U.S. patent application number 16/551588 was filed with the patent office on 2020-07-16 for stabilization of radiopharmaceutical compositions using ascorbic acid.
This patent application is currently assigned to Lantheus Medical Imaging, Inc.. The applicant listed for this patent is Lantheus Medical Imaging, Inc.. Invention is credited to James E. Anderson, James F. Castner, Dianne D. Zdankiewicz.
Application Number | 20200222562 16/551588 |
Document ID | / |
Family ID | 42983052 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200222562 |
Kind Code |
A1 |
Castner; James F. ; et
al. |
July 16, 2020 |
STABILIZATION OF RADIOPHARMACEUTICAL COMPOSITIONS USING ASCORBIC
ACID
Abstract
Radiopharmaceutical compositions, and related methods, useful
for medical imaging are provided. The radiopharmaceutical
compositions include one or more radiopharmaceutical compounds,
together with a stabilizer comprising ascorbic acid, wherein the pH
of said composition is within the range of about 3.5-5.5.
Inventors: |
Castner; James F.; (Groton,
MA) ; Zdankiewicz; Dianne D.; (Londonderry, NH)
; Anderson; James E.; (Hudson, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lantheus Medical Imaging, Inc. |
North Billerica |
MA |
US |
|
|
Assignee: |
Lantheus Medical Imaging,
Inc.
North Billerica
MA
|
Family ID: |
42983052 |
Appl. No.: |
16/551588 |
Filed: |
August 26, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15602141 |
May 23, 2017 |
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16551588 |
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13264276 |
Jun 7, 2012 |
9687571 |
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PCT/US2010/001120 |
Apr 15, 2010 |
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15602141 |
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61169353 |
Apr 15, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/375 20130101;
A61K 51/0421 20130101; A61K 51/04 20130101; A61P 39/06 20180101;
A61K 51/0453 20130101; A61K 31/50 20130101; A61K 51/0455 20130101;
A61K 51/0459 20130101; A61K 31/50 20130101; A61K 2300/00 20130101;
A61K 31/375 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 51/04 20060101
A61K051/04; A61K 31/50 20060101 A61K031/50; A61K 31/375 20060101
A61K031/375 |
Claims
1-39. (canceled)
40. A composition, comprising: one or more radiopharmaceutical
compounds of formula: ##STR00008## wherein: X is O, S, or NR; Y is
O, S, NR, or CH.sub.2; R is H or Me; m is 0, 1, 2, or 3; n is 0, 1,
2, or 3; and R.sub.1 and R.sub.2 are hydrogen or C.sub.1-10-alkyl,
together with a stabilizer comprising ascorbic acid, wherein the pH
of said composition is within the range of 3.5 to less than 6; and
wherein the composition comprises greater than 20 mg of ascorbic
acid per milliliter.
41. The composition of claim 40, wherein the composition comprises
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one: ##STR00009## together with a stabilizer
comprising ascorbic acid, wherein the pH of said composition is
within the range of 3.5 to less than 6, and wherein the composition
comprises greater than 40 mg of ascorbic acid per milliliter.
42. The composition of claim 40, wherein said pH is within the
range of 5.5 to less than 6, or wherein said pH is 5.8.
43. The composition of claim 40, wherein said radiopharmaceutical
compound is
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one: ##STR00010##
44. The composition of claim 40, wherein the composition comprises
greater than 30 mg of ascorbic acid per milliliter, or greater than
40 mg of ascorbic acid per milliliter, or greater than 50 mg of
ascorbic acid per milliliter, or greater than 100 mg of ascorbic
acid per milliliter, or greater than 200 mg of ascorbic acid per
milliliter, or between 50 and 200 mg of ascorbic acid per
milliliter, or between 50 and 500 mg of ascorbic acid per
milliliter.
45. The composition of claim 40, wherein the composition comprises
greater than 40 mg of ascorbic acid per milliliter.
46. The composition of claim 40, wherein said radiopharmaceutical
compound is
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one: ##STR00011## wherein the composition comprises
50 mg of ascorbic acid per milliliter.
47. The composition of claim 46, wherein the composition has a pH
of 5.8.
48. The composition of claim 40, wherein the composition comprises
greater than 40 mg of ascorbic acid per milliliter, or greater than
50 mg of ascorbic acid per milliliter, or between 50 and 200 mg of
ascorbic acid per milliliter.
49. A method for preparing a composition of claim 40, comprising:
adding a first solution comprising a radiopharmaceutical compound
of formula: ##STR00012## wherein: X is O, S, or NR; Y is O, S, NR,
or CH.sub.2; R is H or Me; m is 0, 1, 2, or 3; n is 0, 1, 2, or 3;
and R.sub.1 and R.sub.2 are hydrogen or C.sub.1-10alkyl, to a
second solution comprising ascorbic acid within a pH range of 3.5
to less than 6, to form the radiopharmaceutical composition
comprising the compound and ascorbic acid, wherein the
radiopharmaceutical composition comprises greater than 20 mg of
ascorbic acid per milliliter.
50. The method of claim 49, wherein the composition comprises
greater than 40 mg of ascorbic acid per milliliter, or greater than
50 mg of ascorbic acid per milliliter, or between 50 and 200 mg of
ascorbic acid per milliliter, the method further comprising the
step of adjusting the pH of the radiopharmaceutical composition to
less than 6.
51. The method of claim 49, wherein the radiopharmaceutical
compound is (a) purified by chromatography, prior to addition of
the first solution to the second solution, or (b) not purified by
chromatography, prior to addition of the first solution to the
second solution.
52. A method of myocardial imaging a patient, comprising:
administering to the patient the composition of claim 40; and
obtaining a myocardial image of the patient.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/264,276, filed Jun. 7, 2012, which is a national stage
filing under 35 U.S.C. .sctn. 371 of International Application No.
PCT/US2010/001120, filed Apr. 15, 2010, which was published under
PCT Article 21(2) in English, and which claims priority under 35
U.S.C. .sctn. 119 from U.S. Provisional Application Ser. No.
61/169,353, filed Apr. 15, 2009, the entire contents of both of
which are incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention is directed to the stabilization of
radiopharmaceutical compositions, and the protection thereof from
radiolytic and propagative radical decomposition. In particular,
the invention is directed to the use of an antioxidant species in a
radiopharmaceutical formulation via buffering of the composition.
The invention moreover is concerned with the use of the antioxidant
ascorbic acid, under buffered conditions in a particular pH range,
to stabilize a radiopharmaceutical composition useful for medical
imaging, and thereby enhance the shell life of the composition,
while maintaining the composition as suitable for administration to
a human, and other mammalian subjects.
