TW201023874A - Stable solid formulation of a GC-C receptor agonist polypeptide suitable for oral administration - Google Patents

Abstract

Solid, stable formulations of linaclotide suitable for oral administration are described herein as are methods for preparing such formulations. The formulations described herein contain a polypeptide consisting of the 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 formulations described herein are stable and have a sufficient shelf life for manufacturing, storing and distributing the drug.

Description

201023874 VI. Description of the Invention: [Technical Field of the Invention] -c receptor agonism The present disclosure relates to a solid formulation of a tobraric acid cyclase polypeptide suitable for oral administration and a method of preparing the same. [Prior Art]

The therapeutic peptides are formulated in aqueous solutions because they are most active in this form. However, 'most polypeptides are not particularly stable in aqueous solution, so that their formulations often have a short half-life and f is cold t although the aqueous solution of the polypeptide can be dried by freeze drying, spray drying or other methods, but this Dry formulations may also be unstable and have reduced activity relative to aqueous solutions of the polypeptide. Typical decomposition mechanisms that occur in aqueous solutions and dry formulations include aggregation and oxidative or hydrolytic degradation. Therefore, most therapeutic polypeptides are stored under refrigeration conditions due to their limited stability, whether in aqueous solution or dry. Linaclotide has an amino acid sequence Cys Cys Glu Tyr

Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr peptide that activates the guanylate cyclase-C (GC-C) receptor. Linaclotide can be administered orally for the treatment of gastrointestinal disorders and disorders including intestinal irritation (IBs) and chronic constipation (CC). Formulations containing linaclotide need to be frozen to prevent degradation over time. However, freezing is inconvenient for the commercial distribution of drugs and patient storage. Therefore, there is a need for solid linaclotide formulations that are stable for at least 12 months at room temperature. SUMMARY OF THE INVENTION Described herein are solid stable formulations of linaclotide suitable for oral administration 142528.doc 201023874 and methods of making such formulations. The formulations described herein contain the amino acid sequence Cys Cys G1U Tyr Cys Cys Asn Pr〇 AU Cys Thr

A polypeptide consisting of Gly Cys Tyr ("nalopeptide") or a pharmaceutically acceptable salt thereof. The linaclotide formulations described herein are stable and have sufficient shelf life to produce, store and distribute the drug. For example, it is contemplated that the formulations described herein have a shelf life of at least 12 months at room temperature storage conditions (eg, 25 (: / 60% relative humidity (RH)). In a further embodiment, the formulation described herein is contemplated. The shelf life of at least 18 months or at least 24 months at room temperature storage conditions (eg, 25 it/6〇% rh). In some embodiments, this formulation is described wherein When the chromatographic (HPLC) determination is relative to the weight/weight determination of the linaclotide reference standard, when the packaged sample is stored under accelerated conditions (40 ° C / 75% RH) for three months, in the composition Residual 95% of the initial amount of linaclotide. In a further embodiment, when the packaged sample is stored under accelerated conditions (40 C / 75% RH) for at least 6 months, 290% remains in the composition. An initial amount of linaclotide. In other embodiments, the formulation is described wherein when the packaged sample is stored under accelerated conditions (40C/75°/.RH) for a period of at least three months, Chromatographic purity of linaclotide as determined by the area percentage of HpLc Still at 295%. In a further embodiment 'when the packaged sample is stored under accelerated conditions (40 ° C / 75% RH) for a period of at least 6 months, as determined by the area percentage of HPLC The chromatographic purity of nalopeptide is still 290% ° so that 'for example, no more than about 丨〇% of linaclotide undergoes degradation, 142528.doc 201023874 becomes another product, such as the oxidation product of linaclotide, linaclotide Hydrolysate or furfural-mediated imine product of linaclotide ("formaldehyde imine product"). In one embodiment, the invention includes a pharmaceutical composition comprising linaclotide, wherein it is dry The chromatographic purity of linaclotide• is reduced by less than 1% after the pharmaceutical composition in the sealed container of the agent is stored for 18 months or 24 months at a relative humidity of 25 C 6 A. In a further embodiment, The chromatographic purity of linaclotide is reduced by less than 9%, 8% after storage of the pharmaceutical composition in a sealed container containing a desiccant at 25: (:, 60 〇 / 〇 relative humidity for 18 months or 24 months, 7%, 6%, 5%, 4% or 2%. In another In an embodiment, the invention comprises a pharmaceutical composition comprising linaclotide, wherein the pharmaceutical composition in a sealed container containing a desiccant is stored at 4 Torr < t, 75% relative humidity for 3 months or 6 months Thereafter, the chromatographic purity of linaclotide is reduced by less than 10%. In a further embodiment, the pharmaceutical composition in a sealed container containing the desiccant is stored at 4 (rc, 75% relative humidity for 3 φ months or After 6 months, the chromatographic purity of linaclotide is reduced by less than 9%, 8〇/〇, 7%, 6%, 5%, 4% or 2%. In one embodiment, the invention includes Linalol A unit dosage form of a pharmaceutical composition of a peptide, wherein the unit dosage form in a sealed container containing a desiccant is stored at 25 C, 60% relative humidity for 18 months or 24 months, and the chromatographic purity of linaclotide is reduced less than 1〇%. In a further embodiment, the unit dosage form in a sealed container containing a desiccant is at 25t, 6〇0/. After 18 months or 24 months of storage at relative humidity, the chromatographic purity of linaclotide is reduced by less than 9%, 8%, 7%, 6%, 5%, 4% or 2%. In another 142528.doc 201023874 embodiment, the invention comprises a unit dosage form of a pharmaceutical composition comprising linaclotide, wherein the unit dosage form in a sealed container containing a desiccant is stored at 40 C, 75% relative humidity After 3 months or 6 months, the chromatographic purity of linaclotide was reduced by less than 1%. In a further embodiment, the chromatographic purity of linaclotide is reduced less than 3 months or 6 months after storage in a unit containing a desiccant in a sealed container at 4 Torr < t, 75% relative humidity. 9〇/〇, 8%, 7%, 6〇/〇, 5% ' 4% or 20/〇. In one embodiment, the invention comprises a sealed container comprising a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the sealed container containing the desiccant is stored at 25 ° C, 60% relative humidity for 18 months. Or after 24 months, the chromatographic purity of linaclotide is reduced by less than 1%. In a further embodiment, the sealed container containing the desiccant is at 25. (:, 603⁄4 storage at relative humidity for 18 months or 24 months, the chromatographic purity of linaclotide is reduced by less than 9% '8%, 7%, 6%, 5%, 4% or 2%. In another In an embodiment, the invention comprises a sealed container comprising a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the sealed container containing the desiccant is stored for 3 months or 6 at a relative humidity of 40 C 75 /〇 After a month, the chromatographic purity of linaclotide is reduced by less than 10%. In a further embodiment, the sealed container containing the desiccant is stored at 4 (rc, 75% relative humidity for 3 months or 6 months, The chromatographic purity of nalopeptide is reduced by less than 9%, 7%, 6%, 5%, 4% or 2%. In one embodiment, the invention comprises a pharmaceutical composition comprising linaclotide, wherein the drying is contained Determination of linaclotide based on the weight/weight 142528.doc 201023874 after the pharmaceutical composition in the sealed container of the agent is stored at 25 ° C, 60 〇 / 〇 relative humidity for 18 months or 24 months. The value is reduced by less than 丨〇%. In a further embodiment, in a sealed container containing a desiccant After the pharmaceutical composition is stored at 25 C, 60 〇 / 〇 relative humidity for 18 months or 24 months, the measured value of linaclotide determined on a weight/weight basis is reduced by less than 9%, 8%, 7%, 6%, 5%, 4〇/〇, 3%, 2% or 1%. In another embodiment, the invention includes a pharmaceutical composition comprising linaclotide in a sealed container containing a desiccant The pharmaceutical composition is reduced by less than 丨〇% based on weight/weight after storage for 3 months or 6 months at a relative humidity of 4〇〇c, 75〇/. In a further embodiment, the chromatographic purity of linaclotide is reduced by less than 9% after storage of the pharmaceutical composition in a sealed container containing the desiccant at 40 ° C, 75% relative humidity for 3 months or 6 months, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In one embodiment, the invention comprises a unit dosage form of a drug group 3 comprising linaclotide, wherein The unit dosage form in the sealed container of desiccant is stored at 25 C, 60% relative humidity for 18 months or 24 months, and the weight is determined based on the weight/weight. The measured value of the peptide is reduced by less than 丨〇%. In a further embodiment, the unit dosage form in a sealed container containing a desiccant is stored at 25 C, 60 〇 / 〇 relative humidity for 8 months or 24 months. The measured value of linaclotide determined on a weight/weight basis is reduced by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%/〇. In another implementation In the present invention, the invention includes a unit dosage form of a pharmaceutical composition comprising linaclotide, wherein the unit dosage form in a sealed container containing a desiccant is stored at a relative humidity of 40 C 754 for 3 months or 6 months, after weight/ Weight 142528.doc 201023874 The determined value of linaclotide is reduced by less than 1%. ^ In a further embodiment, the unit dosage form in a sealed container containing desiccant is at 40 ° C, 75% relative humidity After 3 months or 6 months of storage, the measured value of the Linilo-type determined on a weight/weight basis is less than 9%, 8%, 7%, 6%, 50/〇, 4%, 3%, 20/〇 or 1%. In one embodiment, the invention comprises a sealed container comprising a plurality of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the sealed container in a sealed container containing a desiccant is at 25 ° C, 60% relative humidity After 18 months or 24 months of storage, the measured value of linaclotide determined on a weight/weight basis was reduced by less than 1%. In a further embodiment, the sealed container containing the desiccant has a decrease in the measured value of linaclotide based on weight/weight after storage for 18 months or 24 months at 25 Torr, 60% relative humidity. %, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In another embodiment, the invention comprises a sealed container comprising a plurality of unit dosage forms comprising a pharmaceutical composition of linaclotide, wherein the sealed container containing the desiccant is stored at 40 ° C, 75% relative humidity for 3 months. Or after 6 months, the measured value of linaclotide determined on a weight/weight basis was reduced by less than 1%. In a further embodiment, the sealed container containing the desiccant is stored at 75% relative humidity for 3 months or (10) months, and the measured value of linaclotide is reduced by less than 9/ on a weight/weight basis. 〇, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. In some embodiments, the linaclotide and the hydrolyzed pharmaceutical composition are contained, the hydrolysate comprising: primaries 142528.doc 201023874

H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH-ss-'-fs-s-1 ss In some embodiments, the hydrolysis The product comprises less than about 15% by weight of the composition - less than about 1% 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 hydrolysate comprises from about 0.01% to about 15% by weight of the composition, from about 0.5% to about 1% by weight of the composition, and the weight of the composition is about 5%. 〇/〇 to about 7°/. The composition or the weight of the composition is from about 5% to about 5%. In a further embodiment, a method of treating a gastrointestinal disorder in a patient in need thereof is provided which comprises administering a pharmaceutical composition comprising linaclotide and a hydrolysate. In some embodiments, a pharmaceutical composition comprising a product of linaclotide and a furfural imine comprising:

H2C=Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH S-S-

S-S

S-S

In some embodiments, the furfural imine product comprises less than about 15% by weight of the composition, less than about 丨〇% by weight of the composition, less than about 7% by weight of the composition, or less than the weight of the composition. About 5%. In other embodiments, the circuitary imine product comprises from about 〇0/〇 to about 15% by weight of the composition, from about 5% to about 1% by weight of the composition, of the composition. From about 0.05% to about 7% by weight, or from about 5% to about 5% by weight of the composition. In a further embodiment, there is provided a method of treating a gastrointestinal disorder in a patient in need thereof which comprises administering a pharmaceutical composition comprising linaclotide and a formaldehyde imine product. 142528.doc 201023874 In a two embodiment, a pharmaceutical composition comprising linaclotide and linaclotide oxidative product is provided. In one embodiment, the linaclotide oxidation product has a molecular weight of 1542.8, which is most likely formed when a single oxygen atom is added to one of the six cysteine sulfurs in linaclotide. The potential structure of the product is described below, although those skilled in the art will appreciate that the oxygen atom can be attached to any of the other five sulfurs:

