecule.
  • Another aspect of the present invention is directed to an analog of mammalian RANTES lacking NH 2 - terminal amino acids corresponding to amino acid residues 1-5, 1-6, 1-7, 1-8, and 1-9 of RANTES, having substantial homology to the native RANTES molecule.
  • a further aspect of the present invention is directed to an analog of mammalian MlP-l ⁇ lacking NH 2 - terminal amino acids corresponding to amino acid residues 1-9 or 1-10, having substantial homology to the native MlP-l ⁇ molecule.
  • a still further aspect of the present invention is directed to a method of inhibiting the biological activity or the in vivo biological activity of native MCP-3, RANTES and MlP-l ⁇ comprising adding to the native MCP-3 RANTES and MlP-l ⁇ (if in vitro) or if in vivo administering to a host, e.g., mammal (for example, human) a therapeutically effective amount of an analog of MCP-3, RANTES or MlP-l ⁇ for a time and under conditions sufficient to inhibit the biological activity of the native molecules.
  • a host e.g., mammal (for example, human) a therapeutically effective amount of an analog of MCP-3, RANTES or MlP-l ⁇ for a time and under conditions sufficient to inhibit the biological activity of the native molecules.
  • Another aspect of the present invention is directed to a method of treating inflammatory diseases in a mammal suffering from said diseases comprising administering to said mammal a therapeutically effective amount of the analog of the present invention.
  • a further aspect of the present invention is directed to a method of treating autoimmune disorders in a* mammal comprising administering to a mammal in need thereof a therapeutically effective amount of an analog of the present invention.
  • Another aspect of the present invention is directed to pharmaceutical compositions comprising an antagonistically effective amount of one or more of the aforementioned analogs and a pharmaceutically acceptable carrier.
  • Figure 1A is a graph showing NH 2 - terminal truncation of RANTES and MCP-3 resulted in a loss of chemotactic activity on THP-1 cells.
  • Figure IB is a graph showing the NH 2 - terminal truncated RANTES analogs (RANTES (6 -68) , RANTES (7 - 68) , RANTES(8-68) , RANTES (9 - 68) , and RANTES ( 10 - 68) ) had no detectable activity.
  • Figure 1C is a graph showing truncation of the residues 1-9 of MlP-l ⁇ (MlP-l ⁇ (10-70)) reduced activity by more than 95% of the native MlP-l ⁇ , further truncation resulted MlP-l ⁇ (11-70) which completely lost chemoattractant activity on THP-1 cells .
  • Figure 2A is a graph showing RANTES antagonist activity of the RANTES (9 -68) analog titrated at the indicated concentrations against RANTES and MCP-3 in a chemotaxis assay using THP-1 cells.
  • Figure 2B is a graph showing MCP-3 antagonist activity of the MCP-3 (10-76) analog titrated at the indicated concentrations against MCP-3 and RANTES in a chemotaxis assay using THP-1 cells.
  • Figure 2C is a graph showing the inhibition of MlP-l ⁇ induced (10 nM or 10 '8 M) THP-1 migration by MIP-l (10- 70) and MlP-l ⁇ (11-70) .
  • Figure 2D is a graph showing the inhibition of RANTES - 1 induced (10 nM or 10 "8 M) THP-1 migration by MIP-l ⁇ (10- 70) and MIP- l ⁇ (11 - 70) .
  • Figure 3A is a graph showing RANTES antagonist activity of RANTES (9-68) titrated at the indicated concentrations against RANTES- induced and MCP- 3- induced N-acetyl - ⁇ -D-glucosaminidase release using human blood monocytes .
  • FIG. 3B is a graph showing MCP-3 antagonist activity of MCP-3 (10-76) titrated at the indicated concentrations against MCP- 3 -induced and RANTES - induced N-acetyl- ⁇ -D-glucosaminidase release using human blood monocytes. 5
  • Figures 3C and 3D are graphs showing MlP-l ⁇ antagonist activity of MIP- l ⁇ (10-70) and MIP- l ⁇ (11 -70) titrated at the indicated concentrations against MIP- l ⁇ - induced and RANTES - induced-N-acetyl - ⁇ -D-glucosaminidase o release using human blood monocytes.
  • concentration of MlP-l ⁇ and RANTES used was 30 nM.
  • Figures 4A and 4B are graphs showing competitive binding to THP-1 cells of unlabeled RANTES and 5 RANTES (9 -68) titrated at the indicated concentrations in the presence of 4 nM labeled RANTES (A) , and 4 nM labeled MCP- 3 (B) .
  • Figures 4C and 4D are graphs showing competitive 0 binding to THP-1 cells of unlabeled MCP- 3 and MCP-
  • Figure 4E is a graph showing competitive binding to THP-1 cells of unlabeled MCP-3 and MCP-3(8-76), MCP- 3(9-76), MCP-3 (10-76) and MCP- 3 (11 - 76) titrated at the indicated concentrations in the presence of 4 nM labeled MCP- 3.
  • Figure 4F is a graph showing competitive binding to THP-1 cells of unlabeled RANTES and RANTES (6-68), RANTES (7-68), RANTES (8-68), RANTES (9-68) and RANTES (10-68) titrated at the indicated concentrations in the presence of 4 nM labeled RANTES.
  • Figure 5 is a graph showing competitive binding to THP-1 cells of unlabeled MlP-l ⁇ and MIP- l (10 -70) and MIP- l ⁇ (11-70) titrated at the indicated concentrations in the presence of 4 nM labeled MlP-l ⁇ .
  • Figure 6 is a chart summarizing the binding and antagonist activities of MlP-l ⁇ , RANTES and MCP- 3 analogs.
