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`UNITED STATES DEPARTMENT OF COMMERCE
`United States Patent and Trademark Office
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`July _10, 2017
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`THIS IS TO CERTIFY THAT ANNEXED IS A TRUE COPE FROM THE
`RECORDS OF THIS OFFICE OF THE FILE WRAPPER AND CONTENTS
`
`OF:
`
`APPLICATION NUNIBER: 10/222,540
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`FILING DATE: August 16, 2002
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`PATENT NUMBER: 6,803,046
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`ISSUE DATE: October 12, 2004
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`By Authority of the
`
`Under Secretary of Commerce for Intelieetual Property
`and Director of the United States Patent and Trademark Office
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`wad/”Z...“
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`JOHN A BURSON
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`Certifying Officer
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`MAIA Exhibit 1002
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`MAIA Exhibit 1002
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`TTORNEY OCKET Nf
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`APPLICATION
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`APPLICANT
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`TITLE
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`:
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`:
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`Edmund C. Metcalie, Jo Anna Monteferrante, Margaret
`Newbom, lrene‘ Ropiak, Ernst Schramm, Gregory W. White,
`Julius P. Zodda
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`SINCALIDE FORMULATIONS
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`F:\50203\50203.017DO1---Utility Cover Sheetwpd
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`24111311111111 ‘1:11.111
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` PATENT
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`ATTORN X DOCKET NO: 50203/017001
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`
`SINCALIDE FORMULATIONS
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`Field ofthe Invention
`_
`.
`The invention relates to pharmaceutically acceptable formulations of sincalide.
`
`Backgr'ound of the Invention I
`KINFV-AC® (‘Sincalid'e for Injection.'USP) is a cholecystopancreatic-
`. T'lgastrointestinalhOrmone peptide for parenteral administration. The active
`'
`j
`'jpharmaceutical ingredient 1—De(5-0Xo-L'-glutamine-5—L—proline)-2-de—L—
`7methion1necaerule1n or “sincalide” (CAS# 25 126—32-3), is a synthetically prepared C-
`7‘ terminaloctapeptide of eholecystokinin (CCK—S), with the followmg amino acid
`‘ sequence: Asp—‘Tyr(SO3H)-Met-Gly-Trp—Met—AspP—he—NH;
`KINFVAC®was first introducedin 1976, and was finished as a sterile,
`inonpyrogenic lyophilized WhitepOWderin a 5-mL (nominal) glass vial to Contain: 5 pg
`l'siiicalide With 45 mg sodium chlorideto provide toniCity;sodium hydroxide or
`' hydrochloricacid may have been addedfor pH adjustment (pH 5.5- 65). The type I
`_V',glass vial Was sealed under anitrogen headSpace with a Tompkins B0849 Closure. This
`I ftwo—ingredientformulation was Incorporated into the U.S. Pharmacopea/National
`V>F0r1nulary, USP 24, NF l9, Ianuaryl, 2000.
`Since its introduction, various draWbacks1n the manufacturing and analysis of
`, KINEVAC® have been identified. For eXample, the tWo--ingredient formulation suffers
`from potenCy variability. This variability was exacerbated by the fact that the
`< formulation Was analyzed using a guinea pig gallbladder contraction bioassay for potency
`Xofboth sincalideand KINFVAC®.-This bioassay was unable todistinguish between
`b10act1v1ty ofsincalide and bioaCtivity of sincalide degradants. Accordingly, a 20%
`. overage- of sincalide was requiredin previoussincalide formulations to compensate for
`.vthe limitations of the bioassay. lhus thereis a need for sincalide formulations having
`improved and consistent potency as established by a sincalide specific assay such as
`finite.
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`Summary of the Invention
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`The present invention satisfies the need for improved sincalide formulations by
`
` - monohydrochloride,andsodiummetabisulfite.
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`t
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`I
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`providing formulatiOns that eliminate theneed for a 20% overage 0f sincalide. The
`sincalide formulations ofthe invention are also purer than prior art formulations, and
`_ have fewer degradants and more consistent potency. In addition the purity ofthese
`formulations maybe assessed bypHPLC,'thus eliminating the need for the bioassay ofthe
`prior art formulations.
`.
`7
`t
`The present inVention provides sincalide formulations adapted for administration
`by'inj ection. These Sincalide formulations are characterized byimproved stability and
`
`fir may bevprepared as a relatively-large volume batch; (a1 00 L),
`‘1
`In oneaspect, the invention features sincalide formulations that include an
`V effective amount0fsmca11de a bulking agent/tonicity adjuster, one Or more stabilizers, a
