United States Patent (10) Patent N0.: US 6,803,046 B2 Metcalfe Et Al

US006803046B2

(12) United States Patent (10) Patent N0.: US 6,803,046 B2 Metcalfe et al. (45) Date of Patent: Oct. 12, 2004

(54) SINCALIDE FORMULATIONS OTHER PUBLICATIONS (75) Inventors: Edmund C. Metcalfe, Hillsborough, NJ SitZmann, et al., “Cholecystokinin Prevents Parenteral (US); J 0 Anna Monteferrante, Raritan Nutrition Induced Biliary Sludge in Humans,” Surgery, Township, NJ (US); Margaret Gynecology & Obstetrics, vol. 170, Jan. 1990, pp. 25—31. Newborn, Hamilton Township, NJ Teitelbaum et al., “Treatment of Parenteral Nutrition—Asso (US); Irene Ropiak, Lawrenceville, NJ ciated Cholestasis with Cholecystokinin—Octapeptide,” (US); Ernst Schramm, North Journal of Pediatric Surgery, vol. 30, No. 7, Jul. 1995, pp. Brunswick, NJ (US); Gregory W. 1082—1085. White, Monmouth Junction, NJ (US); Moss and Amii, “New Approaches to Understanding the Julius P. Zodda, Mercerville, NJ (US) Etiology and Treatment of Total Parenteral Nutrition—Asso ciated Cholestasis,” Seminars in Pediatric Surgery, vol. 8, (73) Assignee: Bracco International B.V., Amsterdam No. 3, Aug. 1999, pp. 140—147. (NL) Teitelbaum, “Parenteral Nutrition—Associated Cholestasis,” Current Opinion in Pediatrics, vol. 9, 1997, pp. 270—275. (*) Notice: Subject to any disclaimer, the term of this Teitelbaum and Tracy, “Parenteral Nutrition—Associated patent is extended or adjusted under 35 Cholestasis,” Seminars in Pediatric Surgery, vol. 10, No. 2, U.S.C. 154(b) by 0 days. May 2001, pp. 72—80. Strickley, “Parenteral Formulations of Small Molecules (21) Appl. No.: 10/222,540 Therapeutics Marketed in the United States (1999) —Part Filed: Aug. 16, 2002 1,” PDA Journal of Pharmaceutical Science & Technology, (22) vol. 53, No. 6, Nov.—Dec. 1999, pp. 324—349. (65) Prior Publication Data Strickley, “Parenteral Formulations of Small Molecules US 2004/0033243 A1 Feb. 19, 2004 Therapeutics Marketed in the United States (1999)—Part II,” PDA Journal of Pharmaceutical Science & Technology, vol. 54, No. 1, Jan.—Feb. 2000, pp. 69—96. (51) Int. Cl.7 ...... A61K 9/00 (List continued on next page.) (52) U.S. Cl...... 424/400; 514/1.65; 514/18; 514/19; 514/951 Primary Examiner—Thurman K. Page (58) Field of Search ...... 424/400; 514/18, Assistant Examiner—Konata M. George 514/19, 1.65, 951 (74) Attorney, Agent, or Firm—Kramer, Levin, Naftalis & Frankel LLP (56) References Cited (57) ABSTRACT U.S. PATENT DOCUMENTS The invention features sincalide formulations that include an 3,723,406 A 3/1973 Ondetti et al...... 260/1125 effective amount of sincalide, a bulking agent/tonicity 5,011,678 A * 4/1991 Wang et al...... 424/45 adjuster, a stabilizer, a surfactant, a chelator, and a buffer. 5,555,610 A 9/1996 Yan et a1...... 424/952 The invention also features kits and methods for preparing 5,567,414 A 10/1996 Schneider et a1...... 424/9.52 improved sincalide formulations, as well as methods for 5,833,948 A 11/1998 Tournier et al...... 424/9.321 treating, preventing, and diagnosing gall bladder-related 6,110,443 A 8/2000 Schneider et al...... 424/9.51 disorders using sincalide formulations. 6,306,905 B1 10/2001 KurZ et al...... 514/551

6,326,406 B1 12/2001 De Tommaso ...... 514/731 6,358,528 B1 3/2002 Grimmett et a1...... 424/474 108 Claims, 12 Drawing Sheets

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

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U.S. Patent 0a. 12, 2004 Sheet 12 0f 12 US 6,803,046 B2

