U.S. patent application number 10/598874 was filed with the patent office on 2007-12-20 for compositions of allosteric hemoglobin modifiers and methods of making the same.
This patent application is currently assigned to ALLOS THERAPEUTICS, INC.. Invention is credited to Alexandre J.G. G. Carvalho, Jeffrey B. Etter, Douglas G. Johnson, Chris Murray, Al Quick, Antonio M. Santos.
Application Number | 20070293698 10/598874 |
Document ID | / |
Family ID | 35197486 |
Filed Date | 2007-12-20 |
United States Patent
Application |
20070293698 |
Kind Code |
A1 |
Quick; Al ; et al. |
December 20, 2007 |
Compositions of Allosteric Hemoglobin Modifiers and Methods of
Making the Same
Abstract
The present invention provides novel compositions of allosteric
hemoglobin modifiers which are substantially free of impurities,
specifically polymeric impurities. In one embodiment, the novel
compositions contain an allosteric hemoglobin modifier compound and
less than 100 ppm of the polymeric impurities generated during the
preparation of this compound. Included in the present invention are
novel methods for preparing allosteric hemoglobin modifiers that
are substantially free of polymeric impurities. Also included in
the present invention are improved methods for the purification of
the product formed by the method of this invention. The novel
methods of purification comprise extracting the crude composition
with a water immiscible or partially immiscible solvent such as
methylisobutyl ketone (MIBK) to lower amounts of impurities,
specifically polymeric impurities. Also included are methods to
reduce impurities by recrystallization of the crude synthesized
product, followed by filtration of the recrystallized product. The
present invention also includes the products made by the processes
of the invention and methods for analyzing compositions comprised
of these products.
Inventors: |
Quick; Al; (Aurora, CO)
; Santos; Antonio M.; (Columbus, NJ) ; Carvalho;
Alexandre J.G. G.; (Lisboa, PT) ; Johnson; Douglas
G.; (Arvada, CO) ; Etter; Jeffrey B.;
(Boulder, CO) ; Murray; Chris; (Arlington,
MA) |
Correspondence
Address: |
SWANSON & BRATSCHUN, L.L.C.
8210 SOUTHPARK TERRACE
LITTLETON
CO
80120
US
|
Assignee: |
ALLOS THERAPEUTICS, INC.
11080 Circle Point Road, Suite 200
Westminster
CO
|
Family ID: |
35197486 |
Appl. No.: |
10/598874 |
Filed: |
April 22, 2005 |
PCT Filed: |
April 22, 2005 |
PCT NO: |
PCT/US05/13876 |
371 Date: |
May 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60564721 |
Apr 22, 2004 |
|
|
|
Current U.S.
Class: |
562/455 ;
436/161 |
Current CPC
Class: |
A61K 31/195
20130101 |
Class at
Publication: |
562/455 ;
436/161 |
International
Class: |
C07C 233/01 20060101
C07C233/01; C07C 231/00 20060101 C07C231/00; G01N 30/00 20060101
G01N030/00; C07C 233/00 20060101 C07C233/00 |
Claims
1. A composition comprising an allosteric effector compound, which
is substantially free of impurities wherein said allosteric
effector compound is selected from the group of compounds having
the following formula: ##STR23## wherein X and Z are independently
selected from the group consisting of CH.sub.2, CO, NH or O, and Y
is selected from the group consisting of CO or NH, with the caveat
that X, Y, and Z must all be different from each other; R.sub.5 and
R.sub.6 are independently selected from the group consisting of
hydrogen, halogen, substituted or unsubstituted C.sub.1-12 alkyl
groups, carboxylic acid and ester groups, substituted or
unsubstituted aromatic or heteroaromatic groups, or alkyl moieties
of part of an aliphatic ring connecting R.sub.5 and R.sub.6;
R.sub.7 is a selected from the group consisting of hydrogen, a
cationic counterion, selected from the group consisting of sodium,
potassium or ammonium, a metal, or a substituted or unsubstituted
C.sub.1-6 alkyl group; and R.sub.8-12 are independently selected
from the group consisting of hydrogen, halogen, substituted or
unsubstituted C.sub.1-3 alkyl groups, or alkyl moieties of an
aromatic or aliphatic ring incorporating two of the R.sub.8-12
sites.
2. The composition of claim 1, wherein said impurities are selected
from the group consisting of polymeric impurities and other related
impurities selected from the group consisting of poly (ethyl
methacrylate) (PEM), 3-monomethyl efaproxiral (3MMRS13),
.alpha.-desmethyl efaproxiral (DDMRS13), monomethyl a to COOH
(DMRS13), 3,4-dimethyl efaproxiral (3,4DMRS13),
.alpha.-ethyl-efaproxiral, diacid (DA), 3,5-dimethyl aniline
(3,5-DMA), amidophenol and the ethyl ester of efaproxiral.
3. The composition of claim 2, wherein said impurity is a polymeric
impurity selected from the group of compounds having the following
structure: ##STR24## wherein R, R', R'' and R''' are independently
selected from the group consisting of substituted or unsubstituted
C.sub.1-12 allyl group, hydrogen, halogen, a carboxylic acid or
ester group, and a substituted or unsubstituted heteroaromatic
group; and n is any number of units appropriate for a polymer of
repeating units.
4. The composition of claim 3, wherein R' is selected from a
substituted or unsubstituted C.sub.1-3 allyl group and R is a
methyl or ethyl group.
5. The composition of claim 2, wherein said polymeric impurity is
poly (ethyl methacrylate) (PEM).
6. The composition of claim 3, wherein said polymeric impurity is
present in the composition at less than about 500 ppm.