`‘_ surfactant, a chelator, and a buffer. TheinVention also features kits and methods for
`preparing improvedSincalide formulations, as Well as methods fortreating, preventing,
`and diagnosing gall bladder—related disorders using sincalide formulations
`The formulat1ons ofthe inventionpreferably have a pH betWieen 6.0 and 8.0.
`.Suitable buffers include, but are not limited to, phosphate, citrate, sulfOSalicylate, borate,
`‘ acetate and amino acidbuffer's; Phosphate buffers, such as dibasic potassium phosphate,
`are preferred.- ,
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`In various embodiments ofthe invention, the surfactant1s a nonionic surfactant,
`preferably a polysorbate, Such as polysorbate 20 Or polysorbate 80; the chelator1s
`:I'pentetic acid (DTPA); and the stabilizer1s an antioxidant and/or amino acid In a
`‘_I particularly desirable embodiment ofthe invention, the formulation includes a plurality of
`'. stabilizers, preferably L—arginine monohydrochloride, L—methionine, L—lysine
`
`Suitable bulkingagents/tonicity adjusters include, but are not limited to, mannitol,
`lactose, sodium chloride, maltose, sucrose, PEG’s, Cyclodextrins, dextran, polysucrose
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`The sincalide formulations of the invention may also be administered to patients
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`receiving total parenteral nutrition (TPN), in order to treat and/or prevent TPN—related
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`disorders.
`
`Other features and advantages of the invention will be apparent from the following
`
`detailed description thereof and from the claims.
`
`
`Brief Description of the Drawings
`FIG. 1‘ is a drawing illustrating the chemical structure of 1—De(5-oxo-L—g1utamine—
`
`S—L—methioninecaerulein or “sincalide” (CAS# 25126—32—3). The amino acid residues
`
`“Met 3" and “Met 6” are outlined by dashed lines.
`
`FIG. 2 is a drawing illustrating the chemical structure of sincalide (Met 3)
`
`FIG. 3 is a drawing illustrating the chemical structure of sincalide (Met 6)
`
` FIG. 4 is a drawing illustrating the chemical-structure of sincalide (Met 3, 6)
`
`monosulfoxide.
`
`monosulfoxide.
`
`disulfoxide.-
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`FIG. 5 is a graphical representation of the effect of pH on the recovery of sincalide
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`in 35 mM phosphate buffer over 24 hours. At each pH for which data is shown, the bars
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`' represent 0, 6, and 24 hours, from left to. right. I
`
`FIG. 6 is a graphical representation of the effect of pH on the recovery of sincalide
`in a formulation of the invention over 8 hours. At each pH for which data is shown, the
`' bars represent 0, 4, and 8 hours, from left to right.
`
`FIG. 7 is a graphical representation of the percent sincalide Met 3 and Met 6
`
`monosulfoxides (vs sincalide), in the presence and absence of pentetic acid (DTPA).
`
`FIG. 8 is a chromatogram of KINEVAC® experimental formulation (no DTPA) spiked
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`with 0.63mM of".
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`FIG. 9 is a chromatogram of KJNEVAC® experimental formulation (lULVI DTPA) spiked
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`with 0.63mM 0112*.
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`(Ficoll), and polyvinylpyr'rolidine 3(PVP). D—Mannitol is a preferred bulking
`pagent/tOnicity adjuster.
`V
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`IIn a paiticularly preferred embodiment, the reconstituted formulation includes
`:0.0008 to 0.0012 mg/mL actiVeingredient (i.e,sincalide); 20.0 to 50.0 mg/mL mannitol,
`12.O to 7.0 mg/mL ar'ginine; 0.2 to 1.0 mg/mL methiOnine; 2.0 to 30.0 mg/rnL lysine,
`0.002 to 0.012 mg/mL sodium metabisulfite; 0.000001 to 0.003 mg/mL p01ysorbate 20,
`10.1to 3.0 mg/mL pentetic acid (DTPA); and 5.4 to 12.0 mg/mL potassium phosphate
`'(diba51c) In a mOre preferredembodiment the reconstituted formulation includes about
`’0001 mg/mL sincalide; about 34 mg/mL D—mannitol, about 6 mg/mL L-arginine
`monohydrochloride about 08 mg/mLL—methionine; about 3 mg/mL L—lysine
`monohydrochloride;_about 0.008 mg/mLsodium metabisulfite; less than about 0.01
`"f‘mg/mL polysorbate 20, about 04 mg/mL pentetic acid (DTPA); and about 1.8 mg/mL
`g potass1umphosphate (diba31c)
`'
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`The kits ofthe invention may, for eXample, include the various components ofthe
`formulation as a rriiXture inpowderfOrinalongWith a container (eg. a vial) to hold the
`powder mixture and a physrologically acceptable fluid fOr reconstitution of the
`‘formulat1on The components of theformulation may be present in the kit eitherin the
`,poWder miXture or in the fluidportion Kitsofthe inventionmay also include all
`coinponents in a liquid mixture or Some components in a liquid form and some in the
`form of a poWder.
`.
`1
`,.
`The formulations ofthe invention have improved stability and potency compared
`to previous sincalideformulations, and are useful as diagnostic aids forimaging the
`.hepatobiliarysystem of apatient When used as adiagnostic aid, the sincalide
`formulations may, for eXample, be co——administered with a radiopharmaceutical agent