H-Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH

In another embodiment, more than one oxygen atom can be added to linaclotide' each additional oxygen atom will increase its molecular weight by M16au. In some embodiments, the linaclotide oxidation product comprises less than about 15% by weight of the composition, less than about 9% by weight of the composition, less than about 7% by weight of the composition, or the weight of the composition. Less than about 5. In other exemplary embodiments, the linaclotide oxidation product comprises from about 0.01% to about 15% by weight of the composition, and the composition comprises from about 5% to about 1% by weight of the composition, combined From about 0.05% to about 7% by weight of the article, or from about 0.05% to about 5% by weight of the composition. In a further embodiment, there is provided a method of treating a gastrointestinal disorder in a patient in need thereof, comprising administering A pharmaceutical composition of linaclotide and linaclotide oxidation products. Weight/weight based measurements can be determined by comparing the amount of linaclotide in the sample to the linaclotide reference standard (eg, by HPLC). ("Weight/Weight Determination"). As used herein, at a specific point in time after storage at room temperature or accelerated conditions (eg, under accelerated conditions [4〇°c / 75% RH] 142528.doc -10 - 201023874 The weight and start time of linaclotide in a composition for one or two months, or stored at room temperature [25〇C/60% RH] for 12, 18 or 24 months) Or Linalo in the composition of the time when the pharmaceutical composition is released ("release period") Peptide weight comparison to provide weight/reciprocal enthalpy. For example, the weight of linaclotide in the composition is measured after accelerated storage (40 C /75 / RH) for a specific period of time, and with the release period Depends on the weight comparison of linaclotide in the sample. In another example, the weight of linaclotide in the composition is measured after storage for a specific time at room temperature (25. 〇 / 6 〇 % rh) And compared with the weight of linaclotide present in the sample on the release date. Therefore, the short sentence "packaged samples are stored in accelerated conditions (40 C / 75% RH) for at least 6 months, residual in the composition > 〇9〇% of the initial amount of linaclotide means that the weight of the weight/weight based linaclotide measured in the assay, as determined by HPLC, after storage for at least 6 months under accelerated conditions is The starting time (e.g., the release period of the linaclotide composition) is > 90 〇 / 存在 of the amount of linaclotide present in the composition. φ can be evaluated by HPLC under the conditions described herein. Chromatographic purity of nalopeptide. Measurement of peak area of linaclotide and desolvation peak Total area comparison of all peaks except any non-polypeptide related peaks (ie, peaks associated with excipients that may be observed in placebo), as used herein, after storage at room temperature or accelerated conditions At a specific point in time (eg for three or six months under accelerated conditions [40 C /75% RH] or 12, 18 or 24 months at room temperature [25 °C/60% RH]) The peptide purity of the linaclotide in the composition and the starting time (for example, the clinical or patient release time of the pharmaceutical composition when the drug is released ("release date")) in the composition of Linalol 142528.doc 11 201023874 peptide The chromatographic purity is compared to provide chromatographic purity values. For example, the chromatographic purity of linaclotide in the composition is measured after accelerated storage conditions (40 ° C / 75 ° R / RH) for a specific period of time 'and chromatogram of linaclotide in the release date composition' In another example, the chromatographic purity of linaclotide in the composition is measured after storage for a specific period of time under room temperature conditions (25 ports of 6% RH) and is in combination with the release date. Linalo-like chromatographic purity comparison. The present disclosure features a method of preparing a drug containing linaclotide or a pharmaceutically acceptable salt thereof, and "a method of alpha", the method comprising: (4) providing a solution, such as a water solution (coating solution), which comprises: (1) linaclotide or a pharmaceutically acceptable salt thereof; (ii) a cation selected from the group consisting of Mg2+, Ca2+, Ζη2+, 2+, κ+, δ+ or A, and/or sterically hindered primary amine (for example, white And/or (b) applying the coating solution to the pharmaceutically acceptable filler to produce a filling of the polypeptide coating "for example by using a coating ♦ liquid Spraying, mixing or coating a pharmaceutically acceptable filler) The method may optionally comprise one or more of the following steps: (1): a polypeptide-coated filler and a pharmaceutically acceptable slip agent (coffee) ―, a pharmaceutically acceptable lubricant or a pharmaceutically acceptable additive that acts as both a slip agent and a lubricant; (H) a polypeptide-coated filler and a #peptide & clothing@filler Mixing '((1)) to mix the polypeptide-coated filler with other additives, (4) taking the medicine An acceptable coating additive is applied to the polypeptide coated filler. The final pharmaceutical composition can be encapsulated (eg, a gelatin capsule) or used to form a tablet. It has been found to be selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+. The cation of dAi3+ is useful for inhibiting the formation of oxidation products of linaclotide during storage by 142528.doc 201023874. It has also been found that steric hindrance of primary amines, such as leucine, against linaclotide during storage The formation of a road imine adduct ("formic acid imine product") is useful. Therefore, it contains a cation selected from the group consisting of Mg2+, Ca2+, Zn2+, Μη, Κ, Na or Α1 (for example, selected from Zn2+, Mg2+ or Ca2+). A divalent cation) and/or a linaclotide formulation that sterically hinders a primary amine (such as an amino acid) has a sufficient shelf life (as measured by chromatographic purity and/or by weight/weight measurement) for production, storage And distribute drugs.

Furthermore, although only the sterically hindered amine can increase the formation of the hydrolysate of linaclotide during storage, the steric hindrance of the combination of primary amine and cation (eg, a combination of leucine and Ca 2+ ) inhibits the sap during storage The hydrolysate and the formation of the oxidation product of linaclotide produce even better overall stability as measured by weight/weight and/or by chromatographic purity. In some embodiments, 'provided with a pharmaceutically acceptable carrier, linaclotide and one or more agents selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+, K+, Na, A13, stereopsis and primary amines A pharmaceutical composition wherein the agent is at least one of the characteristics of the composition relative to the pharmaceutical composition without the agent. In an advanced embodiment, the agent is ton 2+, ah or Zn 2+ . In a further camp, Gu's / Vision, the agent is Ca2+. In some embodiments 'providing cations with cerium, but not limited to, acyl acetate, magnesium sulphate, magnesium sulphate, magnesium sulphate, samarium red & η Α ^ calcium acetate, calcium carbonate, calcium phosphate, sulphate , zinc acetate, zinc sulphate, zinc sulphate, zinc sulphate, manganese acetate, manganese chloride, manganese phosphate, manganese sulfate, acetic acid clock, τ gasification, potassium silicate, sulfuric acid _, sodium acetate, Sodium chloride, sodium sulphate, sodium sulphate, aluminum acetate, aluminum sulphate, aluminum sulphate or aluminum sulphate. In a further enrichment scheme, the cation 142528.doc 201023874 is provided as magnesium gasification, calcium carbonate, calcium phosphate, calcium sulfate, acetic acid, manganese chloride, potassium carbonate, sodium vaporification or aluminum sulfide. In other embodiments, the cation is provided as calcium chloride, magnesium chloride or acetate. In another embodiment, the agent is a sterically hindered primary amine. In a further embodiment, the steric hindrance to the primary amine is an amino acid. In still a further embodiment, the amino acid is a naturally occurring amino acid. In yet a further embodiment, the naturally occurring amino acid is selected from the group consisting of histidine, phenylalanine, alanine, glutamic acid, aspartic acid, acetoic acid, leucine, methyl sulfide a group consisting of aminic acid, aspartic acid, tyrosine, sulphate, isoleucine, tryptophan, methionine and valine; further, the naturally occurring amine group The acid is leucine, isoleucine leucine, alanine or guanidine thiocyanate; in another embodiment, the naturally occurring amino acid is leucine or guanidine thioglycol; further, the The naturally occurring amino acid is leucine. In another embodiment, the sterically hindered primary amine is a non-naturally occurring amino acid or amino acid derivative (e.g., hydrazine-aminocyclohexanecarboxylic acid, lanthionine or theanine). In a further embodiment, the steric hindrance primary amine is cyclohexylamine, 2-methylbutylamine or polyglucosamine. In other embodiments, a cation comprising a pharmaceutically acceptable carrier, linaclotide, selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+, K+, Na+, or Al3+ (eg, selected from the group consisting of Zn2+, Mg2+, or Ca2+) is provided. Divalent cations) and pharmaceutical compositions that sterically hinder primary amines. In one embodiment, the cation is Ca2+. In another embodiment, the cation is a mixture of two or three of Mg2+, Ca2+, Zn2+, Mn2+, K+, Na+, or Al3+ 142528.doc 201023874 (eg, Zn2+, M 2+$ - a mixture of two or three of the steps (four) & In the formula, the pharmaceutical composition further comprises a pharmaceutically acceptable dry agent, and, or a pharmaceutically acceptable slip agent, an additive that acts as both a kinetic agent and a lubricant, and/or an antioxidant In an advanced embodiment, the steric hindrance of the primary amine is an amino acid. In yet another embodiment of the advancement, the amino acid is a naturally occurring amino acid. In a further embodiment of the fork, the naturally occurring

The amino acid is selected from the group consisting of histidine, alanine alapropionate, alanine, amylate, amylin, leucine, methionine, asparagine, tyrosine, sulphate Acid, isoleucine leucine, tryptophan, methionine and proline "and" & έβ, ed, and, further, the naturally occurring amino acid leucine Acid, isoleucine leucine, alanine or methionine; in another embodiment 'the naturally occurring amino acid is leucine or methionine, progressing further, the naturally occurring amine The base acid is leucine. In another embodiment, the steric hindrance primary amine can be a mixture of more than one steric hindrance primary amine. For example, the steric hindrance primary amine may be a mixture of two or more sterically hindered primary amines, such as a mixture of two or more amino acids, under certain circumstances, the cations applied to the aqueous solution of the carrier. : Stereoscopic hinderance of primary amine: The molar ratio of linaclotide (eg Ca2+: leucine: linaclotide) is 5-100:5-50:1. It is expected that the cation: the steric hindrance of the primary amine (e.g., Ca2+: leucine) has a molar ratio equal to or greater than 2:1 (e.g., between 5:1 and 丨). Thus 'in some cases, the cation applied to the carrier: sterically hindered primary amine: linaclotide (eg Ca2+: leucine: linaclotide) Mo 142528.doc -15· 201023874 ear ratio is 100:50 :1, ι〇〇:3〇:ι 60:30:1, 60:20:1,5〇:3〇·ΐ 80:40:1,80:30:1,80:20:1, 50: 20:1, 40:20:1, 20:20:1, 10:10:1, 10:5:1 or 5:l〇.i. The person in charge, ^,, 0, 1 w dry mixture (such as methyl cellulose) is present in the linaclotide solution applied to the carrier, which can be 5% to 5% by weight ( For example, Ο.?%."% or 〇7%]% or 15% or 〇(d) exists. The weight of linaclotide applied to a given weight of filler (eg microcrystalline cellulose) can be from about 〇· 〇2:1〇0 to about 2.67·1〇〇. Therefore, about 5' mg to about 6.0 mg of linaclotide can be applied to 225 〇^ of filler. In embodiments, the weight of linaclotide applied to a given weight of filler is from about 0.05 mg to about 2.0 mg of linaclotide (eg, for a 225 mg filler, (M, 〇.2, 〇) 3, 〇4, 〇5, 〇6, 〇7 mg peptide) 不同 In various embodiments: sterically hindered primary amine is an amino acid (such as a naturally occurring amino acid or selected from histidine, amphetamine alaninate) Acid, alanine, amygic acid, aspartic acid, glacial acid, methionine, aspartic acid, tyrosine, threonine, leucine, isoleucine leucine, Tryptamine _ acid or guanamine A naturally occurring amino acid of an acid. In other instances, the tertiary amine is a non-naturally occurring amino acid or amino acid derivative (eg, lanthionine, theanine or 1-aminocyclohexanide). In other cases, the sterically hindered primary amine is an amino sugar (eg, polyglucosamine or glucosamine). r2 In some cases, the sterically hindered primary amine has the formula: Rl\s|^R3, wherein Nh2 R!, R2*R3 are independently selected from: Η, C(0)0H, C1-C6 alkyl, C1- 142528.doc -16- 201023874 C6 alkyl ether, Cl·(10) base money, C1_C6 county (four), Ci c6 alkyl carboxy guanamine and alkyl aryl, any group in # may be substituted by a single or multiple halogen or amino group, and provided that no more than two of R1, R#R3 are H. In a progressive implementation In the scheme, no more than one of 'R, R> R3 is Η. Under different conditions, the antioxidant is selected from the group consisting of hydrazine (butylated hydroxyanisole), and (butylated toluene), and vitamins. Ε, gallic acid, vinegar, anti-ascorbic acid and its salts or esters, tocopherol and its esters, α-lipoic acid, b carrot

The pharmaceutically acceptable binder is polyvinyl alcohol or polyvinylpyrrolidone, and the pharmaceutically acceptable binder is selected from the group consisting of starch (for example, corn starch, pre-gelatinized potato powder, rice starch, wheat flour and丨 粉 powder sodium glycolate), maltodextrin or cellulose ether (such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, propyl Cellulose and hydroxypropyl fluorenyl cellulose); pharmaceutically acceptable filler 疋 cellulose (for example, micro cellulose (micr〇fjne ceUul〇se) or microcrystalline cellulose, such as Celphere CP_3 〇5 or Avicel); Pharmaceutically acceptable fillers are sugars or sugar alcohols (eg, mannitol, is maltitol, sorbitol, dextrose, xylitol, sucrose, and lactose); Particles of average diameter between 50 μm and 1000 μm; lubricants and/or slip agents selected from the group consisting of: talc, leucine, magnesium stearate, stearic acid and polyvinyl alcohol; and lubricants and/or slip agents Selected from: stearic acid, dish oil, vegetable oil, poly Ethylene glycol (PEG; for example, PEG which is liquid or solid at room temperature), sodium benzoate and sodium lauryl sulfate. In some cases, the linaclotide solution used in the method of preparing the formulation has a pH below 7 (eg, a pH between 1 and 3, or about 1.5 and about 142528.doc -17. 201023874 2.5 PH). The pH can be adjusted, for example, by phosphoric acid. In some cases, the solution is buffered. Different pharmaceutically acceptable buffers (e.g., phosphate buffers) can be used. In some cases, the linaclotide solution used in the method of preparing the formulation comprises a cation (e.g., CaCl2) and a sterically hindered primary amine (e.g., leucine). In some cases, the linaclotide solution comprises CaCl2 and leucine; the binder is sulfhydryl cellulose; the filler is microcrystalline cellulose; the slip agent and/or lubricant comprises talc or leucine. Pharmaceutical compositions prepared by any of the methods described herein are also provided. Another aspect 'discloses a pharmaceutically acceptable carrier, linaclotide and a cation selected from (i) selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+, K+, Na+ or Al3+, or (ii) a sterically hindered primary amine A pharmaceutical composition of one or more agents. In some embodiments, the pharmaceutical composition contains at least one cation and at least one sterically hindered primary amine. Methods of treating various gastrointestinal disorders using pharmaceutical compositions are also described. Oral compositions containing linaclotide can be used to treat a variety of gastrointestinal conditions. In various embodiments, the patient has a gastrointestinal disorder; the patient has a condition selected from the group consisting of gastrointestinal motility disorder, chronic intestinal pseudo-occlusion, colon pseudo-obstruction, Crohn's disease, duodenogastric reflux , dyspepsia, functional dyspepsia, non-ulcer dyspepsia, functional gastrointestinal disorders, functional heartburn, gastroesophageal reflux disease (GERD), gastroparesis, intestinal irritation, postoperative intestinal obstruction, ulcer Colitis, chronic 142528.doc 201023874 Secret, constipation, pain associated with constipation, and conditions and conditions associated with constipation (eg constipation associated with the use of opioid painkillers, post-surgical constipation, psychosis-related Constipation, as well as other conditions and conditions described herein; patients with gastrointestinal motility disorders, chronic intestinal pseudo-occlusion, colonic pseudo-obstruction, Crohn's disease, duodenogastric reflux, dyspepsia, dysfunctional dyspepsia , non-ulcer dyspepsia, functional gastrointestinal disorders, functional heartburn, gastroesophageal reflux disease (GERD), gastroparesis, inflammatory Disease, intestinal irritation (eg diarrhea-type intestinal irritation (d_I)BS), constipation-type intestinal irritation (c-IBS) and/or alternating intestinal irritation (a_IBS)), surgery Post-intestinal obstruction, ulcerative colitis, chronic constipation, constipation, pain associated with constipation, and conditions and conditions associated with constipation (eg constipation associated with the use of sputum painkillers, constipation after surgery, psychosis) Constipation, as well as other conditions and conditions described herein; patients have been diagnosed with functional gastrointestinal disorders according to Roman guidelines (eg Rome II); patients have been diagnosed with intestinal irritation according to Roman guidelines (eg Rome II) (eg abdominal gastrointestinal tract irritable bowel syndrome, constipation-type intestinal irritation and/or alternating intestinal motility

