Stable solid formulations of GC-c receptor agonist polypeptide suitable for oral administration

Abstract

To provide stable solid formulations of GC-C receptor agonist polypeptide suitable for oral administration.SOLUTION: Described herein are a stable solid formulation of linaclotide suitable for oral administration, and a method for preparing such formulations. The formulation described herein comprises a polypeptide comprising an amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (linaclotide) or a pharmaceutically acceptable salt thereof. The linaclotide formulation described herein is stable and has an effective period sufficient for preparation, storage and distribution of the drug.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to solid formulations of guanylate cyclase-C receptor agonist polypeptides suitable for oral administration and methods for preparing such formulations.

[0002] Claims of Priority This application claims priority to U.S. Patent Application No. 61 / 089,422, filed Aug. 15, 2008, and U.S. Patent Provisional Application, titled “Stable Solid Formulation of a GC-C Receptor Agonist Polypeptide Suitable for Oral Administration,” filed Aug. 3, 2009. The entire contents of the above applications are incorporated herein by reference.

Background Art

[0003] Many therapeutic polypeptides are formulated in aqueous solution form because they are most active in this form. However, most polypeptides are not particularly stable in aqueous solution, and as a result, formulations often have a short half-life and require refrigeration. Aqueous solutions of polypeptides can be dried by lyophilization, spray drying, or other methods, but such dry formulations are also unstable and may have lower activity compared to aqueous solutions of polypeptides. Typical degradation mechanisms that occur in both aqueous solutions and dry formulations include aggregation and oxidative or hydrolytic degradation. Thus, many therapeutic polypeptides are stored under refrigeration conditions because their safety is limited, whether they are in aqueous solution or dried.

[0004] Linaclotide is a peptide having the amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr that activates guanylate cyclase-C (GC-C) receptor. Linaclotide may be administered orally and is useful for the treatment of gastrointestinal disorders or conditions including irritable bowel syndrome (IBS) and chronic constipation (CC). Preparations containing linaclotide need to be refrigerated to avoid degradation over time. However, refrigeration is inconvenient for both drug distribution and patient storage. Therefore, a solid linaclotide preparation that is stable at room temperature for at least 12 months is needed.

Summary of the Invention

Means for Solving the Problems

[0005] The present invention provides, for example, the following: (Item 1) A pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein after storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months, or (b) after storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 6 months, the decrease in chromatographic purity of the linaclotide is less than 10%. (Item 2) (a) After storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months, or (b) after storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 6 months, the decrease in chromatographic purity of the linaclotide is less than 9%, 8%, 7%, 6%, 5% or 4%, the pharmaceutical composition according to item 1. (Item 3) A unit dosage form of a pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein after storing the unit dosage form in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the unit dosage form in a sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of the linaclotide is less than 10%. (Item 4) (a) The unit dosage form according to item 3, wherein after storing the unit dosage form in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the unit dosage form in a sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of the linaclotide is less than 9%, 8%, 7%, 6%, 5% or 4%. (Item 5) A sealed container containing a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein after storing the sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of the linaclotide is less than 10%. (Item 6) (a) The sealed container according to item 5, wherein after storing the sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of the linaclotide is less than 9%, 8%, 7%, 6%, 5% or 4%. (Item 7) A pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein after storing the pharmaceutical composition in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the pharmaceutical composition in a sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 10%. (Item 8) (a) After storing the pharmaceutical composition in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the pharmaceutical composition in a sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of the linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, the pharmaceutical composition according to item 7. (Item 9) A unit dosage form of a pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein (a) after storing the unit dosage form in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the unit dosage form in a sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 10%, the unit dosage form. (Item 10) (a) After storing the unit dosage form in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the unit dosage form in a sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of the linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, the unit dosage form according to item 9. (Item 11) A sealed container containing a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein (a) after storing the sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of linaclotide in the unit dosage form determined on a weight / weight basis is less than 10%, the sealed container. (Item 12) (a) After storing the sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months, or (b) after storing the sealed container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of the linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%, the sealed container according to item 11. (Item 13) A unit dosage form according to any one of items 3 to 4 or 9 to 10, wherein each unit dosage form contains 50 μg to 2 mg of linaclotide. (Item 14) An airtight container according to any one of items 5 to 6 or 11 to 12, wherein each unit dosage form contains 50 μg to 2 mg of linaclotide. (Item 15) A pharmaceutical composition according to any one of items 1 or 2, wherein after storing the pharmaceutical composition in an airtight container containing a desiccant at 25°C and 60% relative humidity for 24 months, or (b) after storing the pharmaceutical composition in an airtight container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of linaclotide is less than 10%. (Item 16) A unit dosage form according to any one of items 3 or 4, wherein after storing the unit dosage form in an airtight container containing a desiccant at 25°C and 60% relative humidity for 24 months, or (b) after storing the unit dosage form in an airtight container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of linaclotide is less than 10%. (Item 17) An airtight container according to any one of items 5 or 6, wherein after storing the pharmaceutical composition in an airtight container containing a desiccant at 25°C and 60% relative humidity for 24 months, or (b) after storing the pharmaceutical composition in an airtight container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the chromatographic purity of linaclotide is less than 10%. (Item 18) A pharmaceutical composition according to any one of items 7 or 8, wherein after storing the pharmaceutical composition in an airtight container containing a desiccant at 25°C and 60% relative humidity for 24 months, or (b) after storing the pharmaceutical composition in an airtight container containing a desiccant at 40°C and 75% relative humidity for 6 months, the decrease in the assay value of linaclotide is less than 10%. (Item 19) (a) After storing the unit dosage form in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 24 months, or (b) after storing the unit dosage form in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 6 months, the unit dosage form according to any one of items 9 or 10, wherein the decrease in the assay value of linaclotide is less than 10%. (Item 20) (a) After initially storing the sealed container containing a desiccant at 25 °C and 60% relative humidity for 24 months, or (b) after initially storing the sealed container containing a desiccant at 40 °C and 75% relative humidity for 6 months, the sealed container according to any one of items 11 or 12, wherein the decrease in the assay value of linaclotide is less than 10%. (Item 21) A pharmaceutical composition comprising a pharmaceutically acceptable carrier, linaclotide, and (i) a cation selected from Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ , or (ii) one or more agents selected from sterically hindered primary amines, wherein the agent (a) after initially storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months, or (b) after initially storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 6 months, improves at least one attribute of the pharmaceutical composition compared to a pharmaceutical composition without the agent, and the attribute is selected from a decrease in the degradation rate of linaclotide measured by linaclotide content, a decrease in the degradation rate of linaclotide measured by the chromatographic purity of linaclotide, a decrease in the amount of linaclotide oxidation product relative to the amount of linaclotide, a decrease in the amount of linaclotide hydrolysis product relative to the amount of linaclotide, or a decrease in the amount of linaclotide linaclotaldehyde imine product relative to the amount of linaclotide. (Item 22) The agent is Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K+ and Na + or Al 3+ The pharmaceutical composition according to item 21, which is (Item 23) The Mg 2+ and Ca 2+ and Zn 2+ and Mn 2+ and K + and Na + or Al 3+ is provided as magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. The pharmaceutical composition according to item 22. (Item 24) The Mg 2+ and Ca 2+ and Zn 2+ and Mn 2+ and K + and Na + or Al 3+ is provided as magnesium chloride, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, manganese chloride, potassium chloride, sodium chloride, or aluminum chloride. The pharmaceutical composition according to item 23. (Item 25) The agent is Mg 2+ and Ca 2+ or Zn 2+ The pharmaceutical composition according to item 22, which is (Item 26) The Mg 2+ and Ca 2+ or Zn 2+ is provided as magnesium chloride, calcium chloride, or zinc acetate. The pharmaceutical composition according to item 25. (Item 27) The agent is Ca 2+The pharmaceutical composition according to item 22, which is as described above. (Item 28) The Ca 2+ The pharmaceutical composition according to item 27, wherein the Ca is provided as calcium chloride. (Item 29) The pharmaceutical composition according to item 21, wherein the agent is a sterically hindered primary amine. (Item 30) The pharmaceutical composition according to item 29, wherein the sterically hindered primary amine is an amino acid. (Item 31) The pharmaceutical composition according to item 30, wherein the amino acid is a natural amino acid. (Item 32) The pharmaceutical composition according to item 31, wherein the natural amino acid is histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan, or valine. (Item 33) The pharmaceutical composition according to item 32, wherein the natural amino acid is histidine, phenylalanine, leucine, methionine, isoleucine, tryptophan, or valine. (Item 34) The pharmaceutical composition according to item 33, wherein the natural amino acid is leucine, isoleucine, alanine or methionine. (Item 35) The pharmaceutical composition according to item 34, wherein the natural amino acid is leucine or methionine. (Item 36) The pharmaceutical composition according to item 35, wherein the natural amino acid is leucine. (Item 37) The pharmaceutical composition according to item 30, wherein the sterically hindered primary amine is an unnatural amino acid or an amino acid derivative. (Item 38) The pharmaceutical composition according to item 37, wherein the unnatural amino acid is 1-aminocyclohexanecarboxylic acid, lanthinine or theanine. (Item 39) The sterically hindered primary amine is represented by the following formula: [Chemical formula] having, wherein R1, R2 and R3 are independently selected from H, -C(O)OH, optionally -CO2H, -CONH2 or C1-C6 alkyl, C1-C6 alkoxyalkyl, or C1-C6 thioalkoxyalkyl substituted with 5-10 membered aryl or heteroaryl, wherein any of the above alkyl or aryl groups may be mono- or polysubstituted with halogen or -NH2, provided that no more than two of R1, R2 and R3 are H, the pharmaceutical composition according to item 29. (Item 40) The pharmaceutical composition according to item 39, wherein the sterically hindered primary amine is cyclohexylamine or 2-methylbutylamine. (Item 41) The pharmaceutical composition according to item 29, wherein the sterically hindered primary amine is a polymeric amine. (Item 42) The pharmaceutical composition according to item 41, wherein the polymeric amine is chitosan. (Item 43) Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ further comprising, the pharmaceutical composition according to any one of items 29-42. (Item 44) said Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+The pharmaceutical composition according to item 43, which is provided as magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. (Item 45) Said Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ The pharmaceutical composition according to item 44, wherein said Mg, Ca, Zn, Mn, K, Na or Al is provided as magnesium chloride, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, manganese chloride, potassium chloride, sodium chloride or aluminum chloride. (Item 46) Mg 2+ , Ca 2+ or Zn 2+ The pharmaceutical composition according to any one of items 29 to 42, further comprising Mg, Ca or Zn. (Item 47) Said Mg 2+ , Ca 2+ or Zn 2+ The pharmaceutical composition according to item 46, wherein said Mg, Ca or Zn is provided as magnesium chloride, calcium chloride or zinc acetate. (Item 48) Ca 2+ The pharmaceutical composition according to item 46, further comprising Ca. (Item 49) Said Ca 2+ The pharmaceutical composition according to item 48, wherein said Ca is provided as calcium chloride. (Item 50) The pharmaceutical composition according to any one of items 21 to 49, further comprising an antioxidant. (Item 51) The pharmaceutical composition according to item 50, wherein the antioxidant is BHA, vitamin E or propyl gallate. (Item 52) A pharmaceutical composition comprising a pharmaceutically acceptable carrier, linaclotide, Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ selected from the group consisting of cations, and a sterically hindered primary amine. (Item 53) The Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ is provided as magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate, the pharmaceutical composition according to item 52. (Item 54) The Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ is provided as magnesium chloride, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, manganese chloride, potassium chloride, sodium chloride or aluminum chloride, the pharmaceutical composition according to item 53. (Item 55) The pharmaceutical composition according to item 52, wherein the cation is selected from Mg 2+ , Ca 2+ or Zn 2+ selected from the group consisting of. (Item 56) The Mg 2+ , Ca 2+ or Zn 2+ is provided as magnesium chloride, calcium chloride or zinc acetate, and the pharmaceutical composition according to Item 55. (Item 57) The cation is Ca 2+ and the pharmaceutical composition according to Item 55. (Item 58) The Ca 2+ is provided as calcium chloride, and the pharmaceutical composition according to Item 57. (Item 59) The sterically hindered primary amine is an amino acid, and the pharmaceutical composition according to any one of Items 52 to 56. (Item 60) The amino acid is a natural amino acid, and the pharmaceutical composition according to Item 59. (Item 61) The natural amino acid is histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine , isoleucine, tryptophan, methionine or valine, and the pharmaceutical composition according to Item 60. (Item 62) The natural amino acid is histidine, phenylalanine, leucine, methionine, isoleucine, tryptophan, or valine, and the pharmaceutical composition according to Item 61. (Item 63) The natural amino acid is leucine, isoleucine, alanine or methionine, and the pharmaceutical composition according to Item 62. (Item 64) The natural amino acid is leucine, and the pharmaceutical composition according to Item 63. (Item 65) The sterically hindered primary amine is an unnatural amino acid or an amino acid derivative, and the pharmaceutical composition according to Item 52. (Item 66) The pharmaceutical composition according to item 65, wherein the unnatural amino acid is 1-aminocyclohexanecarboxylic acid, lanthanine or theanine. (Item 67) The sterically hindered primary amine is of the following formula:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0006] The linaclotide formulations described herein are stable and have a shelf life sufficient for manufacturing, storing, and distributing the drug. For example, the formulations described herein are predicted to have a shelf life of at least 12 months under room temperature storage conditions (e.g., 25°C / 60% relative humidity (RH)). In further embodiments, the formulations described herein are predicted to have a shelf life of at least 18 months or at least 24 months under room temperature storage conditions (e.g., 25°C / 60% RH).