  • Figure 7 is a graph showing competitive binding to THP-1 cells of unlabeled PJ lTES and RANTES (9 -68) and RANTES (9-68, P9T) titrated at the indicated concentrations in the presence of 4 nM labeled MCP-1.
  • Figure 8 is a graph which shows that human RANTES (9- 68) was able to bind to the receptor on mouse peritoneal macrophages. Native human RANTES did not significantly bind to mouse peritoneal macrophages.
  • Figures 9A and 9B are graphs showing that daily i.p. injections of RANTES (9-68) inhibited the swelling and the incidence of arthritis in treated mice.
  • Figure 10 is a chart showing untreated control animals and animals administered a peptide control developed rheumatoid arthritis, substantial infiltration of mononuclear cells into the synovial tissue, extensive hyperplasia of the synovial lining, pannus formation and bone and cartilage damage.
  • animals treated with RANTES (9 -68) exhibited no invasive pannus, no bone or cartilage damage and insignificant inflammatory infiltration.
  • Figure 11 is a graph which shows that human MCP- 3 (9- 7 6) was able to bind to the receptor on mouse monocytes. Native human MCP- 3 did not significantly bind to mouse monocytic cells.
  • Figure 12 is a graph showing that daily i.p. injections of MCP-3 (9-76) inhibited the swelling and the incidence of arthritis in treated mice.
  • One aspect of the present invention is directed to NH 2 - terminally truncated analogs of three human chemokines, MCP-3, RANTES and MlP-l ⁇ , useful for treating inflammatory conditions and autoimmune disorders.
  • Inflammatory conditions contemplated by the present invention include both acute and chronic inflammatory diseases. Examples include, but are not limited to, arthritis, asthma, colitis, psoriasis, atheromas and the like. Examples of autoimmune conditions include rheumatoid arthritis and multiple sclerosis and the like.
  • an NH 2 - terminally truncated chemokine analog acts as an antagonist to a corresponding native chemokine.
  • the antagonistic activity of the chemokine analogs of the present invention includes inhibition of biological activity induced by corresponding native chemokines .
  • biological activity refers to the biological activity of the native chemokines, as measured by standard assays, including but not limited to receptor binding, chemotaxis, calcium mobilization and exocytosis characterized by N-Acetyl - ⁇ -D-glucosaminidase release 1 and elastase release.
  • Preferred MCP- 3 analogs of the present invention include: MCP-3 (7-76), (8-76), (9-76), (10- 76) and (11-76) .
  • Preferred RANTES analogs of the present invention include: RANTES (6-68), (7-68), (8- 68), (9-68) and (10-68).
  • Preferred MIP- l ⁇ analogs of the present invention include: MlP-l ⁇ (9-70), (10-70) and (11-70) .
  • any 0 reference by number to an amino acid in an analog e.g., MCP-3 analog will be a reference to the corresponding residue number from the amino acid sequence of the native molecule shown in Table 1.
  • the analogs will be referred to MCP- 3 (8-76), MCP-3 (9-76), MCP-3(10-76) and MCP- 3(11-76), respectively.
  • the analogs of the present invention o comprise an amino acid sequence that is identical to the corresponding region of the native molecule, or a polypeptide fragment having a region that is substantially homologous to all or part of a region of the corresponding native molecule while still 5 maintaining the antagonist activity to the corresponding native molecule.
  • the analogs of the present invention maintain the ability to competitively bind at the receptor site of the corresponding chemokine. Analogs which are 0 "substantially homologous" to all or part of a region
  • the MCP- 3 analogs of the present invention are at least about 80% homologous to native MCP-3 and can have as much as 100% homology to native MCP- 3, inclusive. More preferably, MCP- 3 analogs of the present invention are at least about 85% homologous to native MCP- 3. Still more preferably, MCP- 3 analogs of the present invention are at least about 90% homologous to native MCP- 3.
  • MCP- 3 analogs of the present invention are at least about 95% homologous to native MCP- 3.
  • RANTES and MlP-l ⁇ analogs of the present invention are preferably at least about 60% homologous to the corresponding native polypeptides and can extend to as much as 100% homology to the corresponding native polypeptides, inclusive.
  • the RANTES analogs and MlP-l ⁇ analogs of the present invention are preferably at least about 60% homologous to the native polypeptides and more preferably at least about 75%, even more preferably at least about 85% and most preferably at least about 90% and 95% homologous to the native chemokine.
  • Polypeptides having truncation at both the N-terminal end and the C- terminal end are also contemplated to be within the scope of the present invention.
  • up to 10 amino acids can be truncated from the C- terminal end of the analogs described hereinabove.
  • P9T denotes a substitution of the amino acid Proline for the amino acid Threonine at position 9 of the native molecule. Similar modifications denoted by the single amino acid code followed by a molecule position number and the substituting amino acid are contemplated by the present invention (see Figure 9) .
  • the present inventors have found that there are certain portions of the polypeptides of the present invention that must be maintained and cannot be modified. More specifically, deletion of certain amino acids from the native chemokines and the antagonists of the present invention will result in a loss of receptor binding capability. For example, deleting the disulfide bridge in MCP- 3 or MCP- 3 analogs (i.e. the cysteines at positions 12 and 52) will result in conformational changes and will preclude receptor binding. Similar results have been 1 discovered in RANTES (i.e. deletion of cysteine 11 and 50) and MlP-l ⁇ (i.e. deletion of cysteine 12 and 51).
  • the analogs are synthesized using art 0 recognized techniques in peptide chemistry. For example, they may be synthesized by adding one amino acid at a time to an amino acid or peptide.
