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`APPLICANT
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`TITLE
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`:
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`Edmund C. Metcalfe, Jo Anna Monteferrante, Margaret
`Newborn, Irene Ropiak, Ernst Schramm, Gregory W. White,
`Julius P. Zodda
`
`SINCALIDE FORMULATIONS
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`F:\50203\50203.017001---Utility Cover Sheet.wpd
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`SDeeceercs
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` PATENT
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`ATTORNEY DOCKETNO: 50203/017001
`
` fa improved and consistent potency as established by a sincalide specific assay such as
`
`Backeround of the Invention
`KINEVAC® (Sincalide for Injection,USP) is a cholecystopancreatic-
`-gastrointestinalhormone peptide for parenteral administiation. The active
`me ‘pharmaceutical ingredient, 1-De(5-oxo-L+glutamine-5-L-proline)-2-de-L-
`‘methioninecaerulein or “sincalide” (CAS# 25126-32-3), is a synthetically prepared C-
`: terminaloctapeptide ofcholecystokinin (CCK-8), with the following amino acid
`sequence: Asp-~Tyt(SOsH)-Met-Gly-Txp-Met-Asp--Phe“NED.
`:
`KINEVAC®was first introduced iin 1976, and was finished as a sterile,
`“nonpyrogenic, lyophilized whitepowsder ina 5mL (nominal) glass vial to contain: 5 pg
`‘sincalide with 45 mg sodium chlorideto provide tonicity;‘sodium hydroxide or
`ehydrochloric’acid may have been added:for pH adjustment (pH 5.5-6.5). The type I
`“plas vial was sealed undet anitrogen headspace with a Tompkins B08&49 closure. This
`. -two-ingredientformulation was incorporatedininto the U.S. Pharmacopea/National
`Formulary, USP 24, NEF 19, January 1, 2000.°
`Since its introduction, various drawbacks iin the manufactiring and analysis of
`KINEVAC® have been identified. For example, the two--ingredient formulation suffers
`from potencyvariability. Thisvariability was exacerbated by thefactthat the
`os - formulationwas analyzedusing a guinea pig gallbladder contraction bioassay for potency
`“ofboth sincalideand KINEVAC®-Thisbioassay was unable to.distinguish between
`5 bioactivity ofsincalide and bioactivity ofsincalide degradants. Accordingly, a 20%
`overageof sincalidé was required iin previouss‘sincalide formulations to compensate for
`7 : -the limitationsofthe bioassay, Thus, there is a need for sincalide formulations having
`
`SINCALIDE FORMULATIONS
`
`Field of the Invention
`7
`The invention relates to pharmaceutically acceptable formulationsof sincalide.
`
`“HPLC,
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` monohydrochloride,andsodium:metabisulfite,
`
`Summary of the Invention
`The present invention satisfies the need for improved sincalide formulations by
`providing formulations that éliminate the neéd for a 20% overage of sincalide. The
`sincalide formulations ofthe invention are also purerthan prior art formulations, and
`_ have fewer degradants andmote consistent potency. In addition, the purity ofthese
`formulations maybe assessed byHPLC,thuseliminating the needforthe bioassay ofthe
`prior art formulations.
`|
`Thepresentinvention provides sincalide formulations adapted for administration
`byinijeection. These sincalide formulationsate characterized by.improved stability and
`may beprepared as a relativelylarge volume batch.(~100 L).
`In one.aspect, the invention features siticalide formulations that include an
`_ effective amountofsincalide; a bulking agerit/tonicity adjuster, one or more stabilizers, a