[0007] In some embodiments, when evaluated by a weight / weight based assay determined by high performance liquid chromatography (HPLC) relative to a linaclotide reference standard, formulations are described in which when package samples are stored under accelerated conditions (40°C / 75% RH), after 3 months, at least 95% of the original amount of linaclotide in the composition remains. In further embodiments, When the package sample is stored under accelerated conditions (40 °C / 75% RH), at least 90% or more of the original amount of linaclotide in the composition remains after at least 6 months. In other embodiments, when the package sample is stored under accelerated conditions (40 °C / 75% RH), there are described formulations in which the chromatographic purity of linaclotide, determined as area % by HPLC, remains at 95% or more over a period of at least 3 months. In a further embodiment, when the package sample is stored under accelerated conditions (40 °C / 75% RH), the chromatographic purity of linaclotide, determined by area % by HPLC, remains at 90% or more over a period of at least 6 months. Thus, for example, linaclotide that undergoes decomposition to other products, such as oxidation products of linaclotide, hydrolysis products of linaclotide, or formaldehyde-mediated imine products of linaclotide (the "formaldehyde imine products"), is about 10% or less.

[0008] In one embodiment, the present invention is a pharmaceutical composition comprising linaclotide, wherein after storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, the decrease in the chromatographic purity of linaclotide is less than 10%. In a further embodiment, after storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4% or 2%. In another embodiment, the present invention is a pharmaceutical composition comprising linaclotide, wherein after storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 10%. In a further embodiment, after storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4% or 2%.

[0009] In one embodiment, the present invention is a unit dosage form of a pharmaceutical composition comprising linaclotide, wherein after storing the unit dosage form in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months or 24 months, the decrease in the chromatographic purity of linaclotide is less than 10%. In a further embodiment, after storing the unit dosage form in a sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months or 24 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4% or 2%. In another embodiment, the present invention is a unit dosage form of a pharmaceutical composition comprising linaclotide, wherein after storing the unit dosage form in a sealed container containing a desiccant at 40°C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 10%. In a further embodiment, after storing the unit dosage form in a sealed container containing a desiccant at 40°C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4% or 2%.

[0010] In one embodiment, the present invention is a sealed container containing a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein after storing the sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months or 24 months, the decrease in the chromatographic purity of linaclotide is less than 10%. In a further embodiment, after storing the sealed container containing a desiccant at 25°C and 60% relative humidity for 18 months or 24 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4% or 2%. In another embodiment, the present invention is a sealed container containing a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein after storing the sealed container containing a desiccant at 40°C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 10%. In a further embodiment, after storing the sealed container containing a desiccant at 40°C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4% or 2%.

[0011] In one embodiment, the present invention is a pharmaceutical composition comprising linaclotide, which, after storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, has a decrease in the assay value of linaclotide determined on a weight / weight basis of less than 10%. In a further embodiment, after storing the pharmaceutical composition in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In another embodiment, the present invention is a pharmaceutical composition comprising linaclotide, which, after storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, has a decrease in the assay value of linaclotide determined on a weight / weight basis of less than 10%. In a further embodiment, after storing the pharmaceutical composition in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, the decrease in the chromatographic purity of linaclotide is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0012] In one embodiment, the present invention is a unit dosage form of a pharmaceutical composition comprising linaclotide, which, after storing the unit dosage form in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, has a decrease in the assay value of linaclotide determined on a weight / weight basis of less than 10%. In a further embodiment, after storing the unit dosage form in a sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In another embodiment, the present invention is a unit dosage form of a pharmaceutical composition comprising linaclotide, which, after storing the unit dosage form in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, has a decrease in the assay value of linaclotide determined on a weight / weight basis of less than 10%. In a further embodiment, after storing the unit dosage form in a sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0013] In one embodiment, the present invention is a sealed container containing a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the sealed container containing a desiccant is stored at 25 °C and 60% relative humidity for 18 months or 24 months, and the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 10%. In a further embodiment, after storing the sealed container containing a desiccant at 25 °C and 60% relative humidity for 18 months or 24 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In another embodiment, the present invention is a sealed container containing a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the sealed container containing a desiccant is stored at 40 °C and 75% relative humidity for 3 months or 6 months, and the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 10%. In a further embodiment, after storing the sealed container containing a desiccant at 40 °C and 75% relative humidity for 3 months or 6 months, the decrease in the assay value of linaclotide determined on a weight / weight basis is less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0014] In some embodiments, linaclotide and the following:

[0015] [Chemical formula]

[0016] A pharmaceutical composition is provided that includes a hydrolysis product containing the same.

[0017] In some embodiments, the hydrolysis product comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, or less than about 5% by weight of the composition. In other embodiments, the hydrolysis product comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition. In further embodiments, there is provided a method of treating a gastrointestinal disorder in a patient in need thereof, comprising administering a pharmaceutical composition comprising linaclotide and a hydrolysis product.

[0018] In some embodiments, linaclotide and the following:

[0019]

Chemical formula

[0020] There is provided a pharmaceutical composition comprising a formaldehyde imine product comprising the same.

[0021] In some embodiments, the formaldehyde imine product comprises less than about 15% by weight of the composition, less than about 10% by weight of the composition, less than about 7% by weight of the composition, or less than about 5% by weight of the composition. In other exemplary embodiments, the formaldehyde imine product comprises from about 0.01% to about 15% by weight of the composition, from about 0.05% to about 10% by weight of the composition, from about 0.05% to about 7% by weight of the composition, or from about 0.05% to about 5% by weight of the composition. In further embodiments, there is provided a method of treating a gastrointestinal disorder in a patient in need thereof, comprising administering a pharmaceutical composition comprising linaclotide and a formaldehyde imine product.