  • the amino acids or peptide contain the appropriate protecting groups on the side chains and on the N-terminal 5 portions thereof.
  • the first amino acid containing the appropriate protecting group on the N- terminus and the side chain is coupled to a second amino acid having a protecting group on the side chain and the C- terminal end in the presence of a peptide coupling reagent, o such as DCC to form the resulting peptide.
  • the C- terminal end is bound to a resin so that the peptide is built from the C- terminal end to the N-terminal end thereof.
  • a resin so that the peptide is built from the C- terminal end to the N-terminal end thereof.
  • another N ⁇ -protected amino acid having side 5 chain protection is coupled to the peptide formed previously. This process is continued until the desired peptide is formed; then the protecting groups are removed by art recognized techniques and the peptide is removed from the resin by art -recognized Q techniques.
  • An exemplary procedure for polypeptide synthesis of the compounds of the present invention is as follows.
  • the analogs are preferably synthesized using tBoc chemistry on a peptide synthesizer and purified using reverse phase HPLC.
  • the synthesis is started with a protected C- terminal amino acid linked to a cross -linked polystyrene resin via a 4- (carboxamidomethyl) benzyl ester linkage (the so- called pam resin) (0.4 mmol of 0.8 mmol/g of aminoacyl resin).
  • N ⁇ -t-Boc acids with appropriate side chain protecting groups are added in a stepwise fashion until the entire protected polypeptide chain is formed.
  • the groups utilized for side chain protection are those commonly used in this art.
  • Examples include: benzyl (Asp, Gly, Ser and Thr); 4- methylbenzyl (Cys); toluenesulfenyl (Arg); 2- chlorobenzyloxycarbonyl (Lys) ; 2- bromobenzyloxycarbonyl (Tyr) ; formyl (Trp) ; benzyloxy ethyl (His) ; and none (Ala, Asn, Gly, Gin, lie, Leu, Met, Phe, Pro, Val) . Samples may be taken after each step to retrospectively monitor the amino acid coupling yields using a ninhydrin-based reaction following the procedures of Sarin, et al . (1981) Anal . Biochem.
  • the resin is dried and cleaved using the "lo -high" hydrogen fluoride method as described by Tarn, et al . (1984) J. Am. Chem. Soc . 105 :6442 -6485 (the contents of which are incorporated herein by reference) , except for the following modifications: after the 25% hydrogen fluoride step, the partially protected peptide resin is filtered from the reaction mixture by using an all -Teflon filtration apparatus fitted with a Zitex filter and washed with dichloromethane and dried before the high 90% hydrogen fluoride step.
  • the ethyl acetate precipitate of the material released from the resin is dissolved in 50 ml of 6 M guanidine hydrochloride, 0.1 M Tris- acetate, pH 8.5, and 10% 2 -mercaptoethanol . This mixture is the crude peptide product.
  • histidine may be protected with p-benzyloxymethyl instead of dinitrophenyl .
  • the p- benzyloxymethyl group is acid labile, thus eliminating the need for thiolysis of the dinitrophenyl group before and after hydrogen fluoride deprotection.
  • Acetylation is carried out on the N « deprotected, but otherwise fully protected peptide resin, using acetic anhydride (50%) in dimethylformamide.
  • the crude peptide product may be purified and folded by art recognized techniques.
  • three different C-18 silica HPLC columns may be used in the purification and analysis of the peptide, including a preparative column (22.4 x 250 mm column with a 22.4 x 100 mm guard column) packing with 12 ⁇ m, 300 -A pore size packing (Dynamax, Rainin Instrument Co., Woburn, MA); a semipreparative column (10 x 250 mm) Vydac C018 column, with 5- ⁇ m particle, 300-A pore-size packing (Separations Group, Hesperia, CA) ; and an analytical column (4.6 x 250 mm) (Vydac) containing the same packing.
  • a preparative column (22.4 x 250 mm column with a 22.4 x 100 mm guard column) packing with 12 ⁇ m, 300 -A pore size packing (Dynamax, Rainin Instrument Co., Woburn, MA)
  • the crude peptide product is first acidified to pH 4.0 with 20% acetic acid and filtered.
  • the crude peptide product is then loaded onto the preparative column and the retained material eluted with a 0-60% water- acetonitrile gradient in 0.1% trifluoracetic acid over 4 hours at a flow rate of 15 ml/min.
  • a sample (25 ⁇ l ) of fractions containing 225- nm UV- absorbing material is rerun on the analytical column and by comparison with the profile of the crude material, fractions containing the major peak are pooled and lyophilized.
  • the material is reconstituted in 1 M guanidine hydrochloride and Tris- acetate, pH 8.5 , in 10% dimethyl sulfoxide (DMSO) at a concentration of 0.2 mg/ml and stirred overnight in a covered beaker.
  • MlP-l ⁇ is folded as above in the absence of DMSO.
  • MlP-l ⁇ (10-70) and MlP-l ⁇ (11-70) are folded in 10% DMSO in water. This procedure promotes formation of the disulfide bridges by oxidation of the appropriate half -cysteines .
  • the materials are acidified with 2 ml of acetic acid, and half is loaded onto the semipreparative column and the retained material is eluted with the same gradient as described above at a flow rate of 3 ml/min. Samples °f each fraction are run on the analytical column.
  • Fractions containing only material with the retention time of the major peak in the folded material are pooled and lyophilized.
  • An assay for free sulfhydryls using Ellman reagents may be used to determine the extent of folding.
  • folding may be monitored on the analytical HPLC column by observing the appearance of a peak corresponding to the folded form that has a retention time approximately 3 minutes earlier than the reduced form.