`having rapid hepatic uptake, such as 991115—mebrofenin, or similar hepatobiliaryimaging
`agents, to assist in the diagnosis of gallbladder diseases and related disorders.
`_ Additionally, the formulations may be administered before and/or after diagnostic
`imaging (including for eXample, magneticresonanCe imaging, scintigraphic imaging,
`ultrasound imaging, etc.)
`
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`“1.11111111131 1:15LL11:1 31 1.1.1 1:111:11 11:31 1:1111‘
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`fl. filly-WEHEE 11$?th 111111111 1111111113‘
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`‘ FIG. 10 is a chromatogram of KINEVAC® experimental formulation (no DTTPA) spiked
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`-
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`, with 0.18mM M12+.
`,
`FIG 11 is a chromatogram of K1NBVAC® experimental formulation ‘(lmM DTPA)
`, spiked With 0 18mM Mn2+.
`_
`_
`FIG. 12 shows representative fiill—scale and expanded scale chromatograms of a
`» lyophilized reformulation ofK11§EVAC® upon reconstitution With SinL Water, resultingin a
`. sincalide concentration of lug/mL. "
`
`
`Detailed Description of the Invention
`Inorderto develop an improved Sincalide formulation a series of studies,
`‘ ,Eidescnbedin theExamplesbelow, Were conducted to determine the effects of various
`exc1pients on formulations of sinCalide. Through these studies, we discovered that the
`-.potencyand stability of s1nca11de formulations can be significantlyenhanced through the
`careful selection of excipients that provide certain desired functions. AccOrdingly, the
`., Ipresent inVentionprov1des novel sincalide formulations having improved stability and/or
`potency oVer previous formulations
`‘
`I: As used herein the term‘sincalide” includes the synthetically—prepared C-
`_ terminal octapeptide ofcholecystokiniii (CCK-8), with the amino acid sequence: Asp—
`. Tyr(SO3H)Met—Gly—Trp—Met-Asp—Phe—NH2, as well as derivatives thereofWhich have
`» been optimized or medified (toimprovestability, potency, pharmacokinetics, etc) but
`I: retain the biological actiVity ofthe originaloctapeptide. For example peptidesin which
`, themethionineand/or aspartic acid residues have been replaced Without significantly
`. affecting the biologicalactiVity are included Within‘‘sincalide” as the teim is used herein.
`Similarly, the term‘smcalide encompasses not only monomeric, but multimeric forms
`L " ofthe peptide as well as phySiologically actiVe degradants orportions ofthe peptide and
`'
`its derivatives.
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`The sincalide formulations of the inVention can include a variety of excipients,
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`‘ 5,such as, for example, antioxidants, buffers, bulking agents/tonicity adjusters, chelating
`
`agents, complexing agents, crosslinking agents, co-solvents, osmolality adjustors,
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`H tin-'3” it]:-. 1:114:1_1E3: 131" 15.11)
`i119) :
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`solubilizers, surfactants, stabilizers, pH adjusters, lyoprotectants/cryoprotectants,
`
`f:
`
`
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`air/liquid and/or ice—liquid interface protectantstprotectants against surface induced
`_ denaturation), freeze—thaw protectants, protectants against protein/peptide denaturation,
`.
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`protectants for rehydration, andwetting agents. In preferred embodiments the
`
`iv formulations include eXci'pients that perform the functions of at least: (i) a bulking
`
`agent/tonicity adJ'u'Ster, (ii) a stabilizer, (iii) a Surfactant (iv) a chelator, and (v) a buffer.
`
`Typically, each ofthesefunctionsis performed by a different excipient. However, in
`
`_ Esome embodiments ofthe invention a single exoipient may perform more than one
`
`‘function. For example, a single errcipien't may be multi—functional, e.g. amino acids may
`
`I
`' function as bulking agents, stabilizers and/or buffers and other excipients may function,
`
`~ for example, as botha stabilizer and a chelator or as both a bulking agent and a tonicity
`
`irradjuster Alternatively, multiple excipients serving the same function may be used. For
`_ example, the formulationmay contain more than one eXcipientthat flinctions as a
`stabilizer
`Table 1 below shows _the concentration ranges for various eXcipient's that were
`investigated. In general, the range studies Werebased 011 a 2-mL fill ofbulk solution per
`“Iii'vialbefore lyoph111zat1on Afterrecenstitution W1th 5 mL ofWater for injection the final
`sincalide formulation resultsin an isotonic solution. The concentration ranges ofthe
`'- various ingredients provided111 Table 1 can be adjusted upwardor downward, if
`'_ necessary in conjunction with: increasingor decreasmg the fill volume per vial, obtaining
`the desired pH, obtaining the desired reCOnstitution volume, and the desirability of
`_. achieving tonicity in the final reconstitutedsolution. For example, as indicated above,
`; the concentrations provided in Table 1‘ were developed toprovide an isotonic solution;
`however, one skilled in the art Would recognize that a broader range of concentrations
`could be used if an isotonic solution was not required.
`
`
`
`
`-
`
`25
`
`
`
`-13-
`
`-13-
`
`
`
`
`

