(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`
`
`111111111110110101011111010111110 01111111111111010111111111111110111011111
`
`(43) International Publication Date
`18 February 2010 (18.02.2010)
`
`PCT
`
`(10) International Publication Number
`WO 2010/019266 A2
`
`(51) International Patent Classification:
`A61K 38/10 (2006.01)
`A61P 1/00 (2006.01)
`
`(21) International Application Number:
`PCT/US2009/004675
`
`(22) International Filing Date:
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`14 August 2009 (14.08.2009)
`
`English
`
`English
`
`(30) Priority Data:
`61/089,422
`61/273,332
`61/231,725
`
`15 August 2008 (15.08.2008)
`3 August 2009 (03.08.2009)
`6 August 2009 (06.08.2009)
`
`US
`US
`US
`
`(71) Applicants (for all designated States except US): IRON-
`WOOD PHARMACEUTICALS, INC. [US/US]; 320
`Bent Street, Cambridge, MA 02141 (US). FOREST
`LABORATORIES INC. [US/US]; 909 Third Avenue,
`New York, NY 10022 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): FRETZEN, Angeli-
`ka [DE/US]; 7 Avon Street, Somerville, MA 02143 (US).
`WITOWSKI, Steven [US/US]; 171 Laurel Street, Mel-
`rose, MA 02176 (US). GROSSI, Alfredo [US/US]; 102
`Ten Hills Road, Somerville, MA 02145 (US). ZHAO,
`Hong [CN/US]; 74 Cragie Street, Apt. 31, Somerville,
`MA 02143 (US). DEDHIYA, Mahendra [US/US]; 1 Lea
`Court, Pomona, NY 10970-3219 (US). MO, Yun
`[CN/US]; 60 Fairfield Way, Apt. 1, Commack, NY 11725
`(US).
`
`(74) Agent: O'BRIEN, Jonathan, P.; Honigman Miller
`Schwartz & Cohn LLP, 444 West Michigan Avenue,
`Kalamazoo, MI 49007 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI,
`NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT,
`TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ,
`TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE,
`ES, FI, FR, GB, GR, HR, HU, 1E, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report (Rule 48.2(g))
`
`(54) Title: STABLE SOLID FORMULATION OF A GC-C RECEPTOR AGONIST POLYPEPTIDE SUITABLE FOR ORAL
`ADMINISTRATION
`
`(57) 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 GIu Tyr Cys Cys Asn Pro Ala Cys Thr GIy Cys Tyr ("linaclotide") or a pharmaceutically acceptable salt thereof. The lina-
`clotide formulations described herein are stable and have a sufficient shelf life for manufacturing, storing and distributing the
`drug.
`
`WO 2010/019266 A2
`
`MYLAN - EXHIBIT 1012
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`STABLE SOLID FORMULATION OF A GC-C RECEPTOR AGONIST
`
`POLYPEPTIDE SUITABLE FOR ORAL ADMINISTRATION
`
`5
`
`This disclosure concerns solid formulations of a guanylate cyclase-C receptor agonist
`polypeptide suitable for oral administration and methods for preparing such formulations.
`
`FIELD
`
`10
`
`15
`
`20
`
`25
`
`30
`
`PRIORITY CLAIM
`
`This application claims priortiy to United States Application Serial No. 61/089,422,
`filed August 15, 2008 and to the United States Provisional Application filed August 03, 2009
`and entitled, "Stable Solid Formulation of a GC-C Receptor Agonist Polypeptide Suitable for
`Oral Administration". The entire contents of the aforementioned applications are
`incorporated herein by reference.
`
`BACKGROUND
`
`Many therapeutic polypeptides are formulated in aqueous solution because they are
`most active in this form. However, most polypeptides are not particularly stable in aqueous
`solution, such that the formulations often have a short half-life and require refrigeration.
`Although aqueous solutions of polypeptides can be dried by freeze-drying, spray-drying or
`other methods, such dried formulations may also be unstable and have reduced activity
`relative to an aqueous solution of the polypeptide. Typical break-down mechanisms that
`occur both in aqueous solution and in dried formulations include aggregation and oxidative or
`hydrolytic degradation. Thus, the majority of therapeutic polypeptides, whether in aqueous
`solution or dried, are stored under refrigerated conditions due to their limited stability.
`Linaclotide is a peptide having the amino acid sequence Cys Cys Glu Tyr Cys Cys
`Asn Pro Ala Cys Thr Gly Cys Tyr that activates the guanylate cyclase-C (GC-C) receptor.