  • Analog purity may be assessed on an analytical HPLC column or by other means such as isoelectric focusing.
  • An exemplary protocol for isoelectric focusing is as follows.
  • Mini polyacrylamide gels (Pharmacia PHAST gels, IEF 3-9; Pharmacia, Uppsala, Sweden) are washed in 8 M urea and then in 8 M urea containing pH 9-11 Ampholytes (Pharmacia) , for 30 minutes each, either with or without 10 ⁇ M dithiothreitol .
  • Gels are prerun for 15 V-h at 200-V, 2.0-mA, 3.0-mW maximum settings, and the samples are loaded and run for 410 V-h at 1000-V, 5.0- mA, 3.0-mW maximum settings on the Pharmacia PHAST systems for a total of 500 -V with maximum settings of 2.0-mW, 5.0-mA and 1000-V.
  • the pH gradient may be determined by using a surface pH electrode.
  • the gels may be stained with silver by using the PHAST developing systems as described in the Pharmacia manual .
  • Sequencing of analogs may be determined by protein sequencing, for example by using the following protocol . Protein sequences are determined by Ed an degradations using either solid-phase or gas -liquid- phase methods.
  • reduced and carboxymethylated protein or proteolytic cleavage fragments are coupled to arylamine- functionalized poly (vinylidenedifluoride) membranes (Sequelon AA; Milligen/Biosearch, Burlington, MA) using the water-soluble carbodiimide 1- ethyl -3-3 [3- (dimethylamino) propyl] carbodiimide hydrochloride and sequenced in a Milligen/Biosearch Model 6600 sequencer 1 using standard protocols.
  • polypeptides may be applied to Polybrene- coated glass fibre disks and sequenced in an Applied Biosystems Model 477 protein sequencer using standard protocols.
  • Sequencing of protected peptide resins may be carried out on N ⁇ -deprotected samples by using the same methods.
  • N-terminal solid-phase sequencing runs usually reveal a major portion of the sequence.
  • the remaining sequence is obtained by runs 0 of the HPLC -fractionated fragments, derived either by proteolytic cleavage with Asp -N-endoprotease (Boehringer Mannheim Canada, Laval, Quebec) or by chemical cleavage, through preferential hydrolysis of the Asp- Pro peptide bond in dilute formic acid.
  • Molecular weight of the synthetic proteins prepared as described above are determined by art- recognized techniques, such as electrospray mass spectrometry on a SCIEX triple quadruple Mass Spectrometer equipped with a liquid delivery o apparatus.
  • the molecular mass from the peaks corresponding to the charge to mass ratios of the different multiple ionized species of the protein can also be analyzed as described by Convey, et al . (1988) Rapid Co mun. Mass. Snectrom. 2:249-256.
  • the chemokine analogs of this invention may also be prepared through recombinant means, with expression in mammalian or non-mammalian systems. Portions of a DNA sequence encoding, e.g., MCP-3, are appropriately modified to produce the desired analog Q when the DNA sequence is expressed.
  • the inventors have discovered that, in contrast to the full-length native forms, the analogs of the present invention were antagonists to the corresponding native molecules. Moreover, they lacked detectable chemoattractant activity for the receptor carrying cells. For example, RANTES (9-68), MCP-3 (10-76) and MlP-l ⁇ (10-70) lacked detectable chemoattractant activity for human THP-1 monocytic cells. RANTES (6- 68), (7-68), (8-68) and (10-68) also lacked chemoattractant activity for T- cells, monocytes, NK cells, basophils and eosinophils.
  • MCP-3 (8-76), (9-76) and (11-76) also lacked activity for human CD4+ and CD8+ T lymphocytes, NK cells, eosinophils and basophils.
  • MlP-l ⁇ similarly lacked chemoattractant activity for monocytes and human T- cells.
  • the inventors have observed certain regions within the native chemokines which are essential for their functional activities. Specifically, NH 2 - terminal amino acid residues within the 1-5 region of native RANTES, residues within the 1-7 region of native MCP-3 and residues within the 1-9 region of native MlP-l ⁇ are critical for chemoattractant activity and receptor activation, for example. However, by truncating the 1-5 region of native RANTES, 1-7 region of native MCP- 1 3 and 1-9 region of native MlP-l ⁇ , the resulting molecules become antagonists to the native chemokines.
  • RANTES -68
  • MCP-3(10-76) and MlP-l ⁇ (10-70) inhibited the biological activity (e.g. chemoattractant activity and N-acetyl - ⁇ -D- 0 glucosaminidase release, elastase release and intracellular calcium mobilization) induced by the corresponding native forms.
  • biological activity e.g. chemoattractant activity and N-acetyl - ⁇ -D- 0 glucosaminidase release, elastase release and intracellular calcium mobilization
  • the inventors have assessed the receptor-binding 5 specificity of the truncated analogs.
  • the inventors have discovered significant cross -receptor binding among the identified chemokine analogs of the present invention.
  • RANTES RANTES
  • RANTES -induced, MCP- 1- induced, calcium mobilization, monocyte N-acetyl- -D-glucosaminidase release and chemoattractant activity RANTES -induced, MCP- 1- induced, calcium mobilization, monocyte N-acetyl- -D-glucosaminidase release and chemoattractant activity.
  • the present inventors have determined that the polypeptides (analogs) of the present invention act as antagonists to the effects of native RANTES, MlP-l ⁇ and MCP- 3 in various tests, such as chemotaxis, calcium mobilization, receptor binding in THP-1 cells (a monocytic cell line) . Based upon these observations, the inventors concluded that the polypeptides of the present invention are useful in blocking the effects of native RANTES, MlP-l ⁇ and MCP- 3 molecules and thus are useful as therapeutic agents. A preferred use of the polypeptides of the present invention is in blocking the effects of RANTES, MlP-l ⁇ and MCP- 3 in the recruitment and/or activation of pro- inflammatory cells.