BACKGROUND OF THE INVENTION
[0003] Radiopharmaceuticals are drugs containing a radionuclide.
Radiopharmaceuticals are used routinely in nuclear medicine for the
diagnosis or therapy of various diseases. They are typically small
organic or inorganic compounds with a definite composition. They
can also be macromolecules, such as antibodies or antibody
fragments that are not stoichiometrically labeled with a
radionuclide.
Radiopharmaceuticals form the chemical basis for the diagnosis and
therapy of various diseases. The in vivo diagnostic information can
be obtained by intravenous injection of the radiopharmaceutical and
determination of its biodistribution using a gamma camera or a PET
camera. The biodistribution of the radiopharmaceutical typically
depends on the physical and chemical properties of the radiolabeled
compound and can be used to obtain information about the presence,
progression, and state of disease.
[0004] Radiopharmaceuticals can generally be divided into two
primary classes: those whose biodistribution is determined
exclusively by their chemical and physical properties, and those
whose ultimate distribution is determined by their receptor binding
or other biological interactions. The latter class is often
described as being target-specific.
[0005] Recently, much effort has been expended on the discovery and
development of radiopharmaceuticals for diagnostic imaging which
contain positron emitting isotopes. Positron emitting isotopes
include .sup.82Rb, .sup.124I, .sup.11C, .sup.13N, and .sup.18F,
among others. These isotopes decay by the emission of a positron
from the nucleus. A positron is a particle that has an equivalent
mass of an electron, but a corresponding positive charge. The
positron, after ejection from the nucleus, travels until it
encounters an electron, and the reaction of the two results in a
physical annihilation of the masses. Energy is released in opposing
directions at a value of 511 kEv, and because the annihilation has
no angular momentum, the photons are projected from the point of
annihilation approximately 180 degrees apart, allowing for precise
determination of a line along which the said decomposition
occurred. This property results in exquisite sensitivity and
resolution, and allows for superb image reconstruction and
quality.
[0006] An advantage of the carbon, nitrogen and fluorine isotopes
is that they may be incorporated into small organic molecules, such
as known or investigational pharmaceuticals that could be used to
determine biodistribution of the agent, as well as diagnose the
presence, absence or extent of disease. They may conveniently be
inserted into these molecules by a variety of methods known to
organic chemists and radiochemists ordinarily skilled in the art.
Widespread use in investigational research has been made of
.sup.11C-methyl iodide (.sup.11CH.sub.3I), methylating an alcohol
or an amine to produce the corresponding ether or alkyl amine.
These compounds are then appropriately sterilized, formulated and
injected into a subject.
[0007] The primary drawback to the widespread use of many PET
radiopharmaceuticals is the relatively short half lives associated
with many of the isotopes. Rubidium-82, carbon-11, and nitrogen-13
have half-lives of 1.27, 20.3, and 9.97 minutes, respectively.
Rubidium is administered as the chloride salt from a
.sup.82Sr--.sup.82Rb generator, and is not synthetically modified
or manipulated. Nitrogen-13 is typically administered as ammonia
(.sup.13NH.sub.3) produced in a cyclotron adjacent to an imaging
center with appropriate proximity to a camera. Both .sup.11C- and
.sup.13N-based reagents have been used in the radiolabeling of
imaging agents. Significant engineering and logistical challenges
need to be met to allow for the use of the compounds as
radiopharmaceuticals given the short half life and the necessary
time to accomplish the required reactions and purification prior to
formulation and administration of the drug.
[0008] Correspondingly longer-lived positron emitting isotopes may
be incorporated into new radiotracers for imaging. These include
the aforementioned .sup.131I and .sup.18F, with half-lives of 4.2
days and 107.9 minutes, respectively. The most prevalent use of
late has been .sup.18F, as the decay is entirely through the
emission of positrons and has a favorable half life. The
approximate two hours allows for synthetic incorporation into a
molecule, purification and subsequent distribution from a centrally
located radiopharmacy, obviates the requirement/investment in
either an on-site cyclotron or the monthly purchase of a
.sup.82Sr--.sup.82Rb generator.
[0009] During the course of manufacture, formulation, release, and
delivery of doses, the isotope typically decays at a zero-order
rate dictated by the physics of each particular isotope. However,
this decay can also trigger chemical decay of the dose, by
radiolysis. This can propagate via radical reaction and seriously
diminish the quality of the composition.
[0010] Decomposition of the radiopharmaceutical composition prior
to or during administration can dramatically decrease the targeting
potential and increase the toxicity of the therapeutic
radiopharmaceutical composition. Thus, in some cases, it is
important to ensure that the radionuclide is linked to the
targeting moiety, and to further ensure that specificity of the
targeting agent is preserved.
[0011] Radiolysis is caused by the formation of free radicals such
as hydroxyl and superoxide radicals (Garrison, W. M. Chem. Rev.
1987, 87, 381-398). Free radicals are very reactive towards organic
molecules. The reactivity of these free radical towards organic
molecules can affect the solution stability of a
radiopharmaceutical composition. Stabilization of the
radiopharmaceutical composition is a recurrent challenge in the
development of target-specific radiopharmaceuticals, and radical
scavengers are often employed as a stabilizer to minimize
radiolysis of the radiolabeled molecules. Some stabilizers are
"radical scavenging antioxidants" that readily react with hydroxyl
and superoxide radicals. The stabilizing agent for
radiopharmaceutical compositions may advantageously possess the
following characteristics: low or essentially no toxicity when it
is used for human administration, low or essentially no
interference with the delivery or receptor binding of the
radiolabeled compound to the target cells or tissue(s), and/or the
ability to stabilize the radiopharmaceutical for a reasonable
period of time (e.g., during the preparation, release, storage and
transportation of the radiopharmaceutical).
[0012] Radical scavengers such as ascorbic acid have been used to
stabilize .sup.99mTc (DeRosch, et al, WO95/33757) and
.sup.186/188Re (Anticancer Res. 1997, 17, 1783-1796)
radiopharmaceuticals. U.S. Pat. No. 5,393,512 discloses the use of
ascorbic acid as a stabilizing agent for .sup.186Re and
.sup.131I-labeled antibodies or antibody fragments. U.S. Pat. Nos.
5,093,105 and 5,306,482 disclose the use of ascorbic acid as an
antioxidant for .sup.99mTc radiopharmaceuticals.
[0013] Several strategies have been developed for the use of
antioxidants such as ascorbic acid to terminate decay pathways
prior to significant damage occurring. Ascorbic acid has been used
in various pharmaceutical and radiopharmaceutical compositions.