, Kg, 950 pg or 1 mg). Pg, 750 pg, 800 pg, 850 pg, 9 bark, 67.5 pg, 1〇〇, 133, 266, 300, 350 pg, 0 '600 μδ . 650 μβ > 700 142528.doc •19· 201023874 In a further embodiment, the oral dosage range is from 1 to 600 pg per day. In other embodiments, the oral dose of linaclotide is 50, 67.5, 1 〇〇, 133, 15 〇 μ, 2 每,

266 pg, 300 handsome, 400, 5 or 6. In one embodiment, the linaclotide composition is provided as an isolated unit, unit dosage form (eg, a tablet, capsule, sachet) that is effective at such doses or in multiple suitable doses. of. 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, with no food administration at any time of the day, and with food (e.g., with breakfast) after an overnight fast. In various embodiments, the unit dosage form is administered once a day, twice a day, or three times a day. The unit dosage form can optionally include additional additives. In some embodiments, one, two or three unit dosage forms will contain a daily oral dose of linaclotide. The exact amount of compound administered to the patient will be the responsibility of the attending physician. However, the dosage used will depend on a number of factors, including the age and sex of the patient, the particular condition being treated, and the severity thereof. "

In one embodiment, a method of treating secretory intestinal irritation (IBS-c) in an adult patient in need thereof is provided, comprising administering to a patient an effective amount of a pharmaceutical composition described herein once a day. In a different embodiment, the pharmaceutical composition comprises 133 Å per unit dose per day or the addition of nalopeptide. In other embodiments, the pharmaceutical composition is administered for at least two days, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks or longer. In some embodiments, treatment with a linaclotide composition improves at least a symptom selected from the group consisting of 142528.doc • 20-201023874 remission of abdominal pain, increase in total spontaneous defecation (CSBM) & Increased number of spontaneous defecations (SBM), improved stool consistency, relieved stool remission, relieved abdominal discomfort, relieved bloating, or severed IBS_c symptoms within one week. In one embodiment, a method of treating chronic constipation in an adult patient in need thereof is provided, which comprises administering to the patient an effective amount of a pharmaceutical composition described herein once daily. In various embodiments, the pharmaceutical composition comprises 133 pg or 266 pg of linaclotide per unit dose per day. In other embodiments, the pharmaceutical composition is administered for at least one, two, three, four, five, six, one, two, three, four or more periods. In some embodiments, treatment with a linaclotide composition improves at least one symptom selected from the group consisting of: an increase in the number of complete spontaneous defecation (CSBM) within one week' an increase in the number of spontaneous defecation (SBM) within a week Improved stool consistency, relieved bowel movements, relieved abdominal discomfort, relieved bloating or relieved constipation severity. The fecal consistency of each BM can be passed through the 7-point Bristol Stool Scale (BSFS) (1 = lumps, 2 = multiple sausages, 3 = ruptured sausages, 4 = smooth sausages) 5 = soft blocks 6 = Paste, 7 = water sample) monitoring. Defecation effort can pass the Ease of Passage Scale by 7 points (l = need to manually release the plug / enema, 2 = severe defecation effort, 3 = moderate defecation effort) 4 = slight defecation effort, 5 = no defecation effort, 6 = urgency, 7 = incontinence) to monitor. The CSBM can be measured by the sensation of complete emptying after SBM (yes/no). Abdominal discomfort, bloating, and constipation severity can be used, for example, 142528.doc •21- 201023874 2=slight, 3=moderate 5-point sequential scale (1 = no weight) measurement. 4 = severe, 5 = very severe = a clear cation can be provided as a pharmaceutically acceptable salt, i.e. a cation having a suitable counterion. 7Tenjin & pharmaceutically acceptable salts for use in the present invention, but not limited to magnesium acetate, magnesium sulfate, magnesium phosphate, calcium sulfate sulfate, calcium phosphate, sulfuric acid sulfate, manganese acetate, gas Manganese gasification unloading, potassium silicate, sulfuric acid _ sodium sulphate, aluminum acetate, aluminized magnesium sulphate, acetic acid, sulphate, acetic acid, sodium sulphate, sodium sulphate, sodium sulphate, gasification Acid or sodium aluminum sulfate. In some embodiments, pharmaceutically acceptable salts include calcium carbonated calcium carbonate, calcium acetate, magnesium chloride, magnesium acetate, zinc acetate, and zinc vapor. > In a further embodiment, the pharmaceutically acceptable salts that can be used are gas < good, gasified towns and zinc acetate.

As used herein, the term "adhesive" refers to any grammatically acceptable adhesive that can be used in the practice of the present invention. Examples of pharmaceutically acceptable binders include, but are not limited to, starch (e.g., corn starch, potato starch, and pre-paste).

Huadian powder (such as STARCH 1500® and STAHCH 1500 LM® sold by Colorcon, Ltd.) and other starches, maltodextrin, gelatin, natural and synthetic gums (such as gum arabic), powdered tragacanth, guar gum 'Cellulose and its derivatives (eg sulfhydryl cellulose, hydroxyethyl cellulose, ethyl decyl cellulose, hydroxypropyl cellulose and hydroxypropyl methyl cellulose (hydroxypropyl cellulose), B Cellulose, cellulose acetate, carboxymethylcellulose dance, sodium carboxymethylcellulose, carboxymethylcellulose, microcrystalline weaving (eg FMC Corporation, Marcus Hook, PA, USA sold 142528.doc - 22-201023874 AVICELtm, such as AVICEL pH1〇1TM, 〇3tm and (7)5tm)), polyvinyl alcohol, polyvinylpyrrolidone (for example polyvinylpyrrolidone κ3〇) and mixtures thereof. As used herein, the term "filler" refers to a pharmaceutically acceptable filler that can be used in the practice of the present invention. Examples of pharmaceutically acceptable binders include, but are not limited to, talc, calcium carbonate (e.g., granules or powders), hydrogen phosphate, tristearate, sulfuric acid (e.g., granules or powders), microcrystalline cellulose ( For example, AWcel® 101 or CP-305), powdered cellulose, glucose binder, kaolin, mannitol, money, sorbitol, starch (eg SUrch 1500), pregelatinized starch, lactose, glucose, fructose, galactose, algae Sugar, liga, maltose, isomaltitol, raffinose, maltitol, melezitose, stachyose, lactitol, palatinite, xylitol, inositol, and mixtures thereof. Examples of pharmaceutically acceptable fillers that may be particularly useful for coating with linaclotide include, but are not limited to, talc, microcrystalline cellulose (eg, fine (pH) (8) or "Celphere CP_3G5"), powdered cellulose, glucose binder , kaolin 'mannitol, aspartic acid, sorbitol, starch, pre-gelatinized powder, lactose, glucose, fructose, galactose, trehalose, sucrose, maltose, isomalt, acid hydrogen mother, Raffinose, maltose, pine fructose, lactitol, bismuth-based rock, xylitol, mannitol, a mixture. Heroes = used in this article, the term "additives" - pharmaceutically acceptable additives. Pharmaceutically acceptable additives, including but not limited to disintegrants, eight-dispersion additives, lubricants, slip agents, antioxidants, coating additives, 142,528.doc -23-201023874 release agents, surfactants, flavoring additives, Wetting agents, absorption enhancing additive controlled release additives, anti-caking additives, antimicrobial agents (eg preservatives), colorants, desiccants, plasticizers and dyes. As used herein, "excipient" is any pharmaceutically acceptable additive, filler, binder or agent. As used herein, purified linaclotide is a linaclotide having a purity greater than or equal to 90% or a purity greater than or equal to 95%, or a pharmaceutically acceptable salt thereof, in an H embodiment as described herein. The granule peptide used in the purification is the purity of the purified narlocone, for example, using the reversed-phase Hpu described in Example 2, as measured by the chromatographic purity of linaclotide. The linaclotide assay [w/w] can be determined, for example, by using a reverse phase muscle having a reference standard external calibration as described in Example 2A. >7 \S)

The hydrazine 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: U) providing a solution, such as an aqueous solution ("coating solution"), comprising 2: + (1) linaclotide or a pharmaceutically acceptable salt thereof; (8) selected from _ +, : - (for example leucine) and 'optionally (10) pharmaceutically acceptable and (b) applying the coating solution to a pharmaceutically acceptable filler with:: a coated filler (eg By spraying and mixing a chemically acceptable filler with a coating solution, the method may optionally comprise one or more of the following: (1) filling the polypeptide with a filler and pharmacy 142528.doc •24· 201023874 Acceptable sliding sword, pharmacy, learned lubricant, or a combination of a slip agent and a pharmaceutically acceptable additive that acts as both, (9) a polypeptide-coated filler with no polypeptide coating Filling with a filler, (iv) mixing the filler of the polypeptide & garment with other additives; and (iv) applying a pharmaceutically acceptable coating additive to the filler of the polypeptide coating. The final drug. The composition can be loaded Capsules (such as gelatin capsules) or used to form tablets. In one embodiment, the pharmaceutical composition is prepared by spray drying, which is a technique for preparing drug particles (eg, microcapsules or microspheres). Spray dried peptides generally retain their biological activity when dissolved, And may have useful physical properties 'including uniform particle size and spherical shape. The particles prepared by spray drying are usually free-flowing, which facilitates the drug production process, such as forming tablets and filling capsules. The spray drying process is also Useful as they can be easily scaled up: clinically and commercially produced. Thus, the present disclosure features a method of preparing a pharmaceutical composition comprising linaclotide or a pharmaceutically acceptable salt thereof, the method Including, for example, an aqueous solution or an organic solution ("coating seam"), which comprises: (=nalopeptide or a pharmaceutically acceptable salt thereof; and (π) is selected from the group consisting of Mg2, Ca2+, 11 Mn, K, Na or 八13 a cationic and/or sterically hindered primary amine (such as leucine), and (b) a spray-dried solution containing linaclotide to produce microparticles. A solution containing linaclotide can be Optionally comprising 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 of the following additional steps (1) mixing the linaclotide microparticles with a pharmaceutically acceptable slip agent 142528.doc -25-201023874, a pharmaceutically acceptable lubricant or a pharmaceutically acceptable additive acting as both a slip agent and a lubricant. (ii) mixing the microparticles with the filler, and/or (iu) mixing the microparticles with other additives. The final pharmaceutical composition can be encapsulated (eg, a gelatin capsule) or used to form a tablet.

In other embodiments, the pharmaceutical composition is prepared by spray freeze drying, supercritical fluid processing, or lyophilization of a solution (eg, an aqueous solution or an organic solution) comprising: (1) linaclotide or a pharmaceutically acceptable thereof Accepting salt 3; and (9) a cation selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+, κ+, or Al3+ and/or sterically hindering primary amines (eg, leucine), in some embodiments, for oral administration The linaclotide composition in solid form. Examples of such forms include, but are not limited to, tablet H capsules, pills, capsules or powders. In some embodiments, the compositions can be used to produce unit dosage forms, such as tablets. A composition for oral administration of a capsule, a sachet or a pill can be used, and the substance 7 includes, for example, a binder, a lubricant, an inert diluent, a lubricant, a surface active or a public, and a And a knife-dispersing additive, a flavoring additive and a stimulating agent. The formulation for oral administration, f 绂 "μ, ', (such as a tablet) can optionally be coated or engraved

The trace' can be formulated to provide a sustained release, delayed release or controlled release of linaclotide. The Linalo shape can be co-administered or co-formulated with the fungus. In one embodiment, the Lina η "peptide can be co-administered with other musical compositions for the treatment of gastrointestinal disorders. Linalotol can also be used to treat disorders outside the gastrointestinal tract, such as congestive heart palpitations Exhaustion and benign prostatic hypertrophy. The composition may include, for example, other solvents such as hydrazine, dispersing agents, coating materials, absorption enhancing additives, batches; t-forces and one or more inert additives (including, for example, 'starch' Rong Xifan Alcohol, granulation additives, microcrystalline 孅 142528.doc -26 - 201023874 vitamins, thinners, lubricants, aging agents, disintegrating additives and similar additives), etc., if necessary, tablet dosage forms of the disclosed compositions The coating may be coated by an aqueous technique or an anhydrous technique. The composition may also include, for example, anti-caking additives, preservatives, sweetening additives, colorants, flavoring agents, desiccants, plasticizers, dyes, and the like. Disintegrators include 'eg agar-lime, calcium carbonate, microcrystalline fiber, vitamins, cross-linked methylcellulose, crospovidone, povidone, polac Potassium, sodium starch glycolate, potato or tapioca starch, other powders, pre-gelatinized m, other alginate, other celluloses, gums and mixtures thereof. Suitable lubricants include, for example, stearic acid Calcium, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other monools, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (eg peanut oil) , cottonseed oil, sunflower oil, sesame oil, eucalyptus oil, corn oil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar, e syloid tannin (aerosil 2〇〇, wr Grace co., Baltimore, MD USA), a coagulated aerosol of synthetic cerium oxide (Ev〇nik Degussa c〇.,