`Linaclotide, which may be administered orally, is useful for the treatment of gastrointestinal
`disorders and conditions, including irritable bowel syndrome (IBS) and chronic constipation
`(CC). Formulations comprising linaclotide have needed to be refrigerated in order to avoid
`degradation over time. However, refrigeration is inconvenient both for commercial
`distribution of the drug and for storage by patients. Thus, there is a need to have a solid
`linaclotide formulation that is stable at room temperature for at least 12 months.
`
`1
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`SUMMARY
`
`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
`
`5
`
`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. For example, formulations described
`
`herein are expected to have a shelf life of at least 12 months at room temperature storage
`
`conditions (e.g., 25°C/60% relative humidity (RH)). In further embodiments, the
`
`10
`
`formulations described herein are expected to have a shelf life of at least 18 months or at least
`24 months at room temperature storage conditions (e.g., 25°C/60% RH).
`
`In some embodiments, formulations are described wherein > 95% of the original
`
`amount of linaclotide in the composition remains after three months when packaged samples
`
`are stored at accelerated conditions (40°C/75% RH) when assessed in an assay on a
`
`15 weight/weight basis as determined by high pressure liquid chromatography (HPLC) against a
`
`linaclotide reference standard. In further embodiments, > 90% of the original amount of
`
`linaclotide in the composition remains after at least 6 months when packaged samples are
`
`stored at accelerated conditions (40°C/75% RH). In other embodiments, formulations are
`
`described wherein chromatographic purity of the linaclotide as determined as area percent by
`20 HPLC remains at > 95% over the course of at least three months when packaged samples are
`
`stored at accelerated conditions (40°C/75% RH). In further embodiments, the
`
`chromatographic purity of the linaclotide as determined by area percent by HPLC remains at
`> 90% over the course of at least 6 months when packaged samples are stored at accelerated
`conditions (40 °C/75% RH). Thus, for example, no more than about 10% of the linaclotide
`
`25
`
`undergoes degradation to other products such as an oxidation product of linaclotide, a
`
`hydrolysis product of linaclotide or a formaldehyde-mediated imine product of linaclotide
`
`("formaldehyde imine product").
`
`In one embodiment, the invention comprises a pharmaceutical composition
`
`30
`
`comprising linaclotide, wherein the chromatographic purity of the linaclotide decreases by
`less than 10% after 18 months or 24 months of storage of the pharmaceutical composition at
`25°C at 60% relative humidity in a sealed container containing a desiccant. In a further
`embodiment, the chromatographic purity of the linaclotide decreases by less than 9%, 8%,
`7%, 6%, 5%, 4% or 2% after 18 months or 24 months of storage of the pharmaceutical
`
`2
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`composition at 25°C at 60% relative humidity in a sealed container containing a desiccant. In
`
`another embodiment, the invention comprises a pharmaceutical composition comprising
`
`linaclotide, wherein the chromatographic purity of the linaclotide decreases by less than 10%
`
`after 3 months or 6 months of storage of the pharmaceutical composition at 40°C at 75%
`
`5
`
`relative humidity in a sealed container containing a desiccant. In a further embodiment, the
`
`chromatographic purity of the linaclotide decreases by less than 9%, 8%, 7%, 6%, 5%, 4% or
`
`2% after 3 months or 6 months of storage of the pharmaceutical composition at 40°C at 75%
`
`relative humidity in a sealed container containing a desiccant.
`
`In one embodiment, the invention comprises a unit dosage form of a pharmaceutical
`
`10
`
`composition comprising linaclotide, wherein the chromatographic purity of the linaclotide
`
`decreases by less than 10% after 18 months or 24 months of storage of the unit dosage form
`
`at 25°C at 60% relative humidity in a sealed container containing a desiccant. In a further
`
`embodiment, the chromatographic purity of the linaclotide decreases by less than 9%, 8%,
`
`7%, 6%, 5%, 4% or 2% after 18 months or 24 months of storage of the unit dosage form at
`
`15
`
`25°C at 60% relative humidity in a sealed container containing a desiccant. In another
`
`embodiment, the invention comprises a unit dosage form of a pharmaceutical composition
`
`comprising linaclotide, wherein the chromatographic purity of the linaclotide decreases by
`
`less than 10% after 3 months or 6 months of storage of the unit dosage form at 40°C at 75%
`
`relative humidity in a sealed container containing a desiccant. In a further embodiment, the
`
`20
`
`chromatographic purity of the linaclotide decreases by less than 9%, 8%, 7%, 6%, 5%, 4% or
`
`2% after 3 months or 6 months of storage of the unit dosage form at 40°C at 75% relative
`humidity in a sealed container containing a desiccant.