Unlike other buffering agents such as succinic acid and
aminocarboxylates, ascorbic acid contains no amino or carboxylic
groups. PCT/US94/06276 discloses stabilizing agents such as
ascorbic acid and water soluble salts and esters of ascorbic
acid.
[0014] U.S. Pat. No. 6,066,309 discloses the use of ascorbic acid
and derivatives thereof in stabilizing radiolabeled proteins and
peptides against oxidative loss of radiolabels and autoradiolysis.
In some cases, ascorbic acid is added after radiolabeling,
including any required incubation period, but prior to patient
administration. In addition, derivatives of ascorbic acid are
defined as salts of ascorbic acid, esters of ascorbic acid, or
mixtures thereof.
[0015] Although the use of ascorbic acid/ascorbate as a stabilizer
has been disclosed for a variety of diagnostic and therapeutic
radiopharmaceutical compositions (see, e.g., Deausch, E. A. et
al./U.S. Pat. No. 5,384,113/1995; Vanderheyden, J.-L., et al./U.S.
Pat. No. 5,393,512/1995; Flanagan, R. J. and Tartaglia, D./U.S.
Pat. No. 5,093,105/1992; Tartaglia, D. and Flanagan, R. J./U.S.
Pat. No. 5,306,482/1994; Shochat, D. et al./U.S. Pat. No.
5,961,955/1999; and Zamora, P. O. and Merek, M. J./U.S. Pat. No.
6,066,309/2000), there has been little or no disclosure regarding
the use of ascorbate within a specified range of pH to enhance the
antioxidant action of the compound for clinical applications.
[0016] While significant use of antioxidants such as ascorbic acid
have been exemplified in the literature, little attention has been
paid to the state of the antioxidant, e.g., as when adding it into
a buffered solution for stability studies at low pH or at higher pH
for material suitable for injection.
[0017] Material suitable for injection in humans may be selected to
have a pH higher than 4.0 to reduce the risk of localized
irritation and pain associated with a strongly acidic solution at
an injection site. Typically, solutions for injection have been
buffered by phosphate (phosphate buffered saline (PBS)) in the pH
range of 6-8. However, the employment of ascorbic acid/ascorbate in
buffered solutions at typical biological pH ranges (6-8) often
exhibits a lower ability to stabilize radiopharmaceutical
solutions. Conversely, while previous work may demonstrate
stability of radiopharmaceutical preparations using ascorbic acid
at low pH values (2-3), such formulations are generally not
suitable for use in animal models or humans due to localized
reactions, as noted above. In addition, previous work may set forth
a broad acidic pH range for the ascorbic acid than is useful, or
specify no particular range at all. To date, it is believed that
there has been little guidance for the skilled artisan in selecting
pH when using ascorbic acid for clinical applications of
radiopharmaceuticals.
[0018] Accordingly, improved compositions and methods are
needed.
SUMMARY OF THE INVENTION
[0019] The present invention provides for the use of ascorbic acid
as a stabilizer in a pH range. The agents and stabilizers are
formulated in ethanol-aqueous or aqueous buffer such that the
solution is preferably in the acidic pH range of about 3.5-5.5,
more preferably in the range of about 4-5, and most preferably in
the range of about 4-4.5.
[0020] Thus, in some embodiments, the invention provides a
composition, comprising one or more radiopharmaceutical compounds,
together with a stabilizer comprising ascorbic acid, wherein the pH
of said composition is within the range of about 3.5-5.5. The
radiopharmaceutical compounds as part of the composition of the
invention may be selected from the group consisting of rotenone,
pyridaben, fenazaquin, fenpyroximate, tebufenpyrad, piericidins,
and 2-substituted chromones, and analogs thereof. In some
embodiments, said radiopharmaceutical compound is at least one
member selected from the group consisting of pyridaben and analogs
thereof. In some embodiments, said radiopharmaceutical compound is
at least one member selected from the group consisting of compounds
containing a 2-alkyl-4-chloro-2H-pyridazin-3-one with a lipophilic
side chain substituted at the 5-position. In some embodiments, said
radiopharmaceutical compound is
2-tert-Butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one.
[0021] In some embodiments, said radiopharmaceutical compound is
labeled with a radioisotope, such as a radioisotope is selected
from the group consisting of .sup.11C, .sup.13N, .sup.18F,
.sup.86Br, .sup.124I, .sup.125I, and .sup.131I. In some
embodiments, said radioisotope is selected from the group
consisting of .sup.11C, .sup.13N, and .sup.18F. In some
embodiments, said radioisotope is .sup.18F.
[0022] In any of the foregoing embodiments, the radiopharmaceutical
composition comprises between about 5 and 100 mg/mL of ascorbic
acid, more preferably between about 25 and 500 mg/mL, and more
preferable between about 50 and 200 mg/mL. In some embodiments,
there is greater than about 5 mg, greater than about 10 mg, greater
than about 20 mg, greater than about 30 mg, greater than about 40
mg, greater than about 50 mg, greater than about 100 mg, or greater
than about 200 mg of ascorbic acid per milliliter.
[0023] The invention also provides a method for preparing a
composition as described in any of the foregoing embodiments, which
comprises adding a first solution containing a radiopharmaceutical
compound to a second solution containing ascorbic acid within the
pH range of about 3.5-5.5, more preferably within the range of
about 4-5, and even more preferably within the range of about
4-4.5, to form a third solution comprising the radiopharmaceutical
compound and ascorbic acid. In some embodiments, the
radiopharmaceutical compound is purified by chromatography, prior
to addition of the first solution to the second solution. In some
embodiments, the radiopharmaceutical compound is not purified by
chromatography, prior to addition of the first solution to the
second solution. In some embodiments, the method further comprises
the step of adjusting the pH of the third solution to about 6-8,
after addition of the first solution to the second solution and
prior to using the composition in a patient.
[0024] Further as part of the invention there is a method which
comprises administering to a patient a radiopharmaceutical
composition containing ascorbic acid, such that the composition has
a pH within the range of about 3.5-5.5, more preferably within the
range of about 4-5, and even more preferably within the range of
about 4-4.5.
[0025] The present invention is directed to these, as well as other
important ends, hereinafter described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 shows a plot of radiochemical purity of various
compositions of the invention, as a function of time.
[0027] FIG. 2 shows a plot of the rate of radiochemical impurity
formation for various compositions of the invention at a pH of (a)
4.0, (b) 8.2, (c) 6.3, (d), 5.4, (e) 6.0, and (f) 4.5.