7. The composition of claim 3, wherein said polymeric impurity is
present in the composition at less than about 200 ppm.
8. The composition of claim 3, wherein said polymeric impurity is
present in the composition at less than about 100 ppm.
9. The composition of claim 3, wherein said polymeric impurity is
present in the composition at less than about 80 ppm.
10. The composition of claim 3, wherein said polymeric impurity is
present in the composition at less than about 10 ppm.
11. The composition of claim 2, wherein said related impurities are
each present in the composition at less than about 1000 ppm.
12. The composition of claim 2, wherein said related impurities are
each present in the composition at less than about 500 ppm.
13. The composition of claim 1, wherein said allosteric effector
compound is selected from the group of compounds having the
following chemical structure: ##STR25## wherein R.sub.5 and R.sub.6
are independently selected from the group consisting of hydrogen,
halogen, substituted or unsubstituted C.sub.1-12 alkyl groups,
carboxylic acid and ester groups, substituted or unsubstituted
aromatic or heteroaromatic groups or alkyl moieties of part of an
aliphatic ring connecting R.sub.5 and R.sub.6; R.sub.7 is a
selected from the group consisting of hydrogen, a cationic
counterion, selected from the group consisting of sodium, potassium
or ammonium, a metal, or a substituted or unsubstituted C.sub.1-6
alkyl group; and R.sub.8-12 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
14. The composition of claim 13, wherein R.sub.5 and R.sub.6 are
independently selected from H or CH.sub.3 and R.sub.7 is selected
from hydrogen or a cationic counterion.
15. The composition of claim 1, wherein said allosteric effector
compound is
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (efaproxiral) (5) ##STR26## wherein X is selected
from the group consisting of H or a cationic counterion selected
from the group consisting of sodium, potassium or ammonium.
16. A method for the preparation of a composition comprising an
allosteric effector compound that is substantially free of
polymeric impurities said method comprising the steps of: a)
coupling a substituted aniline with 4-hydroxyphenylacetic acid to
yield the corresponding substituted phenol; b) adding the product
of step (a) to an alkyl ester halide to yield a substituted ethyl
ester; and c) saponifying the substituted allyl ester to provide
the salt of the acid, wherein all steps are performed in a reaction
vessel that does not contain metals that promote the formation of
polymeric byproducts.
17. The method of claim 16, wherein said polymeric impurity is
selected from the group of compounds having the following
structure: ##STR27## wherein R, R', R'' and R''' are independently
selected from the group consisting of substituted or unsubstituted
C.sub.1-12 alkyl group, hydrogen, halogen, a carboxylic acid or
ester group, and a substituted or unsubstituted heteroaromatic
group; and n is any number of units appropriate for a polymer of
repeating units.
18. The method of claim 17, wherein R' is independently selected
from a substituted or unsubstituted C.sub.1-3 allyl group and R is
a methyl or ethyl group.
19. The method of claim 16, wherein said polymeric impurity is poly
(ethyl methacrylate) (PEM).
20. The method of claim 16, wherein said polymeric impurity is
present in the composition at less than about 500 ppm.
21. The method of claim 16, wherein said polymeric impurity is
present in the composition at less than about 200 ppm.
22. The method of claim 16, wherein said polymeric impurity is
present in the composition at less than about 100 ppm.
23. The method of claim 16, wherein said polymeric impurity is
present in the composition at less than about 80 ppm.
24. The method of claim 16, wherein said polymeric impurity is
present in the composition at less than about 10 ppm.
25. The method of claim 16, wherein said reaction vessel is
selected from the group consisting of glass lined stainless steel
(SS), passivated stainless steel, Hastelloy.RTM. or similar
alloys.
26. The method of claim 16, wherein said reaction vessel is
selected from a Hastelloy 276.RTM. reactor or a SS (316)
reactor.
27. The method of claim 16, wherein said allosteric effector
compound is selected from the group of compounds having the
following formula: ##STR28## wherein X and Z are independently
selected from the group consisting of CH.sub.2, CO, NH or O, and Y
is selected from the group consisting of CO or NH, with the caveat
that X, Y, and Z must all be different from each other; R.sub.5 and
R.sub.6 are independently selected from the group consisting of
hydrogen, halogen, substituted or unsubstituted C.sub.1-12 allyl
groups, carboxylic acid and ester groups, substituted or
unsubstituted aromatic or heteroaromatic groups, or alkyl moieties
of part of an aliphatic ring connecting R.sub.5 and R.sub.6;
R.sub.7 is a selected from the group consisting of hydrogen, a
cationic counterion, selected from the group consisting of sodium,
potassium or ammonium, a metal, or a substituted or unsubstituted
C.sub.1-6 alkyl group; and R.sub.8-12 are independently selected
from the group consisting of hydrogen, halogen, substituted or
unsubstituted C.sub.1-3 alkyl groups, or alkyl moieties of an
aromatic or aliphatic ring incorporating two of the R.sub.8-12
sites.
28. The method of claim 27, wherein said allosteric effector
compound is selected from the group of compounds having the
following chemical structure: ##STR29## wherein R.sub.5 and R.sub.6
are independently selected from the group consisting of hydrogen,
halogen, substituted or unsubstituted C.sub.1-12 alkyl groups,
carboxylic acid and ester groups, substituted or unsubstituted
aromatic or heteroaromatic groups or alkyl moieties of part of an
aliphatic ring connecting R.sub.5 and R.sub.6; R.sub.7 is a
selected from the group consisting of hydrogen, a cationic
counterion, selected from the group consisting of sodium, potassium
or ammonium, a metal, or a substituted or unsubstituted C.sub.1-6
alkyl group; and R.sub.8-12 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
29. The method of claim 28, wherein R.sub.5 and R.sub.6 are
independently selected from H or CH.sub.3 and R.sub.7 is selected
from hydrogen or a cationic counterion.
