MYLAN EXHIBIT - 1068
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`US 11,319,346 B2
`Page 2
`
`(56)
`
`References Cited
`
`OTHER PUBLICATIONS
`
`Desai, et al., “Acetonitrile shortage: use of isopropanol as an
`alternative elution system for ultrahigh performance iquid chroma-
`tography”, Analytical Methods, vol. 3, No. 1, 2011, pp. 56-58.
`Pitari, et al., “Pharmacology and clinical potential of guanylyl
`cyclase C agonists in the treatment of ulcerative colitis”, Drug
`Design Development and Therapy, vol. 7, 2013, pp. 351-360.
`
`* cited by examiner
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet1 of 16
`
`US 11,319,346 B2
`
`Particle Size (um)
`
`FIG, I
`
`==a® =go
`
`m 4
`
`Particle Size Distribution of Plecanatide
`
`oPlecanatide
`(Precipitated)
`Lot # 120210
`
`mPlecanatide
`(Lyophilized)
`Lot # 101221
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet2 of 16
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`US 11,319,346 B2
`
`
`
`FIG,2
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet3 of 16
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`US 11,319,346 B2
`
`
`
`

`

`U.S. Patent
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`May3, 2022
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`Sheet 4 of 16
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`US 11,319,346 B2
`
`ate
`
`sey
`
`
`
`aeercnoutetanaeneteeearannenemn
`
`6 MO
`
`“6 #0 £0 £8 OR Zo
`onus
`
`ateeaten
`
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 5 of 16
`
`US 11,319,346 B2
`
`Peptide, e.g., dicyclized SP-304 or SP-333
`| Load to the RP-HPLC cloumn
`| Elute with ACN/H,0
`—
`
`Pool qualified fractions, e.g., >95% HPLC pure
`
`Salt exchange
`
`Pool qualified fractions, e.g., >95% HPLC pure
`
`Load the main pool to column packaged with polymeric adsorbent
`for desalination
`
`Wash the column with deinoized water
`
`aaaaa Add an ether to the dewatered peptide, e.g., cold diethyl ether
`
`Elute the peptide with an alcohol aqueous solution (e.g., isopropanol/water) and
`collect the eluate
`
`Remove water, e.g., by azeotropicdistillation
`
`Remove alcohol, e.g., under reduced pressure
`
`Dry the peptide under vacuum
`
`Purified peptide for further drug manufacturing process
`
`FIG. 5
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 6 of 16
`
`US 11,319,346 B2
`
`Frrac-Giy-2ClTrityl
`
`i Solution Phase
`
`(Fmoe-Cys{Acm}-OQH
`
`
`:
`:
`
`Residues 7-14
`Resin boundpeptide:
`
`i
`
`Residues 1-16
`
`Residues 7-16
`Protected peptide
`
`
`Residues 15-16
`
`{Fragment
`
`
`e:
`|
`Residues 1-6
`
`
`esin boundpeptide _
`
`
`
`
`
`
`
`iMonocyclic Plecanatid
`» (monocyclization with
`
`
`Lo
`peroxide in air Cys4
`
`
`
`
`
`
`
`
`|
`Dicyclic Plecanatide
`| (cyclization with iodine.
`Cys7 and Cys15 to form.
`ond
`disulfide
`
`
`
`
`
`
`
`Primary recycle
`1.
`‘purification by RP-HPLC
`1%
`
`
`
`Secondary recycie
`‘purification by RP HPLC |
`
`(O2% aceticacia in
`radient acetonitrile)
`
`ISOLATION PROCESS 1
`CURRENT
`ISOLATION PROCESS
`
`
`ISOLATION PROCESS 2
`
`REVISED SOLVENT EXCHANGE
`ISOLATION PROCESS.
`
`
`
`
`Solvent exchange
`
`
`
`Reconstitution with
`
`acetonitrile-wate
`
`
`
`
`Final tyophilizatio
`
`Reconstitution with
`buffer
`
`
`
`
`Final tyophilization
`
`
`FIG.6
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 7 of 16
`
`US 11,319,346 B2
`
`FIG, 7
`
`Scheme 1
`
`“
`
`-
`
`Ze
`
`DBU,HOBt
`a,
`
`NH»
`
`O
`bee OH
`0
`NHFmoc piperidine
`
`\ Cc)
`DME/DCN
`6-6 a 3
`
`
`
`2-chioratrity! chloride resin (2-CITrt)—Fmoc-Leu-2-ClTrt resin H-Leu-2-CiTrt resin
`
`STrt
`
`I
`
`)
`
`aN
`
`cl —
`BocHN,,, Pah yn
`Boc-Asn(Trt}-Asp(OtBu)-Giu(OlBu)}+Cyattayct(O%8u).-Leu-OH,(Fragment AorBocAAt-
`
`TrtHN.
`
`60H)
`
`Ou
`
`oO”
`
`“OtBu
`
`{amino acid residues 1-6 of
`SEQ ID NO: 1)
`
`0
`
`DBU,HOBt
`NHFmoc piperidine
`in DMF
`
`NH»
`oO
`eet
`
`2-chiorotrity! chloride resin (2-ClTrt} Fmoc-Gly-2-CiTrt resin
`
`H-Gly-2-ClTrt resin
`
`Oeeen
`
`.