Plano, TX USA), igneous cerium oxide (CAB_0_SIL, cab〇t Co.,

Boston, MA USA) and mixtures thereof Suitable slip agents include, for example, leucine, colloidal cerium oxide, magnesium tricaprate, powdered cellulose, starch, talc, and tricalcium linum. Suitable anti-caking additives include, for example, albino, citric acid, strontium dioxide, colloidal cerium oxide, talc, and mixtures thereof. For example, suitable antimicrobials useful as preservatives for linaclotide compositions 142528.doc • 27, 201023874 Additives include, for example, benzalkonium chloride, benzethyl chloride, benzoic acid, benzyl alcohol, p-hydroxybenzoic acid Butyl ester, ceflurane, cresol, butyl alcohol, dehydroacetic acid, ethyl p-hydroxybenzoate, methyl p-hydroxybenzoate, 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. Suitable coating additives include, for example, sodium carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, gelatin, pharmaceutical glaze, propyl cellulose, propyl fluorenyl Cellulose, hydroxypropyl decyl cellulose phthalate, methyl cellulose, polyethylene glycol, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, carnauba wax, microcrystalline wax and Its mixture. Suitable protective coatings include Aquacoat (eg Aquacoat ethylcellulose aqueous dispersion, 15% w/w, FMC Biopolymer, ECD-30), Eudragit (eg Eudragit E PO PE-EL, Roehm Pharma Polymers) and Opadry ( For example Opadry AMB dispersion, 20% w/w, Colorcon). In certain embodiments, suitable additives for the linaclotide composition include one or more of sucrose, talc, magnesium stearate, crospovidone or BHA. In some embodiments, the term "95%" may be 95.0%, the term "90%" may be 90.0%, the term "10%" may be 10.0%, the term "9%" may be 9.0%, and the term "8%" It 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 142528. Doc -28 - 201023874 may be 2.0%, the term 3%" may be 3.0%, and the term r 2% 1%" may be 1.0%. In certain embodiments, a linaclotide composition in unit dosage form is provided. In some embodiments, the unit dosage form is a capsule, tablet, sachet, pill or powder. In one such embodiment, the unit dosage form is a capsule or tablet. Such unit dosage forms can be enclosed in containers such as, but not limited to, paper or cardboard boxes, glass or plastic bottles or cans, resealable packages (eg,

For example, to accommodate "refilled" tablets for loading into different containers) or blister packs with individual doses to be pressed into the package according to the treatment regimen. More than one container can be used together in a single package to provide a single dosage form. feasible. For example, a tablet or capsule can be contained in a bottle which is instead placed in a box. In some embodiments, the unit dosage form is provided in a container further containing a desiccant. In a further embodiment, unit dosage forms, such as some tablets or capsules, are provided in a container containing a desiccant, such as a bottle, can or a resealable package. In a further embodiment, the container containing the unit dosage form is packaged with the administration or dosage instructions. In certain embodiments 'providing a flavonoid composition in a kit towel^ The linaclotide composition described herein and the combination therapeutic agent can be packaged as a kit comprising two or more doses of two or more medicaments, They are individually packaged or formulated, or a single or multiple doses of two or more agents are packaged or formulated. Thus, the linaclotide composition can be present in the first container, and the kit can optionally include one or more agents in the second container. One or more containers are packaged in the package' and the package can optionally include an administration or dosage instruction. 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 the invention includes many variations and equivalents The solution will be apparent to those skilled in the art. The linaclotide or a pharmaceutically acceptable salt thereof can be prepared and purified using standard techniques known in the art, such as chemical synthesis or recombinant expression and subsequent purification using standard techniques.

Formulation A Preparation of a coating solution: mixing about 32 g to 42 g of purified water with hydrochloric acid to prepare a solution having a pH between 1.5 and 2.0. If used, the cation is metered into the solution to provide the desired concentration, The solution is mixed for a sufficient time to produce a clear solution. If used, the sterically hindered primary amine is metered into the solution to provide the desired concentration and the solution is mixed for a sufficient time to produce a clear solution. If necessary, add other additives such as antioxidants. The pH of the solution is measured, and if necessary, hydrochloric acid is added to produce a solution having a pH of between 1. 5 and 2.0. The binder is then added to the solution and the mixture is then allowed to mix for a sufficient time to produce a clear solution. The amount of linaclotide was added to the solution and mixed for 3 〇 丨〇〇 minutes to provide a coating solution. Preparation of Reactive Beads: About 30-36 g of dried microcrystalline cellulose beads were added to a Mini C〇iumri Fluid Bed Coater. The microcrystalline cellulose beads are fluidized and heated prior to iayering. The beads are then coated with a coating solution. The spray temperature was controlled between 2 and 55 C by controlling the inlet temperature, 142528.doc 201023874 spray rate, atomization pressure and air volume. After the entire coating solution was applied to the beads, the beads were dried. The product of this process is called active beads. Preparation of Reactive Beads with Protective Coating: Approximately 35 g of active beads were added to a microcolumn fluidized bed coater. In use (for example, AquaC〇at ethylcellulose aqueous dispersion (AqUaeous Dispersi〇n), 15% w/w, FMC Bi〇p〇lymer, ECD_3〇), ❿ 响 (for example

The active beads are fluidized and heated prior to coating with Eudragit E P〇 PE_EL, Pharma Polymers) or Opadry (eg 〇padry AMB dispersion, 2% w/w, c〇i〇rc〇n). Subsequently, the beads were coated with a coating solution. The spray temperature was controlled at 24 by controlling the inlet temperature, spray rate, atomization pressure, and air volume. (; and 55 between it. After all the coating solution is coated on the beads, the beads are dried.

Formulation B Preparation of the coating solution: Approximately 8.3 kg of purified water was mixed with hydrochloric acid to prepare a solution having a pH between 1.5 and 2. If used, the cation is metered into the solution to provide the desired concentration and the solution is mixed for a sufficient time to produce a clear solution. If used, the steric hindrance of the primary amine is metered into the solution to provide the desired concentration, and the solution is mixed for a sufficient time to produce a clear solution. Then, if necessary, add other additives such as antioxidants. The binder is then added to the solution and the solution is then mixed for a sufficient time to produce a clear solution. The pH of the solution was measured, and if necessary, hydrochloric acid was added to produce a solution having a pH between 1.5 and 2. This is solution 1. About 8.3 kg of purified water was mixed with hydrochloric acid to prepare a solution having a pH of 142528.doc -31 - 201023874 between 1.5 and 2 Torr. The required amount of linaclotide is added to the solution and mixed for 10 to 30 minutes. The pH of the solution is measured, and hydrochloric acid is added as necessary to produce a solution having a pH of between 1.5 and 2.0. This is solution 2. Solution 1 and solution 2 are then mixed together. The pH of the solution is measured, and if necessary, hydrochloric acid is added to produce a solution having a pH of between 1.5 and 2.0. This is the coating solution. Preparation of Reactive Beads: Approximately 24.19 kg of microcrystalline cellulose beads were added to a Wurster column of a Glatt GPCG-30 fluidized bed. The microcrystalline cellulose beads are fluidized and heated to a product temperature of 45-47 °C. The beads are then coated with a coating solution. Subsequently, the beads were coated with a coating solution. The product spray temperature was controlled between 37 ° C and 47 ° C by controlling the inlet temperature, spray rate, atomization pressure and air volume. After the entire coating solution was coated on the beads, the beads were dried at a product drying temperature of 37 ° C to 47 ° C. The product of this process is called active beads. Examples 1-15: Preparation of Linaclotide Formulations The linaclotide formulations of Examples 1-15 were prepared primarily as described in Formulation A, wherein Table 1 provides cations, sterically hindered primary amines, and adhesions. The amount of the agent, linaclotide and beads, and Table 2 provides the coating conditions for the beads: Table 1 Example cation amount fl* Amount of amine H The amount of binder linaclotide ** Bead amount 1 CaCl2 .2H20 0.6740g [60] leucine 0.2005g P〇l hypromellose 1.019g 0.1282g Celphere CP-305 33.38g 2 CaCl2.2H20 0.6740g [60] leucine 0.3007g P〇l Hydroxypropyl Cellulose 0.3063g 0.1329g Celphere CP-305 33.87g 3 CaCl2.2H20 0.2247g [20] Leucine 1.002g "1001 Hydroxypropylcellulose 0.0656g 0.1282g Celphere CP-305 33.86g 142528.doc -32- 201023874 EXAMPLES cation amount Π* amount of amine π amount of binder linaclotide ** beads amount 4 CaCl2.2H20 1.123 g [100] leucine 0.2005 g propylcellulose 1.969 g 0.1282 g Celphere CP-305 32.36g 5 CaCl2.2H20 0.4493g [40] leucine 0.4009g "401 hypromellose 0_5425g 0.1282g Celphere CP-305 33.78g 6 M gCl2.6H20 0.2590g [10] leucine 0.3341g P〇l hypromellose 0.6636g 0.2100g Celphere CP-305 33.83g 7 ZnAc.2H2〇0.2796g [i〇] leucine 0.3341g [201 hydroxy Propylcellulose 0.6636g 0.2100g Celphere CP-305 33.82g 8 N/A leucine 0.8944g P71 Hydroxypropylcellulose 0.6636g 0.43 87g Celphere CP-305 33.40g 9 CaGl2.2H20 0.3745gn〇i N/A Hypromellose 0_6636g 0.4227g Celphere CP-305 33.83g 10 N/AN/A Hypromellose 0.681 lg 0.2114g Celphere CP-305 34.28g 11 N/AN/A Hydroxypropylcellulose 0.6636g 0.4227g Celphere CP-305 34_13g 12 CuC12.2H20 0.4342g il〇] N/A Hydroxypropylcellulose 0.6636g 0.4227g Celphere CP-305 33.79g 13 ZnAc.2H2〇0.5590g [101 N/A Hydroxypropylcellulose 0_6636g 0.4227g Celphere CP-305 33.68g 14 MgCl2.6H20 0.5178gn〇i N/A Hydroxypropyl cellulose 0.6636g 0.4227g Celphere CP-305 33.72g 15 N/A 曱 thioglycol 0.0380g rn Hydroxypropyl cellulose 0.6636g 0.4387g Celphere CP-305 34.08g * "Cation" means a divalent cation contained in the salt used in the present example, and "amine" means steric hindrance Primary amines, [] refer to the molar ratio of cations and/or amines to linaclotide. * The amount of linaclotide in this example and all of the following examples is determined by peptide content and chromatographic purity, as provided for the active pharmaceutical ingredient (API) of each production batch of linaclotide. Analyze the certificate listed. 142528.doc -33 - 201023874 Table 2 Example Product Spray Temperature CC) Inlet Temperature (°C) Spray Rate (mL/min) Atomization Pressure (psig) Air Flow 1 34.0-37.0 55.7-57.7 0.33-0.40 20 Low 2 27.4-32.3 37.01-42.1 0.40 22 Low 3 32.6-34.7 60.0-60.1 0.33-0.40 20 Low 4 35.3-39.3 58.9-59.2 0.40 18 Low 5 27.8-27.9 58.7-59.8 0.35-0.33 20 Low 6 32.1-38.3 42.0- 53.4 0.39-0.75 22 Low 7 31.7-39.3 50.0-52.5 0.27-0.57 22 Low 8 33.3-41.3 50.5-57.0 0.57-0.65 22 Low 9 33.2-40.0 49.5-58.7 0.82-1.00 20 Low 10 42.5 59.5 0.49 22 Low 11 39.7 52.0 0.66 22 Low 12 36.6-40.0 47.2-54.8 0.65-0.75 20-22 Low 13 32.4 57.4 0.65 22 Low 14 34.0 49.0 0.75 20 Low 15 24.1-39.9 48.5-55.9 0.39-0.65 22-23 Low Example 16: Liana Preparation of Lopeptide Peptides The linaclotide formulation of Example 16 was prepared primarily as described in Formulation B, wherein Table 3 provides cationic, sterically hindered primary amines, binders, linaclotide, and beads. The amount of coating, while Table 4 provides the coating conditions of the beads: Table 3 Example cation amount π amine amount [] binder amount Linaro Amount of beads 16 CaCl2.2H20 385.1 g [60] leucine 171.8 g 『301 hypromellose 175.0 g 73.5 g Celphere CP-305 24.19 kg Table 4 Example product spray temperature (°c) inlet temperature CC) Spray rate (g/min) Atomization pressure (bar) Process air volume (cfm) Product drying temperature (°c) 16 64.9-65.1 80 150 2.0 515-564 54.9-55.0 142528.doc 34- 201023874 Example 17: Preparation of Linaclotide Formulation The linaclotide formulation of Example 17 was prepared essentially as described in Formulation A except that the formulation contained 22.96 mg of butylated hydroxyanisole (BHA), Table 5 The amount of cationic, sterically hindered primary amine, binder, linaclotide and beads is provided, while Table 6 provides coating conditions for the beads: Table 5 Example cationic amount 胺 Amine amount 钻 Drilling dose linaclotide Amount of beads 17 CaCl2.2H20 0.3745g 『201 N/A hydroxypropyl hydrazine cellulose 0.6636g 0.2100g Celphere CP-305 33.99g Table 6 Example product spray temperature (°C) inlet temperature rc) spray rate (mL/min) Atomization pressure (psig) Airflow 17 33.5-34.8 47.7-48.6 0.56-0.74 26 Low implementation 18: Linaclotide content of the capsule containing linaclotide formulation of the active preparation or the beads may be measured by other equivalent manner as described in Example 21. ® In order to form a capsule suitable for oral administration, a suitable amount of active beads is used to fill a gelatin capsule (e.g., size 2 gelatin capsule). A suitable amount of active beads may contain from 50 pg to 2 mg of linaclotide per capsule and a range of 5%. In some embodiments, suitable amounts of linaclotide of the active beads can be 50 pg, 67.5 pg, 100 pg, 133 pg, 150 pg, 200 pg, 266 pg, 300, 400 pg, 500 pg, 600 Pg, 700 pg, 800 pg, 900 pg, 1 mg, 2 mg, 4 mg or 6 mg. In a particular embodiment, the suitable amount of linaclotide of the active beads is 67.5 I42528.doc -35 - 201023874 μβ ' 100 ug ___ . ___ "σ , : > υυ pg , ) μ § , 500 ( 4 ) 600. In a more specific embodiment, the appropriate amount of linaclotide of the active beads is 67 5 per capsule, 400 medium, 133 gg, 150 '500 pg 133, 150 pg, 266 pg or 300. In another embodiment towel, a suitable amount of active beads of the desired amount of gelatin capsules is filled into a container. If desired, one or more pharmaceutically acceptable fillers or other may be used. A pharmaceutically acceptable additive is added to the container. 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 Gelatin capsules are filled with a suitable amount of active beads containing a pinnacle (eg, 5 to 2 mg of linaclotide per capsule, and range ± 5%). In an optional embodiment A suitable amount of active beads is used to fill the gelatin capsule and will - Or a plurality of pharmaceutically acceptable fillers or other pharmaceutically acceptable additives are added to the gelatin capsules.Example 19: Preparation of capsules containing linaclotide formulations Preparation of coating solutions: First, 4198 g The purified water is mixed with 13 g of hydrochloric acid to produce a solution having a pH between 5 and 2 Torr. Then, 7.49 g of calcium carbonate dihydrate and 6 68 g of leucine are added to the solution. It was then mixed for 30 minutes to give a clear solution. The cation was measured and 1.70 g of hydrochloric acid was added to give a solution having between 15 and 2 Torr. Then, 13.27 g of hypromellose (hydroxylpropyl group) was added. Base cellulose; d〇w Chemical C〇mpany; Midland, MI) was added to the solution, and the mixture was mixed for 60 minutes to obtain a clear solution. Money added 4 39 § Liana 142528.doc 201023874 Locipeptide to the solution Medium and mixed for 90 minutes. The pH of the solution was 1.73. This is the coating solution. Preparation of Active Beads: 674.5 g of microcrystalline cellulose beads (Celphere CP-305; Ashai Kasei Corporation (Tokyo; Japan)) were added. To