[0022] In some embodiments, a pharmaceutical composition comprising linaclotide and a linaclotide oxidation product is provided. In one embodiment, the linaclotide oxidation product has a molecular weight of 1542.8 and is most likely formed by adding a single oxygen atom to one of the six cysteinyl sulfurs in linaclotide. One possible structure of the product is shown below:

[0023] [Chemical Formula]

[0024] as shown, but those skilled in the art will recognize that the oxygen atom could be attached to any of the other five sulfur atoms.

[0025] In another embodiment, two or more oxygen atoms may be added to linaclotide, whereby its molecular weight will increase by 16 AU per oxygen atom added.

[0026] In some embodiments, the linaclotide oxidation product comprises less than about 15 wt%, less than about 10 wt%, less than about 7 wt%, or less than about 5 wt% of the composition. In other exemplary embodiments, the linaclotide oxidation product comprises from about 0.01 wt% to about 15 wt%, from about 0.05 wt% to about 10 wt%, from about 0.05 wt% to about 7 wt%, or from about 0.05 wt% to about 5 wt% of the composition. In a further embodiment, a method of treating a gastrointestinal disorder in a patient in need thereof is provided, comprising administering a pharmaceutical composition comprising linaclotide and a linaclotide oxidation product.

[0027] The assay value based on weight / weight (“weight / weight assay”) can be determined, for example, by HPLC, by comparing the amount of linaclotide in the sample with a linaclotide reference standard. As used herein, after storage at room temperature or accelerated conditions, at a specific time point (e.g., storage for 3 or 6 months under accelerated conditions [40°C / 75% RH], or storage for 12, 18 or 24 months under room temperature conditions [25°C / 60% RH]), the weight of linaclotide in the composition is compared with the weight of linaclotide in the composition at the initial time (e.g., when the pharmaceutical composition is released for clinical or patient use (release date)) to provide a weight / weight assay value. For example, after storage for a specific time under accelerated conditions (40°C / 75% RH), the weight of linaclotide in the composition is measured and compared with the weight of linaclotide present in the sample at the release date. In another example, after storage for a specific time under room temperature conditions (25°C / 60% RH), the weight of linaclotide in the composition is measured and compared with the weight of linaclotide present in the sample at the release date. Thus, the expression “when the package sample is stored under accelerated conditions (40°C / 75% RH) for at least 6 months, at least 90% of the original amount of linaclotide in the composition remains” means that after storage for at least 6 months under accelerated conditions, the weight of linaclotide in the composition measured in a weight / weight based assay determined by HPLC is at least 90% of the amount of linaclotide in the composition present at the initial time (e.g., the release date of the linaclotide composition).

[0028] The chromatographic purity of linaclotide can be evaluated by performing HPLC under the conditions described herein. Measure the area under the linaclotide peak and compare it to the total area under all peaks excluding the solvent peak and any peaks not associated with the polypeptide (i.e., peaks associated with excipients that can be observed in placebo). As used herein, after storage at room temperature or accelerated conditions, at a specific time point (e.g., 3 or 6 months of storage under accelerated conditions [40°C / 75% RH], or 12, 18, or 24 months of storage under room temperature conditions [25°C / 60% RH]), compare the chromatographic purity of linaclotide in the composition to the chromatographic purity of linaclotide in the composition at the initial time (e.g., when the pharmaceutical composition is released for clinical or patient use (release date)) to provide a chromatographic purity value. For example, after storing for a specific time under accelerated conditions (40°C / 75% RH), measure the chromatographic purity of linaclotide in the composition and compare it to the chromatographic purity of linaclotide in the composition at the release date. In another example, after storing for a specific time under room temperature conditions (25°C / 60% RH), measure the chromatographic purity of linaclotide in the composition and compare it to the chromatographic purity of linaclotide in the composition at the release date.

[0029] The present disclosure relates to (a)(i) linaclotide or a pharmaceutically acceptable salt thereof, (ii) Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+A solution comprising a cation selected from the group consisting of, and / or a sterically hindered primary amine (e.g., leucine), and optionally, (iii) a pharmaceutically acceptable binder, such as an aqueous solution (the "coating solution"), and (b) applying the coating solution to a pharmaceutically acceptable filler to produce a polypeptide-coated filler (e.g., by spraying, mixing or coating the pharmaceutically acceptable filler with the coating solution), a method for preparing a pharmaceutical composition comprising linaclotide or a pharmaceutically acceptable salt thereof. The method may optionally include (i) mixing the polypeptide-coated filler with a pharmaceutically acceptable glidant, a pharmaceutically acceptable lubricant or a pharmaceutically acceptable additive acting as both a glidant and a lubricant, (ii) mixing the polypeptide-coated filler with an uncoated filler, (iii) mixing the polypeptide-coated filler with other additives, (iii) applying a pharmaceutically acceptable coating additive to the polypeptide-coated filler, and may include one or more of the above. The final pharmaceutical composition can be placed in a capsule (e.g., a gelatin capsule) or used to form a tablet. The final pharmaceutical composition can be placed in a capsule (e.g., a gelatin capsule) or used to form a tablet.

[0030] Mg 2+ Ca 2+ Zn 2+ Mn 2+ K + Na + or Al 3+ Cations selected from are found to be useful in suppressing the formation of oxidation products of linaclotide during storage. Sterically hindered primary amines, such as leucine, are also found to be useful in suppressing the formation of formaldehyde imine adducts ("formaldehyde imine products") of linaclotide during storage. Thus, Mg 2+ Ca 2+ Zn 2+ Mn 2+ K + Na + or Al 3+A cation selected from (e.g., Zn 2+ , Mg 2+ or Ca 2+ a divalent cation selected from), and / or a sterically hindered primary amine, e.g., a linaclotide formulation containing an amino acid, has a sufficient shelf life for manufacturing, storing, and distributing the drug (as measured by chromatographic purity and / or by weight / weight assay). Further, the presence of only the sterically hindered amine may increase the formation of hydrolysis products of linaclotide during storage, but a combination of a sterically hindered primary amine and a cation, e.g., the combination of leucine and Ca 2+ , suppresses the formation of hydrolysis products of linaclotide and oxidation products of linaclotide during storage, and the overall stability determined by weight / weight assay and / or chromatographic purity is even greater.

[0031] In some embodiments, a pharmaceutical composition is provided that includes a pharmaceutically acceptable carrier, linaclotide, and one or more agents selected from Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ , and a sterically hindered primary amine, wherein the agent improves at least one attribute of the composition relative to a pharmaceutical composition that does not have the agent. In further embodiments, the agent is Mg 2+ , Ca 2+ or Zn 2+ . In further embodiments, the agent is Ca 2+It is. In some embodiments, the cation is, but not limited to, magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. In further embodiments, the cation is provided as magnesium chloride, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, manganese chloride, potassium chloride, sodium chloride or aluminum chloride. In other embodiments, the cation is provided as calcium chloride, magnesium chloride or zinc acetate.

[0032] In another embodiment, the agent is a sterically hindered primary amine. In further embodiments, the sterically hindered primary amine is an amino acid. In still further embodiments, the amino acid is a natural amino acid. In even further embodiments, the natural amino acid is selected from the group consisting of histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan, methionine and valine. Still further, the natural amino acid is leucine, isoleucine, alanine or methionine. In another embodiment, the natural amino acid is leucine or methionine. Even further, the natural amino acid is leucine. In another embodiment, the sterically hindered primary amine is an unnatural amino acid or an amino acid derivative (e.g., 1-aminocyclohexanecarboxylic acid, lanthionine or theanine). In further embodiments, the sterically hindered primary amine is cyclohexylamine, 2-methylbutylamine or chitosan. In further embodiments, the sterically hindered primary amine is cyclohexylamine, 2-methylbutylamine or chitosan.

[0033] In other embodiments, a pharmaceutically acceptable carrier, linaclotide, Mg 2+ 、Ca 2+, Zn 2+ , Mn 2+ , K + , Na + or Al 3+ A cation selected from (e.g., Zn 2+ , Mg 2+ or Ca 2+ ), and a sterically hindered primary amine is provided. In one embodiment, the cation is Ca 2+ . In another embodiment, the cation is a mixture of two or three of Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ (e.g., a mixture of two or three of Zn 2+ , Mg 2+ or Ca 2+ ). In a further embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable binder and / or an additive that acts as both a pharmaceutically acceptable glidant and lubricant and / or an antioxidant. In a further embodiment, the sterically hindered primary amine is an amino acid. In yet a further embodiment, the amino acid is a natural amino acid. In an even further embodiment, the natural amino acid is selected from the group consisting of histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan, methionine and valine, and yet further, the natural amino acid is leucine, isoleucine, alanine or methionine, in another embodiment, the natural amino acid is leucine or methionine, and yet further, the natural amino acid is leucine. In another embodiment, the sterically hindered primary amine can be a mixture of more than one sterically hindered primary amine. For example, the sterically hindered primary amine can be a mixture of two or more sterically hindered primary amines, e.g., a mixture of two or more amino acids.