`~~) i Ore
`S
`|?Oy
`
`i) Peptide Chain assembly
`ing
`F
`hemist
`
`using Fmocchemistry
`
`7
`ii} 1% TFAIDCM
`
`‘.
`Hoys yy
`otae
`
`{OH
`0
`
`STHtHiN
`
`™wy moc
`
`-
`
`NHF
`
`NHAC
`
`Fmoc-Cys(Acm}-Val-Asn(Trt}-Val-Ala-Cys(Trt}-Thr(tBu)-Gly-OH (Fragment B or FmocAA7-140H)
`
`{amino acid residues 7-14 of SEQ ID NO: 1)
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 8 of 16
`
`US 11,319,346 B2
`
`FIG.8
`
`Scheme 2
`
`H-Leu-CIBu.HO}
`
`DHEA, HOBtL, HBTU
`
`+ Fmoc-Cys{Acn}-OH
`DME
`
`Frooc-Cys(Aom}-Leu-OiBuy
`FrocAA15-16GiBu
`
`DIEA, CRHOBt HBTU
`;
`Piperidine/DCM
`aeetnnmneennannmnnnnntee CSACLOLetennanntnFmocAA?-1801Bu
`HAATS-1GO%Bu
`PMOCAAT=140K,
`TIS/EDTITFA
`Fiperidine/DCM HAAT BOHR DIEA, CLHOBL HBTU
`
`Boos
`1-BOH,
`BocAAt-18O1Bu
`HAA
`
`
`
`sete LA AEARN feet 4-1801R: qu
`
`T-16OH
`
`H-Asn-Asp-Glu-Cys-Giu-Leu-Cys(Acm)-Val-Asr-VabAla-Cys-Thr-Ghy-Cys(Acm-Le@u-OH
`
`SEQ ID NO: 1
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 9 of 16
`
`US 11,319,346 B2
`
`FIG. 9
`
`Scheme 3
`
`H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH
`
`.
`
`| H,0>
`HAA1-160H
`H-Asn-Asp-Glu-ys-Glu-Leu-Cys(Aem)-Val-Asn-Val-Ala-fys-Thr-Gly-Cys(Acm)-Leu-OF
`
`SEQ ID NO:1
`
`monocyclized HAA1-160H
`Io/AcOH
`
`SEQ ID NO: 1
`
`H.Asn-Asp-Glu-Gys-Ghi-Leu-bys-Val-Asn-ValAla-Cys-Thr-Gly-bys-Leu-O#
`
`SEQ ID NO: 1
`
`dicyclized HAA1-160H
`| Load to the polystyrenic absorbent resin cloumn
`| Elute with Ethanol
`H-Asnhep-Gheye-GlsLovbys-abAsnebAle-GyTr-Gly-Cys-Leu-OH
`
`dicyclized HAA1-160H
`
`SEQ ID NO; 1
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 10 of 16
`
`US 11,319,346 B2
`
`FIG.10
`
`Scheme 4
`
`H-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-Leu-OH
`
`-
`
`| HO»
`HAA1-160H
`H-Asn-Asp-Glu-Fys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Gys-Thi-Gly-Cys(Acm)-Leu-OH
`
`SEQ ID NO: 1
`
`monocyclized HAA1-160H
`lofAcCOH
`
`SEQ iD NO: 1
`
`H-Asn-Asp-Glu- ys-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys-Leu-OH
`
`SEQ ID NO: 4
`
`dicyclized HAA1-160H
`| Load to the RP-HPLC column
`| Elute with ACN/H,0
`| Pool qualified fractions
`| Salt exchange and lyophilization
`H-Asn-Asp-Glu- ys-Glu-Leu-bys:ValeAgn-ValeAla-Cys-Thr-Gly-Cys-Leu-OF
`
`SP 304 drug substance
`
`SEQ ID NO: 1
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 11 of 16
`
`US 11,319,346 B2
`
`FIG. 11
`
`Scheme 5
`
`
`
`Solid phase synthesis of the two side-chain-protected fragments on 2-ClTrt resin
`
`RS
`
`Fmoc-Pro-OH
`DIEA in
`DMF/DCM
`
`DBU,HOBt
`fimoe piperidine
`LINDMF pty
`
`|
`
`
`cy
`
`wig
`>-O
`
`H
`ON
`
`oO
`
`2-chloratrity! chloride resin (2-CiTrt)
`
`Fmoc-Pro-2-CiTit resin
`
`H-Pro-2-ClTot resin
`
`vas a
`6
`THN 4 4 PV ug a 0
`NHTrt
`‘
`Bochin NAw NN oy N
`O o
`cS
`j
`° Oru ° Nett oN
`) ms
`cpSo
`
`6
`
`Boc-Asn{Trt)-Phe*-Cys\(Trt}-Cys4{Trt}-Giu*(OtBu}-Ser9(tBu}-Cys{Trt)-Cys9(Trt)-Asn "(Trt}-Pro'-OH,(BocAAt-100H)
`
`
`
`
`
`Fmoc-Gly-OH
`
`DIEA in
`
`DMF/DCM
`
`
`2-chlorotrity! chloride resin (2-CITrt) Fmoc-Gly-2-CITrt resin
`
`{amino acid residues 1-10 of SEQ 1D NO: 58}
`
`NHFmoc piperidine
`
`
`fie
`
` DBU,HOBt
`in DMFObesor
`
`i} Peptide Chain assembly
`
`usingFmocchemistry
`
`i) 1%FFAIDCM
`
`cman
`
`Fmoc-Ma"-Cys'@(Trt)-Thr’*(tBu)}-Gly"+-OH,(FmocAA11-140H)
`
`H-Gly-2-ClTrt resin
`
`iif)
`Mey
`
`“ou
`
`OH
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 12 of 16
`
`US 11,319,346 B2
`
`FIG, 12
`
`Scheme 6
`
`Solution phase synthesis of the side-chain-protected C-terminal dipeptide
`
`QO
`
`OfBu
`
`H-15-160tBu
`
`OtBu
`
`NH,
`:
`OtBu HC!
