US 6,803,046 132
`(10) Patent N0.:
`(12) Unlted States Patent
`Metcalfe et al.
`(45) Date of Patent:
`Oct. 12, 2004
`
`
`USOO6803046B2
`
`(54) SINCALIDE FORMULATIONS
`
`OTHER PUBLICATIONS
`
`(75)
`
`Inventors: Edmund C. Metcalfe, Hillsborough, NJ
`(us); J0 Anna Monteferrante, Raritan
`Township, NJ (US); Margaret
`Newborn, Hamilton TownShip, NJ
`(US); Irene Ropiak, Lawrenceville, NJ
`(US); Ernst Schramm, North
`Brunswick, NJ (US); Gregory W.
`White, Monmouth Junction, NJ (US);
`Julius P. Zodda, Mercerville, NJ (US)
`
`(73) Assignee: Bracco International B.V., Amsterdam
`(NL)
`
`*
`
`Notice:
`
`J
`y
`Sub'ect to an disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) APPL N05 10/2229540
`(22)
`Filed:
`Aug. 16, 2002
`
`(65)
`
`Prior Publication Data
`US 2004/0033243 A1 Feb. 19, 2004
`
`(51)
`
`Int. Cl.7 .................................................. A61K 9/00
`
`(52) US. Cl.
`
`........................ 424/400; 514/1.65; 514/18;
`514/19; 514/951
`(58) Field of Search ............................ 424/400, 514/18,
`514/19, 1.65, 951
`
`(56)
`
`References Cited
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`ournier e a .
`.........
`.
`gagggagg 2
`1171332 ifhnefler ft laL ~~~~~~~~~434239053?
`’
`’
`6,110,443 A
`8/2000 Schneider et al.
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`.........
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`
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`
`311-, “Cholecystokinin Prevents Parenteral
`Sitzmann, Ct
`Nutrition Induced Biliary Sludge in Humans,” Surgery,
`Gynecology & Obstetrics, vol. 170, Jan. 1990, pp. 25—31.
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`Teitelbaum, “Parenteral Nutrition—Associated Cholestasis,”
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`Strickley, “Parenteral Formulations of Small Molecules
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`Strickley, “Parenteral Formulations of Small Molecules
`Therapeutics Marketed in the United States (1999)—Part
`II,” PDA Journal of Pharmaceutical Science & Technology,
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`
`(List continued on next page.)
`
`Primary Examiner—Thurman K. Page
`Assistant Examiner—Konata M. George
`(74) Attorney, Agent, or Firm—Kramer, Levin, Naftalis &
`Frankel LLP
`
`ABSTRACT
`(57)
`The invention features sincalide formulations that include an
`effective amount of sincalide, a bulking agent/tonicity
`adjuster, a stabilizer, a surfactant, a chelator, and a buffer.
`The invention also features kits and methods for preparing
`'
`'
`'
`'
`improved sincalide formulations, as well as methods for
`t
`t
`t
`dd
`llbldd-ltd
`5122:3135 EZZCHSEZHZ: foriflirlgtsibnngs ga
`a
`er re a 6
`g
`‘
`
`108 Claims, 12 Drawing Sheets
`
`Met 3
`
`Met 6
`
`0
`o
`o I
`o E
`o
`o I
`o :
`o
`II
`II
`n
`:
`II
`5
`II
`ll
`5
`II
`:
`II
`HZN—CH—C—N—CH— —:—N—CH—C-fi-N—CH—C—N—CH—C-I—N—CH—C+N-—CH—C—NH—CH—C—NH2
`|
`H
`|
`I H
`|
`EH
`|
`H
`|
`i H
`|
`i H
`|
`I
`('sz
`CH2
`5
`(lez
`:
`H
`CH2
`:
`<|:H2
`I
`(le2
`CH2
`:
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`.
`c=o
`:
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`5
`I
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`I
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`|
`:
`\ :
`|
`I
`|
`OH
`:
`s
`:
`:
`s
`:
`OH
`I
`I
`I
`:
`I
`l
`;
`CH3
`:
`:
`CH3
`:
`
`NH
`
`s
`
`a
`
`a
`
`a
`
`MAIA Exhibit 1001
`
`MAIA V. BRACCO
`
`IPR PETITION
`
`
`
`
`MAIA Exhibit 1001
`MAIA V. BRACCO
`IPR PETITION
`
`