  • the present invention has utility in the treatment of such inflammatory diseases as asthma, allergic rhinitis, colitis, psoriasis, atheromas, dermatitis and the like.
  • the polypeptides are also useful in the treatment of autoimmune diseases, e.g., rheumatoid arthritis.
  • the analogs of the present invention e.g. RANTES antagonists
  • the analogs of the present invention are useful in treating autoimmune disorders (e.g., rheumatoid arthritis) in mammals.
  • the inventors have determined that the analogs of the present invention prevent the onset of rheumatoid arthritis in MRL-lpr mice.
  • RANTES (9-68) significantly inhibited the clinical incidence of adjuvant enhanced arthritis for 30 days in an autoimmune mouse model (MRL-lpr mice have lupus erythematous and rheumatoid arthritis similar to the corresponding human diseases) .
  • a truncated analog of the present invention in another aspect of the present invention, involves administering a therapeutically effective amount of a truncated analog of the present invention to a mammal to treat, e.g. inflammatory diseases and autoimmune diseases .
  • the NH 2 - terminally truncated chemokine analogs of the present invention are administered in pharmaceutical compositions.
  • the analogs of this invention may be administered as a nasal spray for upper respiratory treatments or as an aerosol inhaler for lung conditions.
  • the analogs may also be used in topical applications.
  • the analogs may also be administered via an osmotic pump or in a sustained release formulation.
  • the analogs of the present invention may be delivered by injection. It is preferred that the injection is intramuscular or intraanal. However, the present invention also contemplates intradermal, intraperitoneal or intraarticular injections as well as long term continuous delivery via subcutaneous osmotic pump or sustained release formulations.
  • chemokine analogs of the present invention are present in the various pharmaceutical formulations described hereinabove in an "antagonistically effective amount” or “therapeutically effective” amount.
  • “Antagonistically effective amount” or “therapeutically effective” 5 amount as used herein is defined as an amount of chemokine analog sufficient to significantly inhibit the biological activity of native chemokines but low enough to avoid serious side effects such as toxicity, for example (at a reasonable risk/benefit ratio) 0 within the sound medical/scientific judgment of the skilled artisan.
  • the dose of the formulation contains between 0.01-1 mg/kg for a nasal spray and 0.1-10 mg/kg for other forms of delivery. It is most preferred that the formulation 5 contains between 0.01-0.1 mg/kg for a nasal spray and 0.1-1 mg/kg for other forms of delivery.
  • the dosage schedule of the chemokine analogs will typically be determined (at a reasonable risk/benefit ratio) within the sound medical/scientific judgement of the skilled o artisan. However, it is preferred that the chemokine analogs are administered at least about 3 times per week. It is most preferred that the chemokine analogs are administered on a daily basis.
  • chemokine analog of the 5 present invention to be used in a particular application may be determined by accepted pharmaceutical methods known to the skilled artisan. This is accomplished by conventionally measuring the concentration of analog in the blood and determining Q the analog half -life. Without wishing to be bound, it
  • compositions comprising the analogs of the present invention additionally contain pharmaceutical vehicles, such as carriers and adjuvants described in the literature of pharmaceuticals and related fields.
  • the analogs of the present invention are formulated by the skilled artisan, using art -recognized techniques, taking into consideration the nature of the polypeptide compounds and the desired mode of administration.
  • the product of the present invention are soluble and are therefore readily formulated in physiological buffers, e.g. physiological saline.
  • the inventors have observed that unlike other chemokines, native human RANTES and MlP-l ⁇ form aggregates in physiological conditions (e.g. at about pH 7.2) at concentrations as low as 100 nM. This presents a significant hurdle to in vivo applications because therapeutic efficacy usually requires much higher concentrations of the active compounds (i.e., up to 2 mM) .
  • the present inventors have surprisingly discovered that the truncated chemokine analogs of the present invention exhibit no aggregation at concentrations above about 1 ⁇ M. Therefore, the truncated chemokine analogs (e.g. RANTES and MlP-l ⁇ ) of the present invention are highly soluble, and thus facilitate more in vivo applications than their native counterparts.
  • chemokine analogs of the present invention can also formulated in sustained release delivery systems or topical formulations containing an aqueous component .
  • MCP- 3, RANTES and MlP-l ⁇ analogs of the present invention are assayed for biological activity by use of a cytosolic-free calcium assay, a chemotaxis assay using cells of monocytic origin, or by other conventional assays for MCP- 3, RANTES or MlP-l ⁇ activity including but not limited to: assays to determine exocytosis of leukocytes such as elastase release and N-acetyl - ⁇ -D-glucosaminidase release, superoxide production, histamine release, LTC 4 release and the like.
  • Fluo-3.AM or Fluo-2 Designated 100% saturation for each set of measurements by addition of 5 ⁇ M lonomycin (Sigma Chemical Co., St. Louis, MO).
  • Representative polypeptides of the present invention were tested for their functional activity according to the procedure described hereinabove. The 5 results are as follows. In contrast to the full- length native forms, RANTES (9-68), MCP-3 (10-76) and MlP-l ⁇ (10-70) lacked detectable chemoattractant activity for human THP-1 monocytic cells ( Figures 1A- 1C) . In addition, for human blood- derived monocytes, 0 neither chemotaxis nor N-acetyl - ⁇ -D-glucosaminidase release was detected up to 1 uM.