`
`+
`
`THs
`
`°
`
`NHFmoc
`
`H-Tyr(tBu}-OtBu.HCl
`i} HOBt, HBTU, DIPEA, DMF;
`ii} 5% Piperidine in DOM
`
`Fmoc-Cys(Trt}-OH
`
`H-Cys(Trt}-Tyr"S(#8u)-OrBu
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 13 of 16
`
`US 11,319,346 B2
`
`FIG, 13
`
`Scheme 7
`
`Fragment condensation,disulfide bridges, folding, and purification
`
`
`
`
`
`
`HoyeStnTyrue + PomdlsOpsTa}ThBabealySH
`HARARE
`PracdAl 4d0H
`
`[peruCBOECEPI, OME
`PreteOyereethrteenySousteThreinCre:
`ProohAl ities
`
`(amino acid residues 11-16 of
`SEQ ID NO: 58}
`
`| 2s Piperidinein DAF
`Heaia*LdyeSTTheiecleCeeteTeMeath
`PEASEROME
`
`{amino acid residues 11-16 of
`SEQ ID NO:58)
`
`HBTU ie2G, DIPEA,
`SEQ ID NO: 58
`$ BomAATTRLDRE
`BandenYTPhehyeTeyTtASerieyeCTRCysYTrtsTitPreaiahatesAyTithe
`ThreBWoytyeeT)TyagCae
`
`TRAEDTTISHott BeesSwi, ah
`SEQ ID NO: 8
`™
`:
`"
`fesigyLPCeieSerieaehetpenihetusoreageneSpeCd
`| AMNEHCO,, 1 MGuHCl, 3nhepsteineand 0.4 mMcystine, pHa4
`Oxidativetoiding
`a
`llENBc
`Pears“sayaGiarya!yeaePgSa‘acyeParteaySaasMaryae
`
`4
`;
`;
`———
`SEQ ID NO: 58
`Ppanarstive RP-HPLC,
`Rourtecion exchange
`Lymhiisaiiicrs
`
`
`
`SPE drug subahance:
`
`
`
`
`
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 14 of 16
`
`US 11,319,346 B2
`
`FIG, 14
`
`Fmoc-Leu-OH
`DIEA in
`
`in DME
`
`Q__
`
`DBU,HOBt
`BUH
`AtFmoc piperidine
`
`|
`
`AO NH
`
`DMEDCM CR o: L oN (
`
`2-chiorotrityl chloride resin (2-ClTr)
`
`Fmoc-Leu-2-CiTrt resin
`
`H-Leu-2-ClTrt resin
`
`Q. OtBu
`| ')
`ii)
`neue OH (amino acid residues 1-6 of
`BocHN
`OtBu
`oo1eu
`Boc-D-Asn(Trt)-Asp(OtBu)-Glu(leu.Ogeerty.Glo1BuyLevon(Fragment AorBocAA1-60H)
`
`Trt
`
`O
`
`SEQ ID NO:9)
`
`_STrt
`
`.
`
`Ry
`
`DBU,HOBt
`
`es
`“S
`
`|
`
`pom
`
`)
`
`in
`
`Nie
`Q_fFmee pierce
`7.
`UO DEAin
`b-c)
`PME/OCM. —o~d ooo
`as
`
`
`Le cl
`2
`Cl
`Cl
`|
`~
`.
`a
`2-chiorotrityl chloride resin (2-ClTrt) Fmoc-Giy-2-CiTrt resin
`H-Gly-2-ClTrt resin
`
`[2hh,
`HO.reytywasyh? Qe
`
`.
`
`NHAC
`
`i) Peptide Chain assembly
`using Fmoc chemist
`
`g
`
`ii) 1%TFA/DCM
`
`ry
`
`STethiN
`ibe
`Fmoc-Cys(Acm)-Val-Asn{Tit}-Val-Ala-Cys(Tit)-Thr(iBu}-Gly-OH(Fsagment 8B or FmocAA7-140H)
`
`{amino acid residues 7-14 of SEQ 1D NO: 9)
`
`Scheme 8
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 15 of 16
`
`US 11,319,346 B2
`
`FIG. 15
`
`Scheme 9
`
`+
`
`H-D-Leu-OtBu.HCl
`
`DIEA, HOBt, HBTU
`
`Fmoc-Cys(Acm)-OH
`DMF
`Piperidine/DCM H-Cys(Acm)-D-Leu-OtBu DIEA, CLHOBt, HBTU
`HAA15-160tBu
`COAT1408,
`
`Fmoc-Cys(Acm)-D-Leu-OtBu
`FmocAA15-1601Bu
`
`FmocAA7-160tBu
`
`TIS/EDT/TFA
`Piperidine/DCMcig EA: CHHOBE, HBTU
`BocAAL6OH”_BOCAAI-1601BU
`HAA1-160H
`
`ie 460t8u —-—_——_——>
`
`.