`

`US 6,803,046 132
`
`Page 2
`
`OTHER PUBLICATIONS
`
`Strickley, “Parenteral Formulations of Small Molecules
`Therapeutics Marketed in the United States (1999)—Part
`III,” PDA Journal of Pharmaceutical Science & Technology,
`vol. 54, No. 2, Mar.—Apr. 2000, pp. 152—169.
`Nema et al., “Excipients and Their Use in Injectable Prod-
`ucts,” PDA Journal of Pharmaceutical Science & Technol-
`ogy, vol. 51, No. 4, Jul.—Aug. 1997, pp. 166—171.
`Wang and Hanson “Parenteral Formulations of Proteins and
`Peptides: Stability and Stabilizers, ” Journal of Parenteral
`Science & Technology, vol. 42, Supplement 1988, pp.
`S3—S25.
`
`Carpenter et al., “Freezing—and Drying—Induced Perturba-
`tions of Protein Structure and Mechanisms of Protein Pro-
`
`tection by Stabilizing Addditives,” Drugs and the Pharma-
`ceutical Sciences, vol. 96, 1999, pp. 123—160.
`Pikal, “Mechanisms of Protein Stabilization During Freez-
`e—Drying and Storage: The Relative Importance of Thermo-
`dynamic Stabilization and Glassy State Relaxation Dynam-
`ics,” Drugs and the Pharmaceutical Sciences, vol. 96, 1999,
`pp. 161—197.
`Shah et al., “The Effects of Various Excipients on the
`Unfolding of Basic Fibroblast Growth Factor,” PDAJournal
`of Pharmaceutical Science & Technology, vol. 52, No. 5,
`Sep.—Oct. 1998, pp. 209—214.
`Powell et al., “Compendium of Excipients for Parenteral
`Formulations,” PDA Journal of Pharmaceutical Science &
`Technology, vol. 52, No. 5, Sep.—Oct. 1998, pp. 238—311.
`Zeissman, “Cholecystokinin Cholescintigraphy: Victim of
`Its Own Success?” Journal of Nuclear Medicine, vol. 40,
`No. 12, Dec. 1999, pp. 2038—2042.
`Krishnamurthy and Krishnamurthy, “Gallbladder Ejection
`Fraction: A Decade of Progress and Future Promise,” Jour-
`nal of Nuclear Medicine, vol. 32, No. 4, Apr. 1992, pp.
`542—544.
`
`Krishnamurthy et al., “Quantitative Biliary Dynamics: Intro-
`duction of a New Noninvasive Scintigraphic Technique,”
`Journal of Nuclear Medicine, vol. 24, No. 3, 1983, pp.
`217—223.
`
`Mesgarzadeh et al., “Filling, Postcholecystokinin Emptying,
`and Refilling of Normal Gallbladder: Effects of Two Dif-
`ferent Doses of CCK on Refilling: Concise Communica-
`tion,” Journal of Nuclear Medicine, vol. 24, No. 8, 1983, pp.
`666—671.
`
`Krishnamurthy et al., “The Gallbladder Emptying Response
`to Sequential Exogenous and Endogenous Cholecystoki-
`nin,” Nuclear Medicine Communications, vol. 5, 1984, pp.
`27—33.
`
`Krishnamurthy et al., “Detection, Localization, and Quan-
`titation of Degree of Common Bile Duct Obstruction by
`Scintigraphy,” Journal of Nuclear Medicine, vol. 26, No. 7,
`Jul. 1985, pp. 726—735.
`
`Fink—Bennett et al., “Cholecystokinin Cholescintigraphic
`Findings in the Cystic Duct Syndrome,” Journal of Nuclear
`Medicine, vol. 26, No. 10, Oct. 1985, pp. 1123—1128.
`
`Fink—Bennett, “The Role of Cholecystogogues in the Evalu-
`ation of Biliary Tract Disorders,” Nuclear Medicine Annual
`1985, Lenny Freeman and Heidi Weissman, eds., New York,
`Raven Press, 1985, pp. 107—132.
`
`Newman et al., “A Simple Technique for Quantitative
`Cholecystokinin—HIDA Scanning,” The British Journal of
`Radiology, vol. 56, Jul. 1983, pp. 500—502.
`
`Pickleman et al., “The Role of Sincalide Cholescintigraphy
`in the Evaluation of Patients with Acalculus Gallbaldder
`
`Disease,” Archives of Surgery, vol. 120, Jun. 1985, pp.
`693—697.
`
`Zeissman et al., “Calculation of a Gallbaldder Ejection
`Fraction: Advantage of Continuous Sincalide Infusion Over
`the Three—Minute Infusion Method,” Journal of Nuclear
`Medicine, vol. 33, No. 4, Apr. 1992, pp. 537—541.
`
`Balon et al., Society of Nuclear Medicine Procedure Guide-
`line for Hepatobiliary Scintigraphy.
`
`* cited by examiner
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 1 0f 12
`
`US 6,803,046 B2
`
`2
`
`0 l
`
`0 l
`
`l
`l
`N—CH—C—NH—(IZH—C—NH
`CH2
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`2
`CH
`
`H2
`
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`|0
`
`H
`
`FIG. 1
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 2 0f 12
`
`US 6,803,046 B2
`
`0 |
`
`|
`
`Met 6
`
`(Fl—C—N—CH
`H
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`
`n_u|C|C.||O
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`lek_........w"oneouu_22=3u_HHHH."CICICISIC"m_mu.......finOHCumU_2OHH820¢,
`4HOHCOu.“um:=H
`
`FIG. 2
`
`
`
`
`