[0028] FIG. 3 shows a plot of radiochemical purity of a series of
solutions comprising ascorbic acid at a concentration of (a) 20
mg/mL (|p|>0.001), (b) 50 mg/mL, (c) 100 mg/mL, (d) and 200
mg/mL.
DETAILED DESCRIPTION OF THE INVENTION
[0029] There are several advantages to using ascorbic acid as a
buffering agent. Ascorbic acid has been approved for pharmaceutical
and radiopharmaceutical applications. Ascorbic acid has a pKa of
4.2 and has buffering capacity at pH 3.0-5.0. At higher
concentrations (>50 mg/mL or 0.25 M), it may also have
sufficient buffering capacity at the pH range 5.5-6.0, or higher.
Typically, it is also employed as a primary buffer.
[0030] The invention is generally directed to novel compositions
(e.g., radiopharmaceutical compositions), and to the unforeseen and
dramatic increase in the antioxidant capacity and stabilizing
effect of the antioxidant ascorbic acid in the radiopharmaceutical
compositions at a certain pH range. At this pH, a significant
portion of the antioxidant is protonated, yet the acidity of the
solution is not so great to cause a severe reaction in the subject.
It is particularly suitable to perform manufacturing and storage
protocols under the conditions described herein, and adjusting to a
higher pH within 5, 10, or 15 minutes of administration to a
subject. In some embodiments, radiotracers (e.g., .sup.18F-labeled
radiotracers) utilizing ascorbic acid as a stabilizing agent and/or
as a clinical PET imaging agent are provided.
[0031] The invention advantageously provides radiopharmaceutical
formulations which utilize ascorbic acid as a stabilizer within a
certain pH range. The pH range enhances the stability and
shelf-life of the composition while minimizing severe localized
site reactions upon injection. In addition, some embodiments
utilize ascorbic acid as a stabilizing agent for the preparation of
labeled molecules, in particular .sup.18F-labeled molecules, in
radiopharmaceutical compositions. In some cases, ascorbic acid and
its analogs, within a certain pH range, can serve as a stabilizer
during preparation, release, and transportation of the
radiopharmaceutical composition, and in particular for those
compounds which are labeled with radioisotopes such .sup.18F.
[0032] The pH of the radiopharmaceutical compositions is selected
to lie at or near the pKa of either the primary or, in the case of
dibasic ions, the secondary pKa of the antioxidant. For ascorbic
acid, with a pK of 4.17, the pH may be selected to be in the range
of about 3.5-5.5, about 4-5, or 4-4.5.
[0033] Ascorbic acid is typically utilized as a stabilizing
component of the radiopharmaceutical composition of the invention.
Ascorbic acid is known as vitamin C, and has been used as an
antioxidant to prevent radiolytic decomposition of certain
radiopharmaceuticals (WO95/33757; Anticancer Res. 1997, 17,
1783-1796; U.S. Pat. Nos. 5,093,105, and 5,306,482) or radiolabeled
peptides (U.S. Pat. Nos. 5,393,512; 5,384,113 and 5,961,955). As
used herein, the term "ascorbic acid" includes ascorbic acid itself
as well as analogs and salts of the acid known to those of ordinary
skill in the art. Ascorbic acid is readily available GRAS
(generally recognized as safe) substance and can be used in
pharmaceutical compositions and other formulations used for
biological purposes, at levels as high as 200 mg/mL of the final
formulation. Previous compositions including ascorbic acid were
typically at pH values within biological pH range (e.g., 6-8)
during essentially all processing steps, as well as administration
to a subject, to reduce the risk of irritation and pain associated
with acidic solutions. However, within biological pH range, the
ability of ascorbic acid/ascorbate in buffered solutions to
stabilize radiopharmaceutical solutions is surprisingly
reduced.
[0034] Some advantages of using ascorbic acid or its analogs in a
radiopharmaceutical composition disclosed in this invention
include: (1) the ability to prepare radiopharmaceutical
compositions in high yield (>90%) and (2) the ability to store
the radiopharmaceutical compositions for several hours or even
days, while maintaining the radiochemical purity or RCP (>90%)
of the radiopharmaceutical. In some cases, ascorbate salts may be
added to the formulation. In some cases, ascorbic acid may be used
in the uncharged form, or in compositions in which a higher
percentage of ascorbic acid is protonated at the appropriate pH.
Without being bound by any particular theory, the efficacy of the
antioxidant may, in some cases, be directly related to the
non-ionic nature of the hydrogen-oxygen bonds in the antioxidant,
with enhanced stability at acidity levels wherein a significant
portion of the antioxidant is in protonated form.
[0035] In some embodiments, the radiopharmaceutical compositions
may include ascorbic acid as a stabilizer, in the absence of other
stabilizers compounds.
[0036] The invention contemplates radiopharmaceutical formulations
containing one or more of the hereinafter described myocardial
perfusion imaging agents or radiopharmaceutical compounds, together
with ascorbic acid, in the pH range as heretofore set forth.
[0037] Recently, several series of novel myocardial perfusion
imaging agents have been disclosed (Casebier, et al. U.S.
2007036716A1; Purohit & Casebier, U.S. 2006083681 A1; Radeke,
et al. U.S. 2005244332A1; Casebier, et al. W02005/079391A2) that
have highly desirable properties for potential clinical diagnostic
use. These agents are often prepared as radiotracers, and are often
labeled with the radioisotopes, such as the radioisotope
.sup.18F.
[0038] Some radiopharmaceutical compounds useful in the invention
can be potent inhibitors of mitochondrial complex 1 (MC-1), and
have potential clinical utility. These compounds may be
radiolabeled with a radiotracer (hereinafter described, such as
.sup.18F by way of illustration), and, therefore, stabilization of
the solution in such a manner as to prevent radiolytic initiated
decay may be desired. Several classes of compounds may be useful as
radiopharmaceutical compounds within the context of the invention,
as described more fully below.
[0039] For example, the natural product rotenone is a known
commercial insecticide and is widely used in commerce. The primary
mode of activity is via the inhibition of MC-1. The compound is
convenient for crop use due to its potency as well as its rapid
breakdown to benign products in the environment. Several analogs of
rotenone are known to inhibit MC-1 and some have been used in
non-human models of myocardial perfusion imaging, such as
dihydrofluorotenone (DHFR), for example.
##STR00001##
[0040] Another compound class that may be used for myocardial
perfusion imaging, and the solutions of which may be stabilized by
ascorbic acid is a class of chromone derivatives shown below. These
compounds are synthetic compounds that have shown good utility in
myocardial perfusion in primates, especially the specific compound
shown below.