[0034] In some cases, the molar ratio of cation: sterically hindered primary amine: linaclotide (e.g., Ca 2+ : leucine: linaclotide) in the aqueous solution applied to the carrier is 5 to 100: 5 to 50: 1. The molar ratio of cation: sterically hindered primary amine (e.g., Ca 2+ : leucine) may desirably be 2: 1 or more (e.g., 5: 1 to 2: 1). Thus, in some cases, the molar ratio of cation: sterically hindered primary amine: linaclotide (e.g., Ca 2+ : leucine: linaclotide) applied to the carrier is 100: 50: 1, 100: 30: 1, 80: 40: 1, 80: 30: 1, 80: 20: 1, 60: 30: 1, 60: 20: 1, 50: 30: 1, 50: 20: 1, 40: 20: 1, 20: 20: 1, 10: 10: 1, 10: 5: 1 or 5: 10: 1. When a binder, e.g., methylcellulose, is present in the linaclotide solution applied to the carrier, it can be present at 0.5 wt% to 2.5 wt% (e.g., 0.7% to 1.7% or 0.7% to 1% or 1.5% to 0.7%).

[0035] The weight of linaclotide applied to a given weight of filler (e.g., microcrystalline cellulose) can vary from about 0.02: 100 to about 2.67: 100. Thus, about 0.05 mg to about 6.0 mg of linaclotide can be applied to 225 mg of filler. In a further embodiment, the weight of linaclotide applied to a given weight of filler is about 0.05 mg to about 2.0 mg of linaclotide (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 mg of peptide for 225 mg of filler).

[0036] In various embodiments, the sterically hindered primary amine is an amino acid (e.g., a natural amino acid or a natural amino acid selected from histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, methionine, asparagine, tyrosine, threonine, leucine, isoleucine, tryptophan, or valine). In other cases, the sterically hindered primary amine is an unnatural amino acid or an amino acid derivative (e.g., lanthionine, theanine or 1-aminocyclohexane). In other cases, the sterically hindered primary amine is an aminosugar (e.g., chitosan or glucosamine).

[0037] In some cases, the sterically hindered primary amine has the following formula:

[0038] [Chemical formula]

[0039] wherein R1, R2 and R3 are independently selected from H, -C(O)OH, optionally -CO2H, -CONH2 or C1-C6 alkyl, C1-C6 alkoxyalkyl, or C1-C6 thioalkoxyalkyl substituted with a 5-10 membered aryl or heteroaryl, wherein any of the above alkyl or aryl groups may be mono- or multi-substituted with halogen or -NH2, provided that no more than two of R1, R2 and R3 are H. In a further embodiment, no more than one of R1, R2 and R3 is H.

[0040] As used herein, the term "alkyl" refers to a saturated straight-chain or branched-chain monovalent hydrocarbon group. Unless otherwise specified, an alkyl group contains from 1 to 20 carbon atoms (e.g., from 1 to 20 carbon atoms, from 1 to 10 carbon atoms, from 1 to 8 carbon atoms, from 1 to 6 carbon atoms, from 1 to 4 carbon atoms or from 1 to 3 carbon atoms). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, etc.

[0041] C n-m "Alkoxyalkyl" and C n-m The term "thioalkoxyalkyl" means an alkyl substituted with one or more alkoxy or thioalkoxy groups, and in some cases, the combined total number of carbon atoms of the alkyl group and the alkoxy group, or the alkyl group and the thioalkoxy group, is in some cases a value between n and m. For example, C 4-6 Alkoxyalkyl has a total of 4 to 6 carbons divided into an alkyl part and an alkoxy part, and can be, for example, -CH2OCH2CH2CH3, -CH2CH2OCH2CH3 or -CH2CH2CH2OCH3.

[0042] As used herein, the term "aryl" (such as in the case of "aryl ring" or "aryl group"), when used alone or as part of a larger moiety, refers to a carbocyclic system in which at least one ring is aromatic and has a single point of attachment to the remainder of the molecule. Unless otherwise specified, an aryl group can be monocyclic, bicyclic or tricyclic and contains from 6 to 18 ring members. Examples of aryl rings include, but are not limited to, phenyl, naphthyl, indanyl, indenyl, tetralin, fluorenyl, and anthracenyl.

[0043] The term "heteroaryl" (or "heterocyclic aromatic" or "heteroaryl group" or "aromatic heterocycle"), used alone or as part of a larger moiety, as seen in "heteroalkyl" or "heteroarylalkoxy", refers to a ring system in which at least one ring is aromatic, contains one or more heteroatoms, each ring in the system contains 3 to 7 ring members, and has a single point of attachment to the remainder of the molecule. Unless otherwise noted, the heteroaryl ring system can be monocyclic, bicyclic or tricyclic and contains a total of 5 to 14 ring members. In one embodiment, all of the rings in the heteroaryl system are aromatic. Heteroaryl radicals in which the heteroaryl ring is fused to one or more aromatic or non-aromatic carbocyclic or heterocyclic rings, or combinations thereof, are also included in this definition, provided that the radical or point of attachment is within the heteroaryl ring. A bicyclic 6.5 heterocyclic aromatic system, as used herein, is, for example, a 6-membered heterocyclic aromatic ring fused to a second 5-membered ring, with the radical or point of attachment on the 6-membered ring.

[0044] Examples of the heteroaryl ring include the following monocyclic rings: 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl), 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl), triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (e.g., 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, 1,3,5-triazinyl, and the following bicyclic rings: benzimidazolyl, benzofuryl, benzothiophenyl, benzopyrazinyl, benzopyranonyl, indolyl (e.g., 2-indolyl), purinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl, 4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl), but are not limited thereto.

[0045] In various cases, the antioxidant is selected from BHA (butylhydroxyanisole), BHT (butylhydroxytoluene), vitamin E, propyl gallate, ascorbic acid and its salts or esters, tocopherol and its esters, alpha-lipoic acid, beta-carotene, the pharmaceutically acceptable binder is polyvinyl alcohol or polyvinylpyrrolidone, the pharmaceutically acceptable binder is selected from starch (e.g., corn starch, pregelatinized potato starch, rice starch, wheat starch and sodium starch glycollate), maltodextrin or cellulose ethers (e.g., methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose), the pharmaceutically acceptable filler is cellulose (e.g., microfine cellulose or microcrystalline cellulose, e.g., Celphere CP-305 or Avicel), the pharmaceutically acceptable filler is sugar or sugar alcohol (e.g., mannitol, isomalt, sorbitol, dextrose, xylitol, sucrose and lactose), the filler contains particles having an average diameter of 50 μm to 1000 μm, the lubricant and / or glidant is selected from talc, leucine, magnesium stearate, stearic acid and polyvinyl alcohol, the lubricant and / or glidant is selected from calcium stearate, mineral oil, vegetable oil, polyethylene glycol (PEG, e.g., PEG that is liquid or solid at room temperature), sodium benzoate, and sodium lauryl sulfate.

[0046] In some cases, the linaclotide solution used in the method for preparing the formulation has a pH of less than 7 (e.g., a pH of 1 to 3 or a pH of about 1.5 to about 2.5). The pH can be adjusted, for example, using phosphoric acid. In some cases, the solution is buffered. Various pharmaceutically acceptable buffers (e.g., phosphate buffer) can be used.

[0047] In some cases, the linaclotide solution used in the method for preparing the formulation contains both a cation (e.g., CaCl2) and a sterically hindered primary amine (e.g., leucine).

[0048] In some cases, the linaclotide solution contains CaCl2 and leucine, the binder is methylcellulose, the filler is microcrystalline cellulose, and the glidant and / or lubricant contains talc or leucine.

[0049] Also provided are pharmaceutical compositions prepared by any of the methods described herein.

[0050] In another aspect, a pharmaceutical composition comprising a pharmaceutically acceptable carrier, linaclotide and (i) a cation selected from Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+ or (ii) one or more agents selected from sterically hindered primary amines is disclosed. In some embodiments, the pharmaceutical composition comprises at least one cation and at least one sterically hindered primary amine.

[0051] Also described is a method of using a pharmaceutical composition to treat various gastrointestinal disorders.

Brief Description of the Drawings

[0052]

Figure 1

Embodiments for Carrying Out the Invention

[0053] This figure is provided by way of example and is not intended to limit the scope of the present invention.

[0054] Oral compositions containing linaclotide can be used to treat various gastrointestinal disorders. In various embodiments, the patient has a gastrointestinal disorder, and the patient has a gastrointestinal motility disorder, chronic pseudo-obstruction, pseudo-colonic obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, functional dyspepsia, non-ulcer dyspepsia, functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GERD), gastroparesis, irritable bowel syndrome, postoperative ileus, ulcerative colitis, chronic constipation, constipation, pain associated with constipation, and disorders and conditions associated with constipation (e.g., constipation associated with the use of opioid analgesics, postoperative constipation, and constipation associated with neuropathy, as well as other conditions and disorders described herein), and the patient has a disorder selected from the group consisting of, and the patient has a gastrointestinal motility disorder, chronic pseudo-obstruction, pseudo-colonic obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, functional dyspepsia, non-ulcer dyspepsia, functional gastrointestinal disorder, functional heartburn, gastroesophageal reflux disease (GERD), gastroparesis, inflammatory bowel disease, irritable bowel syndrome (e.g., diarrhea-predominant irritable bowel syndrome (d-IBS), constipation-predominant irritable bowel syndrome (c-IBS) and / or alternating irritable bowel syndrome (a-IBS)), postoperative ileus, ulcerative colitis, chronic constipation, constipation, pain associated with constipation, and disorders and conditions associated with constipation (e.g., constipation associated with the use of opioid analgesics, postoperative constipation, and constipation associated with neuropathy, as well as other conditions and disorders described herein), and the patient is diagnosed with a functional gastrointestinal disorder according to the Rome Criteria (e.g., Rome II), and the patient is diagnosed with irritable bowel syndrome (e.g., (e.g., diarrhea-type IBS, constipation-type IBS and / or alternating-type IBS) according to the Rome Criteria (e.g., Rome II).