`

`US. Patent
`
`US 6,803,046 132
`
`2
`
`O
`N—CH—C—NH—CH—fl—NH
`I
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`
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`
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`
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`FIG. 3
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`
`
`

`

`US. Patent
`
`2
`
`OHC_4WIIH%_
`NH2,u........w2"OHCOu13“kH2H2__H3"Ml“Wmc||c|c|51lcm0m_uu.......fl......................mOHC”ma_2oHHS
`
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`
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`
`B2%wo,“L"mPH28,ale6_swUOHCO
`
`FIG. 4
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 5 0f 12
`
`US 6,803,046 B2
`
`100
`
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`
`pH
`
`FIG. 5
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 6 0f 12
`
`US 6,803,046 B2
`
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`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 7 0f 12
`
`US 6,803,046 B2
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`
`
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`Ni
`Mn
`Mg
`Pb
`Fe
`Cu
`Cr
`
`
`FIG. 7
`
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 8 0f 12
`
`US 6,803,046 B2
`
`
`DAD1 A, Sig=215,5 Ref=345,5 (OB1OMD11\Oa1DMD15.D)
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`with 0.63 mM Cu2+
`
`FIG. 8
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`US. Patent
`
`Oct. 12, 2004
`
`Sheet 9 0f 12
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`US 6,803,046 B2
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`
`FIG. 9
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 10 0f 12
`
`US 6,803,046 B2
`
`
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`
`FIG. 10
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 11 0f 12
`
`US 6,803,046 B2
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`
`
`
`
`
`
`
`
`
`5 10 15 20 25 30 35 40 450 mir
`Chromatogram of Kinevac Experimental Formulation (1 mM DTPA) Spiked with
`0.13 mM Mn2+
`
`
`
`30—
`
`25“
`
`20—
`
`15
`
`‘0
`
`57
`
`
`
`
`
`
`

`

`US. Patent
`
`Oct. 12, 2004
`
`Sheet 12 0f 12
`
`US 6,803,046 B2
`
`
`mAU
`»
`E
`
`
`
`
`
`
`
`
`
`Sincalide (ta = 29 min.)
`
`
`
`Sincalide (tR = 29 min)
`
`Desulfated Sincalide
`
`(tR = 33 min.)
`
`
`
`m
`
`1~
`
`l
`.1
`
`\
`
`1
`
`7_
`
`6—
`5*
`
`41
`
`31
`
`2..
`
`1
`1
`V
`l
`T
`l
`
`10
`15
`20
`25
`30
`35
`0
`5
`
`Typical Full-Scale and Expanded-Scale Chromatograms of
`Reconstituted Kinevac
`
`FIG. 12
`
`
`
`
`

