U.S. patent application number 10/921732 was filed with the patent office on 2005-01-27 for sincalide formulations.
Invention is credited to Metcalfe, Edmund C., Monteferrante, Jo Anna, Newborn, Margaret, Ropiak, Irene, Schramm, Ernst, White, Gregory W., Zodda, Julius P..
Application Number | 20050020502 10/921732 |
Document ID | / |
Family ID | 31714996 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050020502 |
Kind Code |
A1 |
Metcalfe, Edmund C. ; et
al. |
January 27, 2005 |
Sincalide formulations
Abstract
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 treating, preventing, and
diagnosing gall bladder-related disorders using sincalide
formulations.
Inventors: |
Metcalfe, Edmund C.;
(Hillsborough, NJ) ; Monteferrante, Jo Anna;
(Raritan Township, NJ) ; Newborn, Margaret;
(Hamilton Township, NJ) ; Ropiak, Irene;
(Lawrenceville, NJ) ; Schramm, Ernst; (North
Brunswick, NJ) ; White, Gregory W.; (Monmouth
Junction, NJ) ; Zodda, Julius P.; (Mercerville,
NJ) |
Correspondence
Address: |
KRAMER LEVIN NAFTALIS & FRANKEL LLP
INTELLECTUAL PROPERTY DEPARTMENT
919 THIRD AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
31714996 |
Appl. No.: |
10/921732 |
Filed: |
August 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10921732 |
Aug 19, 2004 |
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10222540 |
Aug 16, 2002 |
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6803046 |
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Current U.S.
Class: |
424/489 ;
514/5.5 |
Current CPC
Class: |
A61K 47/10 20130101;
A61P 1/00 20180101; A61K 47/26 20130101; A61K 38/2207 20130101;
A61K 9/0019 20130101; A61K 47/186 20130101; A61K 38/08 20130101;
A61K 47/02 20130101; A61K 51/0402 20130101; Y10S 514/951 20130101;
A61K 47/18 20130101; A61P 1/16 20180101; A61K 47/20 20130101; A61K
47/183 20130101; A61K 49/0002 20130101 |
Class at
Publication: |
514/016 |
International
Class: |
A61K 038/08 |
Claims
We claim:
1-108. (cancelled)
109. A stabilized, physiologically acceptable formulation of
sincalide comprising: (a) an effective amount of sincalide, and (b)
at least one multi-functional excipient.
110. The formulation of claim 109, wherein said at least one
multi-functional excipient performs the function of a stabilizer, a
chelator, a bulking agent/tonicity adjuster, a
surfactant/solubilizer, and a buffer.
111. The formulation of claim 109, wherein said at least one
multi-functional excipient is an amino acid.
112. A stabilized, physiologically acceptable formulation of
sincalide comprising: (a) an effective amount of sincalide, and (b)
one or more excipients which perform the function of a stabilizer,
a chelator, a bulking agent/tonicity adjuster, a
surfactant/solubilizer, and a buffer.
113. A method of making a stabilized formulation of sincalide, said
method comprising the step of mixing: (a) sincalide, and (b) one or
more excipients which perform the function of a stabilizer, a
chelator, a bulking agent/tonicity adjuster, a
surfactant/solubilizer, and a buffer.
114. A kit, comprising: (i) a powder mixture comprising: (a)
sincalide, and (b) one or more excipients which perform the
function of a stabilizer, a chelator, a bulking agent/tonicity
adjuster, a surfactant/solubilizer, and a buffer, and (ii) a
container to hold said powder mixture; and (iii) optionally, a
physiologically acceptable fluid.
115. A method for treating or preventing a medical condition
associated with total parental nutrition (TPN), said method
comprising administering to a patient receiving TPN an effective
amount of a sincalide formulation, said formulation comprising: (a)
sincalide, and (b) one or more excipients which perform the
function of a stabilizer, a chelator, a bulking agent/tonicity
adjuster, a surfactant/solubilizer, and a buffer.
116. A method for imaging the hepatobiliary system of a subject,
said method comprising: (a) administering a hepatobiliary imaging
agent to said subject; (b) before or after step (a), administering
to a subject a sincalide formulation comprising: (i) sincalide, and
(ii) one or more excipients which perform the function of a
stabilizer, a chelator, a bulking agent/tonicity adjuster, a
surfactant/solubilizer, and a buffer, and (c) detecting said
imaging agent in said subject with a detection device.
117. A method for imaging the hepatobiliary system of a subject,
said method comprising: (a) administering to a subject a sincalide
formulation comprising: (i) sincalide, and (ii) one or more
excipients which perform the function of a stabilizer, a chelator,
a bulking agent/tonicity adjuster, a surfactant/solubilizer, and a
buffer, and (b) scanning the subject using a diagnostic imaging
modality.
Description
FIELD OF THE INVENTION
[0001] The invention relates to pharmaceutically acceptable
formulations of sincalide.
BACKGROUND OF THE INVENTION
[0002] KINEVAC.RTM. (Sincalide for Injection, USP) is a
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 cholecystokinin (CCK-8), with the
following amino acid sequence:
Asp-Tyr(SO.sub.3H)-Met-Gly-Trp-Met-Asp-Phe-NH.sub.2.
[0003] KINEVAC.RTM. was first introduced in 1976, and was finished
as a sterile, nonpyrogenic, lyophilized white powder in a 5-mL
(nominal) glass vial to contain: 5 .mu.g sincalide with 45 mg
sodium chloride to provide tonicity; sodium hydroxide 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 U.S. Pharmacopea/National
Formulary, USP 24, NF 19, Jan. 1, 2000.
[0004] Since its introduction, various drawbacks in the
manufacturing and analysis of KINEVAC.RTM. 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 sincalide and KINEVAC.RTM.. This
bioassay was unable to distinguish between bioactivity of sincalide
and bioactivity of sincalide degradants. Accordingly, a 20% overage
of sincalide 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
[0005] The present invention satisfies the need for improved
sincalide formulations by providing formulations that eliminate 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 consistent 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.
[0006] 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 (.apprxeq.100 L).
[0007] In one aspect, the invention features sincalide formulations
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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] In a particularly preferred embodiment, the reconstituted
formulation includes 0.0008 to 0.0012 mg/mL active ingredient
(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 metabisulfite; 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).
[0012] 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 components in a liquid form and some in the form of
a powder.
[0013] The formulations of the invention have improved stability
and potency compared to previous sincalide formulations, and are
useful as diagnostic aids for imaging the hepatobiliary 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 .sup.99 mTc-mebrofenin,
or similar hepatobiliary imaging agents, to assist in the diagnosis
of gallbladder diseases and related disorders. Additionally, the
formulations may be administered before and/or after diagnostic
imaging (including for example, magnetic resonance imaging,
scintigraphic imaging, ultrasound imaging, etc.)
[0014] 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 disorders.
[0015] 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
[0016] FIG. 1 is a drawing illustrating the chemical structure of
1-De(5-oxo-L-glutamine-5-L-methioninecaerulein or "sincalide" (CAS#
25126-32-3). The amino acid residues "Met 3" and "Met 6" are
outlined by dashed lines.
[0017] FIG. 2 is a drawing illustrating the chemical structure of
sincalide (Met 3) monosulfoxide.
[0018] FIG. 3 is a drawing illustrating the chemical structure of
sincalide (Met 6) monosulfoxide.
[0019] FIG. 4 is a drawing illustrating the chemical structure of
sincalide (Met 3, 6) disulfoxide.
[0020] 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.
[0021] 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.
[0022] 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).
[0023] FIG. 8 is a chromatogram of KINEVAC.RTM. experimental
formulation (no DTPA) spiked with 0.63 mM Cu.sup.2+.
[0024] FIG. 9 is a chromatogram of KINEVAC.RTM. experimental
formulation (1 mM DTPA) spiked with 0.63 mM Cu.sup.2+.
[0025] FIG. 10 is a chromatogram of KINEVAC.RTM. experimental
formulation (no DTPA) spiked with 0.18 mM Mn.sup.2+.
[0026] FIG. 11 is a chromatogram of KINEVAC.RTM. experimental
formulation (1 mM DTPA) spiked with 0.18 mM Mn.sup.2+.
[0027] FIG. 12 shows representative full-scale and expanded scale
chromatograms of a lyophilized reformulation of KINEVAC.RTM. upon
reconstitution with 5 mL water, resulting in a sincalide
concentration of 1 .mu.g/mL.
DETAILED DESCRIPTION OF THE INVENTION
[0028] 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 discovered 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.
[0029] As used herein, the term "sincalide" includes the
synthetically-prepared C-terminal octapeptide of cholecystokinin
(CCK-8), with the amino acid sequence:
Asp-Tyr(SO.sub.3H)-Met-Gly-Trp-Met-Asp-Phe-- NH.sub.2, as well as
derivatives 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 acid residues have
been replaced without significantly affecting the biological
activity are included within "sincalide" as the term is used
herein. Similarly, the term "sincalide" encompasses not only
monomeric, but multimeric forms of the peptide, as well as
physiologically active degradants or portions of the peptide and
its derivatives.
[0030] 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, osmolality 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 functions 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 excipient that functions as a
stabilizer.
[0031] 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 desirability of achieving tonicity
in the final reconstituted solution. 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.
1TABLE 1 Concentration ranges for excipients for preferred
sincalide formulations. Final Formulation (mg) Range 1 mL Range
Range (mg per 1 mL 1 mL 1 vial after Excipient Function (mg/mL
Bulk) (mg/vial) after reconst) Bulk Target reconst. (Sincalide)
Active Ingredient 0.0025 0.0050 0.0008-0.0012 0.0025 0.0050 0.0010
Mannitol Bulking Agent/ 50.0-125.0 100-250 20.0-50.0 85 170 34 Cake
Forming Agent/ Tonicity Adjuster TWEEN .RTM.-20 Non-Ionic
Surfactant/ 0.0000025-0.0075 0.0000050-0.0150 0.0000010-0.0030
<0.01 <0.01 <0.01 Solubilizing Agent/ Wetting Agent DTPA
Chelator/Stabilizer/ 1.0 2.0 0.1-3.0 1.0 2.0 0.4 Antioxidant/
Complexing Agent/ Preservative/pH Adjuster Sodium
Antioxidant/Preservative/ 0.005-0.030 0.010-0.060 0.002-0.012 0.020
0.040 0.008 Metabisulfite Stabilizer Potassium Buffer/pH Adjuster/
2.7-4.5 5.4-12.0 1.1-1.8 4.5 9.0 1.8 Phosphate, Dissolution Aid
dibasic Potassium Buffer/pH Adjuster/ 1.0-6.5 9.6-13.0 1.92-2.6 0 0
0 Phosphate, 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/ 5.0-30.0 10.0-60.0 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 .RTM.-80, potassium
metabisulfite, sodium phosphate dibasic, sodium phosphate
monobasic, and potassium chloride. Additional alternatives are
listed below.
[0032] Table 2 shows preferred ranges for preferred excipients in
the bulk solutions, vials and after reconstitution. All
concentrations shown for the bulk solution are based on a 2 mL fill
volume. The ingredient quantities are matched to result in a pH
slightly below neutral and result in an isotonic solution after
reconstitution of the lyophilized vial as indicated 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.