[0055] The dosage range of linaclotide for adults is generally, orally, 25 μg to 6 mg per day. In further embodiments, the dosage range is orally, 25 μg to 2 mg per day. In some embodiments, the dosage range for adults is orally, 50 μg to 1 m g (e.g., 50 μg, 67.5 μg, 100 μg, 133 μg, 150 μg, 200 μg, 250 μg, 266 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg or 1 mg). In a further embodiment, the dosage range is from 100 μg to 600 μg per day orally. In other embodiments, the dosage is 50 μg, 67.5 μg, 100 μg, 133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg or 600 μg of linaclotide per day orally. In one embodiment, the linaclotide composition is provided in separate units, unit dosage forms (e.g., tablets, capsules, sachets) that are effective at such dosages or as multiples of such dosages. In certain embodiments, the unit dosage form and the daily dosage are equal. In various embodiments, the unit dosage form is administered with food at any time of the day, without food at any time of the day, or with food after an overnight fast (e.g., with breakfast). In various embodiments, the unit dosage form is administered once a day, twice a day, or three times a day. The unit dosage form may optionally contain other additives. In some embodiments, one, two or three unit dosage forms contain the daily oral dosage of linaclotide. The exact amount of the compound administered to the patient will be the responsibility of the attending physician. However, the dosage used will depend on many factors including the patient's age and gender, the exact disorder being treated, and its severity.

[0056] In one embodiment, a method for treating irritable bowel syndrome with constipation (IBS-c) in adult patients in need of such treatment is provided, which comprises administering, once daily, an effective amount of the pharmaceutical composition described herein to adult patients in need of such treatment. In various embodiments, the pharmaceutical composition contains 133 μg or 266 μg of linaclotide per unit dose per day. In other embodiments, the pharmaceutical composition is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or a period exceeding that. In some embodiments, treatment with the linaclotide composition improves at least one symptom selected from reduction of abdominal pain, increase in the number of completely spontaneous bowel movements (CSBM) during one week, increase in the number of spontaneous bowel movements (SBM) during one week, improvement in stool consistency, reduction of defecation straining, reduction of abdominal discomfort, reduction of abdominal distension or reduction of the severity of IBS-c symptoms.

[0057] In one embodiment, a method for treating chronic constipation in adult patients in need of such treatment is provided, which comprises administering, once daily, an effective amount of the pharmaceutical composition described herein to adult patients in need of such treatment. In various embodiments, the pharmaceutical composition contains 133 μg or 266 μg of linaclotide per unit dose per day. In other embodiments, the pharmaceutical composition is administered for at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or a period exceeding that. In some embodiments, treatment with the linaclotide composition improves at least one symptom selected from increase in the number of completely spontaneous bowel movements (CSBM) during one week, increase in the number of spontaneous bowel movements (SBM) during one week, improvement in stool consistency, reduction of defecation straining, reduction of abdominal discomfort, reduction of abdominal distension or reduction of the severity of constipation.

[0058] The firmness of feces in each bowel movement can be observed using the 7-point Bristol Stool Form Scale (BSFS) (1 = hard lumps, 2 = sausage-shaped with lumps, 3 = cracked sausage-shaped, 4 = smooth sausage-shaped, 5 = soft lumps, 6 = mushy, 7 = watery). The ease of defecation can be observed using the 7-point Ease of Passage Scale (1 = need for manual disimpaction / enema, 2 = severe difficulty in defecation, 3 = moderate difficulty in defecation, 4 = mild difficulty in defecation, 5 = no difficulty in defecation, 6 = urgency, 7 = fecal incontinence). CSBM can be measured by the feeling of complete emptying after SBM (yes / no). Abdominal discomfort, abdominal distension, and the severity of constipation can be measured, for example, using a 5-point ordinal scale (1 = none, 2 = mild, 3 = moderate, 4 = severe, 5 = very severe).

[0059] The cations of the present invention can be provided as pharmaceutically acceptable salts, i.e., cations having appropriate counterions. Examples of pharmaceutically acceptable salts that can be used in the present invention include, but are not limited to, magnesium acetate, magnesium chloride, magnesium phosphate, magnesium sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate, zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium sulfate, aluminum acetate, aluminum chloride, aluminum phosphate, or aluminum sulfate. In some embodiments, pharmaceutically acceptable salts include calcium chloride, calcium carbonate, calcium acetate, magnesium chloride, magnesium acetate, zinc acetate, and zinc chloride. In further embodiments, pharmaceutically acceptable salts that can be used are calcium chloride, magnesium chloride, and zinc acetate.

[0060] As used herein, the term "binder" refers to any pharmaceutically acceptable binder that may be used in the practice of the present invention. Examples of pharmaceutically acceptable binders include starches (e.g., corn starch, potato starch, and pregelatinized starches (e.g., STARCH 1500® and STARCH 1500 LM® sold by Colorcon, Ltd. or other starches)), maltodextrin, gelatin, natural and synthetic gums such as acacia, tragacanth powder, guar gum, cellulose and its derivatives (e.g., methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, and hydroxypropylmethylcellulose (hypromellose), ethylcellulose, cellulose acetate, calcium carboxymethylcellulose, sodium carboxymethylcellulose, carboxymethylcellulose, microcrystalline cellulose (e.g., AVICEL® sold by FMC Corporation, Marcus Hook, PA, USA, e.g., AVICEL-PH-101®, AVICEL-PH-103®, and AVICEL-PH-105®)), polyvinyl alcohol, polyvinylpyrrolidone (e.g., polyvinylpyrrolidone K30), and mixtures thereof, but are not limited thereto.

[0061] As used herein, the term "filler" refers to any pharmaceutically acceptable filler that may be used in the practice of the present invention. Examples of pharmaceutically acceptable fillers include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g., granules or powder), microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305), powdered cellulose, dextrate, kaolin, mannitol, silicic acid, sorbitol, starch (e.g., Starch 1500), pregelatinized starch, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, xylitol, myo-inositol, and mixtures thereof.

[0062] Examples of pharmaceutically acceptable fillers that may be particularly used for coating with linaclotide include, but are not limited to, talc, microcrystalline cellulose (e.g., Avicel PH101 or Celphere CP-305), powdered cellulose, dextrate, kaolin, mannitol, silicic acid, sorbitol, starch, pregelatinized starch, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, dibasic calcium phosphate, raffinose, maltitol, melezitose, stachyose , lactitol, palatinite, xylitol, mannitol, myo-inositol, and mixtures thereof.

[0063] As used herein, the term "additive" refers to any pharmaceutically acceptable additive. Pharmaceutically acceptable additives include, but are not limited to, disintegrants, dispersion additives, lubricants, glidants, antioxidants, coating additives, diluents, surfactants, flavoring additives, wetting agents, absorption promoting additives, release control additives, solidification inhibiting additives, antibacterial agents (e.g., preservatives), coloring agents, desiccants, plasticizers, and dyes.

[0064] As used herein, "excipient" is any pharmaceutically acceptable additive, filler, binder or agent.

[0065] As used herein, "purified linaclotide" is linaclotide that is 90% or more pure or 95% or more pure, or a pharmaceutically acceptable salt thereof. In some embodiments, the linaclotide used in the methods and compositions described herein is purified. Linaclotide purity can be measured, for example, by the chromatographic purity of linaclotide using reverse-phase HPLC as described in Example 21. The linaclotide assay (w / w) can be determined, for example, by external calibration using a reference standard by using reverse-phase HPLC as described in Example 21.

[0066] In one aspect, a pharmaceutical composition can be prepared by spraying a solution comprising linaclotide or a pharmaceutically acceptable salt thereof onto a pharmaceutically acceptable filler to produce a linaclotide-coated filler. In one embodiment, the method comprises (a)(i) linaclotide or a pharmaceutically acceptable salt thereof, (ii) Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al 3+A solution (a "coating solution") comprising a cation selected from

[0067] and / or a sterically hindered primary amine (e.g., leucine), and optionally (iii) a pharmaceutically acceptable binder, e.g., an aqueous solution, and (b) applying the coating solution to a pharmaceutically acceptable filler to produce a polypeptide-coated filler (e.g., by spraying, mixing or coating the pharmaceutically acceptable filler with the coating solution). The method can optionally include one or more of (i) mixing the polypeptide-coated filler with a pharmaceutically acceptable glidant, a pharmaceutically acceptable lubricant or a pharmaceutically acceptable additive that acts as both a glidant and a lubricant, (ii) mixing the polypeptide-coated filler with an uncoated filler, (iii) mixing the polypeptide-coated filler with other additives, and (iv) applying a pharmaceutically acceptable coating additive to the polypeptide-coated filler. The final pharmaceutical composition can be placed in a capsule (e.g., a gelatin capsule) or used to form a tablet.

[0068] Thus, the present disclosure provides (a) (i) linaclotide or a pharmaceutically acceptable salt thereof, and (ii) Mg 2+ , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Al3+ providing a solution, such as an aqueous solution or an organic solution, containing a cation selected from and / or a sterically hindered primary amine (e.g., leucine), and (b) spray-drying the linaclotide-containing solution to produce microparticles, a method for preparing a pharmaceutical composition comprising linaclotide or a pharmaceutically acceptable salt thereof. The linaclotide-containing solution may optionally contain a polymer, such as one or more of the binders described herein, a lipid or phospholipid, and / or a filler, such as mannitol. The method may optionally include one or more additional steps of (i) mixing the linaclotide microparticles with a pharmaceutically acceptable glidant, a pharmaceutically acceptable lubricant or a pharmaceutically acceptable additive acting as both a glidant and a lubricant, (ii) mixing the microparticles with a filler, and / or (iii) mixing the microparticles with other additives. The final pharmaceutical composition can be placed in capsules (e.g., gelatin capsules) or used to form tablets.