`

`US 6,803,046 B2
`
`1
`SINCALIDE FORMULATIONS
`
`FIELD OF THE INVENTION
`
`The invention relates to pharmaceutically acceptable for-
`mulations of sincalide.
`
`BACKGROUND OF THE INVENTION
`
`is a
`KINEVAC® (Sincalide for Injection, USP)
`cholecystopancreatic-gastrointestinal hormone peptide for
`parenteral administration. The active 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-terminal octapeptide of cholecys-
`tokinin (CCK-8), with the following amino acid sequence: A
`sp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2.
`KINEVAC® was first introduced in 1976, and was fin-
`ished as a sterile, nonpyrogenic, lyophilized white powder in
`a 5-mL (nominal) glass vial to contain: 5 pg sincalide with
`45 mg sodium chloride to provide tonicity; sodium hydrox-
`ide or hydrochloric acid may have been added for pH
`adjustment (pH 5.5—6.5). The type I glass vial was sealed
`under a nitrogen headspace with a Tompkins B0849 closure.
`This two-ingredient formulation was incorporated into the
`US. Pharmacopea/National Formulary, USP 24, NF 19, Jan.
`1, 2000.
`Since its introduction, various drawbacks in the manu-
`facturing 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 of both sin-
`calide and KINEVAC®. This bioassay was unable to dis-
`tinguish between bioactivity of sincalide and bioactivity of
`sincalide degradants. Accordingly, a 20% overage of sin-
`calide was required in previous sincalide formulations to
`compensate for the limitations of the bioassay. Thus, there is
`a need for sincalide formulations having improved and
`consistent potency as established by a sincalide specific
`assay such as HPLC.
`SUMMARY OF THE INVENTION
`
`The present invention satisfies the need for improved
`sincalide formulations by providing formulations that elimi-
`nate the need for a 20% overage of sincalide. The sincalide
`formulations of the invention are also purer than prior art
`formulations, and have fewer degradants and more consis-
`tent potency. In addition, the purity of these formulations,
`may be assessed by HPLC, thus eliminating the need for the
`bioassay of the prior art formulations.
`The present
`invention provides sincalide formulations
`adapted for administration by injection. These sincalide
`formulations are characterized by improved stability and
`may be prepared as a relatively large volume batch (z100 L).
`In one aspect, the invention features sincalide formula-
`tions that include an effective amount of sincalide, a bulking
`agent/tonicity adjuster, one or more stabilizers, a surfactant,
`a chelator, and a buffer. The invention also features kits and
`methods for preparing improved sincalide formulations, as
`well as methods for treating, preventing, and diagnosing gall
`bladder-related disorders using sincalide formulations.
`The formulations of the invention preferably have a pH
`between 6.0 and 8.0. Suitable buffers include, but are not
`limited to, phosphate, citrate, sulfosalicylate, borate, acetate
`and amino acid buffers. Phosphate buffers, such as dibasic
`potassium phosphate, are preferred.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`2
`In various embodiments of the invention, the surfactant is
`a nonionic surfactant, preferably a polysorbate, such as