`
`
`
`_
`
`H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-D-Leu-OH
`
`DMF
`
`(SEQ ID NO: 9)
`
`

`

`U.S. Patent
`
`May3, 2022
`
`Sheet 16 of 16
`
`US 11,319,346 B2
`
`FIG, 16
`
`Scheme 10
`
`H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys(Acm)-D-Leu-OH
`
`(SEQ ID NO: 9)
`| HO,
`HAA1-160H
`H-D-Asn-Asp-Glu-Gys-Glu-Leu-Cys(Acm)-Val-Asn-Val-Ala-Gys-Thr-Gly-Cys(Acm}-D-Leu-OH
`(SEQ ID NO: 9)
`
`monocyclized HAA1-160H
`|,/ACOH
`
`H-D-Asn-Asp-Glu-Cys-Glu-Leu-Cys-Val-Asn-Val-Ala-Cys-Thr-Gly-Cys-D-Leu-OH
`Pod
`
`(SEQ ID NO:9)
`
`dicyclized HAA1-160H
`| Load to the RP-HPLC column
`| Elute with ACN/H,0
`| Pool qualified fractions
`| Salt exchange and lyophilization
`PED-ASIASPGluC¥S-GlurLeu-CYS-WalASn-Val-Ale-CysTrGly-Cys-D-LeuOH
`SP-333 drug substance
`(SEQ ID NO:9)
`
`

`

`US 11,319,346 B2
`
`1
`ULTRA-PURE AGONISTS OF GUANYLATE
`CYCLASE C, METHOD OF MAKING AND
`USING SAME
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a continuation of, and claims priority
`to, U.S. application Ser. No. 17/207,215, filed Mar. 19, 2021,
`now abandoned, which is a continuation of, and claims
`priority to, U.S. application Ser. No. 16/921,450, filed Jul. 6,
`2020, now issued as U.S. Pat. No. 11,142,549, whichis a
`continuation of, and claims priority to, U.S. application Ser.
`No. 16/000,251, filed Jun. 5, 2018, now issued as U.S. Pat.
`No. 10,745,441, which is a continuation of, and claims
`priority to, U.S. application Ser. No. 14/896,019, filed Dec.
`4, 2015, now issued as U.S. Pat. No. 10,011,637, whichis a
`U.S. national stage application of, and claims priority to,
`International Application No. PCT/US2014/041143, filed
`Jun. 5, 2014, which claims priority to U.S. provisional
`application No. 61/831,402, filed Jun. 5, 2013, the contents
`of which are herein incorporated by reference in their
`entireties.
`
`INCORPORATION-BY-REFERENCE OF
`MATERIAL SUBMITTED AS A TEXT FILE VIA
`THE OFFICE ELECTRONIC FILING SYSTEM
`
`The Sequence Listing associated with this application is
`provided intext format inlieu of a paper copy, and is hereby
`incorporated by referenceinto the specification. The name of
`the text file containing the Sequence Listing is “376464-
`2011US6_Sequence_ListingST25.txt.” The text
`file is
`about 105 KB, was created on Sep. 15, 2021, and is being
`submitted electronically via EFS-Web.
`
`FIELD OF THE INVENTION
`
`The present invention relates to processes of purifying
`guanylate cyclase C peptide agonists useful for preparing
`formulations for the treatment and prevention of various
`diseases and disorders.
`
`BACKGROUNDOF THE INVENTION
`
`Guanylate cyclase C is a transmembrane form of gua-
`nylate cyclase that is expressed on various cells, including
`gastrointestinal epithelial cells (reviewed in Vaandrager
`2002 Mol. Cell. Biochem. 230:73-83).
`It was originally
`discovered as the intestinal receptor for the heat-stable toxin
`(ST) peptides scereted by enteric bactcria and which cause
`diarrhea. The ST peptides share a similar primary amino acid
`structure with two peptides isolated fromintestinal mucosa
`and urine, guanylin and uroguanylin (Currie, et al., Proc.
`Nat'l Acad. Sci. USA 89:947-951 (1992), Hamra, et al.,
`Proc. Nat'l Acad. Sci. USA 90:10464-10468 (1993); Forte,
`L., Reg. Pept. 81:25-39 (1999); Schulz, et al., Cell 63:941-
`948 (1990); Guba, et al., Gastroenterology 111:1558-1568
`(1996); Joo,et al., An. J. Physiol. 274:G633-G644 (1998)).