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FIG. 12 US 6,803,046 B2 1 2 SINCALIDE FORMULATIONS 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 FIELD OF THE INVENTION acid (DTPA); and the stabiliZer is an antioxidant and/or The invention relates to pharmaceutically acceptable for amino acid. In a particularly desirable embodiment of the invention, the formulation includes a plurality of stabiliZers, mulations of sincalide. preferably L-arginine monohydrochloride, L-methionine, BACKGROUND OF THE INVENTION L-lysine monohydrochloride, and sodium metabisul?te. Suitable bulking agents/tonicity adjusters include, but are KINEVAC® (Sincalide for Injection, USP) is a 10 not limited to, mannitol, lactose, sodium chloride, maltose, cholecystopancreatic-gastrointestinal hormone peptide for sucrose, PEG’s, cyclodextrins, dextran, polysucrose parenteral administration. The active pharmaceutical (Ficoll), and polyvinylpyrrolidine (PVP). D-Mannitol is a ingredient, 1-De(5-oxo-L-glutamine-5-L-proline)-2-de-L preferred bulking agent/tonicity adjuster. methioninecaerulein or “sincalide” (CAS# 25126-32-3), is a In a particularly preferred embodiment, the reconstituted synthetically prepared C-terminal octapeptide of cholecys formulation includes 0.0008 to 0.0012 mg/mL active ingre tokinin (CCK-8), With the following amino acid sequence: A dient (i.e., sincalide); 20.0 to 50.0 mg/mL mannitol, 2.0 to sp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2. 7.0 mg/mL arginine; 0.2 to 1.0 mg/mL methionine; 2.0 to KINEVAC® Was ?rst introduced in 1976, and Was ?n 30.0 mg/mL lysine; 0.002 to 0.012 mg/mL sodium met ished as a sterile, nonpyrogenic, lyophiliZed White poWder in abisul?te; 0.000001 to 0.003 mg/mL polysorbate 20, 0.1 to a 5-mL (nominal) glass vial to contain: 5 pg sincalide With 20 3.0 mg/mL pentetic acid (DTPA); and 5.4 to 12.0 mg/mL 45 mg sodium chloride to provide tonicity; sodium hydrox potassium phosphate (dibasic). In, a more preferred ide or hydrochloric acid may have been added for pH embodiment, the reconstituted formulation includes about adjustment (pH 5.5—6.5). The type I glass vial Was sealed 0.001 mg/mL sincalide; about 34 mg/mL D-mannitol, about under a nitrogen headspace With a Tompkins B0849 closure. 6 mg/mL L-arginine monohydrochloride; about 0.8 mg/mL This tWo-ingredient formulation Was incorporated into the 25 L-methionine; about 3 mg/mL L-lysine monohydrochloride; US. Pharmacopea/National Formulary, USP 24, NF 19, Jan. about 0.008 mg/mL sodium metabisul?te; less than about 1, 2000. 0.01 mg/mL polysorbate 20, about 0.4 mg/mL pentetic acid Since its introduction, various drawbacks in the manu (DTPA); and about 1.8 mg/mL potassium phosphate facturing and analysis of KINEVAC® have been identi?ed. (dibasic). For example, the tWo-ingredient formulation suffers from 30 The kits of the invention may, for example, include the potency variability. This variability Was exacerbated by the various components of the formulation as a mixture in fact that the formulation Was analyZed using a guinea pig poWder form, along With a container (e. g., a vial) to hold the gallbladder contraction bioassay for potency of both sin poWder mixture and a physiologically acceptable ?uid for calide and KINEVAC®. This bioassay Was unable to dis reconstitution of the formulation, The components of the tinguish betWeen bioactivity of sincalide and bioactivity of 35 formulation may be present in the kit either in the poWder sincalide degradants. Accordingly, a 20% overage of sin mixture or in the ?uid portion. Kits of the invention may also calide Was required in previous sincalide formulations to include all components in a liquid mixture or some compo compensate for the limitations of the bioassay. Thus, there is nents in a liquid form and some in the form of a poWder. a need for sincalide formulations having improved and The formulations of the invention have improved stability consistent potency as established by a sincalide speci?c 40 and potency compared to previous sincalide formulations, assay such as HPLC. and are useful as diagnostic aids for imaging the hepatobil SUMMARY OF THE INVENTION iary system of a patient. When used as a diagnostic aid, the sincalide formulations may, for example, be co-administered The present invention satis?es the need for improved With a radiopharmaceutical agent having rapid hepatic sincalide formulations by providing formulations that elimi 45 uptake, such as 99mTc-mebrofenin, or similar hepatobiliary nate the need for a 20% overage of sincalide. The sincalide imaging agents, to assist in the diagnosis of gallbladder formulations of the invention are also purer than prior art diseases and related disorders. Additionally, the formula formulations, and have feWer degradants and more consis tions may be administered before and/or after diagnostic tent potency. In addition, the purity of these formulations, imaging (including for example, magnetic resonance may be assessed by HPLC, thus eliminating the need for the imaging, scintigraphic imaging, ultrasound imaging, etc.) bioassay of the prior art formulations. The sincalide formulations of the invention may also be The present invention provides sincalide formulations administered to patients receiving total parenteral nutrition adapted for administration by injection. These sincalide (TPN), in order to treat and/or prevent TPN-related disor formulations are characteriZed by improved stability and ders. may be prepared as a relatively large volume batch (@100 L). 