`polysorbate 20 or polysorbate 80; the chelator is pentetic
`acid (DTPA); and the stabilizer is an antioxidant and/or
`amino acid. In a particularly desirable embodiment of the
`invention, the formulation includes a plurality of stabilizers,
`preferably L-arginine monohydrochloride, L-methionine,
`L-lysine monohydrochloride, and sodium metabisulfite.
`Suitable bulking agents/tonicity adjusters include, but are
`not limited to, mannitol, lactose, sodium chloride, maltose,
`sucrose, PEG’s, cyclodextrins, dextran, polysucrose
`(Ficoll), and polyvinylpyrrolidine (PVP). D-Mannitol is a
`preferred bulking agent/tonicity adjuster.
`In a particularly preferred embodiment, the reconstituted
`formulation includes 0.0008 to 0.0012 mg/mL active ingre-
`dient (i.e., sincalide); 20.0 to 50.0 mg/mL mannitol, 2.0 to
`7.0 mg/mL arginine; 0.2 to 1.0 mg/mL methionine; 2.0 to
`30.0 mg/mL lysine; 0.002 to 0.012 mg/mL sodium met-
`abisulfite; 0.000001 to 0.003 mg/mL polysorbate 20, 0.1 to
`3.0 mg/mL pentetic acid (DTPA); and 5.4 to 12.0 mg/mL
`potassium phosphate (dibasic).
`In,
`a more preferred
`embodiment, the reconstituted formulation includes about
`0.001 mg/mL sincalide; about 34 mg/mL D-mannitol, about
`6 mg/mL L-arginine monohydrochloride; about 0.8 mg/mL
`L-methionine; about 3 mg/mL L-lysine monohydrochloride;
`about 0.008 mg/mL sodium metabisulfite; less than about
`0.01 mg/mL polysorbate 20, about 0.4 mg/mL pentetic acid
`(DTPA); and about 1.8 mg/mL potassium phosphate
`(dibasic).
`The kits of the invention may, for example, include the
`various components of the formulation as a mixture in
`powder form, along with a container (e. g., a vial) to hold the
`powder mixture and a physiologically acceptable fluid for
`reconstitution of the formulation, The components of the
`formulation may be present in the kit either in the powder
`mixture or in the fluid portion. Kits of the invention may also
`include all components in a liquid mixture or some compo-
`nents in a liquid form and some in the form of a powder.
`The formulations of the invention have improved stability
`and potency compared to previous sincalide formulations,
`and are useful as diagnostic aids for imaging the hepatobil-
`iary system of a patient. When used as a diagnostic aid, the
`sincalide formulations may, for example, be co-administered
`with a radiopharmaceutical agent having rapid hepatic
`uptake, such as 99”‘Tc-mebrofenin, or similar hepatobiliary
`imaging agents,
`to assist in the diagnosis of gallbladder
`diseases and related disorders. Additionally, the formula-
`tions may be administered before and/or after diagnostic
`imaging (including for example, magnetic resonance
`imaging, scintigraphic imaging, ultrasound imaging, etc.)
`The sincalide formulations of the invention may also be
`administered to patients receiving total parenteral nutrition
`(TPN), in order to treat and/or prevent TPN-related disor-
`ders.
`
`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-glutamine-5-L-methioninecaerulein or “sin-
`calide” (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) monosulfoxide.
`FIG. 3 is a drawing illustrating the chemical structure of
`sincalide (Met 6) monosulfoxide.
`
`
`
`
`