  • RANTES (6-68), (7- 68), (8-68) and (10-68) as well as MCP-3 (8-76), (9- 76) and (11-76) also lacked chemoattractant activity (Figure IB) .
  • MlP-l ⁇ (10-70) had only minor activity (about 3% of that of the corresponding native MlP-l ⁇ ).
  • MlP-l ⁇ (11-70) had no detectable activity.
  • residues within the 1-5 region of native RANTES, 1-7 region of native MCP-3 and 1-9 region of native MlP-l ⁇ are essential for the functional activities of the o native chemokines. Truncation of these regions resulted in a complete loss of e.g. chemoattractant activity of the native chemokines.
  • Inhibition of native MCP- 3, RANTES or MlP-l ⁇ mediated chemotaxis are determined by using the aforementioned chemotaxis assay. Constant amounts of 5 MCP- 3, RANTES or MlP-l ⁇ (e.g. 10 nM) are added to each well, and the analogs are titrated in the assay.
  • Cell preparations for use in the aforementioned assays consist of human monocytes, or monocytic cell lines such as the cell line THP-1. 0 THP-1 were obtained from American Type Culture
  • Human monocytes were isolated from buffy coats of normal donor blood by the following protocol. A cell suspension was loaded onto Ficol1 -Hypaque (Pharmacia, Uppsala, Sweden) and centrifugated at 400 g for 25 minutes followed by density centrifugation on a discontinuous Percoll (Pharmacia) gradient at 500 g o f° r 30 minutes. Cell fractions with a density of 1.051-1.053 (g/ml) were generally greater than 70% monocytes by morphology and were used in the assay.
  • RANTES (9-68) were obtained for migration of peripheral blood monocytes.
  • a 10-fold lower concentration (IC 50 4 nM) of RANTES (9-68) was required to inhibit RANTES - induced enzyme release than was needed for similar inhibition of chemotaxis ( Figure 3A) .
  • MCP- 3 (10-76) was found to inhibit MCP-3 induced monocyte N-acetyl- ⁇ -D-glucosaminidase release with an IC 50 of 37 nM and also chemoattractant activity IC 50 470 nM. Thus it was less potent than RANTES (9- 68) for RANTES -stimulated activities. However, MCP-3 (10-76) also inhibited RANTES - induced and MlP-l ⁇ - induced activities with similar effectiveness to its inhibition of MCP- 3 elicited function ( Figure 3B) . Furthermore, MCP-3 (10-76) blocked enzyme release from monocytes.
  • IC 50 values for inhibiting both RANTES or MCP- 3 induced release were around 10 -fold lower than for chemotaxis of either THP-1 cells or monocytes.
  • Another MCP-3 variant, MCP-3 (9-76) was 2-3 fold more potent than MCP- 3 (10-76) in all the inhibition assays.
  • MlP-l ⁇ (10-70) was found to inhibit MlP-l ⁇ or RANTES induced monocyte-N- acetyl - -D- glucosaminidase release with an IC 50 of 1 nM ( Figures 3C-3D) .
  • MlP-l ⁇ (10-70) was also found to inhibit monocyte/THP-1 cell migration.
  • MlP-l ⁇ (11-70) also inhibited both chemoattractant activity and enzyme release of native MlP-l ⁇ and native RANTES with an IC 50 of 30 nM and 10 nM, respectively. Because MlP-l ⁇ antagonists did not inhibit MCP-3 activity, MlP-l ⁇ antagonists are relatively selective for MlP-l ⁇ and RANTES. Antagonists of native RANTES and native MCP-3 have relatively broad inhibitory spectra.
  • MCP- 3, RANTES or MlP-l ⁇ receptor binding are determined by the following protocol.
  • MCP- 3 (10 ⁇ g) were labeled with monoiodinated Bolton-Hunter reagent (specific activity 2,200 Ci/mmol; DuPont, Wilmington, DB) at 4°C for 20 minutes, to provide specific activity of, e.g. 125 I-labeled MCP-3 of 150 Ci/mmol.
  • RANTES and MlP-l ⁇ were labeled using lactoperoxidase.
  • monocytic cells such as THP-1 at (5 x 10 6 cells) in 200 ⁇ l of binding buffer (RPMI 1640, 0.5 mg/ml BSA, 50 mM Hepes and 0.01% Nal) were incubated with varying concentrations of 125 I-MCP-3 at 4°C for 30 minutes. The cells were pelleted through a mixture of diacetylphthalate and dibutylphthalate and radioactivity that is cell associated was counted (total binding) . Nonspecific binding was determined in the presence of a 100 -fold concentration of unlabeled ligand and subtracted from the total binding. Kinetic parameters (Kd and receptor number) were determined by Scatchard analysis.
  • the competition of the MCP- 3 (10-76) analog for labeled MCP-3 was only 2 -fold weaker than that of full-length MCP- 3.
  • MCP-3 and MCP-3 (10-76) had about the same affinity.
  • the results indicate that MCP- 3 (10-76) had similar affinity for the binding sites of all three chemokines. Similar receptor binding affinity data were obtained with MCP- 3 (9-76) .
  • MlP-l ⁇ (11-70) competed only for RANTES and MlP-l ⁇ receptor binding. No competition for MCP- 3 was observed. Therefore MlP-l ⁇ (10-70) has higher inhibitory potency than MlP-l ⁇ (11-70).
  • a further assay that may be carried out to determine whether a non-chemotactic analog binds to, e.g. MCP- 3 receptors is to measure the ability of an analog to desensitize calcium mobilization by MCP-3.
  • the calcium response is temporally desensitized to a second treatment with a MCP- 3 receptor ligand.