30. The method of claim 28, wherein said allosteric effector
compound is
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (efaproxiral) (5) ##STR30## wherein X is selected
from the group consisting of H or a cationic counterion selected
from the group consisting of sodium, potassium or ammonium.
31. The method of claim 16, wherein said method further comprises
extracting the product of step (c) with a solvent selected from the
group consisting of methyl isobutyl ketone, isopropyl acetate,
ethyl acetate, methyl ethyl ketone, chlorinated solvents selected
from the group consisting of chloroform and methylene chloride at
least once.
32. The method of claim 31, wherein said extraction is performed
two times.
33. The method of claim 16, wherein said method further comprises
recrystallization of the product of step (c) in an appropriate
solvent.
34. The method of claim 33, wherein said solvent is selected from
the group consisting of ethanol and acetone and mixtures of
acetone/ethanol, acetone/methanol and
ethanol/methanol/acetone/water.
35. The method of claim 16, wherein said method further comprises
extracting the product of step (c) with an appropriate solvent,
followed by recrystallization of the extracted product.
36. The method of claim 16, wherein said method further comprises
extracting the product of step (c) with an appropriate solvent,
followed by recrystallization of the extracted product and
filtration of the recrystallized product.
37. The method of claim 36, wherein said filtration is performed
using a polymeric filter selected from the group consisting of
poly(vinylidene difluoride) (PVDF) or a cellulose ester filter.
38. The method of claim 38, wherein said polymeric filter is
PVDF.
39. A product prepared according to the method of claim 16.
40. A product prepared according to the method of claim 35.
41. A product prepared according to the method of claim 36.
42. A method for preparing a composition of the allosteric effector
compound efaproxiral-Na that is substantially free of polymeric
impurities said method comprising the steps of: a) reacting a
solution of 4-hydroxyphenylacetic acid with 3,5-dimethylaniline to
yield amidophenol; b) reacting the product of step (a) with ethyl
2-bromoisobutyrate to yield the ethyl ester; and c) saponifiying
the product of (b) to yield a carboxylic acid salt, wherein all
steps are performed in a reaction vessel that does not contain
metals that promote the formation of polymeric byproducts.
43. The method of claim 42, wherein said reaction vessel is
selected from a Hastelloy 276.RTM. reactor, a SS (316) reactor or a
glass lined SS reactor.
44. The method of claim 42, wherein said method further comprises
extracting the product of step (c) with a solvent selected from the
group consisting of methyl isobutyl ketone, isopropyl acetate,
ethyl acetate, methyl ethyl ketone, chlorinated solvents selected
from the group consisting of chloroform and methylene chloride at
least once.
45. The method of claim 44, wherein said extraction is performed
two times.
46. The method of claim 42, wherein said method further comprises
recrystallization of the product of step (c) in an appropriate
solvent.
47. The method of claim 46, wherein said solvent is selected from
the group consisting of ethanol and acetone and mixtures of
acetone/ethanol, acetone/methanol and
ethanol/methanol/acetone/water.
48. The method of claim 42, wherein said method further comprises
extracting the product of step (c) with an appropriate solvent,
followed by recrystallization of the extracted product and
filtration of the recrystallized product.
49. The method of claim 48, wherein said filtration is performed
using a polymeric filter selected from the group consisting of PVDF
or a cellulose ester filter.
50. The method of claim 49 wherein said polymeric filter is
PVDF.
51. A product prepared according to the method of claim 42.
52. A product prepared according to the method of claim 48.
53. A method for the preparation of efaproxiral-Na (5) compound of
formula that is substantially free of impurities, said method
comprising the steps of: a) reacting a solution of
4-hydroxyphenylacetic acid with 3,5-dimethylaniline to yield
amidophenol; b) reacting the product of step (a) with ethyl
2-bromoisobutyrate to yield the ethyl ester; c) saponifiying the
product of (b) to yield a carboxylic acid salt; and d) extracting
the product of step (c) with an appropriate solvent at least one
time, wherein all steps are performed in a reaction vessel that
does not contain metals that promote the formation of polymeric
byproducts.
54. The method of claim 53, wherein the impurity is a polymeric
impurity.
55. The method of claim 54, wherein said polymeric impurity is
present in the composition at less than about 100 ppm.
56. The product prepared according to the method of claim 53.
57. A method for the preparation of efaproxiral-Na (5) compound of
formula that is substantially free of impurities, said method
comprising the steps of: a) reacting a solution of
4-hydroxyphenylacetic acid with 3,5-dimethylaniline to yield
amidophenol; b) reacting the product of step (a) with ethyl
2-bromoisobutyrate to yield the ethyl ester; c) saponifying the
product of (b) to yield a carboxylic acid salt; d) extracting the
product of step (c) with an appropriate solvent at least one time,
and e) recrystallizing the product of step (d) in an appropriate
solvent, wherein all steps are performed in a reaction vessel that
does not contain metals that promote the formation of polymeric
byproducts.
58. The method of claim 57, wherein the solvent of step (d) is MIBK
and the solvent of step (e) is selected from ethanol and/or
acetone.
59. The method of claim 57, wherein said polymeric impurity is
present in the composition at less than about 100 ppm.