`In the intestines, guanylin and uroguanylin act as regula-
`tors of fluid and electrolyte balance. In response to high oral
`salt intake, these peptides are released into the intestinal
`lumen where they bind to guanylate cyclase C localized on
`the luminal membrane of enterocytes (simple columnar
`epithelial cells of the small
`intestines and colon). The
`binding of the guanylin peptides to guanylate cyclase C
`induces electrolyte and water excretion into the intestinal
`
`20
`
`30
`
`35
`
`40
`
`45
`
`50
`
`5
`
`60
`
`65
`
`2
`lumen via a complex intracellular signaling cascade that is
`initiated by an increase in cyclic guanosine monophosphate
`(cGMP).
`is initiated by the
`The cGMP-mediated signaling that
`guanylin peptides is critical for the normal functioning of the
`gut. Any abnormality in this process could lead to gastro-
`intestinal disorders such as irritable bowel syndrome (IBS)
`and inflammatory bowel diseases. Inflammatory bowel dis-
`ease is a general name given to a group ofdisorders that
`cause the intestines to become inflamed, characterized by
`red and swollen tissue. Examples include ulcerative colitis
`and Crohn’s disease. Crohn’s disease is a serious inflam-
`matory disease that predominantly affects the ileum and
`colon, but can also occur in other sections of the gastroin-
`testinal tract. Ulcerative colitis is exclusively an inflamma-
`tory disease ofthe colon, the large intestine. Unlike Crohn’s
`disease, in whichall layers ofthe intestine are involved, and
`in which there can be normal healthy bowel in between
`patches ofdiseased bowel, ulcerative colitis affects only the
`innermost
`lining (mucosa) of the colon in a continuous
`manner. Depending on which portionof the gastrointestinal
`tract is involved, Crohn’s disease may be referred to as
`ileitis, regional enteritis, colitis, etc. Crohn’s disease and
`ulcerative colitis differ from spastic colonorirritable bowel
`syndrome, which are motility disorders of the gastrointes-
`tinal tract. Gastrointestinal inflammation can be a chronic
`
`condition. It is estimated that as many as 1,000,000 Ameri-
`cans are afflicted with inflammatory bowel disease, with
`male and female patients appearing to be equally affected.
`Mostcases are diagnosed before age 30, but the disease can
`occur in the sixth, seventh, and later decades of life.
`IBS and chronic idiopathic constipation are pathological
`conditions that can cause a great deal of intestinal discom-
`fort and distress but unlike the inflammatory boweldiseases,
`IBS does not cause the serious inflammation or changes in
`bowel tissue and it is not thought to increase the risk of
`colorectal cancer. In the past, inflammatory bowel disease,
`celiac disease, and IBS were regarded as completely sepa-
`rate disorders. Now, with the description of inflammation,
`albeit low-grade, in IBS, and of symptom overlap between
`IBS and celiac disease,
`this contention has come under
`question. Acute bacterial gastroenteritis is the strongest risk
`factor identified to date for the subsequent development of
`postinfective irritable bowel syndrome. Clinical risk factors
`include prolonged acute illness and the absence of vomiting.
`A genetically determined susceptibility to inflammatory
`stimuli may also be a risk factor for irritable bowel syn-
`drome. The underlying pathophysiology indicates increased
`intestinal permeability and low-grade inflammation, as well
`as altered motility and visceral sensitivity. Serotonin (5-hy-
`droxytryptamine [5-HT]) is a key modulator of gut function
`and is knownto play a majorrole in pathophysiology of IBS.
`The activity of 5-HT is regulated by cGMP.
`While the precise causes of IBS and inflammatory bowel
`diseases (IBD) are not known,a disruptionin the process of
`continual renewal ofthe gastrointestinal mucosa may con-
`tribute to disease pathology in IBD and aggravate IBS. The
`renewal process of the gastrointestinal lining is an efficient
`and dynamic process involving the continual proliferation
`and replenishment of unwanted damaged cells. Proliferation
`rates of cells lining the gastrointestinal mucosa are very
`high, second only to the hematopoietic system. Gastrointes-
`tinal homeostasis depends on both the proliferation and
`programmed cellular death (apoptosis) of epithelial cells
`lining the gut mucosa. Cells are continually lost from the
`villus into the lumen ofthe gut and are replenished at a
`substantially equal rate by the proliferation of cells in the
`
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`US 11,319,346 B2
`
`3
`crypts, followed by their upward movementto the villus.
`The rates of cell proliferation and apoptosis in the gut
`epithelium can be increased or decreased in a variety of
`circumstances, e.g.,
`in response to physiological stimuli
`such as aging, inflammatory signals, hormones, peptides,
`growth factors, chemicals and dietary habits. In addition, an
`enhancedproliferation rate is frequently associated with a
`reduction in turnover time and an expansion ofthe prolif-
`erative zone. The proliferation index is much higher in
`pathological states such as ulcerative colitis and other gas-
`trointestinal disorders.
`Intestinal hyperplasia is a major
`promoter of gastrointestinal inflammation. Apoptosis and
`cell proliferation together regulate cell number and deter-
`mine the proliferation index. Reduced rates of apoptosis are
`often associated with abnormal growth, inflammation, and
`neoplastic transformation. Thus, both increased proliferation
`and/or reduced cell death may increase the proliferation
`index of intestinal tissue, which may in turn lead to gastro-
`intestinal inflammatory diseases.