2TABLE 2 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 Calculated
(mg/mL Bulk) mOsm/kg Mannitol (125.0) 292 Dibasic (3.75) DTPA (1.0)
Mannitol (95.0) 244 Dibasic (4.0) Monobasic (2.8) DTPA (1.0)
Mannitol (103.0) 244 Dibasic (3.75) DTPA (1.0) Mannitol (75.0) 244
NaCl (4.5) Dibasic (3.75) DTPA (1.0) Mannitol (85.0) 187 TWEEN
.RTM. 20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine (2.0) Lysine
(15.0) Mannitol (50.0) 247 NaCl (9.0) Dibasic (3.00) DTPA (1.0)
TWEEN .RTM. 20 (0.0075) 264 Mannitol (75.0) KCl (6.0) Dibasic
(3.25) Monobasic (1.0) DTPA (1.0) Methionine (2.0) TWEEN .RTM. 20
(0.005) 264 Mannitol (75.0) KCl (6.0) Dibasic (3.25) Monobasic
(1.0) DTPA (1.0) Methionine (2.0) TWEEN .RTM. 20 (0.0025) 264
Mannitol (75.0) KCl (6.0) Dibasic (3.25) Monobasic (1.0) DTPA (1.0)
Methionine (2.0) TWEEN .RTM. 20 (2.5 ng) 314 Mannitol (85.0)
Dibasic (4.50) DTPA (1.0) Na metabisulfite (0.020) Methionine (2.0)
Lysine (7.50) Arginine (15.0) Na Meta (0.015) 257 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) 257 Mannitol (85.0) Dibasic (2.75)
DTPA (1.0) TWEEN .RTM. 20 (0.005) Methionine (2.0) Lysine (7.50)
Arginine (15.0) Na Meta (0.005) 257 Mannitol (85.0) Dibasic (2.75)
DTPA (1.0) TWEEN .RTM. 20 (0.005) Methionine (2.0) Lysine (7.50)
Arginine (15.0) Na Meta (0.020) 259 Mannitol (85.0) Dibasic (3.00)
DTPA (1.0) TWEEN .RTM. 20 (0.005) Methionine (2.0) Lysine (7.50)
Arginine (15.0) Dibasic (2.75) 257 Mannitol (85.0) Na Meta (0.015)
DTPA (1.0) TWEEN .RTM. 20 (0.005) Methionine (2.0) Lysine (7.50)
Arginine (15.0) Dibasic (3.00) 259 Mannitol (85.0) Na Meta (0.020)
DTPA (1.0) TWEEN .RTM. 20 (0.005) Methionine (2.0) Lysine (7.50)
Arginine (15.0) Dibasic (3.25) 264 Mannitol (75.0) KCl (6.0) TWEEN
.RTM. 20 (0.0025) Monobasic (1.0) DTPA (1.0) Methionine (2.0)
Dibasic (4.50) 314 Mannitol (85.0) TWEEN .RTM. 20 (2.5 ng) DTPA
(1.0) Na metabisulfite (0.020) Methionine (2.0) Lysine (7.50)
Arginine (15.0) Methionine (2.0) 262 Mannitol (75.0) NaCl (5.0)
TWEEN .RTM. 80 (0.025) Monobasic (1.0) DTPA (1.0) Dibasic (3.25)
Methionine (1.5) 262 Mannitol (75.0) NaCl (5.0) TWEEN .RTM. 80
(0.025) Monobasic (1.0) DTPA (1.0) Dibasic (3.25) Methionine (1.0)
262 Mannitol (75.0) NaCl (5.0) TWEEN .RTM. 80 (0.025) Monobasic
(1.0) DTPA (1.0) Dibasic (3.25) Methionine (0.5) 262 Mannitol
(75.0) NaCl (5.0) TWEEN .RTM. 80 (0.025) Monobasic (1.0) DTPA (1.0)
Dibasic (3.25) Methionine (2.5) 262 Mannitol (75.0) NaCl (5.0)
TWEEN .RTM. 80 (0.005) Monobasic (1.0) DTPA (1.0) Dibasic (3.25)
Lysine (5.0) 209 Mannitol (95.0) TWEEN .RTM. 20 (0.005) Dibasic
(2.75) DTPA (1.0) Methionine (2.0) Lysine (15.0) 187 Mannitol
(85.0) TWEEN .RTM. 20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine
(2.0) Lysine (30.0) 245 Mannitol (70.0) TWEEN .RTM. 20 (0.005)
Dibasic (2.75) DTPA (1.0) Methionine (2.0) Arginine (17.5) 245
Mannitol (85.0) TWEEN .RTM. 20 (0.005) Dibasic (2.75) DTPA (1.0)
Methionine (2.0) Arginine (10.0) 232 Mannitol (85.0) TWEEN .RTM. 20
(0.005) Dibasic (2.75) DTPA (1.0) Methionine (2.0) Arginine (5.0)
238 Mannitol (85.0) TWEEN .RTM. 20 (0.005) Dibasic (2.75) DTPA
(1.0) Methionine (2.0) Lysine (7.5) Arginine (8.75) 245 Mannitol
(85.0) TWEEN .RTM. 20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine
(2.0) Lysine (7.5) Arginine (15.0) 257 Mannitol (85.0) TWEEN .RTM.
20 (0.005) Dibasic (2.75) DTPA (1.0) Methionine (2.0) Lysine
(7.5)
[0033] Chelators
[0034] Excipient impurities and/or stopper extractables can
introduce trace metals into pharmaceutical formulations. Sincalide
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 DPTA has an inhibitory
effect on the formation of these monosulfoxides. 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 percentages of Met 3 and Met 6 monosulfoxides
(vs sincalide) of 85.5 and 128.9, respectively.
[0035] In a preferred embodiment, the sincalide formulations
contain between 0.1 and 3.0 mg of DTPA per mL after reconstitution.
In a particularly preferred embodiment, sincalide formulations of
the invention contain 0.4 mg DTPA/mL after reconstitution with 5
mL.
[0036] Buffering Agents
[0037] Buffering agents are employed to stabilize the pH of
sincalide formulations of the invention, and consequently, reduce
the risk of chemical stability at extreme pH values. Buffering
agents useful in the preparation of formulation kits of the
invention include, but are not limited to, phosphoric acid,
phosphate (e.g. monobasic or dibasic sodium phosphate, monobasic or
dibasic potassium phosphate, etc.), citric acid, citrate (e.g.
sodium citrate, etc.), sulfosalicylate, acetic acid, acetate (e.g.
potassium acetate, sodium acetate, etc.), methyl boronic acid,
boronate, disodium succinate hexahydrate, amino acids, including
amino acid salts (such as histidine, glycine, lysine, imidazole),
lactic acid, lactate (e.g. sodium lactate, etc.), maleic acid,
maleate, potassium chloride, benzoic acid, sodium benzoate,
carbonic acid, carbonate (e.g. sodium carbonate, etc.), bicarbonate
(e.g. sodium bicarbonate, etc.), boric acid, sodium borate, sodium
chloride, succinic acid, succinate (e.g. sodium succinate),
tartaric acid, tartrate (e.g. sodium tartrate, etc.),
tris-(hydroxymethyl)aminomethane, biological buffers (such as
N-2-hydroxyethylpiperazine, N'-2-ethanesulfonic acid (HEPES), CHAPS
and other "Good's" buffers), and the like.
[0038] Phosphate is a preferred buffering agent due to its lack of
interaction with sincalide and an ideal buffering capacity in the
physiological pH range. Dibasic potassium phosphate is a
particularly preferred buffer in sincalide formulations of the
invention. As described in Example 1 below, a sincalide formulation
of the invention proved to be stable over a pH range of 5.5-9.1.
Within the pH range of 5.5-8.5, no distinct pH-dependent related
trends in initial sincalide recovery were observed with a sincalide
formulation of the invention. Preferably, a sincalide formulation
of the invention has a pH from 6.0 to 8.0.
[0039] Stabilizers
[0040] The octapeptide, sincalide, contains one tryptophan and two
methionine residues. Methionine has been identified as one of the
most easily oxidizable amino acids, which degrades to its
corresponding sulfoxide and, under more strenuous oxidation
conditions, its sulfone. The mechanisms of oxidation appear to be
highly dependent on the reactive oxygen species under
consideration: peroxide, peroxyl radicals, singlet oxygen, and
hydroxyl radical have all been shown to oxidize methionine residues
to sulfoxides and other products. Therefore, based on the potential
for oxidation of this peptide, it was necessary to identify
functional additives for peptide stabilization.
[0041] Antioxidants/Reducing Agents. In a preferred embodiment of
the invention, the sincalide formulation contains an antioxidant or
reducing agent as a stabilizer. A wide variety of antioxidants or
reducing agents can be used as stabilizers, including but not
limited to, acetylcysteine, cysteine, ascorbic acid, benzyl
alcohol, citric acid, pentetic acid or diethylenetriamine
pentaacetic acid (DTPA), propyl gallate, methylparaben,
sulfoxylate, propylparaben, edetic acid or
ethylenediaminetetraacetic acid (EDTA), disodium EDTA dihydrate,
dithiothreitol, glutathione, monothioglycerol, potassium
metabisulfite, sodium formaldehyde sulfoxylate, sodium sulfite,
sodium succinate, sodium metabisulfite, stannous chloride,
thioacetic acid, thiodiglycerol, thioethanolamine, thioglycolic
acid, 2-aminoethanethiol (cysteamine), butylated hydroxyanisole
(BHT), and sodium sulfate and derivatives thereof, including salts
and sulfurous acid salts. Sodium metabisulfite is a preferred
antioxidant stabilizer. Additionally, DTPA, which is a preferred
chelator, also may be an antioxidant stabilizer.
[0042] 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
aggregate formation, improve solubility or rehydration, inhibit
isomerization, reduce surface adsorption, or act as chelating
agents. They can also increase the product glass transition
temperature (T.sub.g) and thereby increase process stability, as
well as stabilize the product by minimizing overdrying during
secondary drying. Surface exposed residues can react readily with
oxidizing agents at physiological pH, scavenging oxidizing
molecules and protecting critical regions of peptides.
[0043] Various D- and/or L-amino acids can be used as stabilizers
in sincalide formulations. As used herein "amino acid(s)" and the
names of specific amino acids (e.g arginine, lysine, methionine,
etc.) encompass D- and/or L-amino acids, amino acid salts,
derivatives, homologs, dimers, oligomers, or mixtures thereof.
Preferred amino acids for use as stabilizers in the present
invention include methionine, lysine, and arginine. Examples of
other amino acids (and amino acid salts) suitable as stabilizers
include, but are not limited to, arginine glutamate, asparagine,
gamma aminobutyric acid, glycine (and glycine buffer), glutamic
acid, glutamate, sodium glutamate, histidine (and histidine
buffer), lysine glutamate, lysine aspartate, arginine aspartate,
imidazole, serine, threonine, alanine, polyglutamic acid,
polylysine, glycylglycine and the like, including hydroxypropyl and
galactose derivatives. In one particularly preferred embodiment,
L-arginine monohydrochloride, L-methionine and L-lysine
monohydrochloride are used.
[0044] Cryoprotectants/Lyoprotectants
[0045] Various cryoprotectants/lyoprotectants can be used in the
present invention. Suitable cryoprotectants structure water
molecules such that the freezing point is reduced and/or the rate
of cooling necessary to achieve the vitreous phase is reduced. They
also raise the glass transition temperature range of the vitreous
state. These include, but are not limited to: dimethylsulfoxide
(DMSO), dextran, sucrose, 1,2-propanediol, amino acids/salts such
as, glycine, lysine, arginine, aspartic acid, histidine, proline,
etc., glycerol, sorbitol, sodium chloride, fructose, trehalose,
raffinose, stachychose, propylene glycol, 2,3-butanediol,
hydroxyethyl starch, polyvinylpyrrolidone (PVP), PEG's and similar
compounds, protein stabilizers, such as human serum albumin, bovine
serum albumin, bovine gamma globulin, gelatin (or derivatives, such
as Prionex, etc.), dextrose, glucose, maltose, arabinose, lactose,
inositol, polyols (such as sorbitol, xylitol, erithritol, glycerol,
ethylene glycol, etc.), tetramethylglucose, sodium sulfate,
cyclodextrins and combinations thereof. Lysine and arginine are
preferred cryoprotectants/lyoprotectants.
[0046] Surfactants/Solubilizers/Surface Active Agents
[0047] Peptides are susceptible to physical degradation through
denaturation, aggregation, precipitation, container surface
adsorption and/or agitation induced denaturation. The 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/solid interfaces of the product in
solution.
[0048] Surfactants/solubilizers include compounds such as free
fatty acids, esters of fatty acids with polyoxyalkylene compounds
like polyoxypropylene glycol and polyoxyethylene glycol; ethers of
fatty alcohols with polyoxyalkylene glycols; esters of fatty acids
with polyoxyalkylated sorbitan; soaps; glycerol-polyalkylene
stearate; glycerol-polyoxyethylene ricinoleate; homo- and
copolymers of polyalkylene glycols; polyethoxylated soya-oil and
castor oil as well as hydrogenated derivatives; ethers and esters
of sucrose or other carbohydrates with fatty acids, fatty alcohols,
these being optionally polyoxyalkylated; mono-, di- and
triglycerides of saturated or unsaturated fatty acids; glycerides
or soya-oil and sucrose; sodium caprolate, ammonium sulfate, sodium
dodecyl sulfate (SDS), Triton-100 and anionic surfactants
containing alkyl, aryl or heterocyclic structures.
[0049] Examples of preferred surfactants/solubilizers for use in
the present invention include, but are not limited to, pluronics
(e.g., Lutrol F68, Lutrol F127), Poloxamers, SDS, Triton-100,
polysorbates such as TWEEN.RTM. 20 and TWEEN.RTM. 80, propylene
glycol, PEG and similar compounds, Brij58 (polyoxyethylene 20 cetyl
ether), cremophor EL, cetyl trimethylammonium bromide (CTAB),
dimethylacetamide (DMA), NP-40 (Nonidet P-40), and
N-methyl-2-pyrrolidone (Pharmasolve), glycine and other amino
acids/amino acid salts and anionic surfactants containing alkyl,
aryl or heterocyclic structures, and cyclodextrins. TWEEN.RTM. 20
is the most preferred surfactant in formulations of the
invention.