`
`In one embodiment, the invention comprises a sealed container comprising a plurality
`
`of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the
`
`25
`
`chromatographic purity of the linaclotide decreases by less than 10% after 18 months or 24
`
`months of storage of the sealed container containing a desiccant at 25°C at 60% relative
`
`humidity. In a further embodiment, the chromatographic purity of the linaclotide decreases
`
`by less than 9%, 8%, 7%, 6%, 5%, 4% or 2% after 18 months or 24 months of storage of the
`
`sealed container containing a desiccant at 25°C at 60% relative humidity. In another
`
`30
`
`embodiment, the invention comprises a sealed container comprising a plurality of unit dosage
`
`forms of a pharmaceutical composition comprising linaclotide, wherein the chromatographic
`
`purity of the linaclotide decreases by less than 10% after 3 months or 6 months of storage of
`the sealed container containing a desiccant at 40°C at 75% relative humidity. In a further
`
`3
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`embodiment, the chromatographic purity of the linaclotide decreases by less than 9%, 8%,
`
`7%, 6%, 5%, 4% or 2% after 3 months or 6 months of storage of the sealed container
`
`containing a desiccant at 40°C at 75% relative humidity.
`
`In one embodiment, the invention comprises a pharmaceutical composition
`
`5
`
`comprising linaclotide, wherein the assay value for linaclotide determined on a weight/weight
`
`basis decreases by less than 10% after 18 months or 24 months of storage of the
`
`pharmaceutical composition at 25°C at 60% relative humidity in a sealed container
`
`containing a desiccant. In a further embodiment, the assay value for linaclotide determined
`
`on a weight/weight basis decreases by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%
`
`10
`
`after 18 months or 24 months of storage of the pharmaceutical composition at 25°C at 60%
`
`relative humidity in a sealed container containing a desiccant. In another embodiment, the
`
`invention comprises a pharmaceutical composition comprising linaclotide, wherein the assay
`
`value for linaclotide determined on a weight/weight basis decreases by less than 10% after 3
`
`months or 6 months of storage of the pharmaceutical composition at 40°C at 75% relative
`
`15
`
`humidity in a sealed container containing a desiccant. In a further embodiment, the
`
`chromatographic purity of the linaclotide decreases by less than 9%, 8%, 7%, 6%, 5%, 4%,
`
`3%, 2% or 1% after 3 months or 6 months of storage of the pharmaceutical composition at
`
`40°C at 75% relative humidity in a sealed container containing a desiccant.
`
`In one embodiment, the invention comprises a unit dosage form of a pharmaceutical
`
`20
`
`composition comprising linaclotide, wherein the assay value for linaclotide determined on a
`
`weight/weight basis decreases by less than 10% after 18 months or 24 months of storage of
`
`the unit dosage form at 25°C at 60% relative humidity in a sealed container containing a
`
`desiccant. In a further embodiment, the assay value for linaclotide determined on a
`
`weight/weight basis decreases by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% after 18
`
`25 months or 24 months of storage of the unit dosage form at 25°C at 60% relative humidity in a
`
`sealed container containing a desiccant. In another embodiment, the invention comprises a
`
`unit dosage form of a pharmaceutical composition comprising linaclotide, wherein the assay
`
`value for linaclotide determined on a weight/weight basis decreases by less than 10% after 3
`
`months or 6 months of storage of the unit dosage form at 40°C at 75% relative humidity in a
`
`30
`
`sealed container containing a desiccant. In a further embodiment, the assay value for
`
`linaclotide determined on a weight/weight basis decreases by less than 9%, 8%, 7%, 6%, 5%,
`
`4%, 3%, 2% or 1% after 3 months or 6 months of storage of the unit dosage form at 40°C at
`
`75% relative humidity in a sealed container containing a desiccant.