  • This is determined by carrying out the aforementioned cytosolic- free calcium assay with addition of a first ligand, followed by a second treatment after 60 seconds using either the same of a different ligand.
  • a MCP- 3 antagonist will not of itself stimulate calcium induction but when used as the first ligand, will desensitize the cells to subsequent stimulation by MCP- 3.
  • the following example illustrates the result. EXAMPLE 4
  • RANTES (9-68) and MCP- 3 (10-76) were tested for their ability to inhibit the transient rise of [Ca 2+ ] induced in monocytes by native RANTES and MCP- 3. In all cases the truncated analogs inhibited the [Ca 2+ ] rise induced by the native chemokines. The most potent effect was observed with RANTES (9-68), which at 30 nM totally prevented the [Ca 2+ ] changes induced by 10 nM RANTES. For inhibition of the responses to MCP- 3, markedly higher concentrations of the corresponding truncated analogs were required.
  • Human RANTES (9 -68) was able to bind to the receptor on mouse peritoneal macrophages. Native human RANTES did not bind well to mouse peritoneal macrophages. Human RANTES (9-68) bound to mouse peritoneal cells with an affinity (Kd) of 21 nM which is similar to the binding affinity of RANTES (9-68) to human monocytes ( Figure 8) . Daily i.p. injections of RANTES (9-68) inhibited the swelling and the incidence of arthritis in adjuvant treated mice. Treated mice exhibited insignificant swelling.
  • MRL-lpr mice Three month old MRL-lpr mice, both male and female were injected at two thoracic sites with complete Freunds Adjuvant (CFA) only once on day 0. On that same day human MCP-3 antagonist MCP-3 (9-76) was injected intraperitoneally. Injections of the MCP- 3 analog were administered daily for 30 days at 3 mg/kg/day. The mice were visually examined (by a double-blind observation) every five days for appearance of arthritis. An animal was scored as positive for arthritis if erythema and swelling of a fore or hind paw was observed. In addition ankle width of the hind legs was assessed. Histopathology of the lime joints were evaluated on day 30 post- adjuvant injection.
  • CFA complete Freunds Adjuvant
  • Human MCP-3 (9-76) was able to bind to the receptor on mouse monocytic cells (WEHl 265 cells) .
  • Human MCP- 3 (9-76) did not bind well to mouse monocytes.
  • Human MCP-3 (9-76) bound to mouse monocytic cells with an affinity (kd) of 57 nM which is four-fold lower than that for native MCP- 3 (kd 225 nM) ( Figure 11) .
  • the inventors have discovered that NH 2 - terminally truncated analogs of native chemokines lack chemoattractant and other activities.
  • the present inventors have also determined that the analogs of the present invention competitively bind to native chemokine receptors, thereby significantly inhibiting or even precluding binding by the corresponding native molecules.
  • the inventors have also discovered a correlation between the bound analogs and their concomitant lack of biological activity. It has now been discovered by the present inventors that the analogs of the present invention are useful in treating inflammatory diseases and autoimmune diseases in mammals.

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Abstract

La présente invention se rapporte à des analogues, tronqués au niveau de terminaisons NH2, de trois chimiokines d'origine humaine: MCP-3, RANTES et MIP-1α, qui sont dotés d'une activité anti-inflammatoire et d'une activité anti-autoimmune très puissantes. La présente invention se rapporte également à un procédé visant à inhiber les activités biologiques des trois chimiokines mammaliennes natives: MCP-3, RANTES et MIP-1α. Elle se rapporte en outre au traitement de maladies inflammatoires et de troubles auto-immuns tels que la polyarthrite rhumatoïde. La présente invention se rapporte également à des compositions pharmaceutiques contenant des analogues de chimiokines tronqués au niveau de terminaisons NH2.
PCT/US1997/014485 1996-08-16 1997-08-18 Antagonistes des recepteurs de mcp-3, rantes et mip-1alpha WO1998006751A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998051705A1 (fr) * 1997-05-12 1998-11-19 Fondazione Centro San Raffaele Del Monte Tabor Peptides presentant une activite antivirale
WO2000016796A1 (fr) * 1998-09-18 2000-03-30 Applied Research Systems Ars Holding N.V. Antagoniste du recepteur de la chimiokine et cyclosporine en therapie combinee
US6362193B1 (en) 1997-10-08 2002-03-26 Smithkline Beecham Corporation Cycloalkenyl substituted compounds
WO2002028419A3 (fr) * 2000-10-04 2002-06-13 Applied Research Systems Mutants de chimiokines intervenant dans le traitement de la sclerose en plaques
WO2001090361A3 (fr) * 2000-05-26 2002-08-08 Univ British Columbia Modulation des effets de protease sur des substrats de chimiokines
US6562832B1 (en) 1997-07-02 2003-05-13 Smithkline Beecham Corporation Substituted imidazole compounds
US6774127B2 (en) 1997-06-13 2004-08-10 Smithkline Beecham Corporation Pyrazole and pyrazoline substituted compounds