##STR00002##
[0041] Another compound class that may be used for myocardial
perfusion imaging, and the solutions of which may be stabilized by
ascorbic acid are derivatives of a quinalzoline called fenzaquin.
Fenazaquin itself is a strong inhibitor of MC-1 and is used
commercially as an insecticide. Radiolabeled derivatives of
fenazaquin and its analogs have shown good utility in imaging
myocardium perfusion in primates and other mammals. Fenazaquin and
its analogs are shown below, along with an especially preferred
specific compound for myocardial perfusion imaging.
##STR00003##
[0042] Similarly, analogs of other commercially useful MC-1
inhibitors are useful in this invention, such as tebufenpyrad and
analogs thereof, as shown below. The parent structure of these
compounds are commercially used as insecticides, but analogs of
them may be radiolabeled for use as myocardial perfusion imaging
agents.
##STR00004##
[0043] Similarly analogs of other commercially useful MC-1
inhibitors are useful in this invention, such as analogs of
fenpyroximate, as shown below. The parent structure of these
compounds are commercially used as insecticides, but analogs of
them may be radiolabeled for use as myocardial perfusion imaging
agents.
##STR00005##
[0044] Furthermore, analogs of the natural product piericidins, as
shown below are useful as compounds as part of the invention.
Piericidins are a class of compounds with variability in the
substation and side chain, but can generally be characterized as a
2-alkyl-4-hydroxypyridine. Typically, in piericidins the 3, 5, and
6 positions also are substituted with either alkyl or alkoxy
functionalities. Derivatives of these compounds and analogs may be
radiolabeled for use as myocardial perfusion imaging agents.
##STR00006##
[0045] Another class of compounds suitable for use in the invention
is based on the commercial compound pyridaben. In some cases, the
compound comprises a pyridazinone heterocycle attached via a
lipophilic side chain to a radioisotope, such as .sup.18F-fluoride.
These compounds may comprise a potent series of mitochondrial
complex 1 inhibitors. The potency is retained throughout
substitution of the groups X and Y for chalcogens, and the
tolerance of the side chain (groups m, n, and Y) is wide, with
branched and straight-chain groups of up to ten chain atoms still
affording potent activity. In some embodiments, the compound is
2-alkyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2H-pyr-
idazin-3-one. For example, the compound may be
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one.
##STR00007##
[0046] The compounds described herein may be prepared by methods
known to those skilled in the art of organic radiochemistry and
those familiar with the techniques used for the manufacture of such
radiopharmaceuticals as fluorodeoxyglucose (.sup.18F-FDG), for
example, the only currently approved 18-F radiotracer for human
imaging. The compounds may be purified prior to use and such
methods are exemplified within this application. Other methods are
readily available to the skilled artisan.
[0047] In some cases, the radiopharmaceutical compounds may include
an asymmetric center, i.e., an asymmetrically substituted atom.
Compounds containing an asymmetrically substituted atom may be
isolated in optically active or racemic forms. It is well known in
the art how to prepare optically active forms, including methods
such as resolution of racemic forms or synthesis from optically
active starting materials. Many geometric isomers of olefins,
C.dbd.N double bonds, and the like can also be present in the
compounds described herein, and all such stable isomers are
contemplated for use in the present invention. Cis and trans
geometric isomers of the compounds of the present invention are
described and may be isolated as a mixture of isomers or as
separated isomeric forms. All chiral, diastereomeric, racemic forms
and all geometric isomeric forms of a structure are intended,
unless the specific stereochemistry or isomeric form is
specifically indicated. All processes used to prepare compounds of
the present invention and intermediates made therein are considered
to be useful in the present invention.
[0048] As noted, the radiopharmaceutical compounds herein described
may contain alkyl substituents. As that term may be used herein,
"alkyl" and "alk" as may be employed herein alone or as part of
another group includes both straight and branched chain
hydrocarbons containing 1 to 20 carbons, preferably 1 to 10
carbons, more preferably 1 to 8 carbons, in the normal chain, such
as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl,
pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,
2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, the various
branched chain isomers thereof, and the like as well as such groups
including 1 to 4 substituents such as halo, for example F, Br, Cl
or I or CF.sub.3, alkyl, alkoxy, aryl, aryloxy, aryl(aryl) or
diaryl, arylalkyl, arylalkyloxy, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkylalkyl, cycloalkylalkyloxy, hydroxy,
hydroxyalkyl, acyl, alkanoyl, heteroaryl, heteroaryloxy,
cycloheteroalkyl, arylheteroaryl, arylalkoxycarbonyl,
heteroarylalkyl, heteroarylalkoxy, aryloxyalkyl, aryloxyaryl,
alkylamido, alkanoylamino, arylcarbonylamino, nitro, cyano, thiol,
haloalkyl, trihaloalkyl and/or alkylthio.
[0049] As heretofore noted, the radiopharmaceutical compounds used
herein also include "analogs" thereof. The term "analog" is meant
to include any compounds that are substantially similar in
structure or atom connectivity to the referred structure or
compound. These include compounds in which one or more individual
atoms have been replaced, either with a different atom, or with a
different functional group. The term analog implies a high degree
of homology, but also may include compounds that are intellectually
derived from such a structure. Thus, by way of illustration, an
analog of pyridaben may be taken as any compound containing a
2-alkyl-4-chloro-2H-pyridazin-3-one with a lipophilic side chain
substituted at the 5-position.
[0050] The radiopharmaceutical compounds as part of the present
invention are intended to include all isotopes of atoms occurring
in the present compounds. Isotopes include those atoms having the
same atomic number but different mass numbers. By way of general
example and without limitation, isotopes of hydrogen include
tritium and deuterium. Isotopes of carbon include C-13 and
C-14.
[0051] When a bond to a substituent is shown to cross a bond
connecting two atoms in a ring, then such substituent may be bonded
to any atom on the ring. When a substituent is listed without
indicating the atom via which such substituent is bonded to the
rest of the compound of a given formula, then such substituent may
be bonded via any atom in such substituent. Combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds.
[0052] The radiopharmaceutical compounds hereinabove described are
considered pharmaceutically acceptable. The phrase
"pharmaceutically acceptable" is employed herein to refer to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0053] The radiopharmaceutical compounds hereinabove described also
include pharmaceutically acceptable salts. As used herein,
"pharmaceutically acceptable salts" refer to derivatives of the
disclosed compounds wherein the parent compound is modified by
making acid or base salts thereof. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines; and alkali or
organic salts of acidic residues such as carboxylic acids. The
pharmaceutically acceptable salts include the conventional
non-toxic salts or the quaternary ammonium salts of the parent
compound formed, for example, from non-toxic inorganic or organic
acids. For example, such conventional non-toxic salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared
from organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,
hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, and isethionic.