[0050] Bulking Agents/Tonicity Adjusters
[0051] Due to the small amount of sincalide present in the
formulations of the invention, bulking agents/tonicity adjusters
are useful to provide structure and support for the active
ingredient, sincalide, as well as to provide tonicity. Bulking
agents/tonicity adjusters (also called lyophilization aids) useful
in the preparation of lyophilized products of the invention are
known in the art and include mannitol, lactose, potassium chloride,
sodium chloride, maltose, sucrose, PEG's (such as, for example, PEG
300, PEG 400, PEG 3350, PEG 6000, PEG 8000 and the like, etc.),
trehalose, raffinose, dextrose, polygalacturonic acid galacturonic
acid, amino acids (including amino acid salts) such as lysine,
arginine, glycine, galactose, etc.), cyclodextrins, such as
hydroxypropyl-.gamma.-c- yclodextrin (HP-.gamma.-CD), dextran,
Ficoll, and polyvinylpyrrolidone (PVP). Of these, D-mannitol is the
most preferred bulking agent/tonicity adjuster for use with the
invention.
[0052] Other Excipients
[0053] Other excipients, which may optionally be used in the
formulations of the invention include preservatives (e.g.,
benzalkonium chloride), osmolality adjusters (e.g., dextrose),
lyoprotectants (e.g., sodium sulfate), solubilizers, tonicity
adjusters (e.g. sodium chloride), cake forming agents, complexing
agents, and dissolution aids. A listing of various excipients that
can be used in sincalide formulations for parenteral administration
can be found in, for example, The Handbook of Pharmaceutical
Additives, Second Edition, edited by Michael & Irene Ash;
Remington's Pharmaceutical Sciences, (18.sup.th Edition), edited by
A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. and Pollock
et al.; Strickly, Robert G., Parenteral Formulations of Small
Molecules Therapeutics Marketed in the United States (1999)-Part I,
PDA Journal of Pharmaceutical Science and Technology, 53(6):324
(1999); Strickly, Robert G., Parenteral Formulations of Small
Molecules Therapeutics Marketed in the United States (1999)-Part
II, PDA Journal of Pharmaceutical Science and Technology, 54(1):69
(2000); Parenteral Formulations of Small Molecules Therapeutics
Marketed in the United States (1999)-Part III, PDA Journal of
Pharmaceutical Science and Technology, 54(2): 154 (2000); Nema,
Sandeep, et al., Excipients and Their Use in Injectable Products,
PDA Journal of Pharmaceutical Science and Technology, 51(4): 166
(1997); Wang, Y. J., et al., Parenteral Formulations of Proteins
and Peptides: Stability and Stabilizers (Technical Report No. 10),
Journal of Parenteral Science and Technology, Vol.42 (2S),
Supplement 1988; Carpenter, J. et al., Freezing- and Drying-Induced
Perturbations of Protein Structure and Mechanisms of Protein
Protection by Stabilizing Additives, in Drugs and The
Pharmaceutical Sciences, Louis Rey and Joan C. May., eds., Marcel
Dekker, Inc. New York, N.Y. (1999); Michael J. Pikal, Mechanisms of
Protein Stabilization During Freeze-Drying and Storage: The
Relative Importance of Thermodynamic Stabilization and Glassy State
Relaxation Dynamics, in Drugs and The Pharmaceutical Sciences,
Louis Rey and Joan C. May., eds., Marcel Dekker, Inc. New York,
N.Y. (1999); Shah, D., et al., The Effects of Various Excipients on
the Unfolding of Basic Fibroblast Growth Factor, PDA Journal of
Pharmaceutical Science & Technology, 52(5):238 (1998); Powell,
M. F., et al., Compendium of Excipients for Parenteral
Formulations, PDA Journal of Pharmaceutical Science &
Technology, 52(5):238 (1998); and Inactive Ingredient Guide, Div.
Of Drug Information Resources, FDA, CDER, January 1996; Handbook of
Injectable Drugs, Edition 8, Am. Soc. Hospital Pharmacists, 1994,
L. A. Trissel.
[0054] Formulation Kits
[0055] Kits of the present invention preferably comprise one or
more vials containing the sterile formulation of a predetermined
amount of sincalide, a lyophilization aid or bulking agent/tonicity
adjuster, one or more stabilizers, a surfactant, a chelator, and a
buffer. The one or more vials that contain all or part of the
formulation can independently be in the form of a sterile solution
or a lyophilized solid. Buffering agents useful in the preparation
of formulation kits of the invention are discussed herein and
include, for example phosphate, citrate, sulfosalicylate, and
acetate, and amino acids (including amino acid salts). Dibasic
potassium phosphate is a preferred buffer in sincalide formulations
of the invention. The kits may also include a fluid portion, for
example water or saline, for reconstitution of the formulation
prior to injection.
[0056] Lyophilization aids or bulking agent/tonicity adjusters
useful in the preparation of lyophilized kits include those
dicussed above, particularly, mannitol, lactose, sodium chloride,
maltose, sucrose, PEG's, galaturonic acid, polygalcturonic acid,
cyclodextrins, such as hydroxypropyl-.gamma.-cyclodextrin
(HP-.gamma.-CD) and the like, dextran, amino acids (including amino
acid salts), Ficoll, and polyvinylpyrrolidone (PVP). Of these,
mannitol, sodium chloride, maltose, sucrose, PEG's, HP-.gamma.-CD,
and dextran are preferred bulking agents/tonicity adjusters for use
with the invention, with mannitol being the most preferred.
[0057] As discussed, a component in a formulation kit can also
serve more than one function. For example, an excipient which
serves as a stabilizer may also serve as the chelator and an
excipient which serves as a bulking agent may also serve as a
tonicity adjuster. In addition, in some embodiments, the excipients
are all in dry powder form, or all in liquid form while in other
embodiments, some of the excipients are in dry form and others are
in a fluid portion included in or sold separately from the kit.
[0058] A particularly preferred kit of the invention contains:
about 0.005 mg sincalide, about 170 mg D-mannitol, less than or
equal to 0.01 mg TWEEN.RTM. 20, about 2 mg DTPA, about 0.04 mg
sodium metabisulfite, about 9 mg potassium phosphate (dibasic)
about 4 mg L-methionine, about 15 mg L-lysine monohydrochloride,
and about 30 mg L-arginine monohydrochloride.
[0059] Therapeutic/Diagnostic Uses
[0060] Sincalide is a synthetic analog of the endogenously produced
hormone cholecystokinin (CCK-8). CCK-8 acts on receptors within the
gallbladder wall causing it to contract, cleaning out any remaining
sludge or bile that may have accumulated within the gallbladder.
CCK-8 increases bile flow and small and large bowel motility,
causes the pyloric sphincter to contract and increases pancreatic
enzyme secretion. CCK-8 also causes delayed biliary to bowel
transit. Sincalide has a more rapid physiologic effect on the
gallbladder in terms of contraction and relaxation than the
endogenous hormone (CCK-8) produced by the body, making sincalide
formulations useful as diagnostic aids for hepatobiliary imaging,
when administered alone or in conjunction with a hepatobiliary
imaging agent. For example, sincalide may be administered before
and/or after diagnostic imaging (such as, for example, magnetic
resonance imaging, scintigraphic imaging, ultrasound imaging, etc.)
to improve visualization and/or diagnosis of various disease
states.
[0061] In one embodiment, hepatobiliary imaging can be performed
using, for example, hepatobiliary scintigraphy, an instrumental
imaging tool used in the diagnosis and evaluation of hepatobiliary
disease. Detection of diseases, such as acute and chronic
cholecystitis, biliary obstruction, bile leaks, and other forms of
hepatobiliary disease, help the physician to better determine the
appropriate course of treatment and management of the patient
suffering from a suspected hepatobiliary pathology.
[0062] As explained below, the indications for use of sincalide in
conjunction with hepatobiliary imaging include (a) pretreatment of
patients who have not eaten for more than 20 to 24 hours prior to
imaging (in order to empty the gallbladder (GB) of
non-radiolabelled bile) and (b) use in the analysis of gallbladder
motor function, including the determination of GBEF (gallbladder
ejection fraction).
[0063] It is important to properly prepare the patient prior to
hepatobiliary imagingin order to achieve high quality imaging and
reduce the number of false positive and negative results.
Preferably, patients should have nothing to eat for 4 to 12 hours
prior to hepatobiliary imaging. Prolonged fasting, however, may
result in false positive test results (i.e. failure to visualize
the gallbladder). If a patient has not eaten for more than 24
hours, the patient is preferably pretreated with sincalide by
administration of the sincalide formulation described herein prior
to imaging. Typically, the gallbladder contracts within 15 minutes
after sincalide injection and the hepatobiliary imaging agent
(e.g., radiotracer) is injected 30 minutes later. The gallbladder
is then emptied and is better able to take up and accumulate
imaging agent (e.g., radiotracer), which helps to reduce the number
of false positive studies.
[0064] The preferred radiopharmaceuticals used for hepatobiliary
imaging include, but are not limited to, Tc 99m IDA (Iminodiacetic
acid) analogs, such as Tc-99m mebrofenin (CHOLETEC.RTM.), Tc-99m
disofenin (DISIDA), and Tc-99m lidofenin (see also U.S. Pat. No.
4,418,208). Tc-99m mebrofenin is a preferred hepatobiliary imaging
agent. Methods for coadministration of Tc 99m IDA (Iminodiacetic
acid) analogs with CCK and sincalide are known in the art and
described in, for example, Ziessman HA., Cholecystokinin
cholescintigraphy: victim of its own success? J. Nucl. Med. 1999,
40:2038-2042; Krishnamurthy S., et al., Gallbladder ejection
fraction: A decade of progress and future promise. J. Nucl. Med.
1992, 32:542-544; Krishnamurthy G T., et al., Quantitative biliary
dynamics: introduction of a new noninvasive scintigraphic
technique. J. Nucl. Med. 1983;24:217-223; Mesgarzadeh M., et al.,
Filling, post-cholecystokinin emptying and refilling of normal
gallbladder: effects of two different doses of CCK on refilling:
Concise Comm. J. Nucl. Med. 1983, 24:666-671; Krishnamurthy G T.,
et al., The gallbladder emptying response to sequential exogenous
cholecystokinin, Nucl. Med. Com., 1984, 5 (1) pp 27-33;
Krishnamurthy G T., et al., Detection, localization, and
quantitation of degree of common bile duct obstruction by
scintigraphy, J. Nucl. Med. 1985, 26:726-735; Fink-Bennet D., et
al., Cholecystokinin cholescintigraphic findings in the cystic duct
syndrome, J. Nucl. Med. 1985, 26:1123-1128; Fink-Bennet D., The
role of cholecystogogues in the evaluation of biliary tract
disorders. Nucl. Med. Ann. 1985, Lenny Freeman and Heidi Weissman,
eds., New York, Raven Press, 1985, pp. 107-132; Newman P., et al.,
A simple technique for quantitation cholecystokinin-HIDA scanning.
British J. of Radiology, vol. 56, pp. 500-502, 1983; Pickleman J.,
et al. The role of sincalide cholescintigraphy in the evaluation of
patients with acalculous gallbladder disease. Archives of Surgery,
vol. 120, 693-697; Ziessman, H A., et al., Calculation of a
gallbladder ejection fraction: Advantage of continuous sincalide
infusion over the three-minute infusion method. J. Nucl. Med. 1992,
33:537-41; Sitzmann, J V., et al., Cholecystokinin prevents
parenteral nutrition induced biliary sludge in humans, Surg.
Gynecol. Obstet. 170:25-31, 1990; Teitelbaum D H., et al.,
Treatment of parenteral nutrition-associated cholestasis with
cholecystokinin-octapept- ide. J. Pediatr. Surg. 30:1082, 1995.
[0065] After administration of the hepatobiliary imaging agent, the
hepatobiliary system of the patient can be imaged using an
appropriate detection device. When a Tc-99m IDA (Iminodiacetic
acid) analog, such as CHOLETEC.RTM. is used as an imaging agent, a
gamma camera can be employed to scan the body of the patient for
radioactivity. Imaging of the gallbladder allows for the
non-invasive measurement and analysis of various biliary motor
functions, including the gallbladder ejection fraction (GBEF).