The Wurster column of the Glatt GPCG-2 fluidized bed. The microcrystalline cellulose beads were fluidized and heated at a product temperature of 60 ° C for 30 minutes. The beads are then coated with a coating solution. The product temperature was controlled between 45 ° C and 49 ° C by an inlet temperature of 80 ° C, a spray rate of 5.0 - 11 g / min, an atomization pressure of 2.0 bar and an air volume of 40 to 50 m 3 h. After the entire coating solution was applied to the beads, the beads were dried for 1 Torr at a product temperature of 46.9 ° C to 50.9 ° C. The product of this process is called active beads. Reversed-phase liquid chromatography of linaclotide extracted from the formulation prepared as described above demonstrated that the extracted linaclotide and linaclotide reference standards exhibited the same retention time, and the result of the formulation process was purity. No significant changes. To form a capsule, 49.50 g of active beads were added to a clear package. Then 0.25 g of leucine acid screened through a 60 mesh sieve was added to the bag. Tie the bag and mix for 125 rounds to mix all the materials. Then 0.25 g of talc screened through a 60 mesh tweezers was added to the bag. Tie the bag and mix for 125 rounds to mix all the materials. Once all of the material was mixed, the mixture was used to fill a size 2 gelatin capsule with a target weight of 227 mg/capsule (range ± 5%). Example 20: Preparation of capsules containing linaclotide formulation Active beads were prepared according to Example 16. The active beads were assayed for linaclotide 142528.doc -37- 201023874 content. An appropriate amount of active beads (96 mg-123 mg) was filled in a size 2 gelatin hard capsule using an MG2 Futura encapsulation according to the measurement of the active beads to obtain a concentration of 300 pg of linaclotide. Active beads were prepared according to Example 15. The linaclotide content of the active beads was determined. An appropriate amount of active beads (48 mg-62 mg) was filled in a size 2 gelatin hard capsule using an MG2 Futura encapsulation according to the measurement of the active beads to obtain a concentration of linaclotide of 150 pg. Implementation 21: Measurement of linaclotide content and purity Determination of linaclotide content and purity, and measurement of linaclotide-related substances by reversed-phase gradient liquid chromatography using Agilent with Chemstation Rev A.09.03 software Series 1100 LC system or equivalent system to determine. A YMC ProTM C18 column (size: 3.0 x 150 mm, 3.5 um, 120 A; Waters Corp., Milford, ΜΑ) or equivalent column was used and maintained at 40 °C. The mobile phase ΜΡΑ (ΜΡΑ) consists of water containing 0.1% trifluoroacetic acid, while the mobile phase Β (ΜΡΒ) consists of 95% acetonitrile: 5% water containing 0.1% trifluoroacetic acid. Elution of linaclotide and its related substances was completed with the following gradient: from 0% to 47% 28 in 28 minutes, followed by a constant rate of 100% 4 in 4 minutes, followed by 5 minutes at 100% 以 for washing The column. The column was re-equilibrated by returning to 0% hydrazine in 1 minute followed by 10 minutes at 100% ΜΡΑ. The flow rate was 0.6 mL/min and the detection was completed by 220 nm UV. Samples for analysis were prepared by adding the contents of linaclotide capsules to 0.1 N HCl to obtain a target concentration of 20 pg linaclotide/mL. Inject 100 μί of this solution into the column. 142528.doc -38- 201023874 The content of linaclotide was measured according to a similarly prepared Linacol-like external standard by determining the concentration of linaclotide in the prepared sample. An example of the analysis of linaclotide by HPLC is shown in the figure! Wherein "oxidation" refers to the oxidation product of linaclotide, "formaldehyde imine" refers to the product of linaclotide, and "hydrolysis" refers to the hydrolysis product of linaclotide: Example 22: linaclotide formulation Stability Test For the formulations of Examples 1 -15 and 17 , gelatin capsules were filled with about 225 mg of active beads. Load five filled capsules into a plastic bottle. The bottle contains 1 g to 2 g of desiccant' and is inductively sealed. The bottle was stored at 4 °c / 75% RH for six months. The linaclotide content and purity, as well as the amount of linaclotide-related substance, were measured primarily as described in Example 21 or by equivalent methods. The results are provided in Table 7. Table 7 Example Determination 丨w/wj Initial % HPLC Noodle Box % Linaclotide (Initial %) Oxidative Hydrolysis Formaldehyde Imine 1 107.56 96.88 (99.13) 0.11 0.24 0.19 3 98.87 97.36 (99.42) 0.07 0.52 0.15 4 95.67 95.61 (97.83) 0.10 0.16 0.24 5 103.41 95.87 (98.68) 0.07 0.25 0.24 6 99.46 93.64 (95.51) 0.14 0.70 0.55 7 98.64 93.44 (95.36) 0.45 1.45 0.63 8 92.81 88.20 (94.90) 0.37 1.85 0.49 9 93.53 93.81 (96.55) 0.2 0.41 1.06

142528.doc •39- 201023874 Example Determination 丨w/w 】 initial % HPEC area ° / lininotropine (initial %) oxidative hydrolysis furfural imine 10 77.12 84.85 (87.77) 0.37 0.29 4.45 11 85.73 89.09 ( 91.63) 1.18 0.49 1.38 12 33.60 41.98 (43.15) ND ND ND 13 87.69 91.91 (94.01) 1.98 0.74 0.86 14 86.94 90.59 (92.70) 0.25 0.54 1.23 15 87.71 87.54 (93.24) 0.24 0.66 1.67 17 98.94 93.65 (95.16) ND 0.32 0.73 For the formulation of Example 16, gelatin capsules were filled with about 113 mg of total beads. Load 35 filled capsules into a plastic bottle. The bottle contains 2 g of desiccant and is inductively sealed. The bottle was stored at 40 ° C / 75% RH for one month. The linaclotide content and purity, as well as the amount of linaclotide-related substance, can be measured primarily as described in Example 21 or by equivalent methods. The results are shown in Table 8. Table 8 Example Determination [w/wj initial % HPLC area % linaclotide (initial %) oxidative hydrolysis furfural imine 16 97.01 97.12 (99.79) <0.1 < 0.1 0.34 Example 23: Linaclotide Isolation and Preparation of Hydrolysate The linaclotide hydrolysate appeared due to the conversion of Asn at position 7 to Asp (the numbering of linaclotide starts at 1 at the N-terminal Cys). Its structure is described as follows: 142528.doc -40- 201023874 -s-s-

H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH .. ]~~hs-s--1 ss Using standard solid phase peptide synthesis The linaclotide hydrolysate has been synthesized independently for identification. The linaclotide hydrolysate can also be prepared by other methods known in the art, for example by separation from a linaclotide formulation using chromatographic techniques or by recombinant expression encoding a linaclotide hydrolysate (Cys

The nucleic acid of Cys Glu Tyr Cys Cys Asp Pro Ala Cys Thr Gly Cys Tyr), optionally, then oxidizes the cysteine residue to form a disulfide bond. Example 24: Isolation and preparation of linaclotide formaldehyde imine product The metformimine product was produced via a formaldehyde-mediated reaction when an imine was added to the N-terminal Cys (Cysl). A proposed product structure is described as follows:

H2C=Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cvs-Tvr-OH-ss--Hss-1 ss by linaclotide and furfural (1 : 5 molar ratio) The reaction was carried out for 4 days at room temperature in absolute ethanol, and the linaclotide furfural imine product was synthesized independently, and identity was confirmed with -9. The methine imine product can also be prepared by other methods known in the art, such as by separation from a lindino preparation using chromatographic techniques, or by chemical synthesis of a peptide or recombinant expression of a nucleic acid encoding a linaclot, followed by Deuteration is described, or optionally followed by oxidation of a cysteine residue to form a disulfide bond by other methods known in the art. 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 added to the six semi-ammonia sulphur of linaclotide in a single oxygen atom. 142528.doc -41 - 201023874

To support this judgment, linaclotide oxidation products have been prepared by reacting linaclotide with hydrogen peroxide (3% aqueous solution) at room temperature or 40 ° C for up to 24 hours. The resulting product was enriched with 10% of the oxidation product. The Lina-Laloid oxidation product can also be prepared by other methods known in the art, for example, by separation from the linaclotide formulation using chromatographic techniques, or by chemical/amplification or recombinant expression of the peptide encoding the Linac locus. The nucleic acid, which subsequently oxidizes the cysteine residue to form a disulfide bond, is then formed by reacting linaclotide with hydrogen peroxide or a similar oxidizing reagent to form a linaclotide oxidation product. Example 26: Formation of Linaclotide Tablets Fluidized Bed Granulation linaclotide, CaC 2, leucine and polyethylene ketone (pvp) ❺ K30 were dissolved in 0.0001N HCl to form Coating solution (see Table 9). The isomalt was charged to the feed mash (b〇Wl) of the fluidized bed. With fluidization of the isomaltulose powder, the drug solution is at a rate of ~10 g/min, at ~4〇. (At the product temperature, the top spray was applied to coat the powder with a coating solution. When the spray was completed, the linaclotide particles were dried for 30 minutes and the product was taken out. 142528.doc -42·

201023874 Table 9 Example cationic amount 胺 Amount of amine 粘合剂 amount of binder linaclotide filling dose 26A CaCl2.2H20 15.4 g [60] leucine 6.9 g 『301 PVP K30 40 g 3.08 g isomalt 935 g Phytate II or Avicel is also used as a filler for fluidized bed granulation. Wet granulation Weighed linaclotide and dissolved it in 250 g of 0.1 N HCl φ (pH 1.7) with stirring to form solution 1 (see Table 10). CaCl2 and leucine were weighed and dissolved in 1 〇〇 g 0.1 N HCl to form solution 2 with stirring. Solution 1 and Solution 2 were mixed together under agitation to form a coating solution. Avicel was added to the feed of the high shear granulator. The coating solution was added to Avicel with mixing at 500 rpm. When the addition of the solution was completed, the particles were mixed and cut for 1 minute. The obtained wet granules were charged into a screw of a fluidized bed, and dried for 15 minutes, and then linaclotide particles were taken out. Table 10 Example cationic amount 胺 amount of amine H amount of binder linaclotide filling dose 26B CaCl2.2H20 7.68 g [60] leucine 3.42 g [301 N/A 1.54 g isomalt 488 g In the wet granulation scheme, the molar ratio of CaCl2 and leucine to linaclotide is adjusted in the range of 60 to 100 and 30 to 50, respectively. Additionally, in one embodiment sucrose is added. See Table 11. 142528.doc -43 - 201023874 Table 11