`

`Table 1. Concentration ranges for exeipients for preferred sincalide formulations.
`' Excipient
`
`
`
`r 01,131 111:0.$211)]n1
`
`
`
`
`
`I Function
`
`Range
`(mg/vial)
`
`Range
`(1119 per
`1 mL after
`reconstil
`
`Active ingredient
`
`Final Formulation (mg)
`1 vial
`'
`1 mL
`
`0.0050
`
`reconst.
`0.0010
`
`
`
`
`
`
`
`(51119511119)
`
`, Mannitoi
`
`"TWEVEVN®-720 ’
`
`sodium
`"
`" Metabisulfite .-
`s-'Pota‘s}sium '
`:’ Phosphate,
`' dibasic‘
`" Potassium
`. Phosphate, ,
`.. monObasic '
`
`.Methionine .
`
`i Arginine ‘
`
`formulations.
`
`00000025—
`0.0075
`
`0.0000010-
`0.0030
`
`20.0-50.0
`100-250
`50.0—
`'
`. Bulking Agent/Cake
`1'25.0
`
`.Forrning' Agé'ni/Tonicity
`-
`'
`'Adjuster
`-.
`N.0n-_lonic
`0.0000050
`00150
`Surfactant/SolubIlIzmg
`Agent/Wetting Agent
`ChelatorlStabilizér/Antio
`. xidantl
`'
`Compléxing
`_ Agent/PreservatIveIpl-l
`Adjuster
`_Anton1dantIPreservetIve/
`Stabilizer
`'
`"
`.
`.BufferlpH
`Adjuster/DIssolutIon Aid
`
`'
`
`0002-0012
`
`1.1-1.8
`
`
`
`B_Lifferlpl-i
`Adjuster/D1550lutlon ,Nd'
`-' Stabilizér
`10.0—60.0
`-Stabilizer/Lyoprotectantl
`
`'
`'
`. Cryoprbtectant-
`10.0—35.0
`_ Stabilizer/Lyoprotectantl
`.. CrybprotectantlpH> -
`_
`'Adjuster'
`.
`
`03.5)
`
`O
`
`_\ 00
`
`‘SodiumChloride
`Tonicity Adjuster
`,, Alternativee'xeipients include TWEEN®-SO;potassiummetabisulfite, sodium phosphate dibasic sodium phosphate monobasic,
`ii andpotassium chloride. Additional alternatives are listed below,
`
`'
`
`Table 2 shows preferred ranges for.preferred excipients in the bulk solutions, Vials
`and after reconst1tut10n All concentrations shown for the bulk solution are based on a 2
`mLtill volume. The ingredient quantities are matched to resultin a pH Slightly below
`' neutral and result1n an isotonic solution after reconstitution of the1yophilized Via1 as
`indicated by anOsmolality1n therange of 180 to 320 mOsrn/kg,preferably, 240 to 320
`mOsm. The columns titled “Final Formulation’ represent particularly preferred
`
`-14-
`
`-14-
`
`
`
`
`