`
`4
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`In one embodiment, the invention comprises a sealed container comprising a plurality
`of unit dosage forms of a pharmaceutical composition comprising linaclotide, wherein the
`
`assay value for linaclotide determined on a weight/weight basis decreases by less than 10%
`
`after 18 months or 24 months of storage of the sealed container at 25°C at 60% relative
`
`5
`
`humidity in a sealed container containing a desiccant. In a further embodiment, the assay
`value for linaclotide determined on a weight/weight basis decreases by less than 9%, 8%, 7%,
`6%, 5%, 4%, 3%, 2% or 1% after 18 months or 24 months of storage of the sealed container
`
`containing a desiccant at 25°C at 60% relative humidity. In another embodiment, the
`invention comprises a sealed container comprising a plurality of unit dosage forms of a
`
`10
`
`pharmaceutical composition comprising linaclotide, wherein the assay value for linaclotide
`determined on a weight/weight basis decreases by less than 10% after 3 months or 6 months
`of storage of the sealed container containing a desiccant at 40°C at 75% relative humidity. In
`a further embodiment, the assay value for linaclotide determined on a weight/weight basis
`
`decreases by less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% after 3 months or 6 months
`of storage of the sealed container containing a desiccant at 40°C at 75% relative humidity.
`
`15
`
`In some embodiments, there is provided a pharmaceutical composition comprising
`linaclotide and a hydrolysis product comprising:
`
`H-Cys-Cys-Glu-Tyr-Cys-Cys-Asp-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH
`I S- S
`
`S S
`
`
`
`S S
`
`In some embodiments, the hydrolysis product comprises less than about 15% by
`20 weight of the composition, less than about 10% by weight of the composition, less than about
`7% by weight of the composition or less than about 5% by weight of the composition. In
`other embodiments, the hydrolysis product comprises from about 0.01% to about 15% by
`weight of the composition, about 0.05% to about 10% by weight of the composition, about
`0.05% to about 7% by weight of the composition or about 0.05% to about 5% by weight of
`the composition. In further embodiments, there is provided a method of treating a
`
`25
`
`gastrointestinal disorder in a patient in need thereof comprising administering a
`
`pharmaceutical composition comprising linaclotide and a hydrolysis product.
`In some embodiments, there is provided a pharmaceutical composition comprising
`linaclotide and a formaldehyde imine product comprising:
`
`30
`
`H2C=Cys-Cys-Glu-Tyr-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-Tyr-OH
`I S- S
`
`S S
`
`
`
`S S
`
`5
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`In some embodiments, the formaldehyde imine product comprises less than about
`15% by weight of the composition, less than about 10% by weight of the composition, less
`
`than about 7% by weight of the composition or less than about 5% by weight of the
`
`composition. In other exemplary embodiments, the formaldehyde imine product comprises
`
`5
`
`from about 0.01% to about 15% by weight of the composition, about 0.05% to about 10% by
`
`weight of the composition, about 0.05% to about 7% by weight of the composition or about
`0.05% to about 5% by weight of the composition. In further embodiments, there is provided a
`method of treating a gastrointestinal disorder in a patient in need thereof comprising
`
`administering a pharmaceutical composition comprising linaclotide and a formaldehyde
`
`10
`
`imine product.
`
`In some embodiments, there is provided a pharmaceutical composition comprising
`
`linaclotide and a linaclotide oxidation product. In one embodiment, the linaclotide oxidation
`
`product has a molecular weight of 1542.8, which most likely forms as the addition of a single
`
`oxygen atom to one of the six cysteinyl sulfurs in linaclotide. One potential structure of the
`
`15
`
`product is depicted below, although one of skill in the art will recognize that the oxygen atom
`
`may 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
`I s-s .
` S S
`
`I
`
`'
`
`
`
`0
`
`S S
`
`In another embodiment, there may be an addition of more than one oxygen atom to
`linaclotide, which would increase its molecular weight by 16 AU per added oxygen atom.
`
`20
`
`In some embodiments, the linaclotide oxidation product comprises less than about
`15% by weight of the composition, less than about 10% by weight of the composition, less
`than about 7% by weight of the composition or less than about 5% by weight of the
`composition. In other exemplary embodiments, the linaclotide oxidation product comprises
`from about 0.01% to about 15% by weight of the composition, about 0.05% to about 10% by
`25 weight of the composition, about 0.05% to about 7% by weight of the composition or about
`0.05% to about 5% by weight of the composition. In further embodiments, there is provided a
`
`method of treating a gastrointestinal disorder in a patient in need thereof comprising
`
`administering a pharmaceutical composition comprising linaclotide and a linaclotide
`
`oxidation product.