[0069] In other embodiments, the pharmaceutical composition is prepared by spray freeze-drying, supercritical fluid treatment or freeze-drying of a solution containing (i) linaclotide or a pharmaceutically acceptable salt thereof, and (ii) a cation selected from Mg 2+ Ca 2+ Zn 2+ Mn 2+ K + Na + or Al 3+ and / or a sterically hindered primary amine (e.g., leucine), such as an aqueous solution or an organic solution.

[0070] In some embodiments, the linaclotide composition is provided in a solid form for oral administration. Examples of such forms include, but are not limited to, tablets, sachets, pellets, capsules or powders. In some embodiments, the composition can be used to manufacture unit dosage forms, such as tablets, capsules, sachets or pellets. The composition for oral administration can include, for example, binders, lubricants, inert diluents, lubricating additives, surfactant additives or dispersing additives, flavor additives, and wetting agents. Oral dosage forms, such as tablets, can optionally be coated or scored and formulated to provide sustained, delayed or controlled release of the linaclotide therein. Linaclotide can be co-administered with other agents or formulated simultaneously therewith. In one embodiment, the linaclotide composition can be co-administered with other agents used to treat gastrointestinal disorders. The linaclotide composition can also be used to treat disorders other than those of the gastrointestinal tract, such as congestive heart failure and benign prostatic hyperplasia.

[0071] The composition can include, for example, various additional solvents, dispersants, coatings, absorption enhancing additives, release control additives, and one or more inert additives (such as starch, polyols, granulating additives, microcrystalline cellulose, diluents, lubricants, binders, disintegrating additives, etc.). If desired, the tablets of the disclosed composition can be coated by standard aqueous or non-aqueous techniques. The composition can also include, for example, solidification preventing additives, preservatives, sweetening additives, coloring agents, flavoring agents, drying agents, plasticizers, dyes, etc.

[0072] Suitable disintegrants include, for example, agar-agar, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, povidone, polyacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, clays, other algins, other celluloses, gums, and mixtures thereof.

[0073] Suitable lubricants include, for example, calcium stearate, magnesium stearate, mineral oil, light oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and dai z oil), zinc stearate, ethyl oleate, ethyl laurate, agar, colloidal silica gel (AEROSIL 200, W.R. Grace Co., Baltimore, MD USA), coagulated aerosol of synthetic silica (Evonik Degussa Co., Plano, TX USA), pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, MA USA) and mixtures thereof.

[0074] Suitable glidants include, for example, leucine, colloidal silicon dioxide, magnesium trisilicate, powdered cellulose, starch, talc, and tribasic calcium phosphate.

[0075] Suitable anti-caking additives include, for example, calcium silicate, magnesium silicate, silicon dioxide, colloidal silicon dioxide, talc, and mixtures thereof.

[0076] Suitable antibacterial additives that can be used as preservatives for, for example, linaclotide compositions include, for example, benzalkonium chloride, benzethonium chloride, benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol, dehydroacetic acid, ethyl paraben, methyl paraben, phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium sorbate, propyl paraben, sodium benzoate, sodium dehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, and mixtures thereof.

[0077] Suitable coating additives include, for example, sodium carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, methyl cellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax, and mixtures thereof. Suitable protective coatings include Aquacoat (e.g., Aquacoat ethyl cellulose aqueous dispersion, 15% w / w, FMC Biopolymer, ECD-30), Eudragit (e.g., Eudragit E PO PE-EL, Roehm Pharma Polymers) and Opadry (e.g., Opadry AMB dispersion, 20% w / w, Colorcon).

[0078] In certain embodiments, suitable additives for the linaclotide composition include one or more of sucrose, talc, magnesium stearate, crospovidone or BHA.

[0079] In certain embodiments, the term "95%" can be 95.0%, the term "90%" can be 90.0%, the term "10%" can be 10.0%, the term "9%" can be 9.0%, the term "8%" can be 8.0%, the term "7%" can be 7.0%, the term "6%" can be 6.0%, the term "5%" can be 5.0%, the term "4%" can be 4.0%, the term "3%" can be 3.0%, the term "2%" can be 2.0%, and the term "1%" can be 1.0%.

[0080] In certain embodiments, the linaclotide composition is provided in unit dosage forms. In some embodiments, the unit dosage forms are capsules, tablets, sachets, pellets, or powders. In one such embodiment, the unit dosage form is a capsule or a tablet. Such unit dosage forms can be included in a container, e.g., but not limited to, a paper or cardboard box, a glass or plastic bottle or jar, a resealable bag (e.g., to hold “refill” tablets for placement in a different container), or a blister pack having individual dosages for removal from the pack according to a treatment schedule. It is possible to use more than one container together within a single package to provide a single dosage form. For example, tablets or capsules can be placed in one bottle, which is then placed in one box. In some embodiments, the unit dosage form is provided within one container that further contains a desiccant. In further embodiments, a unit dosage form, e.g., a quantity of tablets or capsules, is provided in one container, e.g., a bottle, jar, or resealable bag, that contains a desiccant. In further embodiments, the container containing the unit dosage form is packaged with administration or dosage instructions. In certain embodiments, the linaclotide composition is provided in a kit. The linaclotide compositions and combination therapy agents described herein can be packaged as one kit that includes two or more agents in single or multiple doses, each individually packaged or formulated, or two or more agents in single or multiple doses packaged or formulated together. In this way, the linaclotide composition can be present in a first container, and the kit can optionally include one or more agents in a second container. One or more containers are placed within one package, and the package can optionally include administration or dosage instructions.

Examples

[0081] Examples The following examples are merely illustrative of the invention and are not to be construed as limiting the scope of the invention in any way, as many modifications and equivalents within the invention will be apparent to those skilled in the art upon reading this disclosure.

[0082] Linaclotide or a pharmaceutically acceptable salt thereof can be produced and purified using standard techniques known in the art, such as chemical synthesis or recombinant expression, followed by purification using standard techniques.

[0083] Formulation Scheme A Preparation of Coating Solution Mix about 32 g to 42 g of purified water with hydrochloric acid to produce a solution having a pH of 1.5 to 2.0. If used, the cation is added to the solution in an amount that provides the desired concentration, and the solution is mixed for a time sufficient to produce a clear solution. If used, the sterically hindered primary amine is added to the solution in an amount that provides the desired concentration, and the solution is mixed for a time sufficient to produce a clear solution. Other additives, such as antioxidants, are added thereafter if desired. The pH of the solution is tested and hydrochloric acid is added if necessary to produce a solution having a pH of 1.5 to 2.0. Thereafter, the binder is added to the solution and the mixture is then stirred for a sufficient time to achieve a clear solution. The desired amount of linaclotide is added to the solution and mixed for 30 to 100 minutes to provide a coating solution.

[0084] Preparation of Active Beads Add about 30 to 36 g of dry microcrystalline cellulose beads to a Mini Column Fluid Bed Coater. The microcrystalline cellulose beads are fluidized and heated prior to lamination. Next, the coating solution is laminated onto the beads. The spray temperature is controlled at 24°C to 55°C by controlling the inlet temperature, spray rate, spray pressure, and air volume. After the entire coating solution has been laminated onto the beads, the beads are dried. The product of this process is called active beads.

[0085] Preparation of Active Beads with a Protective Coating Add about 35 g of active beads to a mini-column fluidized bed coater. Fluidize and heat the active beads before coating them with Aquacoat (e.g., Aquacoat ethylcellulose aqueous dispersion, 15% w / w, FMC Biopolymer, ECD-30), Eudragit (e.g., Eudragit E PO PE-EL, Roehm Pharma Polymers), or Opadry (e.g., Opadry AMB dispersion, 20% w / w, Colorcon). Next, laminate the coating solution onto the beads. Control the spray temperature at 24 °C to 55 °C by controlling the inlet temperature, spray rate, spray pressure, and air volume. After laminating the entire coating solution onto the beads, dry the beads.

[0086] Formulation Scheme B Preparation of Coating Solution Mix about 8.3 kg of purified water with hydrochloric acid to produce a solution having a pH of 1.5 to 2.0. If used, add cations to the solution in an amount providing the desired concentration and mix the solution for a time sufficient to produce a clear solution. If used, add the sterically hindered primary amine to the solution in an amount providing the desired concentration and mix the solution for a time sufficient to produce a clear solution. Add other additives, e.g., antioxidants, if desired, thereafter. Then add the binder to the solution and mix the solution for a sufficient time to achieve a clear solution. Test the pH of the solution and add hydrochloric acid if necessary to produce a solution having a pH of 1.5 to 2.0. This is Solution 1. Mix about 8.3 kg of purified water with hydrochloric acid to produce a solution having a pH of 1.5 to 2.0. Add the desired amount of linaclotide to the solution and mix for 10 to 30 minutes. Test the pH of the solution and add hydrochloric acid if necessary to produce a solution having a pH of 1.5 to 2.0. This is Solution 2. Then mix Solution 1 and Solution 2 together. Test the pH of the solution and add hydrochloric acid if necessary to produce a solution having a pH of 1.5 to 2.0. This is the coating solution.

[0087] Preparation of Active Beads Add approximately 24.19 kg of microcrystalline cellulose beads to the Wurster Column of a Glatt GPCG-30 Fluid Bed. Fluidize the microcrystalline cellulose beads, heat them to produce a temperature of 45 - 47 °C. Next, laminate the coating solution onto the beads. Control the product spray temperature to be between 37 °C and 47 °C by controlling the inlet temperature, spray rate, spray pressure, and air volume. After laminating all of the coating solution onto the beads, dry the beads using a product drying temperature of 37 °C - 47 °C. The product of this process is called active beads.