`

`
`
`
`
`
`
`3 , E” “#53331 L 1in 3123: EL Tin Eli Zr“
`
`.
`
`
`.
`Table 2 Osmolality values for variOus sincalide formulations.
`.(An.formu_lations_ contain 0.0025mg CCK-B/mL; “dibasic” and “monobasio” refer to dibasic and monobasic
`‘a’o'tassiunfi' h'osphate; “Na m'eta" refers to sodium me'tabisulfite)
`
`’ Formulation
`Calculated
`
`
`- a 3 EXciPieIits -
`mOsm/kg
`
`
`. ‘(mJiI'lL Bulk
`'
`-_
`MahnitOl (125.0)
`
`
`1 .' 1".f3Dib'as‘ic (3.75) -
`
`".LDTPA:(1.V0)
`_.
`.
`
`:?";Mani1it01 (95.0)
`-
`-
`
`g
`‘
`‘}Di_basic (4.0)
`
`
`
`"DTPMl-O)
`_ ”
`
`’Mafinitm (103.0)
`‘
`'
`
`,, ,f ‘f-DibziSiC (3.75
`
`5‘
`-7f'.1'-1DTPA (1,;0)”:-_.,___ --
`-,
`'
`:Mai‘mi’tol (75.0)
`'
`'
`
`.
`staCI (4.5)
`'
`.,
`
`"Dibasic.(3.75)
`
`
`3; DTPA(1.0) ;
`.
`«
`‘
`
`
`'
`:Méiihito'1(85;0)
`
`
`TWEE '20 (0.005)
`"Diba's‘ic (2.75)
`
`,
`
`K ‘
`
`f
`~
`
`
`
`
`'
`..-.M¢thionine (2.0)
`‘
`
`LYSine (150)
`
`Manhit‘01fi(50'.0)
`-'
`,
`i‘
`
`'
`
`' Dibasic (3.00)»
`.-DTPA(1.0)
`
`‘
`
`
`
`‘
`
`
`
`'
`
`247
`
`
`
`
`
`-15-
`
`-15-
`
`
`
`
`

`

`
`
`
`
`. TWEEN®'ZG'0.0075
`.M'annitolnio)
`
`__
`
`£31 “fil-iEZ-fi
`
`.2. 11:]? 5.3.41.1. 1E.-’51.!E31 Elf
`
`'-
`fl
`.
`31. 1131 2:3 EMF!
`
`
`
`'.
`
`-'
`
`.'
`
`'.Dibasic 3.25)
`
`
`
`_
`_
`.-Mé'thiofij1ie(2-0
`'
`-
`.
`
`
`..M:imiitol‘(85.0'
`'
`-
`
`
`' Dibasic-(450)
`__
`
`?-'DTPA(1.0)
`
`flNa'mEtabiSulfite (0.020)
`
`"
`' Meflfioninaflfl) ’
`
`'.-szsine,(7.50)
`_
`a
`
`- Argnune (15.0)
`.
`.'
`NirMeta 0.015)
`
`i. Mann'i't'olwio
`
`f
`:Dib'asic (2.75)
`
`-
`
`if:
`
`
`
`5
`
`
`
`
`
`
`
`
`
`
`
`AIglflJllfl (15.0)
`
`-16-
`
`-16-
`
`
`
`
`

`

`.231 11E! 115113111! 5311 “111:1
`
`._.. 11:11 11311 .21. 11111 1111 111?
`
`
`
`-I..
`
` Mannit'ol ('85.o)
`Dibasic 2..75)
`.
`
`
`...
`
`
`TWEEN®20 (0 005)-
`‘TMe'thionine (2.0)III
`
`Lyginc; 7.50)-
`
`
`Afgirfifie. _(15___0)
`
`
`_-NaMéta (0. 020)
`
`-Mannitol (35.0
`
`
`leasw (3.00)
`
`DTPA (1.I0)_
`TWEENII®I20 0.005)
`
`Lysine (750)
`
`Argmine.(15 0) .
`
`
`" -.-D.ibaéié (2:75)
`.
`_
`.
`.
`--
`.1. Mannitbl 85.0)
`
`
`.. NaMeta (0.015) .-
`'—
`
`
`' DTPA-(lfl)
`
`
`" 5; Méfllionine' (2.0) -.
`1
`
`'Ly'siiie (7.50) ,_-_
`
`--Arginjne (15.0)
`
`
`'— I
`
`'Diba'sic (3 00
`Mannitol (85._0)
`
`
`
`I.
`.
`DTPA(1.0) '
`.
`
`
`I
`.-1'TWEEN®.-20'(0.005)
`
`31-
`. I-Méthidijine (2.0)
`
`-'
`-_'.Lysi1Iie (7. 50).-
`
`
`
`10
`
`1
`
`1
`
`-17-
`
`-17-
`
`
`
`
`

`

`1 .fi"‘
`...~n Liz-0:31
`" -1121! Hail
`
`.1. mar: .211. {En m 53?!
`
`
`
`
`
`
`
` IBib-ask (325)
`Mannltol (75. 0)
`
`
`
`.
`
`'
`.
`
`'_
`
`
`.Mdn'oba'sic (1.0)
`-'
`
`"’DTPA-(I—D)
`..
`-
`
`
`.4
`.. "‘--'=..i.Dibasic(3.25),
`
`
`. f“? 'Méthio'iiine (0.5)
`
`-
`'Mfifinitol'fisn)
`
`
`m-
`" TWEEN® 80 0.025) :-
`:
`_ "Mofiobasic (1.0
`
`-
`-'. Dib’a'sic (3.25) _
`
`
`262
`
`-13-
`
`-18-
`
`
`
`
`