60. The product prepared according to the method of claim 57.
61. A method for the preparation of efaproxiral-Na (5) compound of
formula that is substantially free of impurities, said method
comprising the steps of: a) reacting a solution of
4-hydroxyphenylacetic acid with 3,5-dimethylaniline to yield
amidophenol; b) reacting the product of step (a) with ethyl
2-bromoisobutyrate to yield the ethyl ester; c) saponifiying the
product of (b) to yield a carboxylic acid salt; d) extracting the
product of step (c) with an appropriate solvent at least one time;
e) recrystallizing the product of step (d) in an appropriate
solvent; and f) filtering the product of step (e) with a filter
that removes polymeric impurities, wherein all steps are performed
in a reaction vessel that does not contain metals that promote the
formation of polymeric byproducts.
62. The method of claim 61, wherein the solvent of step (d) is
MIBK, the solvent of step (e) is selected from ethanol and/or
acetone and the polymeric filter of step (f) is PVDF.
63. The method of claim 61, wherein the solvent of step (d) is
MIBK, the solvent of step (e) is selected from ethanol and/or
acetone and the polymeric filter of step (f) is modified
cellulose.
64. A product prepared according to the method of claim 62.
65. A method for analyzing a composition comprised of an allosteric
hemoglobin modifier compound said method comprising the steps of:
a) pyrolyzing said composition; b) analyzing said pyrolyzed product
by gas chromatography/mass spectrometry (GC/MS).
66. The method of claim 65 further comprising the step of adding an
internal standard to the composition prior to step a).
67. The method of claim 66 wherein said internal standard is
labeled with an isotope.
68. The method of claim 67 wherein said internal standard is
labeled with an isotope of an atom selected from the group
consisting of hydrogen, carbon, oxygen and nitrogen.
69. The method of claim 67 wherein said isotope is selected from
the group consisting of deuterium (D), carbon 13 (.sup.13C), oxygen
18 (.sup.18O) and nitrogen 15 (.sup.15N).
70. The method of claim 67 wherein said isotopically labeled
internal standard is deuterated PEM.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions of allosteric
hemoglobin modifier compounds having low levels of impurities. The
invention also relates to novel methods of preparing such
compositions. Included in the present invention are improved
methods for the purification of compositions of allosteric
hemoglobin modifier compounds. Also included in the present
invention is a method for analyzing compositions of allosteric
hemoglobin modifier compounds, which enables detection and
quantification of impurities.
BACKGROUND OF THE INVENTION
[0002] Hemoglobin is a tetrameric protein which delivers oxygen via
an allosteric mechanism. There are four binding sites for oxygen on
the hemoglobin molecule, as each protein chain contains one heme
group. Each heme group contains a substituted porphyrin and a
central iron atom. The iron atom in heme can be in the ferrous (+2)
or ferric (+3) state, but only the ferrous form binds oxygen. The
ferrous-oxygen bond is readily reversible. The binding of the first
O.sub.2 molecule to hemoglobin enhances the binding of additional
O.sub.2 to the same hemoglobin molecule. In other words, O.sub.2
binds cooperatively to hemoglobin. Thus, binding of the first
oxygen to a heme requires much greater energy than the second
oxygen molecule, binding of the third oxygen requires even less
energy, and the fourth oxygen requires the lowest energy for
binding. Hemoglobin A, the principal hemoglobin in adults consists
of two .alpha. and two .beta. subunits arranged with a two-fold
symmetry. The .alpha. and .beta. dimers rotate during oxygen
release to open a large central water cavity. The allosteric
transition that involves the movement of the alpha beta dimer takes
place between the binding of the third and fourth oxygen.
[0003] Using well-known equipment such as the AMINCO.TM.
HEM-O-SCAN, an oxygen dissociation curve can be plotted to
determine the affinity and degree of cooperativity (allosteric
action) of hemoglobin. In the plot, the Y-axis represents the
percent of hemoglobin oxygenation and the X-axis represents the
partial pressure of oxygen in millimeters of mercury (mm Hg). If a
horizontal line is drawn from the 50% oxygen saturation point and a
vertical line is drawn from the intersection point of the
horizontal line with the curve to the partial pressure X-axis, a
value commonly known as the P.sub.50 is determined. This is the
partial pressure (mm Hg) at which the hemoglobin sample is 50%
saturated with oxygen. Under physiological conditions (i.e.
37.degree. C., pH 7.4, and a partial pressure of carbon dioxide of
40 mm Hg), the P.sub.50 value for normal adult hemoglobin is around
26.5 mm Hg. If a lower than normal P.sub.50 value is obtained for
the hemoglobin being tested, the oxygen dissociation curve is
considered to be "left-shifted" and the presence of high affinity
hemoglobin is indicated. Conversely, if a higher than normal
P.sub.50 value is obtained for the hemoglobin being tested, the
oxygen dissociation curve is considered to be "right-shifted" and
the presence of low affinity hemoglobin is indicated. Such low
affinity hemoglobin will lose oxygen more easily at lower pressures
of oxygen, and therefore may be useful to deliver oxygen to tissues
more efficiently.
[0004] It has been suggested that influencing the allosteric
equilibrium of hemoglobin may be a viable method to treat diseases
that are influenced by oxygen delivery. For example, the conversion
of hemoglobin to a high affinity state is generally regarded to be
beneficial in treating problems associated with deoxyhemoglobin S
(sickle cell anemia.). The conversion of hemoglobin to a low
affinity state is believed to be of general utility in a variety of
disease states in which tissues suffer from low oxygen tension,
such as ischemia, radio-sensitization of tumors, carbon monoxide
poisoning, fetal oxygen delivery and the restoration of the oxygen
affinity of stored blood.
[0005] FIGS. 1A-1D depict the chemical structures of a variety of
compounds which have a "right-shifting" allosteric effect on
hemoglobin (referred to herein as "allosteric hemoglobin modifier
compounds" or "allosteric effector compounds"). The family of
compounds represented by the general structure illustrated in FIG.