Example concentration (linalotide/filler) Filler CaCl2: Leu: Linaclotide sucrose HC1 26C 600 pg/225 mg Avicel ^ 60:30:1 None 0.1N 26D 600 pg/225 mg Avicel 80:40 :1 No 0.1N 26E 600 μ^225 mg Avicel 100:50:1 No 0.1N 26F 600 pg/225 mg Avicel 60:30:1 5% 0.1N Tablets to prepare linaclotide particles with the following shape Mix (see Table 12) and compress to a tablet with a hardness of ~4 kp. Table 12 Functionality 150 pg of linaclotide, 200 mg of tablets with 300 ng of linaclotide, 400 mg of tablets Tablet with 600 linaclotide, 800 mg weight, 1200 Mg linaclotide, 1600 mg tablet linaclotide granule API 53.4 mg 106.8 mg 213.6 mg 427.2 mg isomaltulose tablet Filler 134.1 mg 268.2 mg 536.4 mg 1072.8 mg crospovidone disintegrant 10 mg 20 mg 40 mg 80 mg Magnesium stearate lubricant 1.5 mg 3 mg 6 mg 12 mg talc slip 1 mg 2 mg 4 mg 8 Mg total amount of dry material 200 mg 400 mg 800 mg 1600 mg According to the above scheme, isomalt, starch 1500 or dicalcium phosphate A filler for tablets (see Table 13). Table 13 Narrative filler CaCl2: leucine: linaclotide tablet filler fluidized bed isomalt 60:30:1 isoamyl ketol starch 1500 bismuth citrate fluidized Avicel 60:30:1 Starch 1500 Wet Process Avicel 100:50:1 Starch 1500 Wet Granulation Avicel 60:30:1 + 5% Sucrose Starch 1500 After storage at 40 ° C and 75% relative humidity for 2 weeks, Table 13 All of the tablets described in the figure showed that the measured value of linaclotide was over 90%. 142528.doc • 44· 201023874 Examples 27-53: Preparation of Linaclotide Formulations The linaclotide formulations of Examples 27-53 were prepared essentially as described in Formulation A and Examples 1-15. . The linaclotide coating solution contained 0.7% binder (w/v) and the coating solution was sprayed onto Celphere CP-305 beads as described in Examples 1-15. Table 14 provides the types of cations, amines, and/or other excipients, along with their molar ratios relative to linaclotide, and the type of binder used, while Table 15 provides a package of beads. Clothing conditions: Table 14 Example Cationic amine molar ratio binder additive 27 CaCl2*2H20 — 20:0:1 Hydroxypropylcellulose ~ 28 MnCl2*4H20 — 20:0:1 Hydroxypropylcellulose - 29 KCl — 20:0:1 Hypromellose ~ 30 A1C13.6H20 A 20:0:1 Hydroxypropylcellulose ~ 31 CaCl2.2H20 Amino Acid 60:30:1 Hydroxypropylcellulose - 32 Calcium Alginate Amino acid 60:30:1 Hydroxypropyl cellulose ~ 33 CaHP04 leucine 60:30:1 Hydroxypropyl cellulose - 34 Calcium stearate leucine 60:30:1 Hypromellose - 35 CaS04 -2H20 leucine 60:30:1 hypromellose - 36 Zn(OAc)2 leucine 60:30:1 hydroxypropionin ~ 37 CaCl2.2H20 isoleucine leucine 60:30 :1 Hydroxypropylcellulose ~ 38 CaCl2*2H20 Proline 60:30:1 Hydroxypropylcellulose - 39 CaCl2.2H20 Methionine 60:30:1 Hydroxypropylcellulose - 40 CaCl2-2H20 Amphetamine-alanine 60:30:1 hydroxypropionin- 41 — Histamine 0:20:1 Hypromellose - 42 — Tryptophan 0:20:1 Hydroxypropylcellulose - 43 CaCl2*2H20 ~ 0:20:1:20 (Vit. E) Hydroxyl Propylcellulose Vitamin E 44-1-aminocyclohexanecarboxylic acid 0:20:1 Hydroxypropylcellulose - 45 - Cyclohexylamine 0:20:1 Hydroxypropylcellulose ~ 46 - 2-methylbutyl Amine 0:20:1 Hydroxypropylcellulose ~ 47 - Polyglucosamine 0:20:1 Hydroxypropylcellulose - 48 CaCl2.2H20 Amino Acid 60:30:1 Polyvinylpyrrolidone ~ 49 CaCl2*2H20 White Amino acid 60:30:1 mercapto cellulose (Methocel A15) — 50 CaCl2-2H20 leucine 60:30:1 hydroxypropyl cellulose ~ 51 NaCl - 20:0:1 hypromellose ~ 52 CaCl2 .2H20 leucine 60:30:1 gelatin - 53 CaCl2.2H20 glycine 60:30:1 hydroxypropionin cellulose

-45- 142528.doc 201023874 * "Cation" means the cation contained in the salt used in the examples, "Amine" means steric hindrance to the primary amine '"Mohr ratio" refers to the cation: amine: linaclotide: additive (if applicable ) Moerby. Table 15 Example Product Spray Temperature Inlet Temperature Spray Rate Atomized Pressure Gas Flow (°C) (°C) (g/min) (psig) 27 25.1-35.1 37.0-50.1 0.44-0.62 20 Low 28 24.1-35.8 37.3 -50.9 0.30-0.61 18-20 Low 29 28.1-34.7 37.6-47.8 0.50-0.63 18 Low 30 29.8-35.0 33.9-50.2 0.32-0.47 20 Low 31 25.5-35.1 34.6-50.4 0.40-0.61 20 Low 33 30.4-35.2 38.7 -51.0 0.48-0.52 20 Low 35 29.9-34.9 37.8-50.4 0.37-0.76 20 Low 36 29.9-35.4 38.0-50.1 0.38-0.50 21 Low 37 27.3-34.9 36.2-50.1 0.45-0.54 20 Low 38 27.6-36.2 36.9-47.3 0.43-0.66 20 Low 39 30.1-35.8 40.6-47.1 0.30-0.48 20 Low 40 31.7-37.5 41.3-51.0 0.40-0.67 18 Low 41 29.4-36.2 41.7-49.5 0.48-0.53 20 Low 42 31.0-38.6 42.4-51.2 0.52- 0.64 20 Low 44 31.0-37.6 ^ 39.5-48.8 0.40-0.46 18 Low 45 28.7-36.5 37.1-49.2 0.49-0.61 18 Low 46 28.6-35.2 37.1-47.2 0.39-0.53 18 Low 47 33.4-38.7 40.6-48.5 0.48-0.47 18-26 Low 48 31.6-36.1 41.6-46.7 0.36-0.72 18 Low 49 28.5-36.5 36.8-48.1 0.45-0.51 18 Low 50 27.9-36.4 37.1-48.6 0.35-0.60 18 Low 51 29.3-37.9 36.7-49. 2 0.42-0.55 18 Low 52 29.8-36.3 36.1-49.1 0.44-0.54 18 Low 53 28.9-35.8 36.5-47.7 0.45-0.52 18 Low For Examples 32 (calcium alginate), 34 (calcium stearate) and 43 ( CaCl2: Vitamin E) 'Processing problems occur during spraying on beads. Therefore, the coating solution was mixed with Celphere beads and the beads were dried on a tray. Example 5 4: Linalol-like formulation stability test For the formulations of Examples 27-53, 225528.doc -46-201023874 filled gelatin capsules (600 pg of linaclotide) were filled with about 225 mg of active beads. /capsule). Load the five filled capsules into a plastic bottle. The bottle contains 1 g of desiccant and is inductively sealed. The bottle was stored at 40 ° C / 75% RH for three months or six months. Linaclotide content (gg/mg) and percent chromatographic purity (% CP) were measured primarily as described in Example 21 or by comparable methods. The results are provided in Table 16A (three month stability) or Table 16B (six month stability).

Table 16A Example Determination [w/w] Initial % * % CP %CP [Initial %] 27 96.30 93.98 % 98.07 28 96.82 93.59 % 96.07 29 101.56 92.71 % 95.40 30 109.06 93.07 % 95.76 31 103.59 95.98 % 99.12 32 66.53 82.66 % 85.27 33 96.81 91.94% 93.55 34 30.75 55.47 % 56.88 35 101.37 93.07 % 95.02 36 105.27 91.49% 93.45 37 109.22 95.73 % 97.99 38 99.24 95.79 % 97.59 39 95.22 95.76 % 97.82 40 102.98 95.68 % 97.60 41 110.92 94.03 % 96.30 42 120.05 88.57 % 91.65 43 58.51 70.99 % 74.06 44 98.83 93.84 % 96.88 45 91.72 90.07 % 93.71 46 90.17 89.45 % 91.67 47 105.70 88.59 % 91.31 48 106.92 95.11 % 97.62 49 96.48 94.62 % 96.60 50 112.30 95.86 % 98.98 51 102.92 91.80% 99.79 52 108.12 83.10% 86.80 53 104.22 95.25 % 97.95 *The variability of the measured values [w/w initial %] reflects the uncontrolled release of the uniformity of these capsule batches produced in small scale. 142528.doc -47· 201023874 It is believed that the difficulties encountered during processing and the resulting improved processing of Examples 32, 34 and 43 (see above) may explain the low stability observed in these samples.

Table 16B Example Determination [w/w] Area % of initial Vo HPLC linaclotide (initial %) oxidative hydrolysis furfural imine 27 91.58 89.68 (93.58) 0.09 0.60 1.59 28 93.36 88.44 (90.78) 0.24 0.41 1.55 29 93.73 87.79 (90.34) 0.18 0.53 1.82 30 108.63 93.93 (96.65) 0.39 1.11 0.44 31 94.53 86.83 (89.67) - 0.41 0.98 32 69.28 73.15 (75.46) 0.97 1.93 1.69 33 88.91 85.96 (87.46) 0.97 3.86 0.17 34 77.37 70.42 (72.21) 0.67 0.99 1.78 35 95.34 88.85 (90.71) 0.39 1.80 0.33 36 102.83 87.27 (89.14) 3.31 1.86 0.21 37 99.33 87.23 (89.29) - 0.59 0.25 38 93.97 86.27 (87.89) - 0.42 0.45 39 87.78 85.23 (87.07) - 0.40 0.31 40 94.36 86.28 (88.01) - 0.46 0.41 41 104.28 90.04 (92.22) 0.33 1.61 0.52 42 117.92 76.85 (79.52) 0.14 1.21 0.10 43 54.21 59.54 (62.12) 5.92 4.44 1.83 44 92.56 90.24 (93.17) 0.16 1.47 0.54 45 76.23 79.57 (82.78) 0.17 0.87 1.22 142528.doc -48- 201023874 Example Determination [w/w] Initial % HPLC area % Linaclotide (initial %) Oxidative hydrolysis of formaldehyde imine 46 73.07 78.92 (80.88) 0.51 0.6 6 0.65 47 97.65 82.73 (85.27) 0.92 0.60 2.68 48 93.94 85.24 (87.49) 0.05 0.69 0.20 49 51.65 63.46 (64.79) 0.96 0.58 2.24 50 104.75 92.61 (95.62) - 0.38 0.48 51 94.15 88.19 (92.01) - 0.58 1.35 52 100.06 72.81 (75.62) 0.06 0.49 0.41 53 95.74 89.80 (92.35) 0.06 0.36 1.40 The chromatographic purity values of the examples 27-53 at the 6-month time point showed an abnormally low, especially relative to the three-month time point at these samples. At the office. The relative tendency of the stabilizing or destabilizing effects can be established by comparing Example 27 and Example 31 as internal reference experiments for which the chromatographic purity values are compared to other studies that have been performed (see, for example, Examples 2 and 9). The values observed throughout are about 6-8% lower. The three month old stock of the same formulation provided in Table 1 6A shows a more typical chromatographic purity value. Therefore, the low chromatographic purity values at six months may be insufficient due to the drying ability at these six months for these special storage conditions. This assumption is supported by peaks observed and indicating exposure to moisture. Example 55: 24 month linaclotide formulation stability test at 25 ° C / 60% RH For the formulations of Examples 8-15 and 17, gelatin capsules were filled with about 225 mg of active beads. Load five filled capsules into a plastic bottle. The bottle contains 142528.doc -49- 201023874 with 1 g of desiccant and is inductively sealed. The bottle was stored at 25 ° C / 6 ° ° / ° RH for 24 months. The linaclotide content and purity, as well as the amount of linaclotide-related substance, were measured primarily as described in Example 21 or by equivalent methods. The results are provided in Table 17. Table 17 Determination of 丨w/w] Initial % HPLC Surface _ Example Linaclotide (Initial %) Oxidative Hydrolysis Furfuralimine 8 94.36 94.58 (101.7) 0.21 1.26 0.53 9 94.08 95.09 (97.86) 0.14 0.36 0.93 10 80.80 87.82 (90.84) 0.38 0.26 3.77 10a1) 89.29 91.55 (94.95) 0.50 0.39 1.60 10b 2) 88.41 91.19 (95.02) 0.44 0.34 1.61 10c3) 72.35 72.36 (75.76) 0.30 0.26 19.13 11 87.50 90.25 (92.82) 1.03 0.42 1.94 12 62.82 66.77 (68.62) 2.20 1.24 2.11 13 90.59 93.79 (95.93) 1.21 0.65 0.77 14 91.41 94.88 (97.09) 0.18 0.47 0.65 15 90.91 90.31 (96.18) 0.17 0.56 1.64 17 91.45 92.92 (96.81) 0.71 0.56 0.73 1〗 For Example 10, An additional protective coating Aquacoat (Aquacoat ethylcellulose aqueous dispersion, 15% w/w, FMC Biopolymer, ECD-30). 2) For Example 10, Opadry (Opadry AMB dispersion, 20% w/w, Colorcon) was coated with additional protection. 142528.doc -50- 201023874 3) For Example 10, Eudragit (Eudragit E PO, Degussa, Roehm Pharma Polymers; SLS, stearic acid) was coated with additional protective properties. Example 56: Linaclotide tablet formulation and stability test Active linaclotide particles were prepared by fluid bed granulation, primarily as described in Example 26, using the reagents described in Table 18. Linaclotide particles were mixed with the excipients described in Table 19 and compressed into tablets having a hardness of ~4 kp. Pack 35 tablets in a 60 cc bottle with 5 grams of desiccant and store for up to 3 months at 40 ° C / 75 ° / 〇 RH or up to 3 at 30 ° C / 65% RH Months. The linaclotide content and purity, as well as the amount of linaclotide-related substance, were measured primarily as described in Example 21 or by equivalent methods. The results are provided in Table 20. Table 18 Functional granules, 150 pg linaclotide/53.7 mg granule linaclotide API 0.15 mg mannitol, USP granule filler 50 mg leucine, USP stabilizer 0.64 mg CaCl2-2H20, USP stabilizer 0.72 mg PVP K30, USP Adhesive 2.2 mg HC1 Solution (pH 2.5) — -- Table 19 Ingredients Functional Tablets (200 mg total weight) Linaclotide Granule Actives 53.4 Isomalt, USP Tablet Filler 134.1 Cross-linked sodium carboxymethyl cellulose, USP Disintegrant 10 Magnesium stearate, USP Lubricant 1.5 Talc, USP Slip agent 1.0 142528.doc -51- 201023874 Table 20 Condition time change change % Total degradation 40〇C/ 75 % RH Initial 100 2.27 40〇C/75%RH 1 month 96.2 2.09 40〇C/75 % RH 2 months 102 2.15 40〇C/75 % RH 3 months 99.5 1.52 30〇C/65 % RH 3 Month 100.1 1.19 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 of the complete linaclotide bead drug coating solution, and their theoretical weights (mg/g) and (kg/batch). Table 22 provides the ingredients and theoretical weight (mg/g) and (kg/batch) for the preparation of linaclotide active beads. The linaclotide formulation was encapsulated in a hard gelatin capsule, size 2 (weight 61 mg), primarily as described in Example 20. 1 50 pg linaclotide capsule contains 56 mg linaclotide beads (600 pg linaclotide / 225 mg beads), while 300 pg linaclotide capsules contain 113 mg linaclotide beads (600 Pg linaclotide / 225 mg beads). Table 21 Theoretical Functional Weight (mg/g) Theoretical Weight (kg/batch) Linaclotide API 2.67 0.067 CaCl2*2H20, USP, EP, BP, JP Stabilizer 15.41 0.385 L-Acetylamine, USP Stabilizer 6.87 0.172 Hydroxypropyl methylcellulose, USP (Methocel E5 Premium LV) Adhesive 7.00 0.175 Purified water, USP I — 16.666 HC1 (36.5-38.0), NF — — 0.114 142528.doc • 52- 201023874 Table 22 Compositional functions Theoretical weight (mg/g) Theoretical weight (kg/batch) Linaclotide bead drug coating solution coating solution 31.95 0.799 Microcrystalline cellulose sphere NF (Celphere CP-305) Beads 968.05 24.201 Final total: Nallopeptide Beads, (600 μ^225 mg) Active Beads 1000 25.000 [Simplified Schematic] Figure 1 shows an example of HPLC analysis of linaclotide.