`

`3' _"-.-'-TWEEN®.20 (0.005)
`"
`' Dibaéic_(2.75
`"'3'DTPA(1.0)
`Methiphifia (2.0)
`-Lysi_he.(15.0
`..
`mantra-"350
`-_
`__
`_
`.'
`Diba‘sic'(2.‘75 -
`.—
`'- Meflfidiajnean)‘
`'-
`.Lysiu'e_:_(3'0.0
`-
`., ;-.Malinitol 70.0)
`'IWEEN® 20(0005)
`' .: '
`""Dib'aéié 2.75) ..
`-
`.'
`'-
`a-rDTlE'AflD).
`.
`.'
`; 00015100311090)
`=
`'in'in'e‘ 17.5)
`: Ménfiitol (35.0)
`".TWEEN®'20 0.005)
`-'Di‘bas.i¢(2:75)
`
`Dibasic (2.75)
`
`~-
`
`'
`
`-
`
`_
`
`.3
`
`'
`"
`
`'
`
`L:
`
`l
`.35 fl ‘fib
`
`.
`
`:1 0.3.03!
`
`
`
`'
`
`_
`
`'
`
`' -*Me'thi£:injne'(2.0)
`IAi-‘inine' 10.0
`_'M:§1_1-iijt01(85-0)
`TWEEN® 20 (0 005)
`
`.-
`
`_5
`
`':_.1_"WEEN® 20 (0.005)
`
`-19-
`
`-19-
`
`
`
`
`

`

`
`
`
`
`
`
` Arginine (8.75)
`,
`)— Maimitol (85.0)
`' TWEENZO (0.005)
`. Dibasic (2.75)
`
`g
`'
`DTPA
`(1.0).
`
`
`
`,
`:,
`,_7'1'Lysine (7.5).
`
`_J
`7
`‘Arginilie (15.0)
`
`‘
`"Mannitol (85.0) '
`
`
`, .TWEEN 20 (0.005)
`
`. T'Diba'sic'(2.75)
`
`,
`‘
`".WDTPA (1.0)
`
`
`. Methionine (2.0)
`.
`
`
`
`1
`' Chelators .
`'
`‘
`_‘ Eicipientimpurities and]or stopper extractables Can introduce tracemetals into _
`
`} phannaCeutical formulations. Sincalide contains two methionine residues (Met 3 and
`
`'
`:Met 6) that are susceptible to oxidation by free metals. Thus,:the sincalide formulations
`
`ofthe invention contain chelators to’inhibitthe oxidation ofthe two methionine residues '
`
`;;_present in sincalide (Met 3 'andMet 6). Preferred ohelators include pentetic acid (DTPA),
`
`I
`edetic acid '(EDTA) and derivatives thereof, including Salts. DTPA is a preferred
`
`Qhelator. As described in Example 2 below, the amounts of the degradants, sincalide Met
`
`j ‘ 3and sincalide/Met 6 mondsulfoxides, increase in the preSerice of certain metals and in
`
`the absenceof DTPA, While the presence ofDPTA has an} inhibitory effect on the
`
`’7 formation of these‘monosulfoxides. In particular, copper and manganese, in the absence
`
`Qf DTPA, have the greatest oxidatiVe effect on the methionine residues of sincalide
`
`resulting in combined height percentages of Met 3 and Met 6 monosulfoiides (vs
`
`' sincalide)'of 85.5 and 128.9, respectively.
`
`In a preferred embodiment, the sincalide formulations contain between 0.1 and 3.0
`
`mg of DTPA per mL after reconStitution. In a particularlyppreferred embodiment,
`' Sincalide formulations of the intention contain 0.4 mg DTPA/mL after reconstitution
`
`'
`
`,y,
`
`i 7
`
`,With 5mL.
`
`
`
`13
`
`-20-
`
`-20-
`
`
`
`
`