55 Other features and advantages of the invention Will be In one aspect, the invention features sincalide formula apparent from the folloWing detailed description thereof and tions that include an effective amount of sincalide, a bulking from the claims. agent/tonicity adjuster, one or more stabiliZers, a surfactant, BRIEF DESCRIPTION OF THE DRAWINGS a chelator, and a buffer. The invention also features kits and methods for preparing improved sincalide formulations, as 60 FIG. 1 is a draWing illustrating the chemical structure of Well as methods for treating, preventing, and diagnosing gall 1-De(5-oxo-L-glutamine-5-L-methioninecaerulein or “sin bladder-related disorders using sincalide formulations. calide” (CAS# 25126-32-3). The amino acid residues “Met The formulations of the invention preferably have a pH 3” and “Met 6” are outlined by dashed lines. betWeen 6.0 and 8.0. Suitable buffers include, but are not FIG. 2 is a draWing illustrating the chemical structure of limited to, phosphate, citrate, sulfosalicylate, borate, acetate 65 sincalide (Met 3) monosulfoxide. and amino acid buffers. Phosphate buffers, such as dibasic FIG. 3 is a draWing illustrating the chemical structure of potassium phosphate, are preferred. sincalide (Met 6) monosulfoxide. US 6,803,046 B2 3 4 FIG. 4 is a drawing illustrating the chemical structure of acid residues have been replaced Without signi?cantly sincalide (Met 3, 6) disulfoxide. affecting the biological activity are included Within “sin FIG. 5 is a graphical representation of the effect of pH on calide” as the term is used herein. Similarly, the term the recovery of sincalide in 35 mM phosphate buffer over 24 “sincalide” encompasses not only monomeric, but multim hours. At each pH for Which data is shoWn, the bars eric forms of the peptide, as Well as physiologically active represent 0, 6, and 24 hours, from left to right. degradants or portions of the peptide and its derivatives. FIG. 6 is a graphical representation of the effect of pH on The sincalide formulations of the invention can include a the recovery of sincalide in a formulation of the invention variety of excipients, such as, for: example, antioxidants, over 8 hours. At each pH for Which data is shoWn, the bars 10 buffers, bulking agents/tonicity adjusters, chelating agents, represent 0, 4, and 8 hours, from left to right. complexing agents, crosslinking agents, co-solvents, osmo FIG. 7 is a graphical representation of the percent sin lality adjustors, solubiliZers, surfactants, stabiliZers, pH calide Met 3 and Met 6 monosulfoxides (vs sincalide), in the adjustors, lyoprotectants/cryoprotectants, air/liquid and/or presence and absence of pentetic acid (DTPA). ice-liquid interface protectants (protectants against surface FIG. 8 is a chromatogram of KINEVAC® experimental 15 induced denaturation), freeZe-thaW protectants, protectants formulation (no DTPA) spiked With 0.63 mM Cu2+. against protein/peptide denaturation, protectants for FIG. 9 is a chromatogram of KINEVAC® experimental rehydration, and Wetting agents. In preferred embodiments, formulation (1 mM DTPA) spiked With 0.63 mM Cu2+. the formulations include excipients that perform the func FIG. 10 is a chromatogram of KINEVAC® experimental tions of at least: a bulking agent/tonicity adjuster, (ii) a formulation (no DTPA) spiked With 0.18 mM Mn2+. 20 stabiliZer, (iii) a surfactant, (iv) a chelator, and (v) a buffer. FIG. 11 is a chromatogram of KINEVAC® experimental Typically, each of these functions is performed by a different formulation (1 mM DTPA) spiked With 0.18 mM Mn2+. excipient. HoWever, in some embodiments of the invention FIG. 12 shoWs representative full-scale and expanded a single excipient may perform more than one function. For scale chromatograms of a lyophiliZed reformulation of example, a single excipient may be multi-functional, e.g. KINEVAC® upon reconstitution With 5 mL Water, resulting 25 amino acids may function as bulking agents, stabiliZers in a sincalide concentration of 1 pg/mL. and/or buffers and other excipients may function, for example, as both a stabiliZer and a chelator or as both a DETAILED DESCRIPTION OF THE bulking agent and a tonicity adjuster. Alternatively, multiple INVENTION excipients serving the same function may be used. For In order to develop an improved sincalide formulation a 30 example, the formulation may contain more than one excipi series of studies, described in the Examples beloW, Were ent that functions as a stabiliZer. conducted to determine the effects of various excipients on Table 1 beloW shoWs the concentration ranges for various formulations of sincalide. Through these studies, We discov excipients that Were investigated. In general, the range ered that the potency and stability of sincalide formulations studies Were based on a 2-mL ?ll of bulk solution per vial 35 can be signi?cantly enhanced through the careful selection before lyophiliZation. After reconstitution With 5 mL of of excipients that provide certain desired functions. Water for injection the ?nal sincalide formulation results in Accordingly, the present invention provides novel sincalide an isotonic solution. The concentration ranges of the various formulations having improved stability and/or potency over ingredients provided in Table 1 can be adjusted upWard or previous formulations. doWnWard, if necessary in conjunction With: increasing or 40 As used herein, the term “sincalide” includes the decreasing the ?ll volume per vial, obtaining the desired pH, synthetically-prepared C-terminal octapeptide of cholecys obtaining the desired reconstitution volume, and the desir tokinin (CCK-8), With the amino acid sequence: Asp-Tyr ability of achieving tonicity in the ?nal reconstituted solu (SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2, as Well as deriva tion. For example, as indicated above, the concentrations tives thereof Which have been optimiZed or modi?ed (to provided in Table 1 Were developed to provide an isotonic 45 improve stability, potency, pharmacokinetics, etc.), but solution; hoWever, one skilled in the art Would recogniZe that retain the biological activity of the original octapeptide. For a broader range of concentrations could be used if an example, peptides in Which the methionine and/or aspartic isotonic solution Was not required.