`" surfactant, achelator, and a buffer. Theinvention also features kits and methods for
`preparing improved §sincalide formulations, as well as methods fortreating, preventing,
`and diagnosinggall bladder-telated disordets using sincalide formulations.
`The formulations‘ofthe inventionpreferably have a pH between 6.0 and8.0.
`Suitable buffers include, but ate not limited to, phosphate,citrate, sulfosalicylate, borate,
`‘acetate and amino acidbuffers: Phosphate buffers, such as dibasic potassium phosphate,
`are preferred.
`-
`|
`7
`|
`Invariouss embodiments ofthe invention, the surfactantiis a nonionic surfactant,
`: preferablya polysorbate, such as polysorbate 20 or polysorbate 80; the chelatoriis
`- pentetic acid (DTPA); and the stabilizer is an antioxidantand/or amino acid. In a
`- particularly desirable embodimient ofthe invention, the formulation includes a plurality of
`5. stabilizers,preferably L-arginine monobydrochloride, L-methionine, L-lysine
`
`Suitablebulking.agents/tonicity adjusters include, but are not limited to, mannitol,
`~ factose, sodiumchloride, maltose, sucrose, PEG’s, cyclodextrins, dextran, polysucrose
`
`an
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`The sincalide formulations of the invention may also be administered to patients
`
`receiving total parenteral nutrition (TPN), in orderto treat and/or prevent TPN-related
`
`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 drawingillustrating the chemical structure of 1-De(5-oxo-L-glutamine-
`5-L-methioninecaerulein or “‘sincalide” (CAS# 25 126-32-3). The amino acid residues
`
`“Met 3” and “Met 6” are outlined by dashedlines.
`
`FIG. 2 is a drawingillustrating the chemical structure of sincalide (Met 3)
`
`monosulfoxide.
`
`FIG. 3 is a drawing illustrating the chemical structure of sincalide (Met 6)
`
`=<. monosulfoxide.
`FIG. 4 is a drawing illustrating the chemicalstructure of sincalide (Met3, 6)
`
`
`
`disulfoxide..
`
`FIG, 5 is a graphical representation of the effect of pH on the recovery of sincalide
`
`in 35 mM phosphate buffer over 24 hours. At each pH for which data is shown, the bars
`‘ represent 0, 6, and 24 hours, from left to right.
`FIG. 6is a graphical representation ofthe effect ofpH 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
`with 0.63mM Cu”.
`FIG, 9 is a chromatogram of KINEVAC®experimental formulation (lmM DTPA)spiked
`with 0.63mM Cu’”.
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`(Ficoll), and polyvinylpyrfolidine (PVP). D-Mannitol is a preferred bulking
`agent/tonicity adjuster.
`os
`Ina particularly 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,
`2.0to 7.0 mg/mLarginine; 0.2 to‘1,0 mg/ml. methionine; 2.0 to 30.0 mg/mL lysine;
`0.002to 0.012 mg/mL sodium metabisulfite; 0.000001 to 0.003 mg/mL polysorbate 20,
`“¢0.J.to 3.0 mg/mL pentetic acid (DTPA); and5.4 to 12.0 mg/mLpotassium phosphate
`(dibasic). Inamorepreferredembodiment, the reconstituted formulation includes about
`0.001 me/mL sincalide; about 34 mg/mL D-manhitol, about 6 mg/mL L-arginine
`monohydrochloride: about 0.8 mg/mL|L-methionine: about 3 mg/mL L-lysine
`monohydrochloride;about0.008 mg/mLsodium metabisulfite; less than about 0.01
`“mg/mL,polysorbate20;about 0.4mg/mLpentetic acid (DTPA); and about 1.8 mg/mL
`potassium‘phosphate(dibasic).
`. The kits ofthe invention may, for example,“inchide the various components ofthe
`formulation asa mixture inpowderform,alongwitha container (e.g., a vial) to hold the
`powder mixtureanda physiologically acceptable fluid for reconstitution ofthe
`‘formulation. Thecomponents oftheformulation may be present in the kit eitherin the