1D (referred to as "RSR compounds"), are representative of a large
family of compounds having a strong allosteric effect. For example,
one compound in this family,
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (efaproxiral, also referred to as RSR13), which has
the following structure, when X.sup.+is H.sup.+: ##STR1## is an
allosteric effector of hemoglobin, and has been shown to enhance
tissue oxygenation in vivo. In general, efaproxiral is administered
as a physiologically acceptable salt, such as the monosodium salt;
that is, X.sup.+is Na.sup.+. Efaproxiral induces allosteric
modification of hemoglobin, such that its binding affinity for
oxygen is decreased, resulting in increased oxygen distribution to
tissues by erythrocytes. Efaproxiral has been reported to enhance
fractionated radiation therapy in mice bearing the Lewis lung
carcinoma. See Teicher (1996) Drug Dev. Res. 38:1-11. Enhancement
of the effect of radiation was observed in EMT6 mouse mammary
tumors by treatment with efaproxiral plus oxygen breathing, with
the absence of enhanced radiation effects in normal tissues.
Rockwell and Kelley (1998) Rad. Oncol. Invest. 6:199-208.
Additionally, mouse fibrosarcoma tumor growth has been shown to be
reduced by the combination of efaproxiral and radiation relative to
radiation alone. See Teicher (1996) Drug Dev. Res. 38:1-11;
Khandelwal et al. (1996) Rad. Oncol. Invest. 4:51-59. This family
of compounds, together with their utility and methods for using
them are described in a number of patents including, U.S. Pat. No.
5,661,182, issued Aug. 26, 1997, U.S. Pat. No. 5,290,803, issued
Mar. 1, 1994, U.S. Pat. No. 5,382,680, issued Jan. 17, 1995, U.S.
Pat. No. 5,432,191, issued Jul. 11, 1995, U.S. Pat. No. 5,648,375,
issued Jul. 15, 1997, U.S. Pat. No. 5,677,330, issued Oct. 14,
1997, U.S. Pat. No. 5,731,454, issued Mar. 24, 1998, U.S. Pat. No.
5,122,539, issued Jun. 16, 1992, U.S. Pat. No. 5,927,283, issued
Jul. 27, 1999, U.S. Pat. No. 5,827,888, issued Oct. 27, 1998, U.S.
Pat. No. 5,049,695, issued Sep. 17, 1991, U.S. Pat. No. 5,591,892,
issued Jan. 7, 1997, U.S. Pat. No. 5,049,695, issued Sep. 17, 1991,
U.S. Pat. No. 5,250,701, issued Oct. 5, 1993, U.S. Pat. No.
5,248,785, issued Sep. 28, 1993, U.S. Pat. No. 5,705,521, issued
Jan. 6, 1998, and U.S. Pat. No. 5,525,630, issued Jun. 11, 1996.
Each of these references is specifically incorporated herein by
reference in its entirety.
[0006] As a result of the general utility and importance of these
compounds a number of methods have been developed to synthesize
them. Two of the principal methods developed to date are compared
in FIG. 2 using the synthesis of the sodium salt of
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (also referred to herein as efaproxiral sodium and
efaproxiral-Na) (5), for purposes of illustration. With reference
to FIG. 2, in the first method developed (referred to as Process
A), efaproxiral-Na (5) was synthesized as the free acid (6), which
was then treated with base to provide the sodium salt (5). (Randad
et al. (1991) J. Med. Chem. 34:752-757). In the second method
(referred to as Process B), this compound was synthesized as the
ethyl ester (4), which was then saponified to provide the sodium
salt (5). (Witt et al., DE 2,432,560, published Jan. 22, 1976).
Process A is highly exothermic, not easily amenable to commercial
scale manufacture and uses a halogenated hydrocarbon solvent.
Process B eliminates the use of a halogenated hydrocarbon solvent
and is more amenable to commercial scale manufacture and thus is
the preferred method. The primary drawback of Process B, however,
is the unexpected generation of the polymeric impurity poly (ethyl
methacrylate) and precursors to this compound, which are referred
to herein collectively as PEM, which is formed in Step 2 via the
following mechanism. ##STR2##
[0007] In the manufacture of the efaproxiral sodium (5) via Process
B poly (ethyl methacrylate) is typically formed in concentrations
of from approximately 0.5% (5000 parts per million (ppm)) to 9%
(90,000 ppm) by weight.
[0008] Despite the general utility and importance of these
compounds in treating disease, problems remain in generating
pharmaceutical grade compositions. Specifically, the compounds are
administered to patients in a sterile intravenous (IV) solution
preparation. In the process of testing these compounds, difficulty
with the drug product manufacturing (IV solution formation) has
been traced to the PEM byproduct generated during their synthesis
as outlined above. Thus, there is a need for methods to reduce the
level of polymeric impurity in preparations of allosteric
hemoglobin modifier compounds in order for use in patients. There
also remains a need for compositions of allosteric hemoglobin
modifying compounds with lower amounts of impurities in general.
The present invention provides an improved process for making
highly pure allosteric effector compounds.
[0009] Another problem associated with the prior art methods is
that there is currently no effective way to measure the low levels
of impurities capable of causing failure in the IV solutions
prepared from compositions, which are comprised of these compounds.
As noted above, a particularly undesirable impurity is PEM. Prior
art methods for measuring PEM are deficient in several respects,
particularly in that they are unable to detect very low levels of
PEM. Current methods for detecting and measuring PEM include
.sup.1H NMR, gel permeation chromatography (GPC) or size exclusion
chromatography (SEC); MALDI-TOF mass spectrometry; ultraviolet (UV)
analysis; and infrared (IR) analysis. The latter two techniques are
accurate for mixtures containing >2-4% PEM w/w in efaproxiral
ethyl ester (4) or efaproxiral sodium (5). The former techniques
can be used for determining the concentration of >0.5% w/w PEM.