142528.doc -53- 201023874 Sequence Listing <11〇> American Aino House Pharmaceuticals <120> Stable solid formulation of a suitable GC-C receptor agonist polypeptide for oral administration

<130> 223355/-057TW <140> 098127484 <141> 2009-08-14 <150>61/089,422; 61/273,332 <151>2009-8-14; 2010-08-03 <160> 13 <170> Patentln version 3.5 <210> 1 <211> 14 <212> PRT <213> Artificial sequence <220>

<223> Sequence generated by synthesis <4〇〇> 1

Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1 5 10 <210> 2 <211> 13' <212> PRT <213> Artificial Sequence <220><223> Synthesis Peptide <400> 2

Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys Thr Gly Cys 1 5 10 <210> 3 <211> 14 <212> PRT <213>

<220><223> Synthesis of peptide produced <400> 3

Cys Cys Glu lie Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 15 10 <210> 4 <211> 33 <212> PRT <213>Artificial Sequence <220><223><400> 4

Cys Cys Glu lie Cys Cys Asn Pro Ala Cys Thr Gly Cys 1 5 10 <210> 5 <211> 14 <212> PRT <213>Artificial Sequence <220> 142528 - Sequence Listing.doc 201023874 <223> Synthesis of peptides produced <400> 5

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220><223> Peptide <400> 6

Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys 1 5 10

<210> 7 <211> 14 <212> PRT <213> Artificial sequence <220><223> Synthesis of peptide produced <400>

Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1 5 10 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220><223> Peptide <400> 8

Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys Thr Gly Cys 1 5 10

<210> 9 <211> 13 <212> PRT <213> Artificial sequence <220><223> Synthesis of peptide produced <400>

Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys 1 5 10 <210> 10 <211> 15 <212> PRT <213>Artificial Sequence <220><223> Synthesis of Peptide <400> 10

Pro Gly Thr Cys Glu lie Cys Ala Tyr Ala Ala Cys Thr Gly Cys 142528 · Sequence Listing. doc -2- 3 201023874 15 10 15 <210> 11 <211> 16 <212> PRT <213><220>

<223> Synthesis of peptides produced <400> II

Asn Asp Asp Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu 15 10 15 <210> 12 <211> 16 <212> PRT <213:>Artificial Sequence <220>

<223> Synthesis of peptides produced <400> 12

Asn Asp Glu Cys Glu Leu Cys Val Asn Val Ala Cys Thr Gly Cys Leu 15 10 15 <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220><223> Produced peptide <400> 13

Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr 1 5 10

142528 - Sequence Listing.doc

Claims (1)