`powder mixture or in the fluid‘portion. Kits:ofthe inventionmay also include all
`‘components ina liquid mixture orsome components in aYiquid form and somein the
`form ofa powder.
`The formulations ofthe invention have imptoved stability and potency compared
`to previous sincalide,fotmulations, and are useful as diagnostic aids for imagingthe
`hepatobiliary,system ofapatient. When used as a‘diagnostic aid, the sincalide
`formulations may, for example, be co--administered with a radiopharmaceutical agent
`having rapidhepatic uptake, such as 99m.mmebrofenin, or similarhepatobiliary imaging
`agents, to assist in the diagnosis ofgallbladder diséases 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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`FIG. 10 is a chromatogram of KINEVAC® experimental formulation (no DTPA) spiked
`- with 0.18mM Mn”.
`_FIG. 11 is a chromatogram of KINEVAC®experimental formulation (ImM DTPA)
`spiked with 0.18mM Mn”,
`. FIG. 12 shows represeniative full-scaleand expanded scale chromatograms ofa
`: lyophilized reformulation ofKINEVAC® uponreconstitution with SmI water, resulting in a
`. ‘gincalide concentration of LygimL. -
`"Detailed Desctiption ofthe Invention
`“Inorder to‘develop animproved sincalide formulation a series ofstudies,
`. “describediin theExamplesbelow,‘were conduciedto determinethe effects ofvarious
`aexcipients on formulations ofsincalide, Through these studiés, we discovered thatthe
`:“potencyand stabilityofsincalide formulations canbe significantlyenhanced through the
`~ carefulselection ofexcipients that provide certain desited functions. Accordingly, the
`i ‘present inventionprovides novelsincalideformulations havirig improved stability and/or
`- potencyover previotis fotmulations.
`: “As used herein, the term ‘‘sincalide” includes the synthetically-prepared C-
`co ‘terminal octapeptide ofcholecystokinin (CCK-8), with the amino acid sequence: Asp-
`.: Tye(SO3H)-Met-Gly-Trp-Met-Asp-Phe--~NH3, as well as derivatives thereofwhich have
`ae been optimized or modified (toiimprovestability, potency, pharmacokinetics, ete.ds but
`ms retain thebiological activity ofthe originaloctapeptide. For example, peptidesiin which
`: ‘themethionineand/or aspartic acid residues have béeri replaced without significantly
`: affectingthe biologicalactivityare inchided within ‘‘sincalide” as the term is used herein.
`Similatly, the term ‘‘sincalide” encompasses not only monomeric, but multimeric forms
`oe ofthe peptide, as well asphysiologically activee degradants orr portions ofthe peptide and
`. its derivatives.
`The sincalide formulations ofthe invention can include a variety of excipients,
`~ such as, for example; antioxidants, buffets, bulking agents/tonicity adjusters, chelating
`agents, complexing agents, crosslinking agents, co-solvents, osmolality adjustors,
`
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`a HESt_ Eylie
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`solubilizers, surfactants, stabilizers, pH adjustors, lyoprotectants/cryoprotectants,
`
`ait/liquid and/or ice-liquid interface protectants(protectants against surface induced
`_ denaturation), freeze-thaw protectants,protectants against protein/peptide denaturation,
`
`protectants for rehydration, andwetting agents. In preferred embodiments, the
`
`formulations include excipients that perform the functions ofat least: (i) a bulking
`
`agent/tonicity adjuster, (ii) a stabilizer, (iii) a surfactant, (iv) a chelator, and (v) a buffer.
`
`Typically, each ofthesefunctionsiis performed bya different excipient. However, in
`
`‘some embodiments ofthe invention a singleexcipient may performmore than one
`