`

`
`
`
`
`"31.311 1111111,11113:.“"
`
`Bufferin A ents '
`Buffering agents are employed to stabilize the pH of sincalide formulations of the
`
`
`invention, and cOnsequently, reduce the risk of chemical stability at extreme pH values.
`
`A __:Buffering agents useful.111 the preparation of formulation kits of the invention include, but
`are notlimited to, phosphoric acid, phosphate (6.g. monobasicor dibasic sodium
`
`7 phosphate, monobasic or dibasic potassium phOsphate, etc.), citric acid, citrate (e.g.
`[1”lisod1umcitrate, etc), sulfosalicylate, acetic acid, acetate (eg. pctasSium acetate, sodium
`
`' ’cetate', e‘tc._,) methylboromc acid,boronate, disodium succinate hexahydrate amino
`
`
`
`'
`
`
`
`_ hydroxyethylplperazmeN’:2-ethanesulfonicacid (HEPES), CHAPSand other “Good””
`‘ buffers) and the 11ke_
`'
`
`
`i anIdeal bu feringcapac1ty1nthe phys1olog1ca1 pH iange._D1bas1c potass1um .
`
`p sph‘ate1s apartlcularlypreferredbufferin sincalide forrnulatlons ofthe 1nvent1on As
`
`described1n Example 1 below a Sinca11de forrnulat1on ofthe 1nvent1onproved to be
`’EStableover a pH range of5.5 —9.1 Within the pH range of 5.5—S.5; no distinct pH-
`
`}dependent related trends1n initial sincalide recovery were observed with a sinealide
`
`'iformulationofthe invention Preferably, a sincalide formulation of the invention has a
`i ,prfrorr160t080
`
`
`
`Stabilizers
`The oCtapeptide, sincalide, containsOne tryptophan and two methionine residues
`'y Methionine has been identified as one of the mest easily oxidizable amino acids, which
`
`14
`
`-21-
`
`-21-
`
`
`
`
`

`

`
`
`" degrades to its cOrres'ponding sulfoxide and, under more strenuous oxidation conditions,
`
`sulfone. The mechanisms of oxidation appear to be highly dependent on the reactive
`
`oxygen species under consideration: peroxide, peroxyl radicals, singlet oxygen, and
`hydroxyl radical have all been shown to oxidize methionine residues to sulfoxides and
`
`otherprOducts. Therefore based on the potential for oxidation ofthis peptide, it was
`
`cessai'y to identify functional additives for peptide stabilization.
`
`AntzoxzdantS/Reducmg Agents. In a preferred embodiment ofthe invention, the
`
`
`sincalide.formulation contains an antioxidant or reduCing agent as a stabilizer. A wide
`variety ofanti‘oXidants or reducing agents can be used as stabilizers, including but not
`
`united to, acetylcysteine, cysteine ascorbic acid, benzyl alcohol, citric acid, pentetic acid
`
`
`diethylenetnamine pentaa'ceticacid (DTPA), prOpyl gallate, methylparaben,
`
`sulfoxylate propylparabeii, edetic acid or ethylened1am1netetraacet1c acid (EDTA),
`
`adisbdium EDTA dihydrate, dithiothreitol, glutathione, monothioglyCerol, potassium_
`
`’metabisulfite Sodium formaldehyde sulfoxylate, sodium sulfite, sodium succinate,
`
`
`_ odiummetabisulfite, stannous chloride, thioacetic acid, thiodiglycerol,
`
`,_ lthioethanolarnine thioglycolic acid 2—a1ninoethanethiol (cysteamine), butylated
`
`' hydroxyanisole (BHT), and sodium sulfate and derivatives thereof, including salts and
`
`Sulfurous acid salts. Sodium metabiSulfite1s a preferred antioxidant stabilizer.
`
`
`Additlonally, DTPA Whichis a preferred chelator, also may be an antioxidant stabilizer.
`Amino Acids. Amino acids have also been used as stabilizers or 00--stabilizers of
`
`
`peptides to: act as cryoprotectants during freeze drying, stabilize against heat
`idenaturatlon inhibit aggregateformation, improve solubility or rehydration, inhibit
`
`
`emerization reduce surface adsorption, or-act as chelating agents. They can also
`
`[crease the product glass transition temperature (Tg) and therebyincrease process
`
`ability, as well as stabilize the product by minimizing overdrying during secondary
`
`drying Surface exposed residues can react readily With oxidizing agents at physiological
`
`scavenging oxidizing molecules and protecting critical regions of peptides
`
`7'
`, Various D— and/or L-aInino acids can be used as stabilizers1n sincalide
`
`" Grinulations. As used herein “amino acid(s)”and the names of specific amino acids (e.g
`
`
`
`15
`
`-22-
`
`-22-
`
`
`
`
`