[0054] The pharmaceutically acceptable salts useful in the present
invention can be synthesized from the parent radiopharmaceutical
which contains a basic or acidic moiety by conventional chemical
methods. Generally, such salts can be prepared by reacting the free
acid or base forms of these compounds with a stoichiometric amount
of the appropriate base or acid in water or in an organic solvent,
or in a mixture of the two; generally, nonaqueous media like ether,
ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
Lists of suitable salts are found in Remington's Pharmaceutical
Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.
1418, the disclosure of which is hereby incorporated by
reference.
[0055] As heretofore set forth, the radiopharmaceutical compounds
herein utilized are preferably MC-1 inhibitors. The term "MC-1
inhibitor" refers to specific known compounds, and analogs of those
compounds which have the ability to inhibit MC-1. Specifically
compounds which may be radiolabeled with a suitable radioisotope
such that an image of myocardial tissue may be obtained by
administration of said compound to a patient, followed by scan the
patient in a suitable camera, be it PET, SPECT or planar capable.
Such inhibitors may include, but are not limited to, pyridaben and
its analogs, fenazaquin and its analogs, rotenone and its analogs,
deguelin and its analogs, and substituted chromone derivatives and
their analogs, including those illustrated above.
[0056] The radiopharmaceutical compounds of the invention are
preferably labeled with a suitable radioisotope. The term "suitable
radioisotope" refers to isotopes that may be covalently
incorporated into a molecule without detrimentally effecting the
biological potency, and possessing decay parameters, such as
sufficiently long half life, and suitable particle/emission energy
such that a satisfactory image may be obtained. Such radioisotopes
may include, but are not limited to, .sup.11C, .sup.13N, .sup.18F,
.sup.86Br, .sup.124I, .sup.125I, and .sup.131I. Of these, .sup.18F
is particularly preferred for use with the invention.
[0057] Radiolabeling is accomplished using materials and techniques
available to those skilled in the art. For example, radiolabeling
with fluorine may be accomplished by electrophilic fluorination,
using [.sup.18F] fluorine gas under appropriate conditions, but is
most preferably accomplished via nucleophilic displacement of an
appropriate leaving group by [.sup.18]-fluoride ion. The
[.sup.18F]-fluoride ion is rendered more reactive by the addition
of kryptates to sequester the potassium counterion. The preferred
leaving groups may be selected from those known to practitioners
ordinarily skilled in the art, but are preferably halogens,
including iodide, bromide, chloride and fluoride. Most preferably
the leaving group is a alkyl or aryl sulfonated ester, specifically
a toluenesulfonate ester.
[0058] In one set of embodiments, the radiopharmaceutical
composition comprises
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-be-
nzyloxy]-2H-pyridazin-3-one, together with a stabilizer comprising
ascorbic acid, wherein the pH of the composition is within the
range of about 4-4.5 and the radiopharmaceutical composition
comprises greater than about 50 mg of ascorbic acid per
milliliter.
[0059] The stabilized radiopharmaceutical formulations of the
invention may be prepared by addition of a first solution (e.g., an
aqueous solution or ethanolic solution) comprising a crude (e.g.,
unpurified) or purified radiopharmaceutical compound to a second,
prepared solution comprising ascorbic acid, to form a third
solution comprising the radiopharmaceutical compound and ascorbic
acid. The first solution may be an aqueous solution or an alcohol
solution, such as an ethanolic solution. In some cases, the second
solution is adjusted to the desired pH (e.g., pH in the range of
3.5-5.5) by addition of either an acidic solution (e.g.,
hydrochloric acid solution) or a basic solution (e.g., an aqueous
solution of sodium hydroxide), prior to contact with the first
solution.
[0060] Methods of the invention may include additional processing
steps. For example, after addition of the first solution to the
second solution, the third solution may be adjusted to a different
pH, such as a pH within biological range, i.e., about 6-8. In some
embodiments, the radiopharmaceutical composition comprises greater
than about 50 mg of ascorbic acid per milliliter, and the method
further comprises the step of adjusting the pH of the third
solution to about less than 6, after addition of the first solution
to the second solution.
[0061] In one set of embodiments, the method involves the addition
of a first solution comprising
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one, or a .sup.19F analog thereof, to a second
solution comprising ascorbic acid, wherein the second solution has
a pH within the range of about 4-4.5 and comprises greater than
about 50 mg of ascorbic acid per milliliter, to form a third
solution comprising the
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one and ascorbic acid.
[0062] In some embodiments, the method may include one or more
purification steps, such as purification by chromatography. For
example, the method can include purification of the
radiopharmaceutical compound via chromatography, i.e., prior to
addition to a solution comprising ascorbic acid. The chromatography
can be reverse-phase chromatography, regular-phase chromatography,
and/or ion exchange chromatography. In some embodiments, the
regular-phase chromatography may involve use of an alumina or
silica gel column. In some cases, methods of the invention may
involve use of a reverse phase HPLC column. For reverse phase
chromatography, the HPLC column may be eluted using a mobile phase
comprising water, acetonitrile, a buffer (e.g., ammonium acetate
buffer), an alcohol (e.g., methanol, ethanol) an acid (e.g., formic
acid), or mixtures thereof. In some cases, the HPLC column is a
reverse phase column and the column is eluted using a mobile phase
comprising ammonium acetate buffer, acetonitrile, ethanol, formic
acid, or mixtures thereof.
[0063] The typical radiopharmaceutical composition of the invention
comprises an aqueous solution containing not more than about 0-10%
ethanol by volume, and greater than about 5 mg of ascorbic acid per
milliliter. In some cases, the aqueous solution contains greater
than about 10 mg, greater than about 20 mg, greater than about 30
mg, greater than about 40 mg, greater than about 50 mg, greater
than about 100 mg, or, in some cases, greater than about 200 mg of
ascorbic acid per milliliter of dosage form. In some embodiments,
the aqueous solution also includes not more than about 20 mCi of a
radiopharmaceutical compound (e.g., about 10-20 mCi) and not more
than about 5 .mu.g of the cold, .sup.19F-analog of the radiotracer
(e.g., about 1-5 .mu.g) per each milliliter of dosage form.
Radiolysis is typically initiated by the addition of Na.sup.18F
into the solution.