[0088] Examples 1 - 15: Preparation of Linaclotide Formulations The linaclotide formulations of Examples 1 - 15 were produced essentially as described in Formulation Scheme A, where Table 1 provides the amounts of cation, sterically hindered primary amine, binder, linaclotide, and beads, while Table 2 provides the conditions for coating the beads.

[0089]

Table 1 - 1

[0090]

Table 1 - 2

[0091] * "Cation" refers to the divalent cation contained in the salt used in the example, "amine" refers to the sterically hindered primary amine, and [] indicates the molar ratio of cation and / or amine to linaclotide. ** The amount of linaclotide in this and all subsequent examples is determined based on the peptide content and chromatographic purity as listed in the analytical certificate provided for each manufacturing lot of the linaclotide pharmaceutical active ingredient (API).

[0092]

Table 2

[0093] Example 16: Preparation of Linaclotide Formulation The linaclotide formulation of Example 16 was generated essentially as described in Formulation Scheme B, where Table 3 provides the amounts of cation, sterically hindered primary amine, binder, linaclotide and beads, while Table 4 provides the conditions for coating the beads.

[0094] [Table 3]

[0095] [Table 4]

[0096] Example 17: Preparation of Linaclotide Formulation The linaclotide formulation of Example 17 was generated essentially as described in Formulation Scheme A, except that the formulation contains 22.96 mg of butylated hydroxyanisole (BHA), where Table 5 provides the amounts of cation, sterically hindered primary amine, binder, linaclotide and beads, while Table 6 provides the conditions for coating the beads.

[0097] [Table 5]

[0098] [Table 6]

[0099] Example 18: Preparation of Capsules Containing Linaclotide Formulation The linaclotide content on the active beads can be measured as described in Example 21 or by other equivalent methods.

[0100] To form capsules suitable for oral administration, gelatin capsules (e.g., size 2 gelatin capsules) are filled with an appropriate amount of active beads. The appropriate amount of active beads can contain 50 μg to 2 mg of linaclotide, having a range of ±5% per capsule. In some embodiments, the appropriate amount of linaclotide on the active beads can be 50 μg, 67.5 μg, 100 μg, 133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 4 mg or 6 mg. In certain embodiments, the appropriate amount of linaclotide on the active beads is 67.5 μg, 100 μg, 133 μg, 150 μg, 200 μg, 266 μg, 300 μg, 400 μg, 500 μg, 600 μg. In more certain embodiments, the appropriate amount of linaclotide on the active beads is, per capsule, 67 .5 μg, 133 μg, 150 μg, 266 μg or 300 μg.

[0101] In another embodiment, an appropriate amount of active beads is placed in a container to fill a desired number of gelatin capsules. One or more pharmaceutically acceptable fillers or other pharmaceutically acceptable additives can be added to the container, if desired. In some embodiments, the filler or additive is talc, leucine, microcrystalline cellulose or mannitol. The contents of the container are mixed and the mixture is used to fill gelatin capsules with an appropriate amount of active beads containing linaclotide (e.g., 50 μg to 2 mg of linaclotide per capsule having a range of ±5%).

[0102] In another embodiment, gelatin capsules are filled with an appropriate amount of active beads and one or more pharmaceutically acceptable fillers or other pharmaceutically acceptable additives are added to the gelatin capsules.

[0103] Example 19: Preparation of Capsules Containing Linaclotide Formulation Preparation of Coating Solution First, 41.98 g of purified water was mixed with 1.13 g of hydrochloric acid to produce a solution having a pH of 1.5 to 2.0. Next, 7.49 g of calcium chloride dihydrate and 6.68 g of leucine were added to the solution, which was then mixed for 30 minutes to produce a clear solution. The pH was tested and 1.70 g of hydrochloric acid was added to produce a solution having a pH of 1.5 to 2.0. Next, 13.27 g of hypromellose (hydroxypropyl methylcellulose, Dow Chemical Company, Midland, MI) was added to the solution and the mixture was stirred for 60 minutes to achieve a clear solution. Next, 4.39 g of linaclotide was added to the solution and mixed for 90 minutes. The pH of the solution was 1.73. This was used as the coating solution.

[0104] Preparation of Active Beads 674.5 g of microcrystalline cellulose beads (Celphere CP-305; Asahi Kasei Corporation (Tokyo; Japan)) were added to the Wurster Column of a Glatt GPCG-2 Fluid Bed. The microcrystalline cellulose beads were fluidized and heated at a product temperature of 60 °C for 30 minutes. Next, the coating solution was layered onto the beads. The product temperature was controlled at 45 °C to 49 °C with an inlet temperature of 80 °C, a spray rate of 5.0 to 11 g / min, a spray pressure of 2.0 bar, and an air volume of 40 to 50 m 3 h. After the entire coating solution was layered onto the beads, the beads were dried for 10 minutes using a product temperature of 46.9 °C to 50.9 °C. The product of this process was called the active beads.

[0105] Reverse-phase liquid chromatography of linaclotide extracted from the formulation prepared as described above demonstrated that the extracted linaclotide and the linaclotide reference standard showed the same retention time and that there was no significant change in purity as a result of the formulation process.

[0106] To form the capsules, 49.50 g of activated beads were added to a transparent bag. Next, 0.25 g of leucine, sorted through a 60-mesh screen, was added to the bag. The bag was tied and mixed 125 revolutions to mix all the materials. Next, 0.25 g of talc, sorted through a 60-mesh screen, was added to the bag. The bag was tied and mixed 125 revolutions to mix all the materials. As soon as all the materials were mixed, the mixture was used to fill size 2 gelatin capsules with a target weight of 227 mg / capsule having a range of ±5%.

[0107] Example 20: Preparation of Capsules Containing Linaclotide Formulation Activated beads were prepared according to Example 16. The activated beads were tested for linaclotide content. Based on the assay method of the activated beads, using an MG2 Futura encapsulation machine , an appropriate amount of activated beads (96 mg to 123 mg) was filled into size 2 hard gelatin capsules to achieve a linaclotide concentration of 300 μg.

[0108] Activated beads were prepared according to Example 15. The activated beads were tested for linaclotide content. Based on the assay method of the activated beads, using an MG2 Futura encapsulation machine, an appropriate amount of activated beads (48 mg to 62 mg) was filled into size 2 hard gelatin capsules to achieve a linaclotide concentration of 150 μg.

[0109] Example 21: Measurement of Linaclotide Content and Purity The content and purity of linaclotide, as well as the measured values of linaclotide-related substances, can be determined by reverse-phase gradient liquid chromatography using an Agilent Series 1100 LC system and Chemstation Rev A.09.03 software or equivalent. Use a YMC Pro™ C18 column (dimensions: 3.0×150 mm, 3.5 μm, 120 Å; Waters Corp., Milford, MA) or equivalent and maintain at 40 °C. Mobile phase A (MPA) consists of water with 0.1% trifluoroacetic acid, while mobile phase B (MPB) consists of 95% acetonitrile: 5% water with 0.1% trifluoroacetic acid. Elution of linaclotide and its related substances is achieved with a gradient of 0% to 47% MPB for 28 minutes, followed by a gradient to 100% MPB for 4 minutes and held at 100% MPB for 5 minutes to wash the column. The column is re-equilibrated by returning to 0% MPB in 1 minute and then held at 100% MPA for 10 minutes. The flow rate is 0.6 mL / min and detection is achieved by UV at 220 nm.

[0110] Samples for analysis are prepared by adding the contents of linaclotide capsules to 0.1 N HCl to obtain a target concentration of 20 μg linaclotide / mL. 100 μL of this solution is injected onto the column.

[0111] The linaclotide content is measured by determining the linaclotide concentration in the prepared samples relative to an externally prepared linaclotide standard prepared in the same manner.

[0112] An example of the analysis of linaclotide by HPLC is shown in Figure 1, where "oxidation" indicates the linaclotide oxidation product, "formaldehyde imine" indicates the linaclotide formaldehyde imine product, and "hydrolysis" indicates the linaclotide hydrolysis product.

[0113] Example 22: Linaclotide formulation stability study In the formulations of Examples 1 to 15 and 17, gelatin capsules were filled with approximately 225 mg of active beads. Five filled capsules were placed in a plastic bottle. The bottle contained 1 - 2 g of desiccant and was induction - sealed. The bottle was stored at 40°C / 75% RH for 6 months.

[0114] The linaclotide content and purity, and the amount of linaclotide - related substances were measured essentially as described in Example 21, or by an equivalent method. The results are shown in Table 7.

[0115]

Table 7 - 1

[0116]

Table 7 - 2

[0117] In the formulation of Example 16, gelatin capsules were filled with approximately 113 mg of total beads. Thirty - five filled capsules were placed in a plastic bottle. The bottle contained 2 g of desiccant and was induction - sealed. The bottle was stored at 40°C / 75% RH for 1 month.

[0118] The linaclotide content and purity, and the amount of linaclotide - related substances can be measured essentially as described in Example 21, or by an equivalent method. The results are shown in Table 8.

[0119]

Table 8

[0120] Example 23: Isolation and Preparation of Linaclotide Hydrolysis Product Conversion of Asn at position 7 to Asp (linaclotide numbering starts from 1 for the N - terminal Cys) results in a linaclotide hydrolysis product. Its structure is shown below.

[0121]

Chemical formula

[0122] The linaclotide hydrolysis product has been independently synthesized for identity confirmation using standard solid-phase peptide synthesis techniques. The linaclotide hydrolysis product can also be prepared by other methods known in the art, for example, by isolation from a linaclotide preparation using chromatography techniques, or by recombinant expression of a nucleic acid encoding the linaclotide hydrolysis product (Cys Cys Glu Tyr Cys Cys Asp Pro Ala Cys Thr Gly Cys Tyr), optionally followed by oxidation of cysteine residues to form disulfide bonds.