`

`US 6,803,046 B2
`
`3
`FIG. 4 is a drawing illustrating the chemical structure of
`sincalide (Met 3, 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. 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 sin-
`calide 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 0.63 mM Cu2+.
`FIG. 9 is a chromatogram of KINEVAC® experimental
`formulation (1 mM DTPA) spiked with 0.63 mM Cu2+.
`FIG. 10 is a chromatogram of KINEVAC® experimental
`formulation (no DTPA) spiked with 0.18 mM Mn2+.
`FIG. 11 is a chromatogram of KINEVAC® experimental
`formulation (1 mM DTPA) spiked with 0.18 mM Mn2+.
`FIG. 12 shows representative full-scale and expanded
`scale chromatograms of a lyophilized reformulation of
`KINEVAC® upon reconstitution with 5 mL water, resulting
`in a sincalide concentration of 1 Mg/mL.
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`In order to develop an improved sincalide formulation a
`series of studies, described in the Examples below, were
`conducted to determine the effects of various excipients on
`formulations of sincalide. Through these studies, we discov-
`ered that the potency and stability of sincalide formulations
`can be significantly enhanced through the careful selection
`of excipients that provide certain desired functions.
`Accordingly, the present invention provides novel sincalide
`formulations having improved stability and/or potency over
`previous formulations.
`As used herein,
`the term “sincalide” includes the
`synthetically-prepared C-terminal octapeptide of cholecys-
`tokinin (CCK-8), with the amino acid sequence: Asp-Tyr
`(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, as well as deriva-
`tives thereof which have been optimized or modified (to
`improve stability, potency, pharmacokinetics, etc.), but
`retain the biological activity of the original octapeptide. For
`example, peptides in which the methionine and/or aspartic
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`4
`acid residues have been replaced without significantly
`affecting the biological activity are included within “sin-
`calide” as the term is used herein. Similarly,
`the term
`“sincalide” encompasses not only monomeric, but multim-
`eric forms of the peptide, as well as physiologically active
`degradants or portions of the peptide and its derivatives.
`The sincalide formulations of the invention can include a
`
`variety of excipients, such as, for: example, antioxidants,
`buffers, bulking agents/tonicity adjusters, chelating agents,
`complexing agents, crosslinking agents, co-solvents, osmo-
`lality adjustors, solubilizers, surfactants, stabilizers, pH
`adjustors,
`lyoprotectants/cryoprotectants, air/liquid and/or
`ice-liquid interface protectants (protectants against surface
`induced denaturation), freeze-thaw protectants, protectants
`against protein/peptide denaturation, protectants for
`rehydration, and wetting agents. In preferred embodiments,
`the formulations include excipients that perform the func-
`tions of at least: (i) a bulking agent/tonicity adjuster, (ii) a
`stabilizer, (iii) a surfactant, (iv) a chelator, and (v) a buffer.
`Typically, each of these functions is performed by a different
`excipient. However, in some embodiments of the invention
`a single excipient may perform more than one function. For
`example, a single excipient may be multi-functional, e.g.
`amino acids may function as bulking agents, stabilizers
`and/or buffers and other excipients may function,
`for
`example, as both a stabilizer and a chelator or as both a
`bulking agent and a tonicity adjuster. Alternatively, multiple
`excipients serving the same function may be used. For
`example, the formulation may contain more than one excipi-
`ent that functions as a stabilizer.
`
`Table 1 below shows the concentration ranges for various
`excipients that were investigated.
`In general,
`the range
`studies were based on a 2-mL fill of bulk solution per vial
`before lyophilization. After reconstitution with 5 mL of
`water for injection the final sincalide formulation results in
`an isotonic solution. The concentration ranges of the various
`ingredients provided in Table 1 can be adjusted 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 desir-
`ability of achieving tonicity in the final reconstituted solu-
`tion. For example, as indicated above, the concentrations
`provided in Table 1 were developed to provide 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.
`
`TABLE 1
`
`Concentration ranges for excipients for preferred sincalide formulations.
`
`Range
`(mg/mL
`Bulk)
`
`Range
`(mg/vial)
`
`0.0025
`
`0.0050
`
`50.0—
`125.0
`
`100—250
`
`Range
`
`Final Formulation (mg)
`
`(mg per
`1 mL after
`reconst)
`
`0.0008—
`0.0012
`20.0—50.0
`
`1 mL
`Bulk
`
`1 vial
`Target
`
`1 mL
`after
`reconst.
`
`0.0025
`
`0.0050
`
`0.0010
`
`85
`
`170
`
`34
`
`0.0000025— 0.0000050—
`0.0075
`0.0150
`
`0.0000010— <0.01
`0.0030
`
`<0.01
`
`<0.01
`
`Excipient
`
`Function
`
`(sincalide)
`
`Active Ingredient
`
`Mannitol
`
`Bulking Agent/Cake
`Forming Agent/Tonicity
`Adjuster
`TWEEN ®-20 Non-ionic
`Surfactant/Solubilizing
`Agent/Wetting Agent
`
`
`
`
`