Measurement of GBEF is clinically valuable in the diagnosis and
management of certain gallbladder-related disorders, including
chronic acalclulous cholecystitis (CAC). In particular, low GBEF
has been found to have a >90% positive predictive value for CAC.
Other changes in biliary dynamics may be used in the diagnosis of a
variety of biliary disorders.
[0066] Methods for determining GBEF scintigraphically are known in
the art, and are described in, for example, the references cited
above. Sincalide aids in the analysis of biliary function,
including the measurement of GBEF, through its physiological
effects on the gallbladder, e.g. it ability to induce gallbladder
contraction and emptying. One technique for measuring GBEF is to
administer sincalide slowly as a 1-3 minute infusion and to
calculate GBEF at the end of about 20 minutes. Alternatively,
sincalide may be infused rapidly as a bolus, or as a slower
continuous infusion ranging from 15 to 60 minutes. By inducing
certain biliary functions during hepatobiliary imaging, sincalide
aids in the identification of anomalies in such functions, which
may be indicative of certain hepatobiliary diseases.
[0067] Administration of sincalide formulations can be via IV or IM
injections: For IV administration the dose can be administered as a
bolus or slow injection over time optionally with the aid of an
infusion pump. The dose for IV administration is typically 0.005 to
0.04 .mu.g/kg (bolus injection) or 0.005 .mu.g/kg in a series of
4-three minute injections. A dose of 0.02-0.04 .mu.g/kg IV over 2-3
minutes, but up to 1 hour is described in the art. Injection rates
of 0.58 .mu.g/kg/hour can also be employed with the use of an
infusion pump. Other regimens starting at 10 ng/kg/hr and
increasing to 160 ng/kg/hr are also known in the art. Bolus
injection is not recommended in every case, but injection of 0.02
to 0.04 .mu.g/kg over 2-3 minutes even up to 15 min. can be used to
avoid spasm of the cystic duct or GB.
[0068] Doses for IM administration are generally higher and range
from 0.1 to 0.4 .mu.g/kg. In one embodiment the 0.4 .mu.g/kg IM
dose is generally preferred resulting in the greatest GB response
with the fewest side effects. Further details on administration are
provided in, for example, Mesgarzadeh M., et al., Filling, post
cholecystokinin emptying and refilling of normal gallbladder:
effects of two different doses of CCK on refilling, J. Nucl. Med.
1983, 24:666-671; Ziessmann H A., et al., Calculation of a
gallbladder ejection fraction: Advantage of continuous sincalide
infusion over the three-minute infusion method. J. Nucl. 1992,
33:537-541; Pickleman J, et al., The role sincalide
cholescintigraphy in the evaluation of patients with acalculous
gallbladder disease. Archives of Surgery, vol. 120, 693-697;
Krishnamurthy G T., et al., The gallbladder emptying response to
sequential exogenous cholecystokinin, Nucl. Med. Com., 1984, 5 (1)
pp 27-33; Krishnamurthy G T., et al., Quantitative biliary
dymanics: introduction of a new noninvasive scintigraphic
technique. J. Nucl. Med. 1983, 24:217-223; Fink-Bennet D., The role
of cholecystogogues in the evaluation of biliary tract disorders.
Nucl. Med. Ann. 1985, Lenny Freeman and Heidi Weissman, eds., New
York, Raven Press, 1985, pp. 107-132; Balon H. R., et al. Society
of Nuclear Medicine procedure guideline for hepatobiliary
scintigraphy.
[0069] The sincalide formulations of the invention are also useful
for treating patients receiving total parenteral nutrition (TPN).
TPN induces biliary sludge, the development of cholestasis, and the
formation of gall stones and other gallbladder related
complications. Indeed, TPN associated cholestatis (TPN-AC) can be a
fatal in some instances. The clinical implications of TPN-AC
include increased rates of sepsis, cirrhosis, declined lymphocyte
function, obstructive jaundice, liver failure, and increased
mortality. Although the mechanisms by which these disorders develop
have not been definitely established, biliary stasis, the reduction
in gallbladder emptying, bile flow, and bile acid secretion that
accompanies TPN, has been implicated in the pathogenesis of TPN-AC
and other TPN-associated complications. By promoting biliary
contraction and emptying, the administration of sincalide to a TPN
patient can help to treat and prevent diseases and other
complications associated with prolonged TPN.
[0070] For TPN patients the dose of 0.05 .mu.g/kg is typically
given IV over 10 minutes as a daily infusion. In infants, to treat
high bilirubin levels the dose is 0.02 .mu.g/kg IV or IM twice or 3
times daily with doses increasing up to 0.32 .mu.g/kg. CCK induces
not only GB contraction but also increases intrahepatic bile flow.
Information on the treatment of TPN-patients is provided in, for
example, Sitzmann, J V., et al., Cholecystokinin prevents
parenteral nutrition induced biliary sludge in humans, Surg.
Gynecol. Obstet. Vol. 170:25-31, 1990; Moss R L., et al., New
approaches to understanding the etiology and treatment of total
parenteral nutrition-associated cholestasis, Surg. Gynecol. Obstet.
Vol. 8:140-147, 1999; Teitelbaum D H., et al., Treatment of
parenteral nutrition-associated cholestasis with
cholecystokinin-octapeptide. J. Pediatr. Surg. 30:1082, 1995;
Teitelbaum D H. Parenteral nutrition-associated cholestasis,
Current Opinion in Pediatrics 1997, 9:270-275; Teitelbaum D H., et
al., Parenteral nutrition-associated cholestasis. Seminars in
Pediatric Surgery, Vol. 10, pp. 72-80.
[0071] The present invention is illustrated by the following
examples, which are in no way intended to be limiting of the
invention.
EXAMPLE 1
[0072] Effect of Buffering Agent and Formulation pH on Sincalide
Formulations
[0073] Experiments were conducted to determine the effect of pH on
the chemical stability of sincalide. Chemical instability, or
degradation, may be caused by, for example, oxidation, reduction,
deamidation, hydrolysis, imide formation, racemization,
isomerization, and/or .beta.-elimination. To examine the effect of
pH on sincalide in phosphate buffer solution, solutions of
sincalide (.apprxeq.1.7 .mu.g/mL) were prepared in 35 mM phosphate
buffer and pH-adjusted with either dilute HCl or NaOH for final pH
values ranging from 3.0-9.1. Using reverse-phase HPLC (RP-HPLC)
with gradient elution and UV detection at 215 nm, sincalide
stability in solution was assessed by measuring the recovery of
sincalide at 0, 6, and 24 hours after pH adjustment.
[0074] Results of the 24-hour study on the stability of sincalide
in phosphate buffer over the pH range of 3.0-9.1 are summarized in
Table 3 and also represented graphically in FIG. 5. By measure of
the percentage recovery, sincalide was stable in 35 mM phosphate
buffer solution at pH values ranging from 5.0-9.1 over a 24-hour
period. At pH values <5.0, sincalide degradation was evident
even at the initial time point.
3TABLE 3 Results of pH Study of Sincalide in 35 mM Phosphate Buffer
Average % Sincalide Recovery pH n 0 Hours 6 Hours 24 Hours 3.0 2
95.2 .+-. 0.4 93.4 .+-. 0.4 90.8 .+-. 1.2 4.0 2 93.0 .+-. 0.6 92.6
.+-. 1.6 85.5 .+-. 3.0 5.0 4 100.0 .+-. 2.7 99.8 .+-. 1.3 97.3 .+-.
1.8 5.5 2 100.7 .+-. 0.0 102.1 .+-. 0.3 101.6 .+-. 0.6 6.0 2 97.8
.+-. 0.4 99.8 .+-. 0.2 99.8 .+-. 1.0 6.5 2 98.8 .+-. 0.4 100.7 .+-.
0.3 99.6 .+-. 0.1 7.0 2 101.0 .+-. 0.0 101.0 .+-. 1.8 100.2 .+-.
1.2 7.5 2 101.0 .+-. 0.2 101.2 .+-. 0.8 100.4 .+-. 0.0 9.1 5 101.3
.+-. 2.3 101.1 .+-. 1.6 99.7 .+-. 0.9
[0075] Based on the results shown in Table 3, phosphate was
selected as the buffering agent of choice due to a lack of
interaction with sincalide and an ideal buffering capacity in the
physiological pH range. Subsequently, experiments using phosphate
in the formulation shown in Table 4 over the stable pH range
established above were performed. Briefly, solutions of sincalide
containing the following components (in the concentrations
indicated in Table 4) were prepared: sincalide, D-mannitol,
L-arginine, L-methionine, L-lysine, sodium metabisulfite,
polysorbate 20, pentetic acid and dibasic potassium phosphate.
4TABLE 4 Components of a Sincalide Formulation for Example 1
Concentration Component (mg/vial) Function Sincalide 0.0050 Active
D-Mannitol 170.0 Bulking Agent/ Tonicity Adjuster L-Arginine
Monohydrochloride 30.0 Stabilizer L-Methionine 4.0 Stabilizer
L-Lysine Monohydrochloride 15.0 Stabilizer Sodium Metabisulfite
0.040 Stabilizer Polysorbate 20 (TWEEN .RTM.-20) <0.01
Surfactant Pentetic Acid (DTPA) 2.0 Chelator Dibasic Potassium
Phosphate 9.0 Buffer
[0076] Solutions were pH-adjusted from 5.5-8.5 with dilute HCl or
NaOH, and were evaluated for stability by measuring the sincalide
recoveries at 0, 4, and 8 hours after pH adjustment, using RP-HPLC
with gradient elution and UV detection at 215 nm, as described
above. The results of an 8-hour study on the stability of sincalide
in the above formulation over the pH range of 5.5-8.5 are
summarized in Table 5 and also represented graphically in FIG.
6.
5TABLE 5 Results of pH Study of a Preferred Lyophilized Sincalide
Formulation of the Invention Average % Sincalide Recovery pH n 0
Hours 4 Hours 8 Hours 5.5 2 99.7 .+-. 0.2 98.5 .+-. 0.1 98.1 .+-.
0.0 6.0 2 97.4 .+-. 0.5 98.0 .+-. 0.1 98.0 .+-. 0.2 7.0 2 98.4 .+-.
0.1 98.1 .+-. 0.1 97.5 .+-. 1.3 8.0 2 97.2 .+-. 0.6 95.4 .+-. 0.4
96.4 .+-. 0.2 8.5 1, 2, 2 99.2 98.0 .+-. 0.0 99.5 .+-. 0.9
[0077] No distinct pH-dependent related trends in initial sincalide
recovery were observed over the pH range studied. Any fluctuation
in sincalide recovery over time can be attributed to normal assay
variability and not degradation. Sincalide stability in this
formulation is further supported by analyses of the chromatographic
profiles for the presence of sincalide-related degradants which
were consistent at 1.2-1.6% (impurity index) over the 8-hour study
from pH 5.5-8.5. A bulk batch solution of sincalide formulation was
prepared containing 25 mM phosphate, as a buffering agent, at a
target pH value of 6.8 (range 6.7-6.9). Reconstitution of the
lyophile with 5 mL of water is equivalent to 10 mM phosphate in the
drug product. The data demonstrate solution stability over a
physiologically compatible pH range and support a preferred pH of
6.0-8.0 for reconstituted sincalide.
EXAMPLE 2
[0078] Effect of Chelators on Sincalide Formulations
[0079] As shown in FIG. 1, the amino acid composition of sincalide
includes two methionine (Met) residues which are designated as Met
3 and Met 6 in the structural sequence. Experiments were performed
to determine whether these residues, as present in sincalide, were
susceptible to oxidation by free metals. These experiments also
examined the role of DTPA as a formulation excipient to chelate
metals and thereby inhibit sincalide oxidation. FIGS. 2-4 show the
three oxidized forms of sincalide containing either mono- or
disulfoxides. As shown in Table 6, experimental formulations
(without amino acids) at pH 6.5-7.0, with 1 mM DTPA (0.39 mg
DTPA/mL) and without DTPA were prepared to evaluate potential
oxidative effects due to the presence of metals.
6TABLE 6 Sincalide Formulations Used in Example 2 (without Amino
Acids) Bulk Concentration Concentration Component (mg/vial) (mg/mL)
Sincalide 0.0050 0.0025 D-Mannitol 170.0 85.0 L-Arginine
Monohydrochloride 0 0 L-Methionine 0 0 L-Lysine Monohydrochloride 0
0 Sodium Metabisulfite 0.040 0.02 Polysorbate 20 <0.01 2.5
.times. 10.sup.-6 Pentetic Acid (DTPA) (+)/(-) 1.0/0 Dibasic
Potassium Phosphate 9.0 4.5
[0080] The experimental formulation (25 mL) solution with (+) and
without (-) DTPA were individually spiked with nine metal ions, as
summarized in Table 7.