`In addition to a role for uroguanylin and guanylin as
`modulators of intestinal fuid and ion secretion, these pep-
`tides may also be involved in the continual renewal of
`gastrointestinal mucosa by maintaining the balance between
`proliferation and apoptosis. For example, uroguanylin and
`guanylin peptides appear to promote apoptosis by control-
`ling cellular ion flux. Giventhe prevalence of inflammatory
`conditions in Western societies a need exists to improve the
`treatment options for inflammatory conditions, particularly
`of the gastrointestinal tract.
`Peptide agonists of guanylate cyclase C agonists (“GCC
`agonists”) are described in U.S. Pat. Nos. 7,041,786, 7,799,
`897, and U.S. Patent Application Publication Nos. US2009/
`0048175, US 2010/0069306, US 2010/0120694, US 2010/
`0093635, and US 2010/0221329, and WO2012/118972.
`However, the previous syntheses of peptides for pharma-
`ceutical application present a numberof special problems
`such as an overall low yield (e.g., less than 10%), and/or
`high levels of impurities (e.g., contaminants resulted from
`organic solvents used during syntheses or purification, and
`degradation products or topoisomers created, e.g., during
`purification).
`
`BRIEF SUMMARY OF THE INVENTION
`
`40
`
`45
`
`In one aspect, the present invention is derived [rom the
`effort to solve various unexpected problems encountered
`during the purification processes of peptide GCC agonists
`for pharmaceutical application, such as the lyophilization
`process and precipitation process described in WO2012/
`118972. The methods described herein provide a solution to ;
`those problems.
`In one aspect, the present invention provides a purified
`peptide comprising the GCC agonist sequenceselected from
`the group consisting of SEQ ID NOs: 1, 9, and 104, wherein
`the purified peptide has the following characteristics:
`a) has a bulk density of not greater than 0.1 g/mL;
`b) contains less than 50 ppm acetamide;
`c) less than 0.3% alpha-Asp-9-plecanatide.
`The purified peptide can have one or more ofthe follow-
`ing features.
`For, example, the peptide is stable at 25° C. for at least
`three months.
`For example , the peptide has a particle size distribution
`having a D10 value of between about 2 tp 15 um; a D50
`value of between about 15-50 um; and a D90 value of
`between about 40-80 um when measured by light scattering
`with liquid dispersant.
`
`60
`
`65
`
`4
`For example, the purified peptide contains no more than
`35 ppm acetamide (e.g., =18 ppm).
`For example,
`the purified peptide contains less than
`0.15% alpha-Asp-9-plecanatide (which has a Relative
`Retention Time (RRT) of ~1.33 from the ultra-performance
`liquid chromatography (UPLC) analysis described herein).
`For example, the purified peptide has a bulk density of not
`greater than 0.09 g/mL, not greater than 0.08 g/mL, not
`greater than 0.07 g/mL, not greater than 0.06 g/mL, not
`greater than 0.05 g/mL, not greater than 0.04 g/mL, or not
`greater than 0.03 g/mL.
`For example, the purified peptide is substantially free of
`water (e.g., water content not exceeding 10%, 9%, 8%, 7%,
`6%, 5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.25%,
`or 0.1%, of the total weight of the peptide).
`For example, the purified peptide has a chromatographic
`purity of no less than 95%, no less than 96%, or no less than
`97%.
`For example,the total content of impurities in the purified
`peptide is less than 3% (e.g., <2% or <1%).
`For example, the purified peptide is further substantially
`free of one or more impurities selected from acetonitrile,
`alcohols, ammonium, acetates, and TFA.
`For example, the purified peptide contains less than 300
`ppm acetonitrile (e.g., <250 ppm).
`For example, the purified peptide contains less than 0.2%
`TPA (e.g., <0.15%, <0.1%, <400 ppm, <300 ppm, <200
`ppm, <100 ppm, or <50 ppm).
`For example, the purified peptide contains less than 0.2%
`isopropanol,
`i.e., IPA (e.g., <0.15%, <0.1%, <1000 ppm,
`<900 ppm<800 ppm, <700 ppm, <600 ppm, <500 ppm,
`<400 ppm, <300 ppm, <200 ppm, <100 ppm, <50 ppm, or
`<20 ppm).
`the purified peptide contains less than
`For example,
`0.25% acetate (e.g., <0.2% or <0.1%).
`For example, the purified peptide is substantially free of
`topoisomers (e.g., <0.4%, <0.3%, <0.2% or <0.1%),.
`For example, the purified peptide is substantially free of
`iso-Asp2-plecanatide (RRT 0.96-0.97) (e.g., <0.4%, <0.3%,
`<0.2% or <0.1%).
`In another aspect, the invention also provides a process of
`purifying/isolating the peptide comprising the GCC agonist
`sequence selected from the group consisting of SEQ ID
`NOs: 1-251. The process includes:
`providing a first peptide solution comprising a peptide
`comprising the GCC agonist sequence selected from the
`group consisting of SEQ ID NOs: 1-251, water, and acetoni-
`trile;
`loading a C18 or polymeric adsorbent column with the
`first peptide solution to adsorb the peptide onto the poly-
`meric adsorbent column,
`eluting the peptide off the C18 or polymeric adsorbent
`column with an alcohol aqueous solution to form a second
`peptide solution,
`reducing the amount of alcohol in the second peptide
`solution, and
`lyophilizing the second peptide solution such that a dry
`peptide is obtained.