`

`US 6,803,046 B2
`
`TABLE 1-continued
`
`Concentration ranges for excipients for preferred sincalide formulations.
`
`Range
`
`Final Formulation mg
`
`Range
`(mg/mL
`Bulk)
`1.0
`
`Range
`(mg/vial)
`2.0
`
`(mg per
`1 mL after
`reconst)
`0.1—3.0
`
`1 mL
`Bulk
`1.0
`
`1 vial
`Target
`2.0
`
`1 mL
`after
`reconst.
`0.4
`
`0.005—
`0.030
`2.7—4.5
`
`0.010—
`0.060
`5.4—12.0
`
`1.1—1.8
`
`0002—0012
`
`0.020
`
`0.040
`
`0.008
`
`1.0—6.5
`
`9. 6—130
`
`1.92—2.6
`
`0.5—2.5
`5.0—30.0
`
`1.0—5.0
`10.0—60.0
`
`0.2—1.0
`2.0—30.0
`
`5.0—17.5
`
`10.0—35.0
`
`2.0—7.0
`
`4.5—9.0
`
`9.0—18.0
`
`1.8—3.6
`
`4.5
`
`0
`
`2.0
`7.5
`
`15
`
`0
`
`9.0
`
`0
`
`4.0
`15.0
`
`30.0
`
`0
`
`1.8
`
`0
`
`0.8
`3.0
`
`6.0
`
`0
`
`Excipient
`DTPA
`
`Sodium
`Metabisulfite
`Potassium
`Phosphate,
`dibasic
`Potassium
`Phosphate,
`monobasic
`Methionine
`Lysine
`
`Arginine
`
`Sodium
`Chloride
`
`Function
`Chelator/Stabilizer/Anti-
`oxidant/
`CompleXing
`Agent/Preservative/pH
`Ad'uster
`An ioxidant/Preservative/
`Staailizer
`Bu: er/pH
`Ad'uster/Dissolution Aid
`
`Bu: er/pH
`Ad'uster/Dissolution Aid
`Staailizer
`Staailizer/Lyoprotectant/
`Cryoprotectant
`Staailizer/Lyoprotectant/
`Cryoprotectant/pH
`Ad'uster
`Tonicity Adjuster
`
`
`
`Alternative excipien s include TWEEN ®-80, potassium metabisulfite, sodium phosphate dibasic, sodium phosphate
`monobasic, and potassium chloride. Additional alternatives are listed below.
`
`30
`
`Table 2 shows preferred ranges for preferred excipients in
`the bulk solutions, Vials and after reconstitution. All con-
`centrations shown for the bulk solution are based on a 2 mL
`fill volume The in redient uantities are matched to result
`.
`’.
`g
`q
`.
`.
`.
`In a pH shghtly below Helltral and reSUIt In an ISOtOHIC 35
`solution after reconstitution of the lyophilized Vial as indi-
`
`TABLE 2-continued
`_
`_
`_
`_
`_
`osmOIahty values for vanous smcahde formulauons'
`(All formulations contain 0.0025 mg CCK—8/mL.; “dibasic”
`and “monobasic” refer to dibasic and monobasic potassium
`
`_P_P_=—Lh05hate' “Na meta” refers to sodium metabisulfite
`
`cated by an osmolality in the range of 180 to 320 mOsm/kg
`, preferably, 240 to 320 mOsm. The columns titled “Final
`Formulation” represent particularly preferred formulations.
`
`
`TABLE 2
`
`Osmola i y values for various sincalide formulations.
`(All formu a ions contain 0.0025 mg CCK—8/mL.; “dibasic”
`and “monoaasic” refer to dibasic and monobasic potassium
`hos hate' “Na meta” refers to sodium metabisulfite
`
`Calculated
`mOsm/kg
`
`292
`
`244
`
`244
`
`244
`
`187
`
`247
`
`)
`
`
`
`Manni 0 (125.0)
`Disasic (3-75)
`DTPA_( -0)
`Manm o (95.0)
`13133510 (4-0)
`Monobasw (2-8)
`DTPA( -0)
`Manni 0 (103.0)
`Dwasw (3-75)
`3W5 (
`'0)75 0
`annio (
`.
`NaCl (4.5)
`Diaasic (3.75)
`DTPA ( 0)
`Manni o (85.0)
`TWEEN ® 20 (0.005)
`Diaasic (2.75)
`DTPA ( _0)
`Methionine (2.0)
`Lysine (15.0)
`Mannito (50.0)
`
`Formu a ion
`Excipients
`(mg/mL Bulk)
`
`
`
`
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Formulation
`Excipients
`(mg/mL Bulk)
`NaCl (9.0)
`Dibasic (3.00)
`DTPA( .0)
`TWEEN ® 20 (0.0075)
`Mannito (75.0)
`KC (60)
`Dibasic (3.25)
`Monobasic (1.0)
`DTPA (
`.0)
`Me hionine (2.0)
`TWEEN ® 20 (0.005)
`Mannito (75.0)
`KC (6.0)
`Dibasic (3.25)
`Monobasic (1.0)
`DTPA( _0)
`Me hionine (2.0)
`TWEEN ® 20 (0.0025)
`Mannito (75.0)
`KC (6.0)
`Dibasic (3.25)
`Monobasic (1.0)
`DTPA( .0)
`h'
`-
`Me
`ionine (2'0)
`TWEEN ® 20 (2.5 ng)
`Mf‘nn}t°
`(85-0)
`leam (450)
`DTPA (
`'0)
`Na meta3isulfite (0.020)
`Methionine (2-0)
`Lysine (7.50)
`Arginine (15.0)
`
`
`
`
`
`
`Calculated
`mOsm/kg
`
`264
`
`264
`
`264
`
`314
`
`
`
`
`