[0064] Some aspects of the invention relate to the discovery that,
during development of radiopharmaceutical compositions according to
the invention for widespread manufacture, distribution and use,
ascorbic acid exhibits an enhanced ability to stabilize
radiopharmaceutical preparations at certain pH values. It was found
that at the pH values set forth herein, the radiopharmaceutical
preparations exhibited significantly higher stability against
decomposition. At higher pH values, the stabilization of these
solutions was markedly less effective. Comparison of the pH of the
ascorbic acid-containing solutions, the stability over a six hour
period, and the pKa of ascorbic acid revealed that the most
efficacious stabilization was in the range in which the oxidative
center on the stabilizer was protonated.
[0065] In some cases, the use of ascorbic acid or its analogs in
radiopharmaceutical compositions described herein can stabilize a
radiopharmaceutical such that high radiochemical purity (e.g.,
>90%, >95%, >97%) can be maintained during the essentially
the total lifetime of the radiopharmaceutical. For example, a
radiopharmaceutical including .sup.18F can be maintained at high
radiochemical purity for 1 hour or greater, 2 hours or greater, or,
in some cases, 5 hours or greater.
[0066] The invention also includes methods for administering a
radiopharmaceutical composition to a subject. In some cases, the
radiopharmaceutical composition contains ascorbic acid and has a pH
within the range of about 3.5-5.5. In some cases,
radiopharmaceutical composition contains ascorbic acid in an amount
greater than about 50 mg of ascorbic acid per milliliter and has a
pH that is less than about 6. In one set of embodiments, the
invention provides a method for administering to a patient a
radiopharmaceutical composition comprising
2-tert-butyl-4-chloro-5-[4-(2-[.sup.18F]fluoro-ethoxymethyl)-benzyloxy]-2-
H-pyridazin-3-one, ascorbic acid in an amount greater than about 50
mg of ascorbic acid per milliliter, wherein the radiopharmaceutical
composition has a pH that is less than about 6.
[0067] The compositions of the invention herein described may be
administered in the following manner, by way of illustration: A
catheter or heparin lock line is prepared into the vein of a
subject, and is flush with the appropriate saline and or heparin
solution. The dose is administered via luer-lock into the catheter
or heparin lock line. The patient is either in situ in a PET
camera, and imaging may commence immediately, or the patient is
allowed to rest for a time prior to being placed in a PET camera.
Alternatively, the patient, is dosed in a similar manner, via a
catheter or heparin lock, under treadmill or pharmacological
stress, using protocols similar to those known in the art.
[0068] The following examples utilize various embodiments of the
invention, but should not be construed as limiting the scope
thereof:
EXAMPLES
[0069] The integrity of a radiopharmaceutical is measured by the
radiochemical purity (RCP) of the radiolabeled compound using ITLC
or more preferably HPLC. The advantage of using HPLC is that
radio-impurities caused by radiolytic degradation can be separated
from the radiopharmaceutical under certain chromatographic
conditions. Improved stability over time for radiopharmaceutical
compositions of this invention can be demonstrated by determining
the change in RCP of the radiolabeled compound in samples taken at
representative time points. The radiopharmaceutical compositions of
this invention are effective in maintaining the stability of
samples for up to ten hours.
[0070] The initial RCP of a radiopharmaceutical is largely
dependent on radiolabeling conditions such as pH, heating
temperature and time. Once a radiopharmaceutical is prepared in
high yield, the stability of the radiopharmaceutical composition is
measured by the RCP change of the radiopharmaceutical over a
certain period of time.
[0071] Acetic acid (ultra-pure), ammonium hydroxide (ultra-pure),
and gentisic acid were purchased from either Aldrich or Sigma
Chemical Co., and were used as received. Hydrochloric acid
purchased from Fisher and sodium hydroxide (1 N solution) from VWR
were used for pH adjustment. Ascorbic acid (500 mg/mL, USP
injectable solution) was purchased from Myoderm Medical and diluted
with sterile water for injection (SWFI) as required. Sodium
[F-18]fluoride (Na.sup.18F) was purchased from Siemens Biomarker
Solutions as a salt deposited on a polymeric column support. The
fluoride was eluted from the column into a reaction flask or vial
using a solution of potassium carbonate (K.sub.2CO.sub.3) and
Kryptofix [222].
[0072] The following HPLC analytical methods may be used. HPLC
method 1 used a HP-1100 HPLC system with a UV/visible detector
(.lamda.=220 nm), an IN-US radio-detector, and a Zorbax C.sub.18
column (4.6 mm.times.250 mm, 80 .ANG. pore size). The flow rate was
1 mL/min with the mobile phase starting with 92% solvent A (0.025 M
ammonium acetate buffer, pH 6.8) and 8% solvent B (acetonitrile) to
90% solvent A and 8% solvent B at 18 min, followed by an isocratic
wash using 40% of solvent A and 60% solvent B from 19 to 25
min.
[0073] HPLC method 2 used a HP-1100 HPLC system with a UV/visible
detector (.lamda.=220 nm), an IN-US radio-detector, and a Zorbax
C.sub.18 column (4.6 mm.times.250 mm, 80 .ANG. pore size). The flow
rate was 1 mL/min with the mobile phase starting with 92% solvent A
(0.025 M ammonium acetate buffer, pH 6.8) and 8% solvent B
(acetonitrile) to 80% solvent A and 20% solvent B at 18 min,
followed by an isocratic wash using 40% of solvent A and 60%
solvent B from 19 to 25 min.
[0074] HPLC method 3 used a HP-1100 HPLC system with a UV/visible
detector (.lamda.=220 nm), an IN-US radio-detector, and a Zorbax
C.sub.18 column (4.6 mm.times.250 mm, 80 .ANG. pore size). The flow
rate was 1 mL/min with an isocratic mobile phase with 92% solvent A
(0.025 M ammonium acetate buffer, pH 6.8) and 8% solvent B
(acetonitrile) over 25 min, followed by an isocratic wash using 40%
of solvent A and 60% solvent B from 26 to 30 min.
[0075] HPLC method 4 used a HP-1100 HPLC system with an EG&G
Berthold Radioflow detector, and a Zorbax C.sub.18 column (4.6
mm.times.50 mm, 1.8 .mu.m particle size). The flow rate was 1
mL/min with the mobile phase of 50% acetonitrile/50% water in 0.1%
formic acid with a run time of 12 min.
[0076] The following examples describe the preparation and
purification of .sup.18F-labeled myocardial perfusion imaging
radiotracers. Using the following general procedure pyridaben,
fenazaquin and chromone analogs were prepared in good yields, and
formulated into stable radiopharmaceutical compositions.