7TABLE 7 Evaluation of Metal Ions for Oxidative Effects on
Sincalide Volume (.mu.L) 1 mM DTPA of Metal Ion Metal Ion (+) with/
Metal Standard Concentration (-) without Aluminum (Al.sup.3+) 100
1.48 mM + 40 ppm - Chromium (Cr.sup.3+) 25 0.19 mM + 10 ppm -
Copper (Cu.sup.2+) 100 0.63 mM + 40 ppm - Iron (Fe.sup.3+) 25 0.18
mM + 10 ppm - Lead (Pb.sup.2+) 100 0.19 mM + 40 ppm - Magnesium
(Mg.sup.2+) 50 0.82 mM + 20 ppm - Manganese (Mn.sup.2+) 25 0.18 mM
+ 10 ppm - Nickel (Ni.sup.2+) 100 0.68 mM + 40 ppm - Zinc
(Zn.sup.2+) 100 0.61 mM + 40 ppm -
[0081] The metal-containing solutions were analyzed within 8 hours
for sincalide and related oxidized forms by RP-HPLC with gradient
elution and UV detection at 215 nm, as described above. FIG. 7
shows the effects of the nine metals in the presence and absence of
DTPA on the formation of sulfoxides (Met 3 and Met 6). These data
show that, with the exception of Cr.sup.3+, the amounts of
sincalide Met 3 and Met 6 monosulfoxides increase in the presence
of certain metals and in the absence of DTPA, while the presence of
DPTA has an inhibitory effect on the formation of sincalide Met 3
and Met 6 monosulfoxides. Copper and manganese, in the absence of
DTPA, have the greatest oxidative effect on the methionine residues
of sincalide resulting in combined weight percentages of Met 3 and
Met 6 monosulfoxides (vs sincalide) of 85.5 and 128.9,
respectively. In addition to the presence of sincalide Met 3 and
Met 6 monosulfoxides (t.sub.R.apprxeq.14.8 min. {doublet} and
t.sub.R.apprxeq.18 min.), formation of sincalide disulfoxide
(t.sub.R.apprxeq.6.5 min.) was also noted in the cases of copper
and manganese, but not with the other metals.
[0082] Chromatograms of formulations spiked with copper or
manganese (FIGS. 8-11) and with or without DTPA also support this
conclusion. The analyses of the chromatographic profiles indicate
that levels of DTPA at 1 mM (0.39 mg DTPA/mL) protect sincalide
from metal-catalyzed oxidation to sulfoxides. As trace metals often
arise in formulations as a result of excipient impurities and/or
stopper extractables, the results of the study support the use of
pentetic acid (DTPA) as a formulation excipient to chelate trace
levels of free metals, thus reducing the formation of sincalide
methionine mono- and disulfoxides and inhibiting the degradation of
sincalide in solution. Sincalide formulations were prepared
containing 2 mg DTPA/vial, equivalent to 1 mM upon reconstitution
with 5 mL.
EXAMPLE 3
[0083] Effect of Surfactants on Sincalide Formulations
[0084] During the preliminary developmental studies of a new
formulation that consisted of bulking agent/tonicity adjuster,
buffer, salt, chelator, and sincalide, it was observed by HPLC
analysis that the recovery of the active pharmaceutical ingredient,
sincalide, in the bulk solution was sensitive to standing open to
air. For example, when using reversed-phase gradient elution HPLC
with UV detection at 215 nm to monitor sincalide potency, a
substantial decrease of 50-60% in sincalide recovery was observed
in unstoppered vials with a 2-mL fill of bulk solution either
stirred or left standing open to air for 17 hours. Although to some
extent, this sincalide decrease can be accounted for by an increase
in the presence of sincalide mono- and disulfoxide degradants,
these represent only a very minor percentage of the decreases
noted. Thus the decrease in recovery is thought to be attributed to
either adsorption/denaturing or air/liquid interface effects. To
minimize sincalide degradation associated with surface adsorption,
surfactants are added as formulation excipients in bulk and
lyophilized formulations of sincalide.
[0085] Sincalide formulations consisting of a bulking
agent/tonicity adjuster (D-mannitol), buffer (mono- and dibasic
potassium phosphate), salt (sodium/potassium chloride) for
tonicity, chelator (pentetic acid), and active ingredient
(sincalide) were prepared using varying concentrations of the
nonionic surfactant, polysorbate 80 (TWEEN.RTM. 80). Bulk solution
and reconstituted lyophilized samples were either stoppered
immediately or left unstoppered for 17 hours, and were assayed for
sincalide recovery by reversed-phase gradient elution HPLC at 215
nm.
[0086] As shown in Table 8, the effect of TWEEN.RTM. 80 is more
apparent in formulations that have been exposed to air. For bulk
and reconstituted lyophilized formulations, the data show decreases
in sincalide recovery of .apprxeq.50% and .apprxeq.20%,
respectively, when compared to corresponding formulations
containing a TWEEN.RTM. 80 concentration of 1 mg/mL. Low sincalide
recoveries in closed bulk and reconstituted lyophilized
formulations without TWEEN.RTM. 80 are also evident, but not nearly
as substantial (4-8%) as the exposed formulations. These
preliminary screening studies on the influence of TWEEN.RTM. 80
concentration indicate that <1 mg/mL bulk may be optimal.
8TABLE 8 Sincalide Recovery in Formulations With and Without TWEEN
.RTM. 80 Formulation TWEEN .RTM. 80 Sincalide Description Test
Conc. % (mg/mL Bulk) Condition (mg/mL) Recovery D-Mannitol (75.0),
Bulk; open 1.0 97.0 KH.sub.2PO.sub.4 (3.25), (.about.17 h) 0.0 47.0
K.sub.2HPO.sub.4 (1.0), Bulk; 1.0 100.0 NaCl (5.0), closed 0.0 96.0
DTPA (1.0), Lyophilized; 1.0 91.3 Sincalide (0.0025), open
(.about.17 h) 0.1 98.2 TWEEN .RTM. 80 (0; 0.1; 1.0) 0.01 98.3 0.0
78.4 Lyophilized; 1.0 90.2 closed 0.1 98.1 0.01 97.8 0.0 92.3
[0087] To compare the effects of two nonionic surfactants,
sincalide formulations (75 mg/mL D-mannitol, 6.0 mg/mL KCl, 3.25
mg/mL KH.sub.2PO.sub.4, 1.0 mg/mL K.sub.2HPO.sub.4, 1.0 mg/mL DTPA,
0.0025 mg/mL sincalide (Bulk formulation)) were prepared using
either TWEEN.RTM. 20 or TWEEN.RTM. 80 in varying amounts. The
results of this experiment are presented in Table 9.
9TABLE 9 Effect of Surfactants on Sincalide Recovery TWEEN .RTM.
Sincalide Sincalide Concentration Recovery Formulation (.mu.g/mL
Bulk) (%) TWEEN .RTM. 80 A 7.5 95.4 B 5.0 96.3 C 2.5 98.6 G 0 94.1
TWEEN .RTM. 20 D 7.5 99.5 E 5.0 101.3 F 2.5 98.7 G 0 94.1
[0088] As shown in Table 9, the data indicate that the presence of
trace levels (2.5-7.5 .mu.g/mL) of either TWEEN.RTM. 80 or
TWEEN.RTM. 20 has a beneficial effect on the recovery of sincalide,
when compared to formulations without surfactant. However, the
sincalide recoveries (98-102%) with formulations containing
TWEEN.RTM. 20 are consistently higher than recoveries (95-98%) with
TWEEN.RTM. 80, and thus TWEEN.RTM. 20 is a preferred
surfactant.
[0089] An additional experiment was performed to confirm the effect
of the concentration of TWEEN.RTM. 20 in terms of sincalide
recovery in both air exposed and sealed bulk formulation. Sincalide
recovery, determined for bulk formulation (75.0 mg/mL D-mannitol,
6.0 mg/mL KCl, 3.25 mg/mL KH.sub.2PO.sub.4, 1.0 mg/mL DTPA, 0.0025
mg/mL sincalide) containing varying trace levels of TWEEN.RTM. 20
stored in open or closed vials using reversed-phase gradient
elution HPLC, is shown in Table 10.
10TABLE 10 Effect of TWEEN .RTM. 20 Concentration on Recovery of
Sincalide in Bulk Formulations TWEEN .RTM. 20 Sincalide
Concentration Sincalide % Recovery Formulation (.mu.g/mL Bulk) Open
Vial Closed Vial D 7.5 100.7 100.8 E 5.0 100.0 100.4 F 2.5 99.0
98.2 G 0 89.8 96.1
[0090] As shown in Table 10, the bulk formulations containing
TWEEN.RTM. 20 have improved sincalide recoveries over formulations
with no TWEEN.RTM. 20 and the sincalide recoveries are independent
of the TWEEN.RTM. 20 concentration range (2.5-7.5 .mu.g/mL bulk)
studied. In addition, the air sensitivity relative to sincalide
recovery was eliminated, as both open and closed formulations
containing TWEEN.RTM. 20 have equivalent sincalide recoveries.
Although these data support the use of TWEEN.RTM. 20, it was noted
that 2-mL filled vials containing a TWEEN.RTM. 20 concentration of
5 .mu.g/mL show slight foaming in the reconstituted product upon
vigorous stirring. To reduce foaming, a lower TWEEN.RTM. 20
concentration was evaluated.
[0091] As summarized in Table 11, an experiment was conducted on
the lyophilized product comparing the recovery of the sincalide in
the formulations with TWEEN.RTM. 20 (2.5 ng/mL) and without
TWEEN.RTM. 20. In this Example and the subsequent Examples,
mannitol refers to D-mannitol, methionine refers to L-methionine,
arginine refers to L-arginine monohydrochloride, and lysine refers
to L-lysine monohydrochloride.
11TABLE 11 Sincalide Recovery in Reconstituted Formulations With
and Without TWEEN .RTM. 20 Formulation Sincalide % Recovery
Description TWEEN .RTM. 20 Concentration (mg/mL Bulk) 0 ng/mL 2.5
ng/mL Mannitol (85.0), 94.8 (n = 5) KH.sub.2PO.sub.4 (4.5), 100.0
(n = 2) DTPA (1.0), 100.0 (n = 2) Methionine (2.0), 99.0 (n = 2)
Lysine (7.5), Arginine (15.0), Sodium metabisulfite (0.02),
Sincalide (0.0025) Average 94.8 99.7 Variance 0.862 0.667 P(T <=
t) two-tail 1.6 .times. 10.sup.-5
[0092] Reducing the amount of TWEEN.RTM. 20 to a minimal trace
concentration (2.5 ng/mL) still produced a significant effect on
the air/liquid interface and eliminated the foaming in the
formulation. A statistical two-tail t-test performed on the results
showed a significant difference (P<0.05) between 2.5 ng/mL and
no TWEEN.RTM. 20 in the formulation. Based on these data, the
effectiveness of TWEEN.RTM. 20, polyoxyetbylene sorbitan
monolaurate, as a surfactant was established by enhancing the
sincalide recovery and thus maintaining sincalide potency in the
formulation. A preferred formulation of sincalide includes the
nonionic surfactant TWEEN.RTM. 20 as a trace excipient at a
concentration of 2.5 ng/mL of bulk formulation equivalent to 1
ng/mL in the final product when reconstituted to 5 mL.
EXAMPLE 4
[0093] Effect of Antioxidants on Sincalide Formulations
[0094] An experiment was performed to evaluate the addition of
antioxidants as stabilizing agents to prevent sincalide oxidation
in formulations of sincalide (formulations for Example 4 contained
85 mg/mL mannitol, 0.005 mg/mL TWEEN.RTM. 20, 2.75 mg/mL
KH.sub.2PO.sub.4, 1.0 mg/mL DTPA, 2.0 mg/mL methionine, 7.5 mg/mL
lysine, 15 mg/mL arginine, 0.0025 mg/mL sincalide (Bulk
formulation), except placebos which contained no sincalide.) The
formation of sincalide methionine (Met 3 or Met 6) monosulfoxides,
desulfated sincalide and unknown degradants was investigated. The
effectiveness of sodium metabisulfite, ascorbic acid, cysteine,
glutathione, sodium sulfate, benzalkonium chloride, and
benzethonium chloride in inhibiting the degradation of sincalide in
terms of their effect on sincalide recovery and sincalide-related
impurities, was evaluated by HPLC.