`The process can include one or more of the following
`features.
`the alcohol aqueous solution comprises
`For example,
`isopropanol (e.g., with isopropanol content in the alcohol
`aqueous solution being about 40%).
`In another embodiment,
`the alcohol aqueous solution
`comprises propanol, tert-butanol, 2-butanol, or ethanol.
`For example,thefirst peptide solution further comprises
`acetamide.
`
`

`

`US 11,319,346 B2
`
`5
`For example, the first peptide solution further comprises
`acetic acid (e.g., 0.2%) or triethylamine phosphate (e.g.,
`1%).
`For example, the amountofalcohol (e.g., isopropanol) in
`the second peptide solution is reduced e.g. by rotoevapora-
`tion to less than 5%.
`For example, the process further comprises dissolving the
`dry peptide in water to form a third peptide solution after
`lyophilization. For example, the third peptide solution fur-
`ther comprises ammoniumacetate or ammonium hydroxide
`(e.g., suchthat the third solution has a pH value ofabout 5).
`For example, the process further comprises lyophilizing
`the third peptide solution such that a purified peptide is
`obtained.
`
`6
`(e.g., <0.2% or <0.1%). The content of contaminants can be
`determined by conventional methods such as gas chroma-
`tography. Preferably, the residual solvents in the purified
`peptide of the inventionare less thanthe limits set in the ICH
`guidelines,
`eg.
`IMPURITIES: GUIDELINE
`FOR
`RESIDUAL
`SOLVENTS
`Q3C(R5)
`(available
`at
`www.ich.org/fileadmin/Public_Web_Site/ICH_Products/
`Guidelines/Quality/Q3C/Step4/Q3C_R5_Step4 pdf).
`For example,the purified peptide contains less than 0.3%
`(e.g., <0.15%) alpha-Asp-9-plecanatide (RRT 1.33).
`For example, the purified peptide has a bulk density of not
`greater than 0.09 g/mL, not greater than 0.08 g/mL, not
`greater than 0.07 g/mL, not greater than 0.06 g/mL, not
`greater than 0.05 g/mL, not greater than 0.04 g/mL, or not
`greater than 0.03 g/mL.
`For example, the purified peptide is substantially free of
`iso-Asp2-plecanatide (RRT ~0.96-0.97).
`In this context
`“substantially” free of iso-Asp2-plecanatide means that the
`iso-Asp2-plecanatide content ofthe peptide at the end of the
`purification process is preferably less than 2%, less than
`1.5%, less than 1.25%, less than 1%, less than 0.9%, less
`than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%,
`less than 0.4%, less than 0.3%, less than 0.2%, or less than
`0.1%, of the total weight of the peptide.
`For example, the purified peptide is substantially free of
`topoisomers. In this context “substantially” free of topoiso-
`mers meansthat the topoisomer content of the peptide at the
`end of the purification process is preferably less than 2%,
`less than 1.5%, less than 1.25%, less than 1%, less than
`0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less
`than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%,
`or less than 0.1%, of the total weight ofthe peptide.
`For example, the purified peptide is substantially free of
`water. In this context “substantially” free of water means
`that the water content of the peptide at
`the end of the
`purification process is preferably less than 10%, 9%, 8%,
`7%, less than 6%, less than 5%, less than 4.5%, less than
`4.25%, less than 4%, less than 3.5%, less than 3%, less than
`2.5%, less than 2%, less than 1.5%, less than 1%, less than
`0.5%, less than 0.25%,or less than 0.1%, of the total weight
`ofthe peptide.
`For example , the peptide has a particle size distribution
`having a D10 value of between about 2 tp 15 um; a D50
`value of between about 15-50 um; and a D90 value of
`between about 40-80 um when measured by light scattering
`with liquid dispersant.
`For example,
`the purified peptide has a particle size
`distribution characterized by a D50 value of about 600 pm
`when measured by light scattering with air dispersant. In
`comparison, the peptides purified from the lyophilization
`and precipitation processes described in WO2012/118972
`have 1D50 values of about 180-250 umand about 300 um,
`respectively.
`For example, the purified peptide prepared by the pro-
`cesses of the invention has a suitable size distribution for
`pharmaceutical formulation. In one embodiment, the peptide
`(e.g., SP-304) has a sizedistribution(e.g., an average size of
`80-120 um) comparable to that of the pharmaceutical excipi-
`ent (e.g., microcrystalline cellulose) used in the formulation,
`for example,
`in the 3 mg/day unit dose form. The size
`distribution of the purified peptide may vary based on the
`unit dose. For example, when unit dose is lower than 3
`mg/day, the purified peptide in the pharmaceutical formu-
`lation has a smaller average size than that in the 3 mg/day
`dose. For example,
`the purified peptide prepared by the
`processesofthe inventionis milled to reachthe suitable size
`distribution.
`
`For example, the peptide in the first peptide solution is
`prepared by the fragment condensation process (i.e., hybrid
`solution- and solid-phase process) as described in WO2012/
`118972. In one embodiment, the first peptide solution is
`obtained froma salt exchanging step in whichthe peptide is
`washed with an aqueous acetonitrile solution comprising
`triethylamine phosphate or acetic acid.