For example, to analyze intermediates with >0.5% w/w PEM,
.sup.1H NMR can be used by comparing the integration of the PEM
methylene proton signal to the ethoxy-methylene proton signal of
the efaproxiral ethyl ester (4). By multiplying the appropriate
molecular weights to the respective signals of the PEM and
efaproxiral ethyl ester one can develop a formula for determining
the percent weight/weight (% w/w) of PEM. Muguruma et al. describe
a method for the quantitative analysis of poly(methylmethacrylate)
(PMMA) in drug substances using pyrolysis-gas chromatography
(PY/GC). Using this method, Muguruma were able to detect levels of
PMMA >0.1 wt % with a precision of approximately 4.5% at a level
of 0.1%. (Mugurma et al. (July 1999) LC-GC International, pp.
432-436).
[0010] For analysis of highly pure compositions of allosteric
hemoglobin modifiers however, none of the prior art techniques can
be used because the limit of detection is not low enough. The
improved processes of the instant invention produce compositions of
allosteric hemoglobin modifiers having very low levels of
impurities (<100 ppm (0.0100% w/w) of PEM in efaproxiral-Na).
Consequently, there remains a need for a method for analyzing these
compounds which has a low detection limit and good specificity to
measure very low levels of PEM, as well as other polymeric
impurities with adequate sensitivity. Since pyrolysis/gas
chromatography/mass spectrometry (PY/GC/MS) has been used for
identification of polymers in relatively intractable matrices, it
was evaluated to determine whether it would be useful for trace
level analysis of polymers in efaproxiral-Na. Extensive development
led to the discovery of a method for quantitation of trace levels
of PEM (limit of quantitation=10 ppm). The PY/GC/MS method
described herein is a novel analytical technique that utilizes
single ion monitoring and an isotopically labeled PEM internal
standard to provide the sensitivity, precision, accuracy and
reproducibility required for the detection and quantitation of a
trace level impurity. This technique can be extended to the
analysis of compositions of allosteric hemoglobin modifiers
containing polymeric impurities other than PEM in the event that
the method of synthesis illustrated in FIG. 3 is modified.
[0011] It is therefore an object of this invention to provide
compositions of allosteric hemoglobin modifying compounds having
lower amounts of polymeric impurities, particularly PEM, as well
as, lower amounts of impurities in general.
[0012] It is also an objective of the present invention to provide
improved methods for the synthesis of compositions of allosteric
hemoglobin modifying compounds having lower amounts of polymeric
impurities.
[0013] It is another object of the present invention to provide
improved methods for purification of compositions of allosteric
hemoglobin modifying compounds prepared by any known synthetic
method, in particular by the method disclosed herein.
[0014] Finally, it is an objective of the present invention to
provide a method for analyzing compositions of allosteric
hemoglobin modifying compounds, which enables detection and
quantification of low levels of impurities, particularly polymeric
impurities.
SUMMARY OF THE INVENTION
[0015] The present invention includes novel compositions of
allosteric hemoglobin modifier compounds that are substantially
free of impurities, particularly polymeric impurities. The
compositions of allosteric hemoglobin modifier compounds included
within the scope of this invention are generally represented by the
following formula: ##STR3##
[0016] wherein
[0017] X and Z are independently selected from the group consisting
of CH.sub.2, CO, NH or O, and Y is selected from the group
consisting of CO or NH, with the caveat that X, Y, and Z must all
be different from each other;
[0018] R.sub.5 and R.sub.6 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-12 alkyl groups, carboxylic acid and ester groups,
substituted or unsubstituted aromatic or heteroaromatic groups, or
alkyl moieties of part of an aliphatic ring connecting R.sub.5 and
R.sub.6;
[0019] R.sub.7 is a selected from the group consisting of hydrogen,
a cationic counterion, selected from the group including, but not
limited to sodium, potassium or ammonium, a metal, or a substituted
or unsubstituted C.sub.1-6 alkyl group; and
[0020] R.sub.8-12 are independently selected from the group
consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups, or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
[0021] In a preferred embodiment of the invention, the allosteric
hemoglobin modifying compounds are generally represented by the
following formula: ##STR4##
[0022] wherein
[0023] R.sub.5 and R.sub.6 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-12 alkyl groups, carboxylic acid and ester groups,
substituted or unsubstituted aromatic or heteroaromatic groups or
alkyl moieties of part of an aliphatic ring connecting R.sub.5 and
R.sub.6;
[0024] R.sub.7 is a selected from the group consisting of hydrogen,
a cationic counterion, including but not limited to sodium,
potassium or ammonium, a metal, or a substituted or unsubstituted
C.sub.1-6 alkyl group; and
[0025] R.sub.8-12 are independently selected from the group
consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups, or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
[0026] More preferably R.sub.5 and R.sub.6 are independently
selected from H or CH.sub.3 and R.sub.7 is selected from hydrogen
or a cationic counterion as defined above.
[0027] In the most preferred embodiment of the invention the
allosteric hemoglobin modifier compound is
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (efaproxiral) (5). ##STR5##
[0028] An impurity includes any substance that does not belong in
the allosteric hemoglobin modifier composition. Typically, the
impurities present in the compositions are a result of the process
employed to produce the allosteric hemoglobin modifier compound.