TABLE 1

Concentration ranges for excipients for preferred sincalide formulations.

Range Final Formulation (mg)

Range (mg per 1 mL (mg/mL Range 1 mL after 1 mL 1 vial after Excipient Function Bulk) (mg/vial) reconst) Bulk Target reconst.

(Sincalide) Active Ingredient 0.0025 0.0050 0.0008- 0.0025 0.0050 0.0010 0.0012 Mannitol Bulking Agent/Cake 50.0- 100-250 20.0-50.0 85 170 34 Forming Agent/Tonicity 125.0 Adjuster TWEEN ®-20 Non-ionic 0.0000025- 0.0000050- 0.0000010- <0.01 <0.01 <0.01 Surfactant/Solubilizing 0.0075 0.0150 0.0030 Agent/Wetting Agent US 6,803,046 B2

TABLE l-continued

Concentration ranges for eXcipients for preferred sincalide formulations.

Range Final Formulation mg

Range (mg per 1 mL (mg/mL Range 1 mL after 1 mL 1 vial after EXcipient Function Bulk) (mg/vial) reconst) Bulk Target reconst.

DTPA Chelator/Stabilizer/Anti- 1.0 2.0 0.1-3.0 1.0 2.0 0.4 oXidant/ CompleXing Agent/Preservative/pH Adjuster Sodium AntioXidant/Preservative/ 0.005- 0.010- 0002-0012 0.020 0.040 0.008 Metabisul?te Stabilizer 0.030 0.060 Potassium Buffer/pH 2.7-4.5 5.4—12.0 1.1-1.8 4.5 9.0 1.8 Phosphate, Adjuster/Dissolution Aid dibasic Potassium Buffer/pH 1.0-6.5 9.6—13.0 192-26 0 0 0 Phosphate, Adjuster/Dissolution Aid monobasic Methionine Stabilizer 0.5-2.5 1.0-5.0 0.2-1.0 2.0 4.0 0.8 Lysine StabiliZer/Lyoprotectant/ 50-300 100-600 2.0-30.0 7.5 15.0 3.0 Cryoprotectant Arginine StabiliZer/Lyoprotectant/ 5.0—17.5 10.0-35.0 2.0-7.0 15 30.0 6.0 Cryoprotectant/pH Adjuster Sodium Tonicity Adjuster 4.5-9.0 9.0-18.0 1.8-3.6 0 0 0 Chloride

Alternative eXcipients include TWEEN ®—80, potassium metabisul?te, 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- TABLE 2-continued centrations shoWn for the bulk solution are based on a 2 mL _ _ _ _ _ ?ll volume The in redient uantities are matched to result osmolahty Values for Vanous Smcahde formulanons' ' '_ g q _ _ _ (All formulations contain 0.0025 mg CCK—8/mL.; “dibasic” In a PH shghtly belDW neutral and result In an ISOtOnlC 35 and “monobasic” refer to dibasic and monobasic potassium solution after reconstitution of the lyophiliZed vial as indi- _P_P_=_hOShate‘ “Na meta” refers to Sodium metabisul?tel cated by an osmolality in the range of 180 to 320 mOsm/kg Formulation , preferably, 240 to 320 mOsm. The columns titled “Final Excipients calculated Formulation” represent particularly preferred formulations. (mg/InL Bulk) mOSrn/kg 40 NaCl (9.0) TABLE 2 Dibasic (3.00) DTPA (1.0) Osmolality values for various sincalide formulations. TWEEN ® 20 (00075) 264 (All formulations contain 0.0025 mg CCK—8/mL.; “dibasic” Mannitol (75_O) and “monobasic” refer to dibasic and monobasic potassium KCl (6_O) phosphate‘ “Na meta” refers to sodium metabisul?te) 45 Dibasic (325) Monobasic (1.0) Formulation DTPA (1.0) EXcipients Calculated Methionine (20) (mg/mL Bulk) mOsm/kg TWEEN ® 20 (0.005) 264 Mannitol (75.0) Mannitol (125.0) 292 50 KC} (60) Dibasic (3-75) Dibasic (3.25) DTPA_(1-O) Monobasic (1.0) Mannitol (95.0) 244 DTPA (10) Dlbaslc Methionine (2.0) Monobaslc (2-8) TWEEN ® 20 (0.0025) 264 DTPA (1-0) 55 Mannitol (75.0) Mannitol (103.0) 244 Kcl (60) Dlbasw (3-75) Dibasic (3.25) (11'O)75 O 244 Monobasic (1.0) annito ( . ) DTPA (LO) NaCl (4.5) h' ' Dibasic (3.75) Met 1on1ne(2.0) DTPA (l0) 60 TWEBN ® 20 (2.5 ng) 314 Mannitol (85.0) 187 MFHHFOI (85-0) TWEEN ® 20 (0.005) Dlbaslc (4'50) Dibasic (2.75) DTPA (1-0) DTPA (10) Na metabisul?te (0.020) Methionine (2.0) Methionine (Z-O) Lysine (15.0) 65 Lysine (7.50) Mannitol (50.0) 247 Arginine (15.0) US 6,803,046 B2 7 8

TABLE 2-c0ntinued TABLE 2-c0ntinued

Osmolality values for various sincalide formulations. Osmolality values for various sincalide formulations. (All formulations contain 0.0025 mg CCK—8/mL.; “dibasic” (All formulations contain 0.0025 mg CCK—8/mL.; “dibasic” and “monobasic” refer to dibasic and monobasic potassium 5 and “monobasic” refer to dibasic and monobasic potassium phosphate‘ “Na meta” refers to sodium metabisul?te) phosphate‘ “Na meta” refers to sodium metabisul?te)

Formulation Formulation EXcipients Calculated EXcipients Calculated (mg/mL Bulk) mOsm/kg (mg/mL Bulk) mOsm/kg 10 Na Meta (0.015) 257 Methionine (1.5) 262 Mannitol (85.0) Mannitol (75.0) Dihasic (2.75) NaCl (5.0) DTPA (1.0) TWEEN ® 80 (0.025) 20 (0.005) Monobasic (1.0) Methionine (2.0) 15 DTPA (1.0) Lysine (7.50) Dibasic (3.25) Arginine (15.0) Methionine (1.0) 262 Na Meta (0.030) 257 Mannitol (75.0) Mannitol (85.0) NaCl (5.0) Dibasic (2.75) TWEEN ® 80 (0.025) DTPA (1.0) 20 Monobasic (1.0) TWEEN ® 20 (0.005) DTPA (1.0) Methionine (2.0) Dibasic (3.25) Lysine (7.50) Methionine (0.5) 262 Arginine (15.0) Mannitol (75.0) Na Meta (0.005) 257 NaCl (5.0) Mannitol (85.0) TWEEN ® 80 (0.025) Dihasic (2.75) 25 Monobasic (1.0) DTPA (1.0) DTPA (1.0) TWEEN ® 20 (0.005) Dibasic (3.25) Methionine (2.0) Methionine (2.5) 262 Lysine (7.50) Mannitol (75.0) Arginine (15.0) NaCl (5.0) Na Meta (0.020) 259 30 TWEEN ® 80 (0.005) Mannitol (85.0) Monobasic (1.0) Dibasic (3.00) DTPA (1.0) DTPA (1.0) Dibasic (3.25) TWEEN ® 20 (0.005) Lysine (5.0) 209 Methionine (2.0) Mannitol (95.0) Lysine (7.50) 35 TWEEN ® 20 (0.005) Arginine (15.0) Dibasic (2.75) Dibasic (2.75) 257 DTPA (1.0) Mannitol (85.0) Methionine (2.0) Na Meta (0.015) Lysine (15.0) 187 DTPA (1.0) Mannitol (85.0) TWEEN ® 20 (0.005) 40 TWEEN ® 20 (0.005) Methionine (2.0) Dibasic (2.75) Lysine (7.50) DTPA (1.0) Arginine (15.0) Methionine (2.0) Dihasic (3.00) 259 Lysine (30.0) 245 Mannitol (85.0) Mannitol (70.0) Na Meta (0.020) TWEEN ® 20 (0.005) DTPA (1.0) 45 Dibasic (2.75) TWEEN ® 20 (0.005) DTPA (1.0) Methionine (2.0) Methionine (2.0) Lysine (7.50) Arginine (17.5) 245 Arginine (15.0) Mannitol (85.0) Dibasic (3.25) 264 TWEEN ® 20 (0.005) Mannitol (75.0) 50 Dibasic (2.75) KCl (6.0) DTPA (1.0) TWEEN ® 20 (0.0025) Methionine (2.0) Monobasic (1.0) Arginine (10.0) 232 DTPA (1.0) Mannitol (85.0) Methionine (2.0) 314 TWEEN ® 20 (0.005) Dihasic (4.50) 55 Dibasic (2.75) Mannitol (85.0) DTPA (1.0) TWEEN ® 20 (2.5 ng) Methionine (2.0) DTPA (1.0) Arginine (5.0) 238 Na metabisul?te (0.020) Mannitol (85.0) Methionine (2.0) TWEEN ® 20 (0.005) Lysine (7.50) 60 Dibasic (2.75) Arginine (15.0) DTPA (1.0) Methionine (2.0) 262 Methionine (2.0) Mannitol (75.0) Lysine (7.5) NaCl (5.0) Arginine (8.75) 245 TWEEN ® 80 (0.025) Mannitol (85.0) Monobasic (1.0) 65 TWEEN ® 20 (0.005) DTPA (1.0) Dibasic (2.75) Dihasic (3.25) DTPA (1.0) US 6,803,046 B2 9 10 the physiological pH range. Dibasic potassium phosphate is TABLE 2-continued a particularly preferred buffer in sincalide formulations of the invention. As described in Example 1 beloW, a sincalide Osmolality values for various sincalide formulations. formulation of the invention proved to be stable over a pH (All formulations contain 0.0025 mg CCK—8/mL.; “dibasic” and “monobasic” refer to dibasic and monobasic potassium range of 5.5—9.1. Within the pH range of 5.5—8.5, no distinct phosphate‘ “Na meta” refers to sodium metabisul?te) pH-dependent related trends in initial sincalide recovery Were observed With a sincalide formulation of the invention. Formulation Preferably, a sincalide formulation of the invention has a pH Excipients Calculated (mg/mL Bulk) mOsm/kg from 6.0 to 8.0. 10 Stabilizers Methionine (2.0) The octapeptide, sincalide, contains one tryptophan and Lysine (7.5) tWo methionine residues. Methionine has been identi?ed as Arginine (15.0) 257 Mannitol (85.0) one of the most easily oxidizable amino acids, Which TWEEN ® 20 (0.005) degrades to its corresponding sulfoxide and, under more Dibasic (2.75) 15 strenuous oxidation conditions, its sulfone. The mechanisms DTPA (1.0) Methionine (2.0) of oxidation appear to be highly dependent on the reactive Lysine (7.5) oxygen species under consideration: peroxide, peroxyl radicals, singlet oxygen, and hydroxyl radical have all been shoWn to oxidize methionine residues to sulfoxides and Chelators 20 other products. Therefore, based on the potential for oxida Excipient impurities and/or stopper extractables can intro duce trace metals into pharmaceutical formulations. Sin tion of this peptide, it Was necessary to identify functional calide contains tWo methionine residues (Met 3 and Met 6) additives for peptide stabilization. that are susceptible to oxidation by free metals. Thus, the Antioxidants/Reducing Agents. In a preferred embodi sincalide formulations of the invention contain chelators to ment of the invention, the sincalide formulation contains an inhibit the oxidation of the tWo methionine residues present 25 antioxidant or reducing agent as a stabilizer. A Wide variety in sincalide (Met 3 and Met 6). Preferred chelators include of antioxidants or reducing agents can be used as stabilizers, pentetic acid (DTPA), edetic acid (EDTA) and derivatives including but not limited to, acetylcysteine, cysteine, ascor thereof, including salts. DTPA is a preferred chelator. As bic acid, benzyl alcohol, citric acid, pentetic acid or dieth described in Example 2 beloW, the amounts of the ylenetriamine pentaacetic acid (DTPA), propyl gallate, degradants, sincalide Met 3 and sincalide Met 6 30 methylparaben, sulfoxylate, propylparaben, edetic acid or monosulfoxides, increase in the presence of certain metals ethylenediaminetetraacetic acid (EDTA), disodium EDTA and in the absence of DTPA, While the presence of DPTAhas dihydrate, dithiothreitol, glutathione, monothioglycerol, an inhibitory effect on the formation of these monosulfox potassium metabisul?te, sodium formaldehyde sulfoxylate, ides. In particular, copper and manganese, in the absence of sodium sul?te, sodium succinate, sodium metabisul?te, DTPA, have the greatest oxidative effect on the methionine 35 stannous chloride, thioacetic acid, thiodiglycerol, residues of sincalide resulting in combined height percent thioethanolamine, thioglycolic acid, 2-aminoethanethiol ages of Met 3 and Met 6 monosulfoxides (vs sincalide) of (cysteamine), butylated hydroxyanisole (BHT), and sodium 85.5 and 128.9, respectively. sulfate and derivatives thereof, including salts and sulfurous In a preferred embodiment, the sincalide formulations acid salts. Sodium metabisul?te is a preferred antioxidant contain betWeen 0.1 and 3.0 mg of DTPA per mL after 40 stabilizer. Additionally, DTPA, Which is a preferred chelator, reconstitution. In a particularly preferred embodiment, sin also may be an antioxidant stabilizer. calide formulations of the invention contain 0.4 mg DTPA/ Amino Acids. Amino acids have also been used as stabi mL after reconstitution 0 With 5 mL. lizers or co-stabilizers of peptides to: act as cryoprotectants Buffering Agents during freeze drying, stabilize against heat denaturation, Buffering agents are employed to stabilize the pH of inhibit aggregate formation, improve solubility or sincalide formulations of the invention, and consequently, rehydration, inhibit isomerization, reduce surface reduce the risk of chemical stability at extreme pH values. adsorption, or act as chelating agents. They can also increase Buffering agents useful in the preparation of formulation kits the product glass transition temperature (Tg) and thereby of the invention include, but are not limited to, phosphoric increase process stability, as Well as stabilize the product by acid, phosphate (e.g. monobasic or dibasic sodium 50 minimizing overdrying during secondary drying. Surface phosphate, monobasic or dibasic potassium phosphate, etc.), exposed residues can react readily With oxidizing agents at citric acid, citrate (e.g. sodium citrate, etc.), sulfosalicylate, physiological pH, scavenging oxidizing molecules and pro acetic acid, acetate (e.g. potassium acetate, sodium acetate, tecting critical regions of peptides. etc.), methyl boronic acid, boronate, disodium succinate Various D- and/or L-amino acids can be used as stabiliz hexahydrate, amino acids, including amino acid salts (such 55 ers in sincalide formulations. As used herein “amino acid(s)” as histidine, glycine, lysine, imidazole), lactic acid, lactate and the names of speci?c amino acids (eg arginine, lysine, (e.g. sodium lactate, etc.), maleic acid, maleate, potassium