US7335350B2 (en) 2002-04-04 2008-02-26 Laboratoires Serono Sa Chemokines mutants having improved oral bioavailability
US8012474B2 (en) 2007-08-02 2011-09-06 Nov Immune S.A. Anti-RANTES antibodies
US20120077733A1 (en) * 2005-10-14 2012-03-29 Rwth Aacehn Antagonists against interaction of pf4 and rantes
WO2018112264A1 (fr) 2016-12-14 2018-06-21 Progenity Inc. Traitement d'une maladie du tractus gastro-intestinal avec une chimoikine/un inhibiteur du récepteur de chimiokine
WO2019229615A1 (fr) 2018-05-28 2019-12-05 Université De Genève Méthodes d'inhibition de l'inflammation cérébrale
WO2020106704A2 (fr) 2018-11-19 2020-05-28 Progenity, Inc. Dispositif ingestible pour administrer un agent therapeutique dans le tractus digestif
WO2021119482A1 (fr) 2019-12-13 2021-06-17 Progenity, Inc. Dispositif ingérable pour administrer un agent thérapeutique dans le tractus gastro-intestinal
EP4252629A2 (fr) 2016-12-07 2023-10-04 Biora Therapeutics, Inc. Procédés, dispositifs et systèmes de détection du tractus gastro-intestinal

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CLARK-LEWIS I ET AL: "STRUCTURE-ACTIVITY RELATIONSHIPS OF CHEMOKINES", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 57, no. 5, May 1995 (1995-05-01), pages 703 - 711, XP000605180 *
GONG E.A.: "RANTES and MCP-3 antagonists bind multiple chemokine receptors", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 271, no. 18, 3 May 1996 (1996-05-03), MD US, pages 10521 - 10527, XP002047804 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998051705A1 (fr) * 1997-05-12 1998-11-19 Fondazione Centro San Raffaele Del Monte Tabor Peptides presentant une activite antivirale
US6774127B2 (en) 1997-06-13 2004-08-10 Smithkline Beecham Corporation Pyrazole and pyrazoline substituted compounds
US6562832B1 (en) 1997-07-02 2003-05-13 Smithkline Beecham Corporation Substituted imidazole compounds
US6362193B1 (en) 1997-10-08 2002-03-26 Smithkline Beecham Corporation Cycloalkenyl substituted compounds
EP1000626A1 (fr) * 1998-09-18 2000-05-17 Applied Research Systems ARS Holding N.V. Antagoniste de récepteur de chemokine et cyclosporine en thérapie combinée
WO2000016796A1 (fr) * 1998-09-18 2000-03-30 Applied Research Systems Ars Holding N.V. Antagoniste du recepteur de la chimiokine et cyclosporine en therapie combinee
US6902728B1 (en) 1998-09-18 2005-06-07 Applied Research Systems Ars Holding N.V. Chemokine receptor antagonist and cyclosporin in combined therapy
WO2001090361A3 (fr) * 2000-05-26 2002-08-08 Univ British Columbia Modulation des effets de protease sur des substrats de chimiokines
WO2002028419A3 (fr) * 2000-10-04 2002-06-13 Applied Research Systems Mutants de chimiokines intervenant dans le traitement de la sclerose en plaques
KR100837898B1 (ko) * 2000-10-04 2008-06-13 라보라토리스 세로노 에스.에이. 다발성 경화증의 치료에서 사용되는 케모킨 변이체
US7402303B2 (en) 2000-10-04 2008-07-22 Laboratoires Serono Sa Chemokine mutants in the treatment of multiple sclerosis
CZ303409B6 (cs) * 2000-10-04 2012-09-05 Merck Serono Sa Zkrácený a mutovaný lidský chemokin
HRP20030215B1 (en) * 2000-10-04 2011-09-30 Laboratoires Serono Sa Chemokine mutants in the tratment of multiple sclerosis
US7335350B2 (en) 2002-04-04 2008-02-26 Laboratoires Serono Sa Chemokines mutants having improved oral bioavailability
US20120077733A1 (en) * 2005-10-14 2012-03-29 Rwth Aacehn Antagonists against interaction of pf4 and rantes
US8501680B2 (en) * 2005-10-14 2013-08-06 Rwth Aachen Antagonists against interaction of PF4 and RANTES
US8012474B2 (en) 2007-08-02 2011-09-06 Nov Immune S.A. Anti-RANTES antibodies
US8673299B2 (en) 2007-08-02 2014-03-18 Novimmune S.A. Anti-RANTES antibodies
EP4252629A2 (fr) 2016-12-07 2023-10-04 Biora Therapeutics, Inc. Procédés, dispositifs et systèmes de détection du tractus gastro-intestinal
US10980739B2 (en) 2016-12-14 2021-04-20 Progenity, Inc. Treatment of a disease of the gastrointestinal tract with a chemokine/chemokine receptor inhibitor
WO2018112264A1 (fr) 2016-12-14 2018-06-21 Progenity Inc. Traitement d'une maladie du tractus gastro-intestinal avec une chimoikine/un inhibiteur du récepteur de chimiokine
WO2019229615A1 (fr) 2018-05-28 2019-12-05 Université De Genève Méthodes d'inhibition de l'inflammation cérébrale
WO2020106704A2 (fr) 2018-11-19 2020-05-28 Progenity, Inc. Dispositif ingestible pour administrer un agent therapeutique dans le tractus digestif
WO2020106757A1 (fr) 2018-11-19 2020-05-28 Progenity, Inc. Dispositif ingérable pour administrer un agent thérapeutique au tube digestif
WO2020106750A1 (fr) 2018-11-19 2020-05-28 Progenity, Inc. Méthodes et dispositifs pour traiter une maladie au moyen d'une biothérapie
WO2020106754A1 (fr) 2018-11-19 2020-05-28 Progenity, Inc. Méthodes et dispositifs pour traiter une maladie à l'aide d'agents biothérapeutiques
WO2021119482A1 (fr) 2019-12-13 2021-06-17 Progenity, Inc. Dispositif ingérable pour administrer un agent thérapeutique dans le tractus gastro-intestinal
EP4309722A2 (fr) 2019-12-13 2024-01-24 Biora Therapeutics, Inc. Dispositif ingérable pour l'administration d'un agent thérapeutique au tractus gastro-intestinal

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