[0095] The effect of various antioxidants on the stabilization of
sincalide was evaluated on open and sealed sincalide formulations
over 15 hours. The antioxidants were separately added at a
concentration of 10 .mu.g/mL to water-reconstituted lyophilized
sincalide formulations containing all formulation ingredients
except antioxidant. Spiked and unspiked solutions were pooled,
subdivided, and either exposed to or protected from air over 15
hours. The sincalide and total sincalide-related impurities were
monitored by reversed-phase HPLC with gradient elution and UV
detection at 215 nm to compare the effectiveness of the
antioxidants.
[0096] As shown in Table 12, the data at these concentrations
indicate that benzalkonium chloride and benzethonium chloride had a
significant destabilizing effect on sincalide, while ascorbic acid,
cysteine, glutathione, and sodium sulfate were essentially
equivalent to the control formulation (no antioxidant). Of all the
sincalide formulation/antioxidant combinations examined, the
formulation with 10 .mu.g sodium metabisulfite/mL showed the
highest sincalide potency (98.3%) over 8 hours, and the lowest
total sincalide-related impurities (1.79%) through 15 hours.
Therefore, sodium metabisulfite is a preferred antioxidant for
formulations of the invention.
12TABLE 12 Effect of Various Antioxidants (10 .mu.g/mL) on
Sincalide Formulation Stability % Sincalide % Total
Sincalide-Related Impurities Antioxidant Sealed Open Sealed Open
(10 .mu.g/mL) 0 h 7 h 14 h 1 h 8 h 15 h 0 h 7 h 14 h 1 h 8 h 15 h
Control (None) 98.1 98.1 98.1 98.1 98.2 98.2 1.94 1.95 1.86 1.90
1.85 1.81 Sodium Metabisulfite 98.3 98.3 98.3 98.2 98.3 98.2 1.67
1.66 1.73 1.76 1.69 1.79 Ascorbic Acid 98.1 98.0 97.8 98.0 98.0
97.8 1.95 2.05 2.25 2.00 2.01 2.16 Cysteine 98.2 98.1 98.1 97.8
97.7 98.0 1.85 1.87 1.91 2.20 2.32 2.05 Glutathione 98.1 98.3 98.2
98.1 98.2 97.9 1.90 1.74 1.82 1.94 1.85 2.13 Sodium Sulfate 98.2
98.1 98.2 98.3 98.2 98.1 1.76 1.90 1.81 1.70 1.78 1.92 Benzalkonium
Chloride 97.8 97.7 97.4 82.7 88.4 82.9 2.21 2.34 2.58 17.3 11.6
17.1 Benzethonium Chloride 97.9 98.0 98.0 92.1 88.0 92.6 2.13 1.98
1.96 7.93 12.0 7.36
[0097] To optimize the level of sodium metabisulfite in the
formulation, lyophilized sincalide formulations were prepared
containing four levels of sodium metabisulfite (0, 10, 30, and 60
.mu.g/vial), as summarized in Table 13. Samples at each
concentration were maintained under unstressed and stressed
(65.degree. C., 64 hours) storage conditions, and were subsequently
assayed by HPLC. The "% sincalide" was determined, and the "% (Met
6) monosulfoxide" (t.sub.R.about.19.7 min.) was monitored as an
indication of sincalide oxidation. The data are presented in Table
14.
13TABLE 13 Sincalide Lyophilized Formulations Formulation No.
Formulation Description 1 complete formulation, no sodium
metabisulfite 2 complete formulation, 10 .mu.g sodium
metabisulfite/vial 3 complete formulation, 30 .mu.g sodium
metabisulfite/vial 4 complete formulation, 60 .mu.g sodium
metabisulfite/vial 5 placebo (no sincalide), 40 .mu.g sodium
metabisulfite/vial 6 complete formulation, no sodium metabisulfite
7 complete formulation, 40 .mu.g sodium metabisulfite/vial
[0098]
14TABLE 14 Effect of Sodium Metabisulfite Concentration on
Sincalide Oxidation Sodium % (Met 6) Monosulfoxide % Sincalide
Metabisulfite Stressed Stressed Concentration Un- (65.degree. C.,
Un- (65.degree. C., Formulation (.mu.g/vial) stressed 64 h)
stressed 64 h) 1 0 0.08 0.20 95.7 95.1 2 10 0.07 0.09 95.1 96.0 3
30 0.07 0.10 95.0 96.9 4 60 0.06 0.08 95.8 96.2
[0099] The addition of sodium metabisulfite up to 60 .mu.g/vial
improved sincalide recovery and inhibited the oxidation of
sincalide to the (Met 6) monosulfoxide derivative under stressed
conditions. Based on this data, as there was no apparent
concentration relationship, 40 .mu.g/vial sodium metabisulfite was
selected as the preferred concentration for the final formulation,
using 30 .mu.g/vial and 60 .mu.g/vial as lower and upper limits,
respectively.
[0100] Another experiment was conducted under longer-term
accelerated storage conditions utilizing a sincalide formulation
with the optimized concentration (40 .mu.g/vial) of sodium
metabisulfite to confirm the protective effect on the degradation
of sincalide. Sincalide lyophilized formulations with and without
the antioxidant from the same batch were heat-stressed at
40.degree. C. and 60.degree. C. for 6 weeks. Also, formulations
without sincalide from the same batch were heat-stressed at
40.degree. C. for 8 months. The results of the HPLC analyses for %
sincalide and % total impurity are presented in Table 15.
15TABLE 15 Effect of Sodium Metabisulfite on Heat Stress-Related
Impurities Sodium Metabisulfite Storage % Concentration Temp. %
Total Formulation (.mu.g/vial) (6 weeks) Sincalide Impurity 7 40
40.degree. C. 96.7 3.30 6 0 40.degree. C. 93.4 6.56 7 40 60.degree.
C. 89.5 10.51 6 0 60.degree. C. 84.0 16.00
[0101] The results of this longer-term accelerated storage
experiment further emphasized the need for the presence of the
excipient sodium metabisulfite. Sincalide formulations with sodium
metabisulfite (40 .mu.g/vial) protected against sincalide heat
stress-related degradant formation (3.30%), as compared without the
antioxidant, which exhibited several elevated sincalide heat
stress-related impurities (6.56%). These impurities were confirmed
to be sincalide heat stress-related (t.sub.R=35 to 44 min.), as
they were not present in chromatograms of formulations without
sincalide. Sodium metabisulfite was chosen as a preferred
antioxidant and stabilizing agent over ascorbic acid, cysteine,
glutathione, sodium sulfate, benzalkonium chloride, and
benzethonium chloride because it provided superior protection in
inhibiting the oxidative and heat stress-related degradation of
sincalide. A preferred concentration in the lyophilized formulation
is 40 .mu.g sodium metabisulfite/vial or 8 .mu.g/mL in the
reconstituted product.
EXAMPLE 5
[0102] Selection of Bulking Agent/Tonicity Adjuster
[0103] Due to the minute amount of the active pharmaceutical
ingredient (API), sincalide (5 .mu.g/vial), in the formulations of
the invention, the use of a bulking agent was considered extremely
beneficial for providing tonicity as well as for providing both
structure and support for the API. Experiments were conducted for
the selection and optimization of bulking agent in the sincalide
formulations of the invention. Criteria for evaluation were: an
efficient lyophilization cycle, a pharmaceutically elegant finished
product, enhanced product solubility and usefulness as an excipient
for isotonicity in the reconstituted product. Various
concentrations of lactose, lactose/sodium chloride and mannitol
were considered, and experimental batches containing these
excipients were evaluated in terms of cake appearance, osmolality,
dissolution rate, and thermal analysis including freeze dry
microscopy, and electrical resistance vs. temperature
measurements.
[0104] Experimental Formulations
[0105] Batch A: ingredients: lactose 375 mg/vial, dibasic sodium
phosphate 12.0 mg/vial, DTPA 2.0 mg/vial, monobasic sodium
phosphate 19.5 mg/vial, and 0.005 mg/vial sincalide.
[0106] Batch C.sub.1-3: ingredients: mannitol 170 mg/vial, dibasic
potassium phosphate 9.0 mg/vial, TWEEN.RTM. 20<0.01 mg/vial,
methionine 4.0 mg/vial, lysine 15.0 mg/vial, arginine 30.0 mg/vial,
sodium metabisulfite 0.04 mg/vial, sincalide 0.005 mg/vial, and
DTPA 2.0 mg/vial.
[0107] Batch D.sub.1: ingredients: lactose 150 mg/vial, dibasic
potassium phosphate 9.1 mg/vial, DTPA 2.0 mg/vial, monobasic sodium
phosphate 9.8 mg/vial, and NaCl 21.0 mg/vial.
[0108] Batch E.sub.1: ingredients: lactose 200 mg/vial, dibasic
sodium phosphate 7.5 mg/vial, DTPA 2.0 mg/vial and NaCl 17
mg/vial.
[0109] Batch F.sub.1-2: F.sub.1: ingredients: mannitol 250 mg/vial,
dibasic sodium phosphate 7.5 mg/vial, DTPA 2.0 mg/vial and
sincalide 0 mg/vial; and
[0110] F.sub.2: ingredients: mannitol 206 mg/vial, dibasic sodium
phosphate 7.5 mg/vial, DTPA 2.0 mg/vial and sincalide 0.005
mg/vial.
[0111] Batch H.sub.1-2: H.sub.1: ingredients: mannitol 180 mg/vial,
dibasic sodium phosphate 6.0 mg/vial, sincalide 0 mg/vial, NaCl 5
mg/vial and DTPA 2.0 mg/vial; and
[0112] H.sub.2: ingredients: mannitol 150 mg/vial, dibasic
potassium phosphate 4.5 mg/vial, sincalide 0.005 mg/vial, NaCl 10
mg/vial and DTPA 2.0 mg/vial.
[0113] Batch I.sub.1-2: I.sub.1: ingredients: mannitol 140 mg/vial,
dibasic potassium phosphate 5.5 mg/vial, TWEEN.RTM. 200.01 mg/vial,
methionine 4.0 mg/vial, lysine 60.0 mg/vial, sincalide 0.005
mg/vial and DTPA 2.0 mg/vial; and
[0114] I.sub.2: ingredients: mannitol 170 mg/vial, dibasic
potassium phosphate 5.5 mg/vial, TWEEN.RTM. 200.01 mg/vial,
methionine 4.0 mg/vial, lysine 30.0 mg/vial, sincalide 0.005
mg/vial and DTPA 2.0 mg/vial.
[0115] Batch J: ingredients: mannitol 170 mg/vial, dibasic
potassium phosphate 8.5 mg/vial, TWEEN.RTM. 200.01 mg/vial,
methionine 4.0 mg/vial, lysine 15.0 mg/vial, arginine 30.0 mg/vial,
Na metabisulfite 0.04 mg/vial, sincalide and DTPA 2.0 mg/vial.
[0116] Methods:
[0117] 1. Appearance: Visual assessment of the freeze-dried
plug.
[0118] 2. Osmolality: Determined by vapor pressure osmometry.
[0119] 3. Dissolution: Dissolution time measured by visual
inspection under an inspection light upon reconstitution with 5 mL
of water.
[0120] 4. Thermal Analysis:
[0121] a. Electrical resistance vs. temperature measurements:
Electrical resistance measured using a proprietary resistance
instrument, temperature measured using a 32-gauge type T
thermocouple.
[0122] b. Freeze drying microscopy: Performed using a freeze dry
microscope an Infinivar microscope and color camera.
[0123] In the initial investigations lactose was used as a bulking
agent/tonicity adjuster. The formulation as listed in table 16 is
based on a 3-mL fill volume with a high concentration of lactose to
achieve isotonicity in the reconstituted product. The osmolality
for this formulation upon reconstitution with 5 mL of water was
.about.300 mOsm/kg.
16TABLE 16 Lactose Containing Sincalide Formulation (Batch A)
Concentration Raw Materials Function (mg/vial) Lactose Bulking
Agent/ 375 Tonicity Adjuster Dibasic Sodium Phosphate Buffer 12.0
Monobasic Sodium Phosphate Buffer 19.5 Pentetic Acid Chelator 2.0
Sincalide Active 0.005
[0124] This experimental formulation, Batch A, with a
lyophilization cycle of 130 hours (.apprxeq.5.4 days) showed
evidence of meltback in the lyophilized cakes and had
reconstitution dissolution times of .gtoreq.9 minutes. The high
number of vials with poor cake formation and the long freeze dry
cycle required were attributed to the high concentration of lactose
(125 mg/mL) in the bulk formulation relative to its solubility and
the high fill volume (3-mL) in a small vial.