`
`30
`
`The assay value on a weight/weight basis ("weight/weight assay") may be determined
`by comparing, e.g., by HPLC, the amount of linaclotide in a sample, to a linaclotide reference
`
`6
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`standard. As used herein, the weight of linaclotide in a composition after storage at room
`temperature or accelerated conditions at a specified time point (e.g., three or six months of
`storage under accelerated conditions [40°C/75% RH] or 12, 18 or 24 months of storage under
`room temperature conditions [25 °C/60% RH]) is compared to the weight of linaclotide in a
`composition at an initial time (e.g., the time when the pharmaceutical composition is released
`for clinical or patient use ("the release date")) to provide the weight/weight assay value. For
`example, the weight of linaclotide in a composition is measured after storage for a specified
`time at accelerated conditions (40°C/75% RH) and compared to the weight of linaclotide that
`was present in the sample at the release date. In another example, the weight of linaclotide in
`a composition is measured after storage for a specified time at room temperature conditions
`(25°C/60% RH) and compared to the weight of linaclotide that was present in the sample at
`the release date. Thus, the phrase "> 90% of the original amount of linaclotide in the
`composition remains after at least 6 months when packaged samples are stored at accelerated
`conditions (40°C/75% RH)" means the weight of linaclotide in the composition measured in
`an assay on a weight/weight basis as determined by HPLC after at least 6 months storage at
`accelerated conditions is > 90% of the amount of linaclotide in the composition present at the
`initial time (e.g., the release date of the linaclotide composition).
`
`Chromatographic purity of linaclotide may be assessed by performing HPLC under
`the conditions described herein. The area under the linaclotide peak is measured and
`compared to the total area under all peaks excluding the solvent peak and any non-
`polypeptide related peaks (i.e., peaks associated with excipients that may be observed in a
`placebo). As used herein, the chromatographic purity of linaclotide in a composition after
`storage at room temperature or accelerated conditions at a specified time point (e.g., three or
`six months of storage under accelerated conditions [40°C/75% RH] or 12, 18 or 24 months of
`storage under room temperature conditions [25 °C/60% RH]) is compared to the
`chromatographic purity of linaclotide in a composition at an initial time (e.g., the time when
`the pharmaceutical composition is released for clinical or patient use ("the release date")) to
`provide the chromatographic purity value. For example, the chromatographic purity of
`linaclotide in a composition is measured after storage for a specified time at accelerated
`conditions (40°C/75% RH) and compared to the chromatographic purity of linaclotide in the
`composition at the release date. In another example, the chromatographic purity of
`linaclotide in a composition is measured after storage for a specified time at room
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`7
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`temperature conditions (25°C/60% RH) and compared to the chromatographic purity of
`
`linaclotide in the composition at the release date.
`
`This disclosure features a method for preparing a pharmaceutical composition
`
`5
`
`comprising linaclotide or a pharmaceutically acceptable salt thereof, the method comprising:
`(a) providing a solution, e.g., an aqueous solution ("the coating solution"), comprising: (i)
`linaclotide or a pharmaceutically acceptable salt thereof; (ii) a cation selected from Mg2+,
`Ca2+, Zn2+, Mn2+, K+, Na+ or Al3+ and/or a sterically hindered primary amine (e.g., leucine)
`and, optionally, (iii) a pharmaceutically acceptable binder; and (b) applying the coating
`
`solution to a pharmaceutically acceptable filler to generate polypeptide-coated filler (e.g., by
`
`10
`
`spraying, mixing or coating the pharmaceutically acceptable filler with the coating solution).
`
`The method can optionally include one or more of: (i) blending the polypeptide-coated filler
`
`with a pharmaceutically acceptable glidant, a pharmaceutically acceptable lubricant or a
`
`pharmaceutically acceptable additive that acts as both a glidant and lubricant; (ii) blending
`
`the polypeptide-coated filler with filler that is not polypeptide-coated, (iii) blending the
`
`15
`
`polypeptide-coated filler with other additives; (iii) applying a pharmaceutically acceptable
`
`coating additive to the polypeptide-coated filler. The final pharmaceutical composition can
`be placed into capsules (e.g., gelatin capsule) or used to form tablets.