For example, polymeric impurities are produced by the
polymerization of one of the starting materials used to synthesize
these compounds via the method exemplified by Process B. In a
preferred embodiment of the invention the allosteric hemoglobin
modifier composition is the sodium salt of efaproxiral (5)
containing less than 0.010% non-polymeric impurities and less than
100 ppm (0.01%) of polymeric impurities, and more specifically the
polymeric impurity PEM. In a most preferred embodiment, the
allosteric hemoglobin modifier composition contains less than 80
ppm (0.008%) of polymeric impurities, specifically the polymeric
impurity PEM.
[0029] The present invention includes methods for the preparation
of compositions of allosteric hemoglobin modifier compounds that
are substantially free of polymeric impurities. The improved method
for preparing allosteric hemoglobin modifier compounds is shown in
FIG. 3, which illustrates the synthesis of efaproxiral sodium (5)
as an exemplary compound. In the most preferred embodiment of the
invention, the allosteric hemoglobin modifier compounds are
synthesized in reaction vessels that do not contain metals that
promote the formation of polymeric byproducts and the crude
synthetic products produced are then purified by extraction with
methyl isobutyl ketone (MIBK) followed by an ethanol/acetone
recrystallization. In this embodiment of the invention, the method
for preparing compositions of allosteric hemoglobin modifier
compounds is comprised of the steps of a) coupling a substituted
aniline with 4-hydroxyphenylacetic acid to yield the corresponding
substituted phenol; b) adding the product of step (a) to an alkyl
ester halide to yield a substituted ester; and c) saponifying the
substituted ester to provide the salt of the acid. As noted above,
in a preferred embodiment all steps are performed in a reaction
vessel that does not contain metals (referred to herein as
"catalytic metals") that promote the formation of polymeric
byproducts. Examples of catalytic metals that promote the formation
of polymeric byproducts include, but are not limited to copper,
iron, nickel, palladium and rhodium. Acceptable materials for
reaction vessels include, but are not limited to glass lined
stainless steel, passivated stainless steel, Hastelloy.RTM. or
similar alloys low in available catalytic metals.
[0030] The present invention also includes improved methods for
purifying crude synthetic compositions of allosteric hemoglobin
modifier compositions containing impurities, particularly polymeric
impurities. The improved methods for purifying these compositions
include, but are not limited to extracting the crude compositions
obtained following step (c) with any water immiscible or partially
immiscible solvent in which the ester formed in step (c) is
soluble, including, but not limited to methyl isobutyl ketone
(MIBK), isopropyl acetate, ethyl acetate, methyl ethyl ketone,
chlorinated solvents selected from the group including, but not
limited to chloroform and methylene chloride and recrystallizing
the crude compositions with solvents including, but not limited to
ethanol, acetone and mixtures of acetone/ethanol, acetone/methanol
and ethanol/methanol/acetone/water. In a preferred embodiment, the
recrystallized product is further purified by filtering through a
polymeric filter, wherein said polymeric filter is selected from
the group including, but not limited to poly(vinylidene difluoride)
(PVDF) or a cellulose ester filter.
[0031] Finally the present invention includes a method for
analyzing compositions of allosteric hemoglobin modifying compounds
for impurities, particularly polymeric impurities and more
particularly PEM. In this embodiment of the invention, the method
for analyzing compositions of allosteric hemoglobin modifier
compounds is comprised of the steps of a) pyrolyzing (PY) a
composition comprised of an allosteric hemoglobin modifier compound
and b) analyzing said pyrolyzed composition by gas
chromatography/mass spectrometry (GC/MS). In order to improve the
sensitivity of the method by the two orders of magnitude needed to
measure trace amounts of polymeric impurity an isotopic internal
standard is added to the sample prior to analysis. It is believed
that this is the first report of the use of an internal standard,
particularly an isotopic internal standard prior to analysis by
PY/GC/MS for trace level analysis of polymers in drugs. The
internal standard is added in an amount so as to produce a
concentration approximately the same as that expected for the
polymeric impurity in the compound being analyzed, which in the
instant application is endogenous PEM. As noted above, the use of
an internal standard, particularly an isotopic internal standard
greatly improves the sensitivity while maintaining precision and
accuracy in the analytical method making quantitative measurements
in the 10-100 ppm range possible. Isotopes of any atom can be used
including, but not limited to isotopes of hydrogen, carbon, oxygen
and nitrogen. Examples of such isotopes include, but are not
limited to deuterium (D), carbon 13 (.sup.3C), oxygen 18 (.sup.18O)
and nitrogen (.sup.15N). The method described herein can be
extended to the identification and quantification of any compound
amenable to analysis by PY/GC/MS.
[0032] Additional advantages and novel features of this invention
shall be set forth in part in the description and examples that
follow, and in part will become apparent to those skilled in the
art upon examination of the following or may be learned by the
practice of the invention. The advantages of the invention may be
realized and attained by means of the instrumentation and in
combinations particularly pointed out in the appended claims. It is
to be understood that both the foregoing general description and
the following detailed description are exemplary and explanatory
only and are not restrictive of the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIGS. 1A-1E depict the chemical structures of a variety of
compounds that have a "right-shifting" allosteric effect on
hemoglobin. The family of compounds illustrated by FIG. 1D
(referred to as RSR compounds) are representative of a large family
of compounds having a strong allosteric effect.
[0034] FIG. 2 illustrates two of the principal prior art methods
developed for the synthesis of the allosteric hemoglobin modifier
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (also known as efaproxiral).
[0035] FIG. 3 depicts the improved method of the invention
developed for the synthesis of the allosteric hemoglobin modifier
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (also known as efaproxiral).
[0036] FIG. 4 depicts a total ion chromatograph (TIC) for a sample
of efaproxiral, showing all of the ions resulting from pyrolysis
and ionization of the drug. Additionally, the sample contains low
levels of PEM (less than 50 ppm) and PEM-d.sub.5 (internal
standard).