[0125] Studies were undertaken to reduce cycle time and improve
product appearance/solubility by modifying the initial lactose
formulation with the use of an additional excipient, sodium
chloride, thereby reducing lactose concentration and the fill
volume from 3 to 2-mL.
17TABLE 17 Lactose/NaCl Containing Sincalide Formulations (Batches
D.sub.1 and E.sub.1-2) Concentration Raw Materials Function
(mg/vial) Lactose Bulking Agent/ 150-200 Tonicity Adjuster Dibasic
Sodium Phosphate Buffer 12.0 Monobasic Sodium Phosphate Buffer 19.5
Pentetic Acid Chelator 2.0 Sodium Chloride Tonicity Adjuster 17-21
Sincalide Active 0.005
[0126] Use of NaCl contributed to the isotonicity of the product
with osmolality values in the range of 240 to 270 mOsm/kg, while
permitting a reduction in the concentration of lactose. Varying the
amounts of lactose, sodium chloride and sodium phosphate decreased
the lyophilization cycle from 130 hours to 96 hours, but did not
consistently improve the appearance of the freeze-dried cake.
[0127] Thermal analysis of two experimental formulations with
varying lactose/sodium chloride ratios (Table 18) confirm that the
relatively long lyophilzation cycles for these formulations were
due to low primary drying temperatures in the range of -38.degree.
C. to -42.degree. C., resulting in slow sublimation rates at these
temperatures. In addition to long lyophilization cycles, the low
primary drying temperatures lead to increased vial-to-vial
variation and an increased risk of poor plug appearance with
associated solubility issues.
18TABLE 18 Thermal Analysis of Experimental Lactose/NaCl
Formulations Lactose/NaCl Freezing Temp. Primary Drying
Concentration Range (.degree. C.) Temp. Range (.degree. C.) Batch
(mg/vial) High Low High Low D.sub.1 150/21 -32 -39 -39 -42 E.sub.1
200/17 -15 -35 -36 -38
[0128] Mannitol, a common excipient for freeze-dried
pharmaceuticals, was selected next for evaluation as bulking agent
because of the high melting temperature of the mannitol/ice
eutectic mixture (about -1.5.degree. C.) and its tendency to
crystallize from frozen aqueous solutions. Ideally, this leads to
shorter primary and secondary drying times, promoting an efficient
freeze-drying cycle and a physically stable, pharmaceutically
elegant freeze-dried solid. Several bench-scale batches were
prepared, replacing lactose with D-mannitol while maintaining
isotonicity with a 2 mL fill volume, to evaluate the parameters of
cycle time and primary drying temperature and the solubility of the
solid cake. The freeze dry cycle parameters along with lyophilized
product reconstitution times with a 5 mL reconstitution volume are
shown in Table 19.
19TABLE 19 Effect of Formulation Bulking Agent/Tonicity Adjuster on
Lyophilization Cycle Optimization and Reconstitution/Dissolution
Time Formulation Bulking Dissolution Description Agent Osmolality
Freeze Dry Time Batch (mg/vial) (mg/vial) (mOsm/kg) Cycle
Parameters (sec) F.sub.1 Na.sub.2HPO.sub.4 (7.5), Mannitol 280
Total Cycle 85 hr 12-48 DTPA (2.0) (250) Primary drying @
-34.degree. C. (n = 10) F.sub.2 Na.sub.2HPO.sub.4(7.5), Mannitol
240 Total Cycle 69 hr 22-71 DTPA (2.0) (206) Primary drying @
-25.degree. C. (n = 30)
[0129] Lyo-cycle time was reduced from >130 hours for lactose
formulations, to .about.69 hours for the mannitol formulation,
Batch F.sub.2. The cakes from both formulations, F.sub.1 and
F.sub.2 dissolved in 5 mL of water in approximately the same time
range of <1 minute. Increasing the primary drying temperature
from .about.-34.degree. C. to -25.degree. C. Batch F.sub.1 vs.
Batch F.sub.2 had the desired effect of reducing the overall cycle
time from 85 to 69 hours.
[0130] Additional studies were conducted to optimize the mannitol
concentration and lyo-cycle time for a 2-mL fill volume. These
studies were carried out concurrently with formulation development
studies to adjust the osmolality to .about.250 mOsm/Kg after
reconstitution and to stabilize the peptide by addition of other
excipients to the formulation (Table 20).
20TABLE 20 The Effect of Mannitol Concentration on Appearance,
Solubility and Freeze Dry Cycle of Sincalide Formulations
Formulation Bulking Freeze Dry Moisture Appearance/ Description
Agent Osmolality Cycle Content Dissolution Batch (mg/vial)
(mg/vial) (mOsm/kg) Parameters (%) Time (sec) H.sub.1
Na.sub.2HPO.sub.4 (6.0), Mannitol (180) 250 Total Cycle: 36 hr ND
Solid cake/ DTPA (2.0), NaCl (5.0) 27 hr primary 22-66 Sincalide
(0) @ -8.degree. C. (n = 30) H.sub.2 K.sub.2HPO.sub.4 (4.5),
Mannitol (150) 240 Total Cycle: 30 hr 1 Solid cake/ DTPA (2.0),
NaCl (10.0) 23 hr primary 11-31 Sincalide (0.005) @ -10.degree. C.
(n = 30) I.sub.1 TWEEN .RTM. (0.01), Mannitol (140) 250 Total
Cycle: 59 hr 1 Solid cake/ K.sub.2HPO.sub.4 (5.5), 50 hr primary
21-69 Methionine (4.0), @ -22.degree. C. (n = 5) Lysine (60.0),
DTPA (2.0), Sincalide (0.005) I.sub.2 TWEEN .RTM. (0.01), Mannitol
(170) 250 Total Cycle: 33 hr 1 Solid cake/ K.sub.2HPO.sub.4 (5.5),
26 hr primary 8-15 Methionine (4.0), @ -12.degree. C. (n = 5)
Lysine (30.0), DTPA (2.0), Sincalide (0.005) ND = Not
Determined
[0131] These results demonstrate that an increase in primary drying
temperature from .about.-25.degree. C. to the -8 to -12.degree. C.
range significantly reduced cycle times from 69 to 30 hours and
produced solid dry cakes that reconstitute within 1 minute.
[0132] Additional optimization studies designed to enhance the long
term stability of sincalide resulted in a preferred sincalide
formulation of 170 mg of D-mannitol/vial with the additional
excipients (in mg/vial): TWEEN.RTM. 20 (0.01), K.sub.2HPO.sub.4
(8.5), methionine (4.0), lysine (15.0), arginine (30.0), DTPA
(2.0), and sodium metabisulfite (0.04). The osmolality of this
optimized formulation was approximately 300 mOsm/kg when
reconstituted with 5 mL of water. Thermal analysis of this
formulation using freeze-dry microscopy and electrical resistance
vs. temperature measurements, indicated an upper limit for product
primary drying temperature of -13.degree. C. to -15.degree. C. to
achieve acceptable product quality.
[0133] To confirm all findings, three scale-up pilot batches,
C.sub.1-3, of a preferred sincalide formulation, in a fill volume
of 2 mL/vial, were prepared and freeze dried in full-scale
production driers to prove process transferability from development
equipment to production equipment. The drying cycle for these
batches incorporated a primary drying temperature of -12.degree. C.
.+-.3.degree. C. and an overall cycle time of 53-61 hours (Table
21).
21TABLE 21 Operating Parameters and Final Product Performance of
Scale-up Pilot Batches Prepared with Mannitol as a Bulking Agent
Lyophilization Primary Total Cycle Osmolality Plug Moisture
Dissolution Batch Temp (.degree. C.) Time (Hrs) (mOsm/kg)
Appearance Content %) Time (sec) C.sub.1 -12 58 300 Solid cake 1 10
C.sub.2 -12 53 300 Solid cake 1 10 C.sub.3 -12 61 300 Solid cake 1
10
[0134] The data from these studies support the selection of
mannitol as a particularly preferred bulking agent, preferably in
an amount of about 170 mg/vial. Using this concentration, the
freeze dry cycle is 53-61 hours when filled as a 2-mL fill. The
finished product is a pharmaceutically elegant, solid white cake,
which is reconstituted within one minute using 5 mL of water,
resulting in a solution with an osmolality of .about.300
mOsm/Kg.
EXAMPLE 6
[0135] Effect of Amino Acids on Sincalide Formulations
[0136] During formulation studies it was observed that both
exposure to air and lyophilization were areas of concern for
scale-up manufacturing due to reduced potency of sincalide in the
formulation. The reduced potency was a result of surface
adsorption/denaturation resulting from exposure of sincalide to
air, and yielding degradants via oxidation. Exposure of sincalide
formulations to thermal stress during lyophilization also resulted
in degradation and reduced recovery of sincalide.
[0137] Experiments were conducted to evaluate several amino acids
as potential stabilizers of sincalide, including the non-polar
(hydrophobic) methionine residue, aspartic acid and glutamic acid,
the polar glycine and cysteine residues, and the basic lysine and
arginine amino acids.
[0138] Except as otherwise indicated, the formulations used in this
example for testing the efficacy of various amino acids contained
the following ingredients (bulk): 75.0 mg/mL mannitol; 3.25 mg/mL
KH.sub.2PO.sub.4; 1.0 mg/mL K.sub.2HPO.sub.4; 1.0 mg/mL pentetic
acid (DTPA); 5.0 mg/mL NaCl; and the active peptide, sincalide
(0.0025 mg/mL). Initially, the non-polar amino acid L-methionine
was evaluated for the reformulation since methionine residues can
act as endogenous antioxidants, or as scavengers by reacting with
hydroxyl free radicals and other reactive oxygen species. Thus,
methionine could improve the processing stability of sincalide
formulations by providing a protectant or antioxidant effect for
sincalide and being preferentially oxidized. Table 22 below
summarizes the results obtained during exposure of experimental
formulations to air when various amounts of L-methionine were added
to a formulation containing mannitol, sodium chloride, potassium
phosphate, and pentetic acid. For these experiments, liquid
formulations in open and closed vials were used to simulate
processing of the product. For formulation in open vials, the
recovery of sincalide was improved approximately 60% and the
concentration of sincalide-related impurities decreased as the
level of methionine was increased from 0.0 to 2.0 mg/mL in the bulk
formulation.
22TABLE 22 Evaluation of Methionine as a Processing Stabilizer for
Bulk Formulations - Open vs Closed Vials. L-Methionine Sincalide
Recovery (%) Related Impurities (%) (mg/mL Bulk) Open Closed Open
Closed 2.0 75.5 95.7 15.0 0.7 0.50 64.7 94.8 19.3 0.8 0.025 35.7
93.9 35.9 1.0 0.00 13.9 95.7 52.7 1.3
[0139] For comparison to the non-polar amino acid methionine as a
potential processing stabilizer, polar amino acids such as glycine
and cysteine were also evaluated. Formulations containing these
amino acids were exposed to air in open vials and compared to
product in closed vials. The efficacy of these amino acids, in
terms of sincalide recovery and sincalide-related impurities, was
compared to the improvements previously observed for the liquid
formulation in the presence of methionine. Table 23 presents the
sincalide recoveries for experimental formulations containing
variable concentrations of methionine, cysteine or glycine.
23TABLE 23 Comparison of Methionine, Glycine and Cysteine as
Processing Stabilizers for Bulk Formulations - Open vs. Closed
Vials Amino Acid Sincalide Recovery (%) Related Impurities (%)
(mg/mL Bulk) Open Closed Open Closed L-Cysteine 2.0 50.0 96.0 31.0
1.0 L-Methionine 2.5 82.4 97.4 10.5 0.7 L-Methionine 2.0 89.9 97.7
6.4 0.7 None 0 37.0 96.0 35.0 1.6 L-Glycine 2.5 31.9 93.2 44.9 1.6
L-Glycine 2.0 22.3 92.5 51.1 1.1
[0140] Results demonstrated that addition of either cysteine or
glycine to a bulk formulation containing mannitol, potassium
phosphate, sodium chloride and pentetic acid did not show a
significant effect in either reduced levels of sincalide impurities
or improved recovery of sincalide when formulations were exposed to
air in open vials.
[0141] Lysine, a basic amino acid, was the next amino acid
evaluated for use in sincalide formulations of the invention. As
shown in Table 24, experimental formulations (70-85 mg/mL mannitol,
0.005 mg/mL TWEEN.RTM. 20, 2.75 mg/mL KH.sub.2PO.sub.4, 1.0 mg/mL
DTPA, 2.0 mg/mL methionine, 0.0025 mg/mL sincalide) were prepared
to contain varying concentrations of lysine and evaluated for
sincalide recovery.
24TABLE 24 Evaluation of Lysine as a Stabilizer in Sincalide
Reconstituted Formulations Sincalide Recovery (%) DL-Lysine 1 Week
3 Weeks 5 Weeks (mg/mL Bulk) 5.degree. C. 40.degree. C. 5.degree.
C. 40.degree. C. 5.degree. C. 40.degree. C. 0.0 99.6 84.3 95.5 51.2
NA 25.4 5.0 98.1 95.4 93.6 98.4 92.0 15.0 97.3 97.0 94.3 99.4 93.2
30.0 96.6 95.0 95.5 97.2 89.7 NA = Not Applicable
[0142] After accelerated storage, lyophilized formulations
containing lysine resulted in significantly improved recoveries of
sincalide compared to a lyophilized control formulation without
lysine. Formulations containing lysine resulted in 50% and 75%
improvements in sincalide recovery after 3 and 5 weeks storage at
40.degree. C., respectively, demonstrating that lysine contributed
to the stability of lyophilized formulations when subjected to
thermal stress.
[0143] The improved sincalide recoveries observed in the presence
of methionine and lysine suggested that other amino acids might
also be suitable as bulk additives in the reformulation. Therefore,
formulation studies continued with the evaluation of two acidic
amino acids, aspartic acid and glutamic acid. Table 25 presents
sincalide recoveries for experimental formulations (85.0 mg/mL
mannitol, 0.005 mg/mL TWEEN.RTM. 20, 2.75 mg/mL KH.sub.2PO.sub.4,
1.0 mg/mL DTPA, 2.0 mg/mL methionine, 0.0025 mg/mL sincalide)
containing the following amounts of either lysine, aspartic acid or
glutamic acid.
25TABLE 25 Comparison of Lysine, Aspartic Acid and Glutamic Acid as
Stabilizers in Sincalide Reconstituted Formulations Formulation
Process Sincalide Recovery (%) Conditions ID Amino Acid (mg/mL) 0
Days 10 Days 30 Days Liquid Bulk A DL-Lysine HCl 15.0 99.9 98.4 NA
Stored 5.degree. C. B L-Aspartic Acid 11.0 98.2 96.3 C L-Glutamic
Acid 12.0 97.3 96.3 Lyophilized A DL-Lysine HCl 15.0 NA 98.2 99.1
Cake B L-Aspartic Acid 11.0 94.6 92.8 Stressed C L-Glutamic Acid
12.0 95.5 95.7 40.degree. C. E Control 0.0 81.7 53.8 NA = Not
Applicable
[0144] The results demonstrated that with increasing storage time
at 40.degree. C., lysine consistently provided better protection
than either aspartic acid or glutamic acid. The results obtained
for lysine also suggested that arginine, another basic amino acid,
or potentially some combination of lysine and arginine, might
further enhance protection during lyophilization and thermal
stress. Experimental formulations (85.0 mg/mL mannitol, 0.005 mg/mL
TWEEN.RTM. 20, 2.75 mg/mL KH.sub.2PO.sub.4, 1.0 mg/mL DTPA, 2.0
mg/mL methionine, 0.0025 mg/mL sincalide) were prepared to contain
varying concentrations of lysine, arginine, or a combination of
lysine and arginine and evaluated for sincalide recovery (Table
26).
26TABLE 26 Evaluation of Lysine and Arginine as Stabilizers in
Sincalide Reconstituted Formulations Sincalide Recovery (%) Amino
Acid 64 hrs. 1 week (mg/mL Bulk) @ 65.degree. C. @ 40.degree. C.
DL-Lysine 15.0 88.4 ND L-Arginine 17.5 93.0 DL-Lysine 7.50 99.8
L-Arginine 8.75 DL-Lysine 7.5 93.8 96.4 L-Arginine 17.5 DL-Lysine
5.0 91.2 ND L-Arginine 11.7 DL-Lysine 7.5 95.1 ND L-Arginine 15.0
N/A (control) 0.0 43.3 ND NA = Not Applicable, ND = Not
Determined
[0145] Results confirmed that after lyophilization and stressing
for 64 hours @ 65.degree. C., approximately 50-70% improvement in
sincalide recovery was observed for formulations containing lysine,
arginine, or a combination of the two. Formulations containing both
lysine and arginine exhibited the highest sincalide recovery
values, indicating that the combination of these two amino acids
provided a particularly stabilizing effect under heat-stressed
storage conditions. The mid-point combination of 7.5 mg/mL of
lysine and 15.0 mg/mL of arginine afforded suitable protection for
the lyophilized and heat-stressed product, resulting in sincalide
recoveries of >95%.
[0146] Methionine, lysine and arginine are preferred over polar
amino acids such as glycine and cysteine and acidic amino acids
such as aspartic acid and glutamic acid for use as stabilizers in
the sincalide formulations of the invention. Methionine improved
the processing stability of the formulation, resulting in improved
recovery of sincalide, and the combination of lysine and arginine
contributed to the stability of the product during lyophilization
and heat-stressing, also resulting in improved recovery of
sincalide. Preferred concentrations in lyophilized formulations of
the invention are: methionine (4.0 mg/vial), lysine (15.0 mg/vial)
and arginine (30.0 mg/vial).
EXAMPLE 7
[0147] Reconstituted Shelf-Life Studies
[0148] A. In-Vial Post-Reconstitution Stability
[0149] Experiments were performed to determine the
post-reconstitution stability of sincalide in terms of appearance,
solubility, particulate matter, color, pH, sincalide assay,
desulfated sincalide assay and other sincalide-related impurities
through 8 hours at ambient temperature. Lyophilized vials from
three 105-L scale-up pilot batches of sincalide formulations were
reconstituted with 5.0 mL of purified water.
[0150] Testing was conducted at 0, 2, 4, 6, and 8 hours
post-reconstitution for appearance, solubility, particulate matter,
color, and pH. Testing was conducted on duplicate vials at 0, 4,
and 8 hours post-reconstitution for sincalide assay, desulfated
sincalide assay and other sincalide-related impurities using
reversed-phase HPLC with gradient elution and UV detection at 215
nm.
[0151] The test results for appearance, solubility, particulate
matter, color, and pH performed at 0, 2, 4, 6, and 8 hours
post-reconstitution for the three sincalide formulation
preparations are shown in Table 27.
27TABLE 27 Post Reconstitution Test Results Time Solubility
Particulate pH Preparation (hr) Appearance (sec.) Matter Color
meter 1 meter 2 A 0 Clear 20 Complies Colorless 7.2 7.0 2 Clear 20
Complies Colorless 7.1 7.0 4 Clear 20 Complies Colorless 7.2 7.0 6
Clear 20 Complies Colorless 7.2 7.0 8 Clear 20 Complies Colorless
7.2 7.0 B 0 Clear 20 Complies Colorless 7.2 7.0 2 Clear 20 Complies
Colorless 7.1 7.0 4 Clear 20 Complies Colorless 7.2 7.0 6 Clear 20
Complies Colorless 7.1 7.0 8 Clear 20 Complies Colorless 7.1 7.0 C
0 Clear 20 Complies Colorless 7.1 7.0 2 Clear 20 Complies Colorless
7.1 7.0 4 Clear 20 Complies Colorless 7.1 7.0 6 Clear 20 Complies
Colorless 7.1 7.0 8 Clear 20 Complies Colorless 7.1 7.0
[0152] For the three preparations examined (referred to herein as
preparations A, B, and C), no changes were observed in the
parameters tested and all results were within specifications
through the 8-hour testing period (85 mg/mL mannitol;
2.5.times.10.sup.-6 mg/mL TWEEN.RTM.20; 4.5 mg/mL KH.sub.2PO.sub.4;
1.0 mg/mL DTPA, 0.02 mg/mL sodium metabisulfite, 2.0 mg/mL
methionine, 7.5 mg/mL lysine, 15.0 mg/mL arginine, 0.0025 mg/mL
sincalide (Bulk formulation). The HPLC test results for sincalide
assay, desulfated sincalide assay and other sincalide-related
impurities performed at 0, 4, and 8 hours post-reconstitution for
the three formulation preparations are shown in Table 28.
28TABLE 28 Post Reconstitution HPLC Test Results Desulfated
Sincalide Related Time Sincalide Sincalide Impurities Preparation
(h) (.mu.g/vial) (w/w % sincalide) (% Impurity Index) A 0 4.99,
4.98 0.32, 0.33 1.41, 1.32 4 4.99, 4.97 0.32, 0.36 1.40, 1.35 8
4.97, 4.97 0.35, 0.39 1.40, 1.34 B 0 5.04, 5.04 0.28, 0.27 1.29,
1.37 4 5.04, 5.03 0.30, 0.29 1.30, 1.39 8 5.03, 5.01 0.31, 0.31
1.44, 1.41 C 0 4.97, 4.94 0.36, 0.36 1.48, 1.33 4 4.97, 4.94 0.39,
0.37 1.41, 1.37 8 4.97, 4.92 0.44, 0.44 1.46, 1.41
[0153] All results were within specifications. The sincalide
potency was unchanged over time. The desulfated sincalide and other
sincalide-related impurities show only relatively minor increases
which are insignificant with respect to their individual
specifications of 2% and 5%, respectively. The study shows that the
initial test values of reconstituted sincalide formulations are
representative of results obtained throughout the 8-hour shelf life
of reconstituted product. The data provided demonstrate the
post-reconstitution stability of the formulation and support a
post-reconstitution shelf-life of 8 hours under ambient
conditions.
[0154] Post-Reconstitution Dilution Study
[0155] An experiment was performed to determine the stability of
sincalide formulations of the present invention that have been
reconstituted and diluted.
[0156] Duplicate vials from three 105-L batches of sincalide
formulations of the invention were reconstituted with 5 mL water.
Vial contents were quantitatively transferred (using Sodium
Chloride Injection USP to rinse) to 100-mL volumetric flasks and up
to 8.4 mL of the formulations were diluted to volume (100 mL) with
Sodium Chloride Injection USP. Sincalide potency, pH and visual
appearance were tested 1-hour post preparation. The results of the
testing are presented in Table 29.
29TABLE 29 Results for Sincalide Formulations to 100 mL With 0.9%
Saline at 1 Hour Post-Reconstitution Sincalide Sample Potency
Particulate Preparation No. (.mu.g/vial) pH Appearance Color Matter
A 1 4.8 6.9 Clear Colorless Free of particles 2 5.0 6.9 Clear
Colorless Free of particles B 1 5.2 6.9 Clear Colorless Free of
particles 2 4.9 6.8 Clear Colorless Free of particles C 1 4.9 6.9
Clear Colorless Free of particles 2 4.9 6.8 Clear Colorless Free of
particles Mean 5.0 6.9 Std. Dev. 0.1 0.1 Confidence Interval
4.8-5.1 6.8-6.9 (p = 0.95 and 5 deg. Freedom) CV (%) 2.8 0.8
[0157] All sincalide potency, pH and appearance results for diluted
samples (reconstituted vial contents further diluted to 100 mL)
measured 1-hour post reconstitution were within the product
specifications for the reconstituted product (vial reconstituted
with 5 mL water).
EXAMPLE 8
[0158] Sincalide Specific Assay Using HPLC
[0159] An HPLC method was developed and validated for the
determination of sincalide potency, quantitation of desulfated
sincalide impurity and determination of a sincalide-related
impurity index in KINEVAC.RTM.. The method is suitable for
determining the reconstituted stability of KINEVAC.RTM. when
reconstituted as per the product package insert. The reversed phase
method employs a C.sub.18 (5 .mu.m, 300 .ANG.) column, stepwise
gradient elution with mobile phase components consisting of 0.15%
trifluoroacetic acid in water (solvent A) and 0.125%
trifluoroacetic acid in acetonitrile (solvent B), and UV detection
at 215 nm.
[0160] FIG. 12 shows representative full-scale and expanded-scale
chromatograms of a lyophilized reformulation of KINEVAC.RTM. upon
reconstitution with 5 mL water, resulting in a sincalide
concentration of 1 .mu.g/mL.
[0161] Other Embodiments
[0162] Although the present invention has been described with
reference to preferred embodiments, one skilled in the art can
easily ascertain its essential characteristics, and without
departing from the spirit and scope thereof can make various
changes and, modifications of the invention to adapt it to various
usages and conditions. Those skilled in the art will recognize or
be able to ascertain using no more than routine experimentation,
many equivalents to the specific embodiments of the invention
described herein. Such equivalents are encompassed by the scope of
the present invention.
[0163] All publications, patents, and applications mentioned in
this specification are herein incorporated by reference.
* * * * *