`‘fuiiction. For example, a singleexcipientmaybe multi-functional, €.g. amino acids may
`
`- function as bulkingagents, stabilizets and/or buffers and otherexcipients may function,
`
`-for example, as bothza stabilizer and a chelator oras both a bulking agent anda tonicity
`“adjuster. Altematively, multiple excipients sery,ing the samefunction may be used. For
`~ example, the formulationmay contain morethan one excipientthat functions as a
`".
`stabilizer.
`Table 1 below shows the concentration ranges for various excipients that. were
`: investigated. Tn general, the range studies werebased ona 2mL, fill ofbulk solution per
`vialbefore lyophilization. Afterreconstitution with 5 mL, ofwater for injection the final
`: sincalideformulation resultsiin anisotonic solution. The concentration ranges ofthe
`~ various ingredients providediin Table 1-cann beadjusted upward|or downward,if
`~ necessary in. conjunction with: increasing.or decreasing the fill volume per vial, obtaining
`the desired pH, obtaining the desired reconstitution volume, and the desirability of
`os achieving tonicity in the final reconstitutedsolution. For example, as indicated above,
`~, the concentrations provided in Table1 were developed toprovide an isotonic solution;
`however, one skilled in the art would recognizethat a broader range of concentrations
`could beusedif an isotonic solution was not required.
`
`n
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`“Metabisulfite ||"Stabilizer. ~~ : v mo
`
`:
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`,
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`formulations.
`
`
`-Table ‘1. Concentration ranges for excipients forpreferredsincalide formulations.
`: Excipient
`Function
`al
`Range
`Range
`Final Formulation (mg)
`a
`j
`(mg/vial) mspe
`n
`dvial
`| tm
`mL after.
`ran . ra oy
`-
`a
`reconst).
`recone
`0.0012
`an
`ee Pa
`.
`i
`.
`~ Mannitol,
`“Buiking Agenticake Lf
`50.0 —
`100-250
`20.0-50.0
`:
`Pose
`‘Forming Agent/Tonicity
`125.0
`ee _ Adjuster.
`ve ‘
`.
`2
`.
`
`: TWEEN®-20 0.0000025-|0.0000050|0.0000010-a -Non-lonic |
`
`
`
`re : Surfactant/Solubilizing
`0.0075
`~0.0150
`~
`0.0030
`Agent/Wetting Agent
`a
`.
`Chelator/Stabilizer/Antio
`. xidantf-*
`:
`Complexing
`. Agent/Préservative/pH ,
`.
`hoe a
`“y Adjuster =:
`we
`o
`0.002-0.012
`“0.
`5
`Sodium —=. “AntioxidantiPresérvative/
`
`
`*Potassium.
`|
`|
`-. Buffer/pH °:
`27-4;
`4-12,
`1.1-1.8
`.Phosphate,
`. AdjusterDissolution Aid
`-.» dibasic
`wet
`“Potassium
`wen “Buffor/p
`: “Phosphate, . Adjustér/Dissolution Aid.
`monobasic ~*
`:
`|
`:
`~ Methionine |. Stabilizer
`30.
`“Lysine
`‘Siabilizer/Lyoprotectant! $0 —
`of}
`6 Fhe
`2. Gryoprotectant ee
`—
`Arginine
`Siahilizer/Lyoprotectant!
`.0
`-17.:
`we ua Cryoprotectant/pH ~
`.
`Les
`“Adjuster °
`:
`“Sodium .-
`‘Tonicity Adluster,
`-
`cnioride
`:
`ce Alternative’excipients Tolude TWEENG-80,potassiumnetabisuliite, sodiumposedibasic, sodiumphosphate monobasic,
`- and.potassium chloride. Additional alternatives are listed below. |
`
`
`
`
`
`
`
`|:
`
`:
`
`.
`
`oP
`
`10.0-60.0
`‘10.0-35.0
`oO
`
`:
`
`‘Table 2 shows preferred ranges for.preferred excipierits inthe bulk solutions, vials
`.
`“andafter recoiistitution.. All concentrations shown for the bulk solution are based on a 2
`: “mbfill voluine. The ingredient quantitiesarematched to result in a pH slightly below
`neutraland result jinan isotonic solution after reconstitution ofthelyophilized vial as
`iridicated by an‘osmolalityin therange of 180 to 320 mOsm/kg,preferably, 240 to 320
`mOsm. The columns titled “Final Formulation” representparticularly preferred
`
`-14-
`
`-14-
`
`
`
`
`

`

` LOSSSug.AS oe
`
`Table 2. Osmolality values for various sincalide formulations.
`-(All-formulations contain 0.0025mg CCK-8/mL.; “dibasic” and “monobasic” refer to dibasic and monobasic
`“potassium phosphate; “Na meta”refers to sodium métabisulfite)
`
`:
`‘Formulation
`Calculated
`
`-. Excipients -
`
`
`
`; - =.’ Monobasic.(2.8) —
`“DTPAG.O)
`
`|Methionine (2.0)
`_- Lysine (15.0)
`Mannitol (60.0)
`NaCl (9.0) -
`Dibasic (3.00)
`{DTPA (1.0) ~
`
`' Mannitol (75.0)
`~ NaCl (4.5)
`
`_-, ~Dibasic(3.75)
`
`
`~ | DTPA (1.0) |
`
`
`--| Mannitol (85:0) |
`
`-|---"TWEEN® 20 (0.005) “
`
`
`“= Dibasic (2.75)
`
`
`
`weWs
`
`
`
`-15-
`
`-15-
`
`
`
`
`

`

`LOS SE SU Og. a0
`
`
`
` | [EWEEN®20 (0.0075
`
`
`
`[= Dibasic (3.25)
`_ |= Monobasic(1.0)
`"DTPA(1.0)
`
`-— [© Mannitol (75.0)
`
`
`
`
`
`_ | Dibasic (2.75)
`LesDTPAGO)|
`-L200.005)
`_[-Methinine2.0),
`
`—[{Tysine(7.50)
`Li_-Arginine(15.0)|
`
`
`
`-16-
`
`-16-
`
`
`
`
`

`

`LH ieak 2Es
`
`
`
`oeEE 21, ty Ta
`
`
`
`
` Na Meta (0.030)
`~ Mannitol (85.om
`ay3 SS__DTPA (0)
`
`“DTPA(1.0)
`
`
`
`
`_TWEEN®:20 (0.005).
`Methionine (2.0).
`“os Tysine. (720) .6.25.
`..
`
`|. Arginine15:0):
`
`|. .NaMeta (0.020)
`ae Dibasie6.00)3
`
`
`TWEEN® 20 (0.005)
`
`* Lysine (7.50): ».
`‘arpnine(15.0)
`
`
`. -Dibasic (2:75)
`Mannitol (85.0)
`
`- Na Meta (0.015)...
`j-
`
`DTPAA1.0)
`bee.
`
`
`_ |TWEEN®(0.005)._|20
`
`
`1-2 °° Methionine(2.0).
`3 Lysine (7.50).
`-Arginine(15.0)
`
`
`
`-17-
`
`-17-
`
`
`
`
`

`

`Se
`
`ERM , CVE Ao OE
`
`
`
`
`
`
`
`"Maunitol (75.0)
` “= Dibasic (3.25)
` Teae(0.0025)
`= _ Monobasic (1.0)...
`“Methionine(2.0).
`
`
`
`‘| Mannitol (85.0).
`x_TWEEN® 20 @.Sng)
`
` Nsmetabisulfite (0.020)
`
`- Methionine(2.0) |.
`Lysine (7.50)
`Arginine (15.0)
`
`
`
` = “Monobasic (1.0)
`:
`
`
`° DTPA(1.0)
`{e.
`
`
`
`‘L-sDibasic (3.25):
`
`|__- -Méthionine(0.5)
`
`
`Mannitol (75.0)
`
`_-Moniobasic (1.0
`_|_ = TWEEN® 80 (0.025)
`
`
` ~Dibasic (3.25) _
`
`-18-
`
`-18-
`
`
`
`
`

`

`d
`
`
`
`TWEEN® 20 (0.005)
`
`_|“Methionine(2.0)
`-.: Arginine (10.0).
`__Mannitol
`(8
`
`ethionine (2.0)
`rginine(17.5)
`nnitol(85.0)
`
`-19-
`
`-19-
`
`
`
`
`

`

`
`
`
`
`
` =e
`
`
`
`
`
`‘MethionineC.0)
`
`
`sal -Lysine (7.5) .
`
`
`Arginine (15.0)
`
`“Mannitol (85.0)
`
`
`
`“Dibasic (2.75)
`“ DTPA (1.0)
`os5 Methionine (2.0).
`
`
`
` ,
`
`TR oe TIMERZl ts Ce
`
`
`
`" Chelators
`' Excipientimpurities and/or stopperextractables can introduce tracemetals into |
`: pharmaceutical formulations. Sincalide contains two methionine residties (Met 3 and
`
`Met6)‘that are susceptible to oxidation by free metals. Thus,the sincalide formulations
`
`ofthe invention contain chelators toinhibitthe oxidation ofthe two methionine residues ©
`
`
`Spresent in sincalide (Met 3-andMet 6).‘Preferred chelators include pentetic acid (DTPA),
`| : edetic acid(EDTA) andderivatives thereof, including salts. DTPAiis a preferred
`
`chelator, Asdescribed inn Example 2 below, the amountsofthe degradants, sincalide Met
`os 3and sincalide‘Met 6 monosulfoxides, increase in the preserice of certain metals and in
`
`‘the absenceofDTPA, while the presence ofDPTA has an‘inhibitory effecton the
`
`
`~ formation of these’‘monosulfoxides. In particular, copper.and manganese, in the absence
`S ofDTPA, havethe greatest oxidative effect on the methionitie residues ofsincalide
`
`“resulting in combined height percentages ofMet 3 and Met 6 monosulfoxides (vs
`
`aSsincalide)of 85.5 and 128.9, respectively.
`.
`
`‘
`.
`Ina préferred embodiment, the sincalide formulations contain between 0.1 and 3.0
`. mg of DTPA per ml. after reconstitution. In a particularly preferred embodiment,
`. gincalide formulationsof the invention contain 0.4 mg DTPA/mL after reconstitution
`0with SmL.
`
`
`
`-20-
`
`-20-
`
`
`
`
`

`

`
`
`
`
`eh BE We
`
`
`Buffering agents are employedto stabilize the pH ofsincalide formulations ofthe
`
`i invention, and consequently, reduce the risk of chemical stability at extreme pH values.
`-Buffering agents useful in the preparation of formulation kits of the invention include, but
`:are notlimited to, phosphoric acid, phosphate(e.g. monobasicor dibasic sodiuin
`
`2 “phosphate, monobasic or dibasic potassium phosphate, etc.), citric acid,citrate (e.g.
`>soilium ¢citrate, eto.), sulfosalicylate, acetic acid, acetate (e.g. potassium acetate, sodium
`
`“8state, etc.)s methylboronicacid,boronate, disodium succinate hexahydrate, aniino
`.cids,including.amitioaciddsalts5uch,ashistidine,&,alycine,Iysine,imidazole),lactic .
`
`
`
`: hydroxyethypiperazine,N2.-cthanesulfonicacid(HEPES),CHAPSandother“Good's”
`
`
`buffers),aandtheHe.
`“3
`
` annidealbu feringoyin‘thephysiolbgiealpHrange.Dibasicpotassium
`
`
`phic sphateiis aparticulatlypreferredbufferin sincalide formulationsofthe invention. As
`
`describediinExample1below,a sincalideformulationoftheinventionprovedto be
`stableoverapH range of5,5 ~9.15 Within the pH range of5.5:“8.5, no distinct pH-
`
`dependent relatedtrends iin initial sincalide recovery were observed with a sincalide
`
`-formulationofthe invention. Preferably, asincalide formulationofthe invention has a
`
`; oHfrom 6.0 to 8.0.
`‘Stabilizers :
`The octapeptide, sincalide, contains ¢one tryptophan andtwo methionine residues.
`
`Methionine has beenidentified as one of the most easily oxidizable amino acids, which
`
`-21-
`
`-21-
`
`
`
`
`

`

`
`
`
`
` LOats
`
`:degrades to its corresponding sulfoxide and, undermore strenuous oxidation conditions,
`
`sulfone. The mechanisms of oxidation appearto be highly dependenton the reactive
`
`oxygen species under consideration: peroxide, peroxylradicals, singlet oxygen, and
`hydroxyl radicalhaveall been shown to oxidize methionine residues to sulfoxides and
`
`otherproduots. ‘Therefore, based on the potential for oxidation ofthis peptide, it was
`
`cessary to identifyfunctional additives for peptide stabilization.
`
`“Antioxidants/Reducing Agents. Ina preferred embodimentofthe invention, the
`
`
`sincalide:formiilation contains an antioxidant or reducing agentas astabilizer. A wide
`variety.ofantioxidants or redacitig agents can be usedas stabilizers, including but not
`
`imited to, acetylcysteine, cysteine, ascorbic acid, benzyl alcohol, citric acid, pentetic acid
`
`diethylenetriamine pentaacetic‘acid (DTPA),propyl gallate, methylparaben,
`
`sulfoxylate, propylparaben, edetic acid or‘éthylenediaminetetraacetic acid (EDTA),
`
`-disodiumEDTA dihydrate, dithiothreitol, glutathione, monothioglycerol, potassium.
`
`‘metabisulfit, sodium formaldehyde sulfoxylate, sodium sulfite, sodium succinate,
`
`sodium:metabisulfite, stannous chloride, thivacetic acid, thiodiglycerol,
`
`a thioethanolamine, thioglycolic acid, 2-aminoethanethiol (cysteamine), butylated
`
`hydroxyanisole (BHT),and sodium sulfite and derivatives thereof, including salts and
`
`sulfurous acid salts. Sodiummetabisulfiteiis a preferred antioxidantstabilizer.
`
`Additionally, DTPA, which iis a preferred chelator, also may be an antioxidantstabilizer.
`
`© Amino Acids. Amino acids have also been used as stabilizers or co--stabilizers of
`
`
`peptides to: act as cryoprotectants during freeze drying, stabilize against heat
`denaturation, inhibit agetegateformiation, improve solubility or rehydration, inhibit
`
`omerization, reduce surface adsorption, or-act as chelating agents. They can also
`
`\creasethe product glass transition temperature (Ts) and therebyiincrease 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
`
`oH, scavenging oxidizing molecules and protecting critical regions ofpeptides.
`
`oo Various D- and/or L-aminoacids can be used as stabilizers in sincalide
`‘formulations. As used herein “amino acid(s)”and the namesof specific amino acids(e.g
`
`
`
`
`
`
`
`
`
`I
`
`-22-
`
`-22-
`
`
`
`
`

`

`
`
`
`
`
`
`
`
`
`
`
`
`
`arginine, lysine, methionine, etc.) encompass D- and/or L-amino acids, aminoacid salts,
`derivatives,homologs, dimers, oligomers, or mixtures thereof. Preferred arninoacids for
`
`useas stabilizers in‘thepresent invention include methionine, lysine, and arginine.
`
`xamples ofother atnino acids (and amino acid salts) suitable as. stabilizers include, but
`
`not limited to, atginine glutamate,‘asparagine, gamma aminobutyric acid, glycine
`
`idglycine buffer), glutaric acid,glutamate, sodium glutamate,histidine (and histidine
`
`butte), lysineglutamate,lysineaspartate, arginineaspartate, imidazole, serine,threonine,
`
`alanine, polyglutarnicacid, polylysine, elycylglycinezand the like, including
`
`Ioxypropyl and galactosederivatives. In one particularly preferred embodiment; L-
`
`né moriohydrochloride,L-methionine and L-lysine monohydrochlorideare used.
`
`
`
`_Cryoprotectants/Lyoprotectants -
`‘Various cryoproteciaats/lyoprotectantscanbe used iifithe present invention.
`
`Suitable cryoprotectant structure water molecules suchthat the freezing point is reduced
`and/ortherate ofcooling necessary toachieve thevitreous phase is reduced. ‘They also
`
`isé the:glass tratisition temperature range ofthe vitreous state. These include, but are
`
`notlimited to:dimethylsulfoxide(DMSO), dextran, suctose,.1,2-propanediol, amino
`
`acids/salts stichas, glycine, lysine, arginine, aspartic acid, histidine, proline;ete.,
`
`elyoes6, sorbitol, sodium chloride, fructose, trehalose, raffinose,stachychose, propylene
`
`glycol;2,3-butanediol,hydroxyethylstarch, polyvinylpyrtolidone (PVP), PEG’s and
`
`milar compounds, protein stabilizers, such as human serum albumin, bovine serum
`
`Ibumin,bovine gamma globulin, gelatin (ordetivatives, such as Prionex, etc.), dextrose,
`
`lucosé; maltose, arabinose, lactose, inositol, polyols (such as sorbitol, xylitol, erithritol,
`
`lyoérol, ethylene glycol,‘etc.), tetramethylglucose, sodiuin sulfate, cyclodextrins and
`
`ombinationstthereof. Lysine and arginine are preferred cryoprotectants/lyoprotectants.
`
`
`
`
`
`
`
`
`Surfactants/Sohibilizers/Sutface Active Agents —
`“Peptides are susceptible to physical degradation through denaturation, aggregation,
`-precipitation, container surface adsorption and/or agitation induced denaturation. The
`
`16
`
`-23-
`
`-23-
`
`
`
`
`