Example 1: Synthetic Procedure for Preparation of .sup.18F
Myocardial Perfusion Imaging Radiotracer for pH Stabilization
Studies
[0077] Potassium carbonate (K.sub.2CO.sub.3, USP grade, 10 mg) was
dissolved in distilled/deionized water (H.sub.2O, 1 mL)and was
added with agitation to a solution of
4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane
(referred to as Kryptofix.TM., K222) in anhydrous acetonitrile
(CH.sub.3CN, 4 mL), and an aliquot of the resulting solution (1 mL)
was used to elute the .sup.18F-bearing resin column. The
radioactivity content of the column eluate was determined and the
elute was transferred to the reaction vessel of the Explora RN
Chemistry Module. This system was controlled by computer using the
GINA-Star software. The eluted complex solution was concentrated to
dryness (70-95.degree. C.), argon bleed; partial vacuum (250-12
mbar)). This afforded a relatively dry, highly activated form of
[.sup.18F] fluoride. The solution of the corresponding
toluenesulfonate ester of the desired radiotracer dissolved in 100%
acetonitrile was then added to the reaction vessel. The mixture was
heated at 90.degree. C. for 10 minutes.
Example 2: Purification of .sup.18F Myocardial Perfusion Imaging
Radiotracers and Preparation of Dose for pH Stabilization
Studies
[0078] After the reaction was complete, the acetonitrile was
evaporated (55.degree. C., argon bleed; partial vacuum (250-15
mbar)) and the reaction mixture was suspended in mobile phase (60%
acetonitrile/40% 50 mM ammonium acetate in water, 1.3 mL). The
mixture was drawn into a sample loop and injected onto a HPLC
column (Phenomenex Synergi 4.mu. Hydro-RP C18, 250.times.10 mm).
The mixture was purified via chromatography under isocratic
conditions (60% acetonitrile/40% 50 mM ammonium acetate in water, 5
ml/min, 36 min. run time). The radiosynthesis module (Explora RN
Chemistry Module) is equipped with both UV (254 nm) and
Geiger-Mueller (GM) detectors.
[0079] The fraction containing the labeled radio-tracer was
collected into a vial. Ascorbic acid solution having an ascorbic
acid concentration of 50mg/mL (10-15 mL) was added, and the
solution was passed through a Sep-Pak.RTM. cartridge (previously
conditioned with 10 mL of ethanol followed by 10 mL of the ascorbic
acid solution). The .sup.18F radiolabeled tracer adsorbs onto the
column and the aqueous eluate is discarded. The Sep-Pak.RTM. was
washed with an additional aliquot of ascorbic acid solution (10mL)
to remove any additional by products and residual acetonitrile. The
radio-tracer was then eluted with ethanol (.ltoreq.0.5 mL) and
added to a vial containing 9.5 mL of ascorbic acid solution.
Example 3: Determination of the Effect of pH Value on the
Stabilization of Radiotracer Dose Solutions
[0080] A series of ascorbic acid solutions at various pH values was
formulated, each solution containing 5 .mu.g/mL of a cold,
.sup.19F-analog of the radiopharmaceutical compound,
2-tert-butyl-4-chloro-5-[4-(2-[18F]fluoro-ethoxymethyl)-benzyloxy]-2H-pyr-
idazin-3-one (e.g., BMS-747158-01 (API)), ethanol/water (5/95), and
50 mg/mL ascorbic acid. The pH of each solution was adjusted by
addition of a stock aqueous solution of either hydrochloric acid or
sodium hydroxide. as required. The list of solutions is shown in
Table 1. Radiolysis was initiated by the addition of Na.sup.18F
into the solution containing the cold, .sup.19F-analog of the
radiopharmaceutical compound, and the solutions were monitored via
the HPLC analysis method for radiochemical purity over a (minimum)
6 hour period. The solutions were analyzed using a C18 RP-HPLC
column with a gradient mobile phase and the elution profile was
monitored using both UV and radiochemical detectors. The results
are shown in FIG. 1.
TABLE-US-00001 TABLE 1 Ascorbic acid solutions used in Example 3.
Solution Lot # pH A 070327 4.0 B 070328 5.8 C 070330 4.0 D 070403
4.0 E 070404 4.5 F 070418 4.6 G 070424 4.6 H 070425 4.6 I 070501
6.5 J 070502 2.4
[0081] As can be seen from the graph in FIG. 1, the purity of the
resultant solutions upon storage was directly dependent upon the pH
of the initial buffered dosage. Solutions at higher pH values
(closer to physiological pH of 7-7.5) had markedly less stability
to storage than did those with relatively more acidic values. This
is illustrated by the plots specifically with the solution pH
values at 5.8 (Solution B) and 6.5 (Solution I), respectively.
These are the two lowest plots on the graph shown in FIG. 1,
respectively.
[0082] Additional studies monitoring the formation of a
radiochemical impurity as a function of solution pH over a range of
4.0 to 8.2, as shown in FIG. 2. For each solution the formation of
radiochemical purity was monitored by HPLC, and the area of the
chromatographic peak corresponding to the radiochemical impurities
was plotted as a function of time. Solutions having a pH range
between 3.5.-5.5 exhibited greater stability relative to solutions
having a pH of 6.0 or greater, demonstrating a much slower rate of
formation of radiochemical impurity. The results shown in FIG. 2
further demonstrate the effect of improved formulation stability
under critical acidic conditions. Over the tested pH range the
observed 1.sup.st order reaction rates for the formation of the
radiochemical impurity is reduced by greater than a factor of
10.
Example 4: Determination of the Effect of Ascorbic Acid
Concentration on the Stabilization of Radiotracer Dose
Solutions
[0083] This example describes the effect of ascorbic acid
concentration on radiochemical purity. In this example, the
radiochemical purity (RCP) of the .sup.18F-labeled drug product
(2-tert-butyl-4-chloro-5-[4-(2-[18F]
fluoro-ethoxymethyl)-benzyloxy]-2H-pyridazin-3-one) was monitored
for solutions having an ascorbic acid concentration range from 200
mg/mL (saturation level) to 20 mg/mL, at pH 5.8. The results shown
in FIG. 3 indicate that the RCP levels do not significantly change
over the 200 to 50 mg/mL range, but an increase in impurities
(i.e., lower RCP level) was observed in the 20 mg/mL sample.
[0084] These examples are intended to illustrate the application of
the invention and are in no way limiting in the intent, application
and utility of the invention as set forth in the following
claims.
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