methionine, etc.) encompass D- and/or L-amino acids, chloride, benzoic acid, sodium benzoate, carbonic acid, amino acid salts, derivatives, homologs, dimers, oligomers, carbonate (e.g. sodium carbonate, etc.), bicarbonate (e.g. or mixtures thereof. Preferred amino acids for use as stabi sodium bicarbonate, etc.), boric acid, sodium borate, sodium 60 lizers in the present invention include methionine, lysine, chloride, succinic acid, succinate (e.g. sodium succinate), and arginine. Examples of other amino acids (and amino tartaric acid, tartrate (e.g. sodium tartrate, etc.), tris acid salts) suitable as stabilizers include, but are not limited (hydroxymethyl)aminomethane, biological buffers (such as to, arginine glutamate, asparagine, gamma aminobutyric N-2-hydroxyethylpiperazine,N‘-2-ethanesulfonic acid acid, glycine (and glycine buffer), glutamic acid, glutamate, (HEPES), CHAPS and other “Good’s” buffers), and the like. 65 sodium glutamate, histidine (and histidine buffer), lysine Phosphate is a preferred buffering agent due to its lack of glutamate, lysine aspartate, arginine aspartate, imidazole, interaction With sincalide and an ideal buffering capacity in serine, threonine, alanine, polyglutamic acid, polylysine, US 6,803,046 B2 11 12 glycylglycine and the like, including hydroxypropyl and active ingredient, sincalide, as Well as to provide tonicity. galactose derivatives. In one particularly preferred Bulking agents/tonicity adjusters (also called lyophiliZation embodiment, L-arginine monohydrochloride, L-methionine aids) useful in the preparation of lyophiliZed products of the and L-lysine monohydrochloride are used. invention are knoWn in the art and include mannitol, lactose, Cryoprotectants/Lyoprotectants potassium chloride, sodium chloride, maltose, sucrose, Various cryoprotectants/lyoprotectants can be used in the PEG’s (such as, for example, PEG 300, PEG 400, PEG present invention. Suitable cryoprotectants structure Water 3350, PEG 6000, PEG 8000 and the like, etc.), trehalose, molecules such that the freezing point is reduced and/or the raf?nose, dextrose, polygalacturonic acid galacturonic acid, rate of cooling necessary to achieve the vitreous phase is amino acids (including amino acid salts) such as lysine, reduced. They also raise the glass transition temperature 10 arginine, glycine, galactose, etc.), cyclodextrins, such as range of the vitreous state. These include, but are not limited hydroxypropyl-y-cyclodextrin (HP-y-CD), dextran, Ficoll, to: dimethylsulfoxide (DMSO), dextran, sucrose, 1,2 and polyvinylpyrrolidone (PVP). Of these, D-mannitol is the propanediol, amino acids/salts such as, glycine, lysine, most preferred bulking agent/tonicity adjuster for use With arginine, aspartic acid, histidine, proline, etc., glycerol, the invention. sorbitol, sodium chloride, fructose, trehalose, raffinose, 15 Other Excipients stachychose, propylene glycol, 2,3-butanediol, hydroxy Other excipients, Which may optionally be used in the ethyl starch, polyvinylpyrrolidone (PVP), PEG’s and similar formulations of the invention include preservatives (e.g., compounds, protein stabiliZers, such as human serum benZalkonium chloride), osmolality adjusters (e.g., albumin, bovine serum albumin, bovine gamma globulin, dextrose), lyoprotectants (e.g., sodium sulfate), solubiliZers, gelatin (or derivatives, such as Prionex, etc.), dextrose, 20 tonicity adjusters (eg sodium chloride), cake forming glucose, maltose, arabinose, lactose, inositol, polyols (such agents, complexing agents, and dissolution aids. A listing of as sorbitol, xylitol, erithritol, glycerol, ethylene glycol, etc.), various excipients that can be used in sincalide formulations tetramethylglucose, sodium sulfate, cyclodextrins and com for parenteral administration can be found in, for example, binations thereof. Lysine and arginine are preferred The Handbook of Pharmaceutical Additives, Second cryoprotectants/lyoprotectants. 