`

`7
`
`8
`
`US 6,803,046 B2
`
`TABLE 2-c0ntinued
`
`TABLE 2-continued
`
`Osmolali y values for various sincalide formulations.
`(All formula ions contain 0.0025 mg CCK—8/mL.; “dibasic”
`and “monobasic” refer to dibasic and monobasic potassium
`phosphate'z “Na meta” refers to sodium metabisulfite
`Formula ion
`Excipients
`(mg/mL Bulk)
`
`Calculated
`mOsm/kg
`
`262
`
`262
`
`262
`
`262
`
`209
`
`187
`
`245
`
`245
`
`232
`
`238
`
`245
`
`Osmolality values for various sincalide formulations.
`(All formulations contain 0.0025 mg CCK—8/mL.; “dibasic”
`and “monobasic” refer to dibasic and monobasic potassium
`phosphate': “Na meta” refers to sodium metabisulfite
`Formulation
`Excipients
`(mg/mL Bulk)
`
`Calculated
`mOsm/kg
`
`257
`
`257
`
`257
`
`259
`
`257
`
`259
`
`264
`
`314
`
`262
`
`Na Meta (0.015)
`Mannitol (85.0)
`Dibasic (2.75)
`DTPA (1.0)
`20 (0.005)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Na Meta (0.030)
`Mannitol (85.0)
`Dibasic (2.75)
`DTPA (1.0)
`TWEEN ® 20 (0.005)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Na Meta (0.005)
`Mannitol (85.0)
`Dibasic (2.75)
`DTPA(1.0)
`TWEEN ® 20 (0.005)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Na Meta (0.020)
`Mannitol (85.0)
`Dibasic (3.00)
`DTPA (1.0)
`TWEEN ® 20 (0.005)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Dibasic (2.75)
`Mannitol (85.0)
`Na Meta (0.015)
`DTPA (1.0)
`TWEEN ® 20 (0.005)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Dibasic (3.00)
`Mannitol (85.0)
`Na Meta (0.020)
`131131500)
`TWEEN ® 20 (0.005)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Dibasic (3.25)
`Mannitol (75.0)
`KCl (6.0)
`TWEEN ® 20 (0.0025)
`Monobasic (1.0)
`DTPA (1.0)
`Methionine (2.0)
`Dibasic (4.50)
`Mannitol (85.0)
`TWEEN ® 20 (2.5 ng)
`DTPA (1.0)
`Na metabisulfite (0.020)
`Methionine (2.0)
`Lysine (7.50)
`Arginine (15.0)
`Methionine (2.0)
`Mannitol (75.0)
`NaCl (5.0)
`TWEEN ® 80 (0.025)
`Monobasic (1.0)
`131131500)
`Dibasic (3.25)
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`
`
`Methionine (1.5)
`Mannito (75.0)
`NaCl (5.0)
`TWEEN ® 80 (0.025)
`Monobasic (1.0)
`DTPA (
`.0)
`Dibasic (3.25)
`Methionine (1.0)
`Mannito (75.0)
`NaCl (5.0)
`TWEEN ® 80 (0.025)
`Monobasic (1.0)
`DTPA( .0)
`Dibasic (3.25)
`Methionine (0.5)
`Mannito (75.0)
`NaCl (5.0)
`TWEEN ® 80 (0.025)
`Monobasic (1.0)
`DTPA( .0)
`Dibasic (3.25)
`Methionine (2.5)
`Mannito (75.0)
`NaCl (5.0)
`TWEEN ® 80 (0.005)
`Monobasic (1.0)
`DTPA( .0)
`Dibasic (3.25)
`Lysine (5.0)
`Mannito (95.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA( .0)
`Methionine (2.0)
`Lysine ( 5.0)
`Mannito (85.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA( .0)
`Methionine (2.0)
`Lysine (30.0)
`Mannito (70.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA( .0)
`Methionine (2.0)
`Arginine (17.5)
`Mannito (85.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA( .0)
`Methionine (2.0)
`Arginine (10.0)
`Mannito (85.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA( .0)
`Methionine (2.0)
`Arginine (5.0)
`Mannito (85.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA (
`.0)
`Methionine (2.0)
`Lysine (7.5)
`Arginine (8.75)
`Mannito (85.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA( .0)
`
`
`
`
`

`

`US 6,803,046 B2
`
`9
`
`TABLE 2-c0ntinued
`
`Osmolality values for various sincalide formulations.
`(All formulations contain 0.0025 mg CCK—8/mL.; “dibasic”
`and “monobasic” refer to dibasic and monobasic potassium
`phosphate; “Na meta” refers to sodium metabisulfite
`Formulation
`Excipients
`(mg/mL Bulk)
`
`Calculated
`mOsm/kg
`
`Methionine (2.0)
`Lysine (7.5)
`Arginine (15.0)
`Mannitol (85.0)
`TWEEN ® 20 (0.005)
`Dibasic (2.75)
`DTPA (1.0)
`Methionine (2.0)
`Lysine (7.5)
`
`257
`
`Chelators
`Excipient impurities and/0r stopper extractables can intro-
`duce trace metals into pharmaceutical formulations. Sin-
`calide contains two methionine residues (Met 3 and Met 6)
`that are susceptible to oxidation by free metals. Thus, the
`sincalide formulations of the invention contain Chelators to
`inhibit the oxidation of the two methionine residues present
`in sincalide (Met 3 and Met 6). Preferred Chelators include
`pentetic acid (DTPA), edetic acid (EDTA) and derivatives
`thereof, including salts. DTPA is a preferred chelator. As
`described in Example 2 below,
`the amounts of the
`degradants, sincalide Met 3 and sincalide Met 6
`monosulfoxides, increase in the presence of certain metals
`and in the absence of DTPA, while the presence of DPTAhas
`an inhibitory effect on the formation of these monosulfox-
`ides. In particular, copper and manganese, in the absence of
`DTPA, have the greatest oxidative effect on the methionine
`residues of sincalide resulting in combined height percent-
`ages of Met 3 and Met 6 monosulfoxides (vs sincalide) of
`85.5 and 128.9, respectively.
`the sincalide formulations
`In a preferred embodiment,
`contain between 0.1 and 3.0 mg of DTPA per mL after
`reconstitution. In a particularly preferred embodiment, sin-
`calide formulations of the invention contain 0.4 mg DTPA/
`mL after reconstitution 0 with 5 mL.
`
`Buffering Agents
`Buffering agents are employed to stabilize th