`
`It has been found that a cation selected from Mg2+, Ca2+, Zn2+, Mn2+, K+, Na+ or
`Al3is useful for suppressing the formation of an oxidation product of linaclotide during
`storage. It has also been found that a sterically hindered primary amine, e.g., leucine, is
`useful for suppressing the formation of a formaldehyde imine adduct of linaclotide
`("formaldehyde imine product") during storage. Thus, a linaclotide formulation comprising a
`
`cation selected from Mg2+, Ca2+, Zn2+, Mn2+, K+, Na+ or Al3+ (e.g., a divalent cation selected
`
`from Zn2+, Mg2+ or Ca2+) and/or a sterically hindered primary amine, such as an amino acid,
`has a sufficient shelf life (as measured by chromatographic purity and/or by a weight/weight
`assay) for manufacturing, storing and distributing the drug. Further, while the presence of a
`sterically hindered amine alone can increase the formation of a hydrolysis product of
`linaclotide during storage, the combination of a sterically hindered primary amine and a
`cation, e.g., the combination of leucine and Ca2+, suppresses the formation of the hydrolysis
`
`product of linaclotide as well as the oxidation product of linaclotide during storage, leading to
`an even greater overall stability as determined by a weight/weight assay and/or by
`chromatographic purity.
`
`20
`
`25
`
`30
`
`8
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`In some embodiments, there is provided a pharmaceutical composition comprising a
`pharmaceutically acceptable carrier, linaclotide and one or more agents selected from Mg2+,
`Ca2+, Zn2+, Mn2-4-, K+, Na+ or Al3+ and a sterically hindered primary amine, wherein the agent
`improves at least one attribute of the composition, relative to a pharmaceutical composition
`5 without said agent. In further embodiments, the agent is Mg2+, Ca2+ or Zn2+. In a further
`
`embodiment, the agent is Ca2+. In some embodiments, the cation is provided as, without
`
`limitation, magnesium acetate, magnesium chloride, magnesium phosphate, magnesium
`
`sulfate, calcium acetate, calcium chloride, calcium phosphate, calcium sulfate, zinc acetate,
`
`zinc chloride, zinc phosphate, zinc sulfate, manganese acetate, manganese chloride,
`
`10 manganese phosphate, manganese sulfate, potassium acetate, potassium chloride, potassium
`
`phosphate, potassium sulfate, sodium acetate, sodium chloride, sodium phosphate, sodium
`
`sulfate, aluminum acetate, aluminum chloride, aluminum phosphate or aluminum sulfate. In
`further embodiments, the cation is provided as magnesium chloride, calcium chloride,
`
`calcium phosphate, calcium sulfate, zinc acetate, manganese chloride, potassium chloride,
`sodium chloride or aluminum chloride. In other embodiments, the cation is provided as
`
`15
`
`calcium chloride, magnesium chloride or zinc acetate.
`
`In another embodiment, the agent is a sterically hindered primary amine. In a further
`
`embodiment, the sterically hindered primary amine is an amino acid. In yet a further
`
`embodiment, the amino acid is a naturally-occurring amino acid. In a still further
`
`20
`
`embodiment, the naturally-occurring amino acid is selected from the group consisting of:
`
`histidine, phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, leucine, methionine,
`
`asparagine, tyrosine, threonine, isoleucine, tryptophan, methionine and valine; yet further, the
`
`25
`
`30
`
`naturally-occurring amino acid is leucine, isoleucine, alanine or methionine; in another
`embodiment, the naturally-occurring amino acid is leucine or methionine; still further, the
`naturally-occurring amino acid is leucine. hi another embodiment, the sterically hindered
`primary amine is a non-naturally occurring amino acid or amino acid derivative (e.g., 1-
`
`aminocyclohexane carboxylic acid, lanthionine or theanine). In a further embodiment, the
`sterically hindered primary amine is cyclohexylamine, 2-methylbutylamine or chitosan.