25 Edition, edited by Michael & Irene Ash; Remington ’s Phar Surfactants/SolubiliZers/Surface Active Agents maceutical Sciences, (18’11 Edition), edited by A. Gennaro, Peptides are susceptible to physical degradation through 1990, Mack Publishing Company, Easton, Pa. and Pollock et denaturation, aggregation, precipitation, container surface al.; Strickly, Robert G., Parenteral Formulations of Small adsorption and/or agitation induced denaturation. The addi Molecules Therapeutics Marketed in the United States (I tion of a nonionic surfactant, such as polysorbate, to the 30 999)-Part I, PDA Journal of Pharmaceutical Science and formulation, may reduce the interfacial tension or aid in Technology, 53(6):324 (1999); Strickly, Robert G., solubiliZation thus preventing or reducing denaturation and/ Parenteral Formulations of Small Molecules Therapeutics or degradation at air/liquid or liquid/solid interfaces of the Marketed in the United States (1999)-Part II, PDA Journal product in solution. of Pharmaceutical Science and Technology, 54(1):69 Surfactants/solubiliZers include compounds such as free 35 (2000); Parenteral Formulations of Small Molecules Thera fatty acids, esters of fatty acids With polyoxyalkylene com peutics Marketed in the United States (1999)-Part III, PDA pounds like polyoxypropylene glycol and polyoxyethylene Journal of Pharmaceutical Science and Technology, 54(2): glycol; ethers of fatty alcohols With polyoxyalkylene gly 154 (2000); Nema, Sandeep, et al.; Excipients and Their Use cols; esters of fatty acids With polyoxyalkylated sorbitan; in Injectable Products, PDA Journal of Pharmaceutical soaps; glycerol-polyalkylene stearate; glycerol 40 Science and Technology, 51(4): 166 (1997); Wang, Y. J., et polyoxyethylene ricinoleate; homo- and copolymers of al.; Parenteral Formulations of Proteins and Peptides: Sta polyalkylene glycols; polyethoxylated soya-oil and castor bility and StabiliZers (Technical Report No. 10), Journal of oil as Well as hydrogenated derivatives; ethers and esters of Parenteral Science and Technology, Vol.42 (2S), Supple sucrose or other carbohydrates With fatty acids, fatty ment 1988; Carpenter, J. et al.; FreeZing- and Drying alcohols, these being optionally polyoxyalkylated; mono-, 45 Induced Perturbations of Protein Structure and Mechanisms di- and triglycerides of saturated or unsaturated fatty acids; of Protein Protection by StabiliZing Additives, in Drugs and glycerides or soya-oil and sucrose; sodium caprolate, ammo The Pharmaceutical Sciences, Louis Rey and Joan C. May., nium sulfate, sodium dodecyl sulfate (SDS), Triton-100 and eds., Marcel Dekker, Inc. NeW York, NY. (1999); Michael anionic surfactants containing alkyl, aryl or heterocyclic J. Pikal, Mechanisms of Protein StabiliZation During FreeZe structures. Drying and Storage: The Relative Importance of Thermo Examples of preferred surfactants/solubiliZers for use in dynamic StabiliZation and Glassy State Relaxation the present invention include, but are not limited to, plu Dynamics, in Drugs and The Pharmaceutical Sciences, ronics (e.g., Lutrol F68, Lutrol F127), Poloxamers, SDS, Louis Rey and Joan C. May., eds., Marcel Dekker, Inc. NeW Triton-100, polysorbates such as TWEEN® 20 and York, NY. (1999); Shah, D., et al.; The Effects of Various TWEEN® 80, propylene glycol, PEG and similar 55 Excipients on the Unfolding of Basic Fibroblast GroWth compounds, Brij58 (polyoxyethylene 20 cetyl ether), cre Factor, PDA Journal of Pharmaceutical Science & mophor EL, cetyl trimethylammonium bromide (CTAB), Technology, 52(5):238 (1998); PoWell, M. F., et al.; Com dimethylacetamide (DMA), NP-40 (Nonidet P-40), and pendium of Excipients for Parenteral Formulations, PDA N-methyl-2-pyrrolidone (Pharmasolve), glycine and other Journal of Pharmaceutical Science & Technology, 52(5) amino acids/amino acid salts and anionic surfactants con 60 :238 (1998); and Inactive Ingredient Guide, Div. Of Drug taining alkyl, aryl or heterocyclic structures, and cyclodex Information Resources, FDA, CDER, Jan. 1996; Handbook trins. TWEEN® 20 is the most preferred surfactant in of Injectable Drugs, Edition 8, Am. Soc. Hospital formulations of the invention. Pharmacists, 1994, L. A. Trissel. Bulking Agents/Tonicity Adjusters Formulation Kits Due to the small amount of sincalide present in the 65 Kits of the present invention preferably comprise one or formulations of the invention, bulking agents/tonicity more vials containing the sterile formulation of a predeter adjusters are useful to provide structure and support for the mined amount of sincalide, a lyophiliZation aid or bulking.