`

`
`
`
`Ee) CUE Ae
`
`
`“addition of a nonionic surfactant, such as polysorbate, to the formulation, may reduce the
`“interfacial tension or aid in solubilization thus preventing or reducing denaturation and/or
`
`degradation at air/liquid or liquid/solidinterfaces oftheproductin solution.
`
`- Surfactants/solubilizers include compoundssuch asfree fatty acids, esters offatty
`
`oswithpolyoxyalkylene compounds|likepolyoxypropylene glycoland
`
`polyoxyethylene glycol; ethers offatty alcohols with polyoxyalkylene glycols; esters of
`
`fatty acids with polyoxyalkylated sorbitan; soaps; glycerol-polyalkylene stearate;
`
`j lycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols;
`
`-polyethoxylated soya-oiland castor oil as; well as hydrogenated derivatives; ethers and
`
`“estersofsucrose‘Or“other carbohydrates with fatty acids, fattyalcohols, these being
`
`ptionally polyoxyalkylated; mono- , di- and triglyceridesofsaturated or unsaturated
`
`fatty.acids; glyceridesor soya-oil and sucrose; sodium caprolate, ammonium sulfate,
`
`“sodiumdodecyl sulfate(SDS), Triton-100 and amonic surfactants containingalkyl, aryl
`
`orlheterocyclic.structures:
`
`_ Examples ofpreferred surfactants/;solubilizers for use in the presentinvention
`
`
`‘include, butare’not limited to, pluronics (e.g.,Lutrol F68, Lutrol F127), Poloxamers,
`SDS, Triton-100,polysorbates such asTWEEN® 20 and TWEEN® 80, propylene glycol,
`
`PEG and similar compounds, Brij58 (polyoxyethylene20 cetyl ether), cremophor EL,
`
`“cetyl trimethylammonium biroinide (CTAB), dimethylacetamide(DMA), NP- 40
`
`i (Nonidet P-40), and N-methyl-2-pytrolidone (Pharmasolve), glycine.and other amino
`
`-acids/amino acid salts and anionic surfactants containing alkyl, aryl or heterocyclic
`
`‘strictures, and cyclodextrins. TWEEN® 20is thé mostprefetred surfactant in
`
`-fortimlations ofthe invention.
`
`BulkingAgents/Tonicity AdAdjusters
`+ Due to the small atnount ofsincalide present in the formulations of the invention,
`
`bulking agents/tonicity adjusters are useful to provide structure and support for the active
`: ingredient, sincalide, as well as to provide tonicity. Bulking agents/tonicity adjusters
`
`(aso called lyophilization aids) useful in the preparation oflyophilizedproducts ofthe
`
`
`
`
`-24-
`
`-24-
`
`
`
`
`

`

`
`
`
`
`
`
`
`
` ALT mele atMTSE
`
`
`vention.are ‘known in the art and include mannitol, lactose, potassium chloride, sodium
`cl oride, maltose, sucrose, PEG’s (such as, for example, PEG 300,PEG 400, PEG 3350,
`
`G66000,PEG 8000 and the like, etc.), trehalose, raffinose, dextrose, polygalacturonic
`
`acidgalacturonic acid, amino acids (inicluding amino acid salts) such as lysine, arginine,
`
`g yeine,galactose, etc.), cyclodextrins,such as hydroxypropyl-y-cyclodextrin (HP-y-CD),
`
`tran,Ficoll, and polyvinylpyrrolidonie (PVP). Ofthese, D-mannitol is the most
`preferredbulking agent/tonicity adjuster for use with the invention.
`
`
`
`
`
`
`
`
`‘OtherExcipients
`Other excipients, which 1may optionallybe used in the formulationsofthe
`
`ventioninclude preservatives (e.g., benizalkoniumchloride), osinolality adjusters (e.g.,
`xtrose), lyoprotectants (e.g., sodium sulfate), solubilizers, tonicity adjusters (e.g.
`
`diumchloride), cake forming agents, complexing agents,and dissolution aids. A listing
`
`ofvarious excipiients that’canbe usediinsincalide formulations forparenteral
`
`5 administrationcan be fouid iin, forexample, The Handbook ofPharmaceutical Additives,
`
`a: Second,Edition, editedby Michael & Irene Ash; Remington’s Pharmaceutical Scienc