`For example, the polymeric adsorbent column is a pre-
`parative C,, RP-HPLC column. In one embodiment, the
`polymeric adsorbent column comprises a polystyrene resin.
`In particular, the resin is selected so that the purified peptide
`eluted or desorbed is not less than 80% of the peptide
`amount adsorbed on the resin, e.g., not less than 85%, not
`less than 90%, or not less than 95%. In one embodiment, the
`resin is formed of crosslinked polystyrene with an average
`pore diameter greater than 5 nm,e.g., about 6-8 nm, 10-15
`nm, 15-20 nm, or 25-30 nm.
`In yet another aspect,
`the invention also provides a
`purified peptide prepared by the purification process of the
`invention. The purified peptides may have one or more of the
`following features.
`For example,
`the purified peptide comprises the GCC
`agonist sequence selected from the group consisting of SEQ
`ID NOs: 1, 9, and 104.
`For example, the purified peptide has a chromatographic
`purity of no less than 96%, no less than 97%, or no less than
`98%. For example, the GCC agonist peptide has chromato-
`graphic impurity content of no greater than 4%, no greater
`than 3.5%, no greater than 3%, no greater than 2.5%, no
`greater than 2%, no greater than 1.5%, or no greater than
`1%. The chromatographic impurity content is determined as
`total area percentages of impurities by HPLC. The chro-
`matographic impurity content includes topoisomer content.
`The impurities do not include any pharmaceutically accept-
`able excipient used for drug formulation.
`For example, the purified peptide is substantially free of ;
`contaminants resulted from the peptide preparation process
`such as organic solvents used in the process, e.g., ammo-
`nium, acetonitrile, acetamide, alcohol (e.g., methanol, etha-
`nol, or isopropanol), TRA, ether or other contaminants. In
`this context “substantially” free of contaminants means that
`the contaminant content of the peptide at the end ofthe
`purification process is preferably less than 0.5%, less than
`0.3%, less than 0.25%, less than 0.1%, less than 0.05%,less
`than 0.04%,
`less than 0.03%, less than 0.02%,
`less than
`0.01%, less than 0.005%, less than 0.003%, or less than
`0.001% of the total weight of the peptide. For example, the
`purified peptide contains <50 ppm acetamide(e.g., =35 ppm
`or =18 ppm), <300 ppm acetonitrile (e.g., <250 ppm), <1000
`ppmTFA(e.g., <400 ppm, <300 ppm, <200 ppm, <100 ppm,
`or <50 ppm), <2000 ppm isopropanol (e.g., <1500 ppm,
`<1000 ppm, <500 ppm, <400 ppm, <300 ppm, <200 ppm,
`<100 ppm, <50 ppm, or <20 ppm), and/or <0.25% acetate
`
`5
`
`20
`
`30
`
`35
`
`40
`
`45
`
`60
`
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`

`US 11,319,346 B2
`
`BRIEF DESCRIPTION OF DRAWINGS
`
`FIG. 1 is a graph showing particle size distribution by
`sieving analysis for lyophilized plecanatide and precipitated
`plecanatide.
`FIG. 2 is an optical microscopic image of lyophilized
`plecanatide.
`FIG. 3 is an optical microscopic image ofprecipitated
`plecanatide.
`FIG. 4 is a UPLC chromatogram of plecanatide isolated
`by one embodimentof the purification process of the inven-
`tion.
`
`7
`Thesize distributionofthe peptide ofthe invention can be
`determined by traditional methods, such as sieve analysis,
`light obscuration or dynamic light scattering analyses.
`The invention also relates to a formulation(e.g., an oral
`formulation) containing the peptides prepared and/or puri-
`fied by the methods described herein and in particular, a low
`dose formulation containing 0.05-10 mg(e.g., 0.1 mg, 0.3
`mg or 0.5 mg) of the purified peptides. The low-dose
`formulation can further have one or more additional features
`as described in WO2012/037380 and US 2012-0237593 and
`can be prepared by the methods disclosed therein, such as
`dry blending.
`Other features and advantages of the invention will be
`apparent
`from and are encompassed by the following
`detailed description and claims.
`
`8
`acetonitrile/water solvent, unexpectedly resulted in enrich-
`mentofresidual acetamide(a trace level impurity in acetoni-
`trile) in the lyophilized plecanatide product with high acet-
`amide content (i.e., ranging from 88 to 453 ppm among
`tested batches, or about 300 ppm on average), which hin-
`dered commercialization of plecanatide with doses higher
`than 3 mg/day.
`In addition,
`the lyophilized plecanatide
`productalso had a high variability of residual salt levels such
`as TFA, acetate, and ammonium salts.
`Onthe other hand, while the plecanatide product purified
`via the precipitation process described in WO2012/118972
`had low residual acetamide content (<50 ppm) and higher
`bulk or tap density (ie., about ten times higher) than the
`lyophilized product,
`the precipitated plecanatide product
`contained high levels of residual solvents (e.g., IPA as high
`as 90,000 ppm) due to difficulty in removing the solvents
`used for peptide precipitation. In addition, while low tem-
`perature heating (45° C.) during vacuumdr