[0123] Example 24: Isolation and Preparation of Linaclotide Formaldehyde Imine Product Addition of an imine to the N-terminal Cys (Cys1) via a formaldehyde-mediated reaction results in the formation of a formaldehyde imine product. The proposed structure of the product is shown below.

[0124]

Chemical formula

[0125] The linaclotide formaldehyde imine product has been independently synthesized for identity confirmation by reacting linaclotide with formaldehyde (1:5 molar ratio) in absolute ethanol at room temperature for 4 days. The formaldehyde imine product can also be prepared by other methods known in the art, for example, by isolation from a linaclotide preparation using chromatography techniques, or by chemical peptide synthesis or recombinant expression of a nucleic acid encoding linaclotide, followed by formylation as described herein, or by other methods known in the art, optionally followed by oxidation of cysteine residues to form disulfide bonds.

[0126] Example 25: Isolation and Preparation of Linaclotide Oxidation Product The linaclotide oxidation product has a molecular weight of 1542.8. The oxidation product is most likely formed by adding a single oxygen atom to one of the six cysteinyl sulfurs in linaclotide. One possible structure of the product is shown below, but one of ordinary skill in the art will recognize that the oxygen atom could be bonded to any of the other five sulfurs.

[0127]

Chemical formula

[0128] To confirm this identification, the linaclotide oxidation product was produced by reacting linaclotide with hydrogen peroxide (3% aqueous solution) at room temperature or 40 °C for up to 24 hours. The resulting product is rich in 1 - 10% oxidation product. The linaclotide oxidation product can also be prepared by other methods known in the art, for example, by isolation from a linaclotide preparation using chromatography techniques, or by chemical peptide synthesis or recombinant expression of the nucleic acid encoding linaclotide, followed by oxidation of cysteine residues to form disulfide bonds, and subsequently reacting linaclotide with hydrogen peroxide or a similar oxidizing reagent to form the linaclotide oxidation product.

[0129] Example 26: Linaclotide Tablet Formulation Fluidized Bed Granulation Linaclotide, CaCl2, leucine, and polyvinylpyrrolidone (PVP) K30 were dissolved in 0.0001N HCl to form a coating solution (see Table 9). Isomalt was added to the bowl of the fluidized bed. While fluidizing the isomalt powder, the drug solution was spray - coated onto the top surface at a rate of about 10 g / min with the product temperature set at about 40 °C, and the powder was coated with the coating solution. When spraying was complete, the linaclotide granules were dried for 30 minutes and the product was discharged.

[0130]

Table 9

[0131] Dicalcium phosphate or Avicel was also used as a filler for fluidized bed granulation.

[0132] Wet granulation The weight of linaclotide was weighed and dissolved in 250 g of 0.1 N HCl (pH 1.7) with stirring to form Solution 1 (see Table 10). The weights of CaCl2 and leucine were weighed and dissolved in 100 g of 0.1 N HCl with stirring to form Solution 2. Solutions 1 and 2 were mixed together with stirring to form a coating solution. Avicel was added to the bowl of a high-shear granulator. The coating solution was added into the Avicel while mixing at 500 rpm. When the addition of the solution was completed, the granules were mixed and sheared for 1 minute. The obtained wet granules were added to the bowl of a fluidized bed and dried for 15 minutes, and then the linaclotide granules were discharged.

[0133] [Table 10]

[0134] In wet granulation formulation, the molar ratios of CaCl2 and leucine to linaclotide were adjusted in the ranges of 60 - 100 and 30 - 50, respectively. Also, sucrose was added as an example. See Table 11.

[0135] [Table 11]

[0136] Tablet formulation The linaclotide granules were mixed with the following excipients (see Table 12) and compressed into tablets having a hardness of about 4 kp.

[0137] [Table 12]

[0138] Isomalt, starch 1500 or dicalcium phosphate was also used as a tablet filler based on the above formulation (see Table 13).

[0139]

Table 13

[0140] After storage at 40 °C and 75% relative humidity for 2 weeks, all the tablets described in Table 13 showed an assay value of more than 90% for linaclotide.

[0141] Examples 27 - 53: Preparation of Linaclotide Formulations The linaclotide formulations of Examples 27 - 53 were essentially produced as described in Formulation Scheme A and Examples 1 - 15. The linaclotide coating solution contained 0.7% binder (w / v), and as described in Examples 1 - 15, the coating solution was sprayed onto Celphere CP - 305 beads. Table 14 provides the type of cation, amine, and / or other excipients, their molar ratios to linaclotide, and the type of binder used, while Table 15 provides the conditions for coating the beads.

[0142]

Table 14 - 1

[0143]

Table 14 - 2

[0144]

Table 14 - 3

[0145] *"Cation" refers to the cation contained in the salts used in the examples, "amine" refers to a sterically hindered primary amine, and [molar ratio] refers to the molar ratio of cation:amine:linaclotide:additive (if applicable).

[0146]

Table 15

[0147] Processing problems were experienced during spraying onto the beads in Examples 32 (calcium alginate), 34 (calcium stearate) and 43 (CaCl2:vitamin E). Thus, the coating solution was mixed with Celphere beads and the beads were dried on a tray.

[0148] Example 54: Linaclotide formulation stability test In the formulations of Examples 27 - 53, gelatin capsules were filled with approximately 225 mg of active beads (600 μg linaclotide / capsule). Five filled capsules were placed in a plastic bottle which contained 1 g of desiccant and was induction - sealed. The bottles were stored at 40°C / 75% RH for 3 or 6 months.

[0149] The linaclotide content (μg / mg) and chromatographic purity % (%CP) were measured essentially as described in Example 21 or by an equivalent method. The results are provided in Table 16A (3 - month stability) or Table 16B (6 - month stability).

[0150]

Table 16A

[0151] * The variation in values for the assay method [% w / w from the start] reflects the imperfect control of content uniformity for these small - scale manufactured capsule lots.

[0152] For Examples 32, 34, and 43, the difficulties encountered during processing and the resulting improved processing procedures (see above) would likely be able to explain the lower stability observed in these samples. It is thought that it would be possible to explain.

[0153]

Table 16B-1

[0154]

Table 16B-2

[0155]

Table 16B-3

[0156] The chromatographic purity values of Examples 27 - 53 at the 6-month time point are atypically low, especially when compared to the 3-month time point for these samples. The relative trends for the stabilization or destabilization effects can be established by comparing Examples 27 and 31 as internal reference experiments, where the chromatographic purity values are approximately 6 - 8% lower than those consistently observed in other studies conducted (see, for example, Examples 2 and 9). For the same formulation, the 3-month data provided in Table 16A show more typical chromatographic purity values. Thus, the low chromatographic purity values at 6 months may be due to insufficient desiccant capacity for these specific storage conditions over 6 months. This hypothesis is supported by the impurity peaks observed indicating exposure to moisture.

[0157] Example 55: Stability Test of Linaclotide Formulation at 25°C / 60%RH for 24 Months For the formulations of Examples 8 - 15 and 17, gelatin capsules were filled with approximately 225 mg of active beads. Five filled capsules were placed in a plastic bottle. The bottle contained 1 g of desiccant and was induction-sealed. The bottle was stored at 25°C / 60%RH for 24 months.

[0158] The linaclotide content and purity and the amount of linaclotide related substances were measured essentially as described in Example 21 or by an equivalent method. The results are provided in Table 17.

[0159]

Table 17

[0160] 1) For Example 10, it has an additional protective coating of Aquacoat (Aquacoat aqueous ethylcellulose dispersion, 15% w / w, FMC Biopolymer, ECD-30). 2) For Example 10, it has an additional protective coating of Opadry (Opadry AMB dispersion, 20% w / w, Colorcon). 3) For Example 10, it has an additional protective coating of Eudragit (Eudragit E PO, Degussa, Roehm Pharma Polymers; SLS, Stearic Acid )

[0161] Example 56: Linaclotide Tablet Formulation and Stability Testing Active linaclotide granules were produced by fluid bed granulation using the reagents described in Table 18, essentially as described in Example 26. The linaclotide granules were mixed with the excipients described in Table 19 and compressed into tablets having a hardness of approximately 4 kp.

[0162] Thirty-five tablets were packaged in a 60 cc bottle with 5 g of desiccant and stored at 40°C / 75%RH for a maximum of 3 months or at 30°C / 65%RH for a maximum of 3 months.

[0163] The content and purity of linaclotide and the amount of linaclotide-related substances were measured essentially as described in Example 21 or by an equivalent method. The results are provided in Table 20.

[0164]

Table 18

[0165]

Table 19

[0166]

Table 20

[0167] Example 57: Linaclotide Capsule Formulation The linaclotide formulation of Example 57 was prepared essentially as described in Example 16. Table 21 provides the coating solution components and their theoretical weights (mg / g) and (kg / batch) relative to the complete linaclotide bead drug layer solution. Table 22 provides the components and theoretical weights (mg / g) and (kg / batch) for the preparation of the linaclotide active beads. The linaclotide formulation was encapsulated in hard gelatin capsules, size 2 (weight 61 mg), essentially as described in Example 20. The 150 μg linaclotide capsules contained 56 mg of linaclotide beads (600 μg linaclotide / 225 mg beads), while the 300 μg linaclotide capsules contained 113 mg of linaclotide beads (600 μg linaclotide / 225 mg beads).

[0168]

Table 21

[0169]

Table 22

Claims

【Claim 1】 Half-life.

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