`
`In other embodiments, there is provided a pharmaceutical composition comprising a
`pharmaceutically acceptable carrier, linaclotide, a cation selected from Mg2+, Ca2+, Zn2+,
`Mn2+, K+, Na+ or Al3+ (e.g., a divalent cation selected from Zn2+, Mg2+ or Ca2+) and a
`sterically hindered primary amine. hi one embodiment, the cation is Ca2+. In another
`embodiment, the cation is a mixture of two or three of Mg2+, ca2+7 zn2+,
`
`mn2+,K+, Na+ or
`
`9
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`Al3+ (e.g., a mixture of two or three of Zn2+, Mg2+ or Ca2+). In a further embodiment, the
`
`pharmaceutical composition further comprises a pharmaceutically acceptable binder and/or a
`
`pharmaceutically acceptable glidant, lubricant or additive that acts as both a glidant and
`
`lubricant and/or an antioxidant. In a further embodiment, the sterically hindered primary
`
`5
`
`amine is an amino acid. In yet a further embodiment, the amino acid is a naturally-occurring
`
`amino acid. In a still further embodiment, the naturally-occurring amino acid is selected from
`
`the group consisting of: histidine, phenylalanine, alanine, glutamic acid, aspartic acid,
`
`glutamine, leucine, methionine, asparagine, tyrosine, threonine, isoleucine, tryptophan,
`
`methionine and valine; yet further, the naturally-occurring amino acid is leucine, isoleucine,
`
`10
`
`alanine or methionine; in another embodiment, the naturally-occurring amino acid is leucine
`
`or methionine; still further, the naturally-occurring amino acid is leucine. In another
`
`embodiment, the sterically hindered primary amine can be a mixture of more than one
`
`sterically hindered primary amines. For example, the sterically hindered primary amine may
`
`be a mixture of two or more sterically hindered primary amines, e.g., a mixture of two or
`
`15 more amino acids.
`
`In some cases the molar ratio of cation:sterically hindered primary amine:linaclotide
`
`(e.g., Ca2+:1eucine:linaclotide) in the aqueous solution applied to the carrier is 5-100:5-50:1.
`
`It can be desirable for the molar ratio of cation:sterically hindered primary amine (e.g.,
`
`Ca2+:1eucine) to be equal to or greater than 2:1 (e.g., between 5:1 and 2:1). Thus, in some
`
`20
`
`cases the molar ratio of cation:sterically hindered primary amine:linaclotide (e.g.,
`
`Ca2+:1eucine:linaclotide) applied to the carrier is 100:50:1, 100:30:1, 80:40:1, 80:30:1,
`
`80:20:1, 60:30:1, 60:20:1, 50:30:1, 50:20:1, 40:20:1, 20:20:1, 10:10:1, 10:5:1 or 5:10:1.
`
`When binder, e.g., methylcellulose, is present in the linaclotide solution applied to the carrier
`it can be present at 0.5% - 2.5% by weight (e.g., 0.7%-1.7% or 0.7% - 1% or 1.5% or 0.7%).
`
`25
`
`The weight of linaclotide applied to a given weight of filler (e.g., microcrystalline
`
`cellulose) can vary from about 0.02:100 to about 2.67:100. Thus, about 0.05 mg to about 6.0
`
`mg of linaclotide can be applied to 225 mg of filler. In a further embodiment, the weight of
`
`linaclotide applied to a given weight of filler is about 0.05 mg to about 2.0 mg of linaclotide
`
`(e.g., 0.1, 0.2, 0.3. 0.4, 0.5, 0.6, 0.7 mg'peptide for 225.mg of filler).
`
`30
`
`In various embodiments: the sterically hindered primary amine is an amino acid (e.g.,
`
`a naturally-occurring amino acid or a naturally-occurring amino acid selected from histidine,
`
`phenylalanine, alanine, glutamic acid, aspartic acid, glutamine, methionine, asparagine,
`
`tyrosine, threonine, leucine, isoleucine, tryptophan, or valine). In other cases the sterically
`
`10
`
`

`

`WO 2010/019266
`
`PCT/US2009/004675
`
`hindered primary amine is a non-naturally occurring amino acid or amino acid derivative
`
`(e.g., lanthionine, theanine or 1-amino cyclohexane). In other cases, the sterically hindered
`
`primary amine is an amino sugar (e.g., chitosan or glucosamine).
`
`NH2
`
`In some cases, the sterically hindered primary amine has the formula:
`
`5 wherein R1, R2 and R3 are independently selected from: H; —C(O)OH; C1-C6 alkyl,
`
`optionally substituted by —CO2H, -CONH2, or a 5-10 membered aryl or heteroaryl; C1-C6
`
`alkoxyalkyl; or C1-C6 thioalkoxyalkyl , wherein any of the alkyl or aryl groups above can
`
`be singly or multiply substituted with halogen or —NH2, and provided that no more than two
`
`of R1, R2 and R3 are H. In a further embodiment, no more than one of R1, R2 and R3 is H.
`
`10
`
`The term "alkyl", as used herein, refers to