201023874 VII. Patent Application Range: ^• A pharmaceutical composition comprising linaclotide and a pharmaceutically acceptable excipient, wherein () the chromatographic purity of the linaclotide is (4) the pharmaceutical composition is dry In a sealed container of 25 ° C, the relative humidity of the sail is 18 months - or (b) the pharmaceutical composition is stored in a sealed container containing desiccant 4 (rc, 75% relative humidity for 6 months, reduced Less than 1% by weight; (8) The chromatographic purity of the Linalol-like form is (4) The pharmaceutical composition is stored for 25 months, at a relative humidity of 25 c, 6% by weight in a barrier state containing a desiccant or (b) the drug combination The product is stored at 75% relative humidity of the sealed container containing the desiccant for 6 months, and is greater than or equal to 9% by weight; (iii) the measured value of linaclotide measured on a weight/weight basis in the unit dosage form is ( a) The pharmaceutical composition is stored in a sealed container containing a desiccant at 25 ° C, 6 % relative humidity for 18 months or sand. The pharmaceutical composition is in a sealed container containing a desiccant in a thief, 75% relative humidity After 6 months of storage, reduce less than 10%; or (IV) based on weight/weight The measured value of the linaclotide measured is (a) the pharmaceutical composition is stored in a sealed container containing a desiccant at 25 C>c, 6%%; relative humidity for 18 months or (b) the pharmaceutical composition is contained 40 in a sealed container of desiccant. 〇, stored at 75% relative humidity for 6 months, greater than or equal to 90%. 2. A unit dosage form' which comprises the pharmaceutical composition of claim 1. 3. A sealed container comprising a plurality of unit dosage forms as claimed in claim 2. 4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, linaclot 142528.doc 201023874 peptide and one or more agents selected from the group consisting of: (i) selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+, K+, Na a cation of A13+, or (ii) a sterically hindered primary amine, wherein the agent is in (a) the pharmaceutical composition in a sealed container containing a desiccant at 25 °, 6 〇 0 / 〇 relative humidity The first 18 months of the error or (b) the pharmaceutical composition is stored in a sealed container containing desiccant at 4 ° C, 75% relative humidity for the first 6 months after the first drug combination And improving at least one property of the composition wherein the property is selected from the group consisting of: a decrease in the degradation rate of linaclotide as measured by the linaclotide content, and a reduction in the chromatographic purity of linaclotide Decrease in the rate of degradation of the loperopeptide, decrease in the amount of linaclotide oxidation product relative to the amount of linaclotide, decrease in the amount of linaclotide hydrolysate relative to the amount of linaclotide or linaclotide formaldehyde The amount of imine product is reduced relative to the amount of linaclotide . The pharmaceutical composition of claim 4, wherein the agent is Mg2+, Ca2+, Zn, Mn2+, κ+, Na+ or A13+, wherein the + y 2+ σ, η +, Κ+, Na+ or Α13+ Provided in the form of calcium chloride, phosphate bow, sulfur _, zinc acetate, chlorine (tetra), gas cut, gasified sodium or gasified knot. A pharmaceutical composition according to the item 4, wherein the agent is Mg2+, Ca2+ or Ζη2+. The pharmaceutical composition of claim 6, wherein the agent is ca2+. The pharmaceutical composition of claim 4, wherein the agent is a sterically hindered primary amine, is selected from the group consisting of an amino acid, a polymeric amine or a compound of the formula: Rl\|^R3, which is independent of Ri, R2 and R3 in 142528.doc 201023874 Selected from: hydrazine, C(0)0H, C1-C6 alkyl, C1-C6 alkyl ether, C1-C6 alkyl sulfide, C1-C6 hospital-based acid, Cl-C6 alkylcarboxy amide An alkylaryl group in which any group may be substituted with a single or multiple halogen or amine groups, and the limitation is that no more than one of Ri, the rulers 2 and 1 is ruthenium. 9. The pharmaceutical composition of claim 8, wherein the steric hindrance to the primary amine is a naturally occurring amino acid selected from the group consisting of histidine, phenylalanine, alanine, glutamic acid, aspartic acid, and branamide a group consisting of acid, leucine, methionine, aspartic acid, tyrosine, threonine, isoleucine, tryptophan and valine. 10. The pharmaceutical composition of claim 9, wherein the naturally occurring amino acid is leucine, isoleucine, alanine or methionine. 11. The pharmaceutical composition of claim 1 wherein the naturally occurring amino acid is leucine. 12. The pharmaceutical composition of claim 8, wherein the steric hindrance primary amine is a non-naturally occurring amino acid or amino acid derivative selected from the group consisting of 1-aminocyclohexanecarboxylic acid, lanthionine ( Lanthanine) and a group of theanine. 13. The pharmaceutical composition of claim 8 which is an amine or 2-methylbutylamine. 14. The pharmaceutical composition of claim 8 which is a glucosamine. Wherein the steric hindrance of the primary amine is cyclohexyl, wherein the steric hindrance of the primary amine is a polyglucose. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is 3 Mg + , Ca 2+ , Zn 2+ , Mn 2+ , K + , Na + or Ai 3 + , Wherein the Mg, Ca2, Zn2+, Mn2+, K+, Na+ or A], magnesium carbide, chlorine 142528.doc 201023874 chemical dance, calcium sulphate, sulfuric acid, sodium or vaporized aluminum is provided in the form of 16. The pharmaceutical composition of 8 contains Mg2+, Ca2+ or zn2+. Acetic acid "vaporized manganese, potassium chloride, chloroquine' wherein the pharmaceutical composition further comprises 17. The pharmaceutical composition of claim 16 comprising Ca2+ oxime wherein the pharmaceutical composition further comprises an antioxidant. The antioxidant is a pharmaceutical composition of claim 4, wherein the pharmaceutical composition of claim 18, wherein the vitamin E or the propyl gallate. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a pinnacle, an ion selected from the group consisting of Mg'Ca2+, Zn2+, Mn2+mAi3+i, and a sterically hindered primary amine. The pharmaceutical composition according to claim 20, wherein the "^10, ca2+, zn2+, Μηκ, Na or Al3 + is made of magnesium gas, calcium carbonate, calcium phosphate, barium sulfate vinegar, feather zinc, gasified turtle, The pharmaceutical composition of claim 20, wherein the agent is Mg2+, ca2+ or Zn2+ 〇23, wherein the pharmaceutical composition of claim 22, wherein 24. The pharmaceutical composition according to claim 20, the base acid, the polymeric amine or the compound of the formula wherein the steric hindrance primary amine is selected from the group consisting of: Rl\^R3 ' wherein R1, R2 nh2 and R3 are independently selected From: η, C(0)〇H, C1-C6 alkyl, C1-C6 alkyl 142528.doc 201023874 Ether, C1-C6 alkyl sulfide, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxyl group Indoleamine and alkylaryl, wherein any group may be substituted by a single or multiple halogen or amine groups, and the restriction is that no more than one of R1, Ruler 2 and r3 is Η. a pharmaceutical composition of 24 which causes the steric hindrance to be a naturally occurring amino acid which is selected from the group consisting of histidine, phenylalanine, alanine, amygic acid, aspartic acid, and Chu a group consisting of proline, leucine, methionine, aspartic acid, tyrosine, threonine, isoleucine, tryptophan and valine. 26 The pharmaceutical composition, wherein the naturally occurring amino acid is leucine. 27. The pharmaceutical composition according to claim 24, wherein the steric hindrance of the primary amine is a non-naturally occurring amino acid or an amine (4) organism, which is selected a group consisting of free oxime-aminocyclohexanecarboxylic acid, lanthionic acid and theanine. 28. The pharmaceutical composition of claim 24, '., 立体 sterically hinders the primary amine to be cyclohexylamine or 2. Methyl butylamine 29. A pharmaceutical composition such as δ luxuries 24, wherein, the steric hindrance of the primary amine is polyglucamine. J 30. 31. ' ^ 7 Bar Taste a slick agent, a lubricant, a sputum, a package as a lubricant and an additive to a lubricant, such as the pharmaceutical composition of claim 20, a pharmaceutically acceptable binder; or pharmacy; a two-package=chemical agent; a drug or a plurality. The filler is in the form of a filler. The pharmaceutical composition of claim 31, wherein the antioxidant From Βαα, 142528.doc 201023874, vitamin E or propyl gallate. 33. 34. 35. 36. 37. 38. 39. 40. 41. The pharmaceutical composition of claim 31, wherein the pharmaceutically acceptable The viscous agent is selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone (polyviyl), powder, maltodextrin or cellulose ether. The pharmaceutical composition of claim 31, wherein the pharmaceutically acceptable viscous person The agent is selected from the group consisting of methyl cellulose, ethyl cellulose, carboxymethyl cellulose, #ethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose and propyl fluorenyl cellulose. Ether ether. The pharmaceutical composition according to claim 31, wherein the pharmaceutically acceptable filler is cellulose, isomah, mannitol or hydrogen hydride. The pharmaceutical composition of claim 35, wherein the cellulose is selected from the group consisting of microfibrils and microcrystalline cellulose. The pharmaceutical composition of claim 35, wherein the pharmaceutically acceptable filler comprises particles having an average diameter between 150 μm and 1 μm. The pharmaceutical composition of claim 31, wherein the pharmaceutical composition comprises a pharmaceutically acceptable filler, and the weight ratio of linaclotide to the pharmaceutically acceptable filler is between 1:25 and 1:2,500. The pharmaceutical composition of claim 38, wherein the weight ratio of linaclotide to the pharmaceutically acceptable filler is between 1:100 and 1:1 Torr. The pharmaceutical composition of claim 20, wherein the sterically hindered amine is leucine and the molar ratio of leucine to linaclotide is at least 1 :1. The pharmaceutical composition of claim 40, wherein the molar ratio of leucine to linaclotide is at least 30:1. The pharmaceutical composition of claim 20, wherein the cation is Ca 2+ and the molar ratio of Ca 2+ to leucine is At least 1:1. The pharmaceutical composition of claim 42, wherein. The molar ratio of 2+ to leucine is at least 1.5:1. A pharmaceutical composition according to claim 20, wherein the cation: sterically hindered primary amine: the molar ratio of linacod is from 40 to 100·20 to 50:1. The pharmaceutical composition of claim 44, wherein the cation is ca 2+ and the stereo cleaves the primary amine to leucine. The pharmaceutical composition according to claim 45, wherein the molar ratio of Ca2+: leucine: linaclotide is 1〇〇: 3〇: 1, 8〇: 4〇: 1, 8〇: 3〇ι, 8 〇:2〇:ι, 60.30.1 60.20:1, 50:30:1 ' 50:20:1 > 40:20:1 ' 20:20:1 ' 10:10:1,10:5:1 , 5: l〇: i or 5: 5: 1. The pharmaceutical composition according to claim 46, wherein the molar ratio of Ca2+: leucine: Linac is 60:30:1. A unit dosage form comprising the pharmaceutical composition of claim 47. s The unit dosage form of item 48, wherein each unit dosage form comprises from 1 mg of linaclotide. The unit dosage form of claim 49, wherein each unit dosage form is a capsule or a tablet, and wherein the unit dosage form comprises 67·5 μ§, ι〇〇μ§, ΐ33μ§, 15〇, 200, 266, 3〇〇盹, 4 〇〇, _ 盹 or 600 Pg linaclotide. A method of preparing a pharmaceutical composition comprising linaclotide or a salt thereof, the method comprising: (a) providing an aqueous solution comprising: 142528.doc 201023874. (i) linaclotide or a pharmaceutically acceptable thereof (ii) one or more selected from the group consisting of Mg2+, ~+, Zn2+, Mn2+, κ+, hexa- or 刈3 + cations or sterically hindered primary amines; and (iii) pharmaceutically acceptable as appropriate Accepting the binder; and (b) applying the aqueous solution to a pharmaceutically acceptable filler to produce a linaclotide coated filler. 52. The method of claim 51, wherein the aqueous solution comprises a sterically hindered primary amine. 53. The method of claim 51, wherein the Mg2+, Ca2+, Zn2+, Mn2+, K, Na or Al3+ is magnesium acetate, magnesium carbonate, magnesium phosphate, magnesium sulfate, calcium vinegar, calcium carbonate, calcium phosphate , calcium sulfate, zinc acetate, gasification, zinc phosphate, zinc sulfate, acetic acid, gasification, acid filling, sulfuric acid, potassium acetate, potassium carbonate, potassium phosphate, potassium sulfate, sodium acetate, sodium carbonate Provided in the form of sodium phosphate, sodium sulphate, acetic acid, gasification, sulphate or sulphate. 54. The method of claim 5, wherein the cation is ca2+. 55. The method of claim 53 wherein the agent is a sterically hindered primary amine selected from the group consisting of r2 10 amino acids, polymeric amines or a compound of the formula: R 1 ^ R 3 , wherein R], n h 2 R 2 and R 3 independently Selected from: η, C(〇)〇H, C1-C6 alkyl, C-C6 alkyl bond, C1-C6 alkyl sulfide, C1-C6 alkyl carboxylic acid, C1-C6 alkyl carboxylate and An alkylaryl group in which any group may be substituted with a single or multiple halogen or amine groups, and the limitation is that no more than one of R1, Ruler 2 and r3 is ruthenium. The method of claim 55, wherein the stereoscopic amine is a naturally occurring aminic acid selected from the group consisting of histidine, albino, and alanine. , glutamine, aspartic acid, glutamic acid, leucine, xje ^ ^ T thiol 'salmonic acid, tyrosine, threonine, isoleic acid, a group of components. A method of claim 56, wherein the naturally occurring amino acid is a method of the white amine Φ 58. = Item 55, wherein the steric hindrance of the primary amine is non-naturally occurring = amino acid or amine A basal organism selected from the group consisting of aminocyclohexanone, lanthionine, and theanine. The method of claim 55 wherein the steric hindrance of the primary amine is cyclohexylamine or 2-methylbutylamine. The method of claim 55, wherein the steric hindrance of the primary amine is polyglucosamine 0. The method of claim 51, wherein the aqueous solution further comprises a selected from the group consisting of A BHT, vitamin E, gallic acid, ascorbic acid, and An antioxidant of a salt or ester, a tocopherol and its ester, a lipoic acid or a p-carrot. The method of claim 51, wherein the aqueous solution comprises the cation selected from the group consisting of Mg2+, Ca2+, Zn2+, Mn2+, K+, Na+ or ai3+ and the steric hindrance of the primary amine. 63. The method of claim 62, wherein the cation is ~+ and is provided in the form of a gasification dance. For example, the method of U.S. Patent 62, wherein the stereoscopic hindrance of the primary amine is leucine. 142528.doc 201023874 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. and a method selected from the group consisting of polymaltodextrin or a fiber such as claim 51, wherein the bonding The agent is present in dilute alcohol, polyethylene "pyrrolidone (poly-dimensional), powder killing, vitamins_. A method of A 65, wherein the binder is cellulose and is selected from the group consisting of cellulose m (tetra) 1 methyl cellulose, base 1 ethyl methyl cellulose, propyl cellulose and vitamins. The method of soil item 51, wherein the filler is a fine cellulose or a crystallite such as the method of claim 51, wherein the weight ratio of linaclotide to the pharmaceutically acceptable filler is 1:100 and 1:1000 between. The method of claim 51, wherein the cation: sterically hindered primary amine: Linole-like molar ratio is from 4 Å to 100:20 to 30:1. The method of claim 69, wherein the cation is Ca 2+ . The method of claim 70, wherein the steric hindrance of the primary amine is leucine. For example, in the method of claim 5, wherein the molar ratio of Ca2+: leucine: Linac is 60:30:1. The method of claim 5 wherein the method further comprises (4) applying a coating to the linaclotide coated filler. The method of claim 73, wherein the coating is selected from the group consisting of Aquac〇at, Eudragit or Opadry. The method of claim 1, wherein the linaclotide coated filler is mixed with one or more pharmaceutically acceptable additives. The method of claim 51, further comprising the linaclotide package 142528.doc •10· 201023874 The filler is tableted or encapsulated. 77. The method of claim 76, wherein each capsule or tablet contains 5 to 1 mg of linaclotide. The method of claim 77, wherein each capsule or tablet contains 67" 盹, 133 pg, 15 〇, 266 pg or 300 pg of linaclotide. 79. Use of a pharmaceutical composition according to claim 4 for the manufacture of a medicament for the treatment of a patient suffering from intestinal motility injury, intestinal irritation, constipation, pain associated with constipation, dyspepsia Gastroparesis, chronic intestinal pseudo-occlusion, Crohn's disease, ulcerative colitis or inflammatory bowel disease. 80. The use of claim 79, wherein the intestinal irritation is constipation-type intestinal irritation or alternating intestinal irritation. 81. The use of item 80, wherein the intestinal irritation is constipation-type intestinal irritation. 82. The use of claim 79, wherein the constipation is chronic constipation, primary constipation, postoperative intestinal obstruction or constipation caused by the use of opiates. 83. The use of claim 82, wherein the constipation is chronic constipation. 84. The use of claim 79, wherein the medicament comprises 5 yang to i mg linaclotide. 85. The use of claim 84, wherein the medicament comprises 67.5 pg, 133 pg, 150 pg, 266 pg or 300 linaclotide. 86. The use of claim 85, wherein the medicament is administered once daily or twice daily. 87. The use of claim 86, wherein the medicament is administered once daily in the form of one or two tablets or capsules 142528.doc 201023874. 88. A composition comprising at least 10%, at least 20%, at least 30%, at least 40% or at least 50% by weight of a hydrolysis product of linaclotide; at least 10% by weight, at least 20 %, at least 30%, at least 40% or at least 50% of the oxidation product of linaclotide; or at least 10%, at least 20%, at least 30%, at least 40% or at least 50% by weight of linaclotide The formaldehyde imine product. 89. The composition of claim 88, wherein the composition has at least 90%, at least 95% or at least 98% by weight of a hydrolysate of linaclotide. 90. The composition of claim 88, wherein the composition has at least 90%, at least 95% or at least 98% by weight of an oxidation product of linaclotide. 91. The composition of claim 88, wherein the composition has at least 90%, at least 95% or at least 98% by weight of the formaldehyde imine product of linaclotide. The pharmaceutical composition according to claim 4, wherein the hydrolysate has the structure H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH SS- SS -1 which is less than 2% by weight based on the weight of the SS linaclotide. 93. The pharmaceutical composition of claim 92, wherein the hydrolysate comprises less than 0.1% by weight or less than 〇0.055% by weight of the linaclotide. 94. The pharmaceutical composition of claim 4, wherein the formaldehyde imine product has the structure H2C-cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH I_ II 142528.doc -12- 201023874 'It accounts for less than 2% by weight of the weight of the linaclotide. 95. The pharmaceutical composition of claim 94, wherein the formaldehyde imine product comprises less than 〇.丨 by weight of the linaclotide. /β or less than 〇 〇5 weight 0 / 〇. 96. The pharmaceutical composition of claim 4, wherein the linaclotide oxidation product having a molecular weight of 1542.8 comprises less than 2% by weight of the weight of the linaclotide. The pharmaceutical composition according to claim 96, wherein the linaclotide oxidation product accounts for less than 〇·1% by weight or less than 0.05% by weight of the linaclotide. 98. Use of a pharmaceutical composition according to claim 2, for the manufacture of a medicament for treating constipation-type intestinal irritation (IBS_c) in an adult patient in need thereof, wherein the medicament is administered once daily. 99. The use of claim 98, wherein the medicament comprises 266 linaclotide. 100. The use of claim 98, wherein the medicament comprises 133 linaclotide. 101. The use of claim 98, wherein the treatment lasts for at least 4 weeks. 102. The use of claim 98, wherein the treatment ameliorates at least one of the following symptoms: relieved abdominal pain, increased number of complete spontaneous defecation (CSBM) within one week, and spontaneous defecation (SBM) within one week Increased number of times, improved stool consistency, relieved bowel movements, relieved abdominal discomfort, relieved bloating or relieved severity of IBS_C symptoms. 103. Use of a pharmaceutical composition according to claim 2, for the manufacture of a medicament for the treatment of chronic constipation in a patient in need thereof, wherein the medicament is administered once daily. 104. The use of claim 1 to 3, wherein the medicament comprises 266 linaclotide. 105. The use of claim 1 to 3, wherein the medicament comprises 丨33盹linalotide. 142528.doc -13· 201023874 106. Use of claim 1 〇3 where the treatment improves at least one symptom selected from the group consisting of an increase in the number of complete spontaneous bowel movements (CSBM) within one week and spontaneous bowel movements within one week Increased number of (SBM), improved stool consistency, relieved bowel movements, relieved abdominal discomfort, relieved bloating, or constipation severity. 107. A pharmaceutical composition comprising: linaclotide; Ca2+; leucine; and hydroxypropyl methylcellulose, wherein Ca2+: leucine: linaclotide molar ratio at 5 to ι 〇0:5 to 50:1. 108. The pharmaceutical composition of claim 107, wherein the 2* is provided in the form of caCl2. 109. A unit dosage form comprising the pharmaceutical composition of claim ι. 110. The unit dosage form of claim 109, wherein the linaclotide is present in the pharmaceutical composition in an amount between 1 and 600. 111. The unit dosage form of claim 110, wherein the linaclotide is present in an amount of 266 (d). 112. The unit dosage form of claim 109 wherein the amount of caci2# 1541 pg is present. 113. The unit dosage form of claim 109, wherein the leucine is present in an amount of 687. 114. The unit dosage form of claim 109, wherein the hydroxypropyl methylcellulose is present in an amount of 142528.doc -14 - 201023874 700 pg. 115. A pharmaceutical composition comprising coated beads, wherein the beads are coated with a coating solution comprising linaclotide. 116. The pharmaceutical composition of claim 115, wherein the coating solution comprises: linaclotide; Ca2+; leucine; and hydroxypropyl methylcellulose, wherein Ca2+: leucine: linaclotide Moerby is between 5 and 100:5 to 50:1. 117. A unit dosage form comprising the pharmaceutical composition of claim 6. 118. The unit dosage form of claim 117, wherein the linaclotide is present in the pharmaceutical composition in an amount between 丨〇〇 and 600 pg. 119. The unit dosage form of claim 118, wherein the linaclotide is present in an amount of 266 tons. 120. The unit dosage form of claim 117, wherein the 2+ is provided in the form of CaCl2 in an amount of 1541 tons. 121. The unit dosage form of claim 117, wherein the leucine is present in an amount of 687. 122. The unit dosage form of claim 11, wherein the hydroxypropyl methylcellulose is present in an amount of 7 〇 0 pg. 123. The pharmaceutical composition of claim 115, wherein the beads comprise microcrystalline cellulose. 124. A pharmaceutical composition comprising: 142528.doc -15. 201023874 Linaclotide; Ca2+; leucine; PVP; and isomalt, wherein Ca. leucine, linaclotide The molar ratio is between 5 and 100:5 to 50.1 'and the weight ratio of linacodol to isomalt is between 1:1000. 125. A unit dosage form comprising the pharmaceutical composition of claim 24. 126. The unit dosage form of claim 125, wherein the linaclotide is present in the pharmaceutical composition in an amount between 1 〇〇 (4) and 6 〇〇 pg. 127. The unit dosage form of claim 126 wherein the linaclotide is present in an amount of 266 cations. 128. The unit dosage form of claim 125, wherein the (^2+ is provided as CaCl2 in an amount of 1541 pg. 129. The unit dosage form of claim 125, wherein the leucine is present in an amount of 687 pg. The unit dosage form of claim 125, wherein the hydroxypropyl decyl cellulose is present in an amount of 700 pg. 142528.doc 16-