`

`
`
`
`
`
`
`
`argmin’e,‘ lysine, methionine, etc.) encompass D— and/or L--amino acids amino acid salts,
`derivatives h'omologs, dimers, oligomers, or mixtures thereof Preferred amino acids for
`
`useas stabilizers1n the present inVention include methionine, lysine, and arginine.
`
`xamples of other amino acids (and amino acid Salts) suitable as stabilizers include, but
`
`notlimited to, arginine glutamate, a's'paragine, gamma aminObutyric acid, glycine
`
`glycine buffer), glutamic acid, glutamate, sodium glutamate,histidine (and histidine
`
`buffer) lysine glutamate, lySine aspartate, arginine aspartate, imidazole, s‘e'rine,threonine,
`
`alanine, polyglutamicacid, polylysine, glycylgly'cineand the like, including
`
`
`
`
`
`
`Cryoprotectants/Lyoprotectant
`Various cryoprotectants/lyoprotectants can be used111 the present invention.
`
`Suitable cryoproteCtant's structure Water molecules such that the freezing point is reduced
`
`and/orthe rate ofcooling necessary to achieve the Vitreous phaseis reduced. They also
`
`
`isc theglass transition temperature range of the Vitreous state These include, but are
`
`not lnmt'ed to: d1methylsulfox1de (DMSO), dextran, sucrose,l,2~propanediol, amino
`
`acids/salts such as,- glycine, lysme, arginiiie, aspartic acid, histidine, proli'ne,etc.,
`
`__lycerol, sorbitol, sodium chloride, fructose, trehalos'e, raffmose,stacl1ychose, propylene
`
`glycol2,3butanediol,hydroxyethylstarch,polyV1nylpyrrolidone (PVP), PEG’s and
`
`
`
`
`
`yeerol, ethylene glycol,et.,c) tetramethylgluc'ose, sodium sulfate, cyclodextrins and
`ombinations thereof. LySine and arginine are preferred cryoprotectants/lyoprotectants
`
`Surfactants/Solubilizers/Surface Active Agents V
`Peptides are susceptible to physical degradatiOn through denaturation, aggregation,
`'fPIECipitation, container surface adSorptiOn and/or agitation induced denaturation. The
`
`
`
`16
`
`1.4
`
`-23-
`
`-23-
`
`
`
`
`

`

`
`
`J
`
`
`lit-{it’ll a U EEK ill: at;
`
`
`[2:5ddition of a nonionic surfactant, such as pelysorbate, to the formulation, may reduce the
`timer-facial tension or aidin solubilization thus preventing or reducing denaturation and/or
`
`idegradation at air/liquid or liquid/solid interfaces ofthe product1n solution.
`
`7,
`_
`Surfactants/solubiliZers include compounds such as free fatty acids, esters of fatty
`
`
`,iacrds With polyoxyalkylene compoundslike polyoxypropylene glycol and
`f polyoxyethylene glyCol; ethers of fatty alcohols with polyoxyalkylene glycols; esters of
`
`';:,fatty acids With polyoxyalkylated sorbitan; soaps, glycerol-polyalkylene stearate;
`
`i: lycerol-polyoxyethylene ricinoleate; homo and cepolymers of polyalkylene glycols;
`
`f’polyethoxylated soya—oiland castor oil as Well as hydrogenated derivatives; ethers and
`
`testersof sucrose Orother carbOhydrates with fatty acids, fatty alcohols, these being
`
`ptionally polyoxyalkylated; mono-, di- and triglyceridesof saturated or unsaturated
`
`fatty.acids; glyceridesor soya-oil and sucrose; sodium Caprolate, ammonium sulfate,
`
`(sodium dodecyl sulfate (SDS), Triton-100 and anionic surfactants containing alkyl, aryl
`
`pf heterocyclicstructures.
`
`, Examples ofpreferred surfactants/solub1l1zers for use in the presentinvention
`
`
`:‘includc, but arenot limited to, pluronics (e.g.,Lutrol F68, Lutrol F127), Poloxamers,
`SDS, T1‘iton-100,Hpolysorbates such as TWEEN® 20 and TWEEN® 80, propylene glycol,
`
`[fP-EG and similar compounds, Brij58 (polyoxyethyle'ne20 eetyl ether), cremophor EL,
`
`:Vcetyl tnmethylammomum hromide (CTAB), dimethylaCetamide (DMA), NP- 4O
`
`‘1 (Nonidet P-40), and N-methyl-2—pyrrolidone (PhérmasOlve), glycineand other amino
`
`9‘ :ac1ds/ammo acid salts and amonic surfactants Containing alkyl, aryl or heterocyclic
`
`Structures, and cyclodextrins. TWEEN® 20is the most preferred surfactant111
`
`,ry-v‘forr'hulations of the invention.
`
`. Bulking—Agents/Tonicifii A_djusters
`'Due to the small amount of sincalide present in the formulations of the invention,
`‘
`,
`
`Bulking agents/tonicity adjusters are useful to provide structure and support for the active
`; 'jingredient, sincalide, as well as to prOVide tonicity. Bulking ag