[0037] FIG. 5 depicts an expansion of the TIC of FIG. 4 in the
region of 4.7 to 5.7 minutes showing the retention time of ions
resulting from chromatographically separated PEM-d.sub.5 (main ion:
m/e 104; 5.25 minutes) and PEM (main ion: m/e 99; 5.38
minutes).
[0038] FIGS. 6A and 6B depict the chromatogram (FIG. 6A) and the
mass spectra single ion monitoring (SIM) (FIG. 6B) of PEM-d.sub.5
using SIM mode analysis.
[0039] FIGS. 7A and 7B depict the chromatogram (FIG. 7A) and the
mass spectra (SIM) (FIG. 7B) of PEM using SIM mode analysis.
[0040] FIG. 8 depicts a standard calibration curve for the PY/GC/MS
method described in Example 4.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0041] The present invention includes novel compositions of
allosteric hemoglobin modifier compounds that are substantially
free of impurities, particularly polymeric impurities. In one
embodiment, the allosteric hemoglobin modifier composition is
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (also known as efaproxiral) containing less than 100
ppm of the polymeric impurity poly (ethyl methacrylate) (PEM). The
present invention includes methods for the preparation of
compositions of allosteric hemoglobin modifier compounds having
very low levels of impurities. In one embodiment, the method of the
invention is comprised of synthesizing the allosteric hemoglobin
modifier compound according to the method described herein using a
reaction vessel that does not contain available metals that promote
the formation of polymeric byproducts, referred to herein as
"catalytic metals."
[0042] The present invention also includes improved methods for
purifying crude synthetic compositions of allosteric hemoglobin
modifier compositions containing impurities, particularly polymeric
impurities. The improved methods for purifying these compositions
include, but are not limited to extracting the crude compositions
with any water immiscible or partially immiscible solvent in which
the polymeric impurity is soluble, including but not limited to
methyl isobutyl ketone (MIBK), isopropyl acetate, ethyl acetate,
methyl ethyl ketone, and chlorinated solvents selected from the
group including, but not limited to chloroform and methylene
chloride and recrystallizing the crude compositions with solvents
selected from the group including, but not limited to ethanol,
acetone and mixtures of acetone/ethanol, acetone/methanol and
ethanol/methanol/acetone/water. In a preferred embodiment, a
solution of the recrystallized product is further purified by
filtering through a polymeric filter, wherein said polymeric filter
is selected from the group including, but not limited to
poly(vinylidene difluoride) (PVDF) or a cellulose ester filter.
[0043] Finally, the present invention includes a method for
analyzing compositions of allosteric hemoglobin modifying compounds
for impurities, particularly PEM. In this embodiment of the
invention, the method for analyzing compositions of allosteric
hemoglobin modifier compounds is comprised of the steps of a)
pyrolyzing a composition comprised of an allosteric hemoglobin
modifier compound and b) analyzing said pyrolyzed composition by
gas chromatography/mass spectrometry (GC/MS) employing an internal
standard, particularly an isotopically labeled version of the
polymeric analyte as the internal standard or an analog of the
polymeric analyte as the internal standard.
[0044] Various terms are used herein to refer to aspects of the
present invention. To aid in the clarification of the description
of the components of this invention, the following definitions are
provided.
[0045] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, an allosteric hemoglobin
modifying compound refers to one or more allosteric hemoglobin
modifying compounds. As such, the terms "a" or "an," "one or more"
and "at least one," are used interchangeably herein.
[0046] As used herein the term "allosteric hemoglobin modifier
compounds" or "allosteric effector compounds" refers to a specific
class of compounds, which can be generally represented by the
following formula: ##STR6##
[0047] wherein
[0048] X and Z are independently selected from the group consisting
of CH.sub.2, CO, NH or O, and Y is selected from the group
consisting of CO or NH, with the caveat that X, Y, and Z must all
be different from each other;
[0049] R.sub.5 and R.sub.6 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-12 alkyl groups, carboxylic acid and ester groups,
substituted or unsubstituted aromatic or heteroaromatic groups, or
alkyl moieties of part of an aliphatic ring connecting R.sub.5 and
R.sub.6;
[0050] R.sub.7 is a selected from the group consisting of hydrogen,
a cationic counterion, selected from the group including but not
limited to sodium, potassium or ammonium, a metal, or a substituted
or unsubstituted C.sub.1-6 alkyl group; and
[0051] R.sub.8-12 are independently selected from the group
consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups, or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
[0052] In a preferred embodiment of the invention, the allosteric
hemoglobin modifying compounds are generally represented by the
following formula: ##STR7##
[0053] wherein
[0054] X and Z are independently selected from the group consisting
of CH.sub.2, NH or O;
[0055] R.sub.5 and R.sub.6 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-12 alkyl groups, carboxylic acid and ester groups,
substituted or unsubstituted aromatic or heteroaromatic groups, or
alkyl moieties of part of an aliphatic ring connecting R.sub.5 and
R.sub.6;
[0056] R.sub.7 is a selected from the group consisting of hydrogen,
a cationic counterion, including but not limited to sodium,
potassium or ammonium, a metal, or a substituted or unsubstituted
C.sub.1-6 alkyl group; and
[0057] R.sub.8-12 are independently selected from the group
consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups, or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
[0058] In another preferred embodiment of the invention, the
allosteric hemoglobin modifying compounds are generally represented
by the following formula: ##STR8##
[0059] wherein
[0060] R.sub.5 and R.sub.6 are independently selected from the
group consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-12 alkyl groups, carboxylic acid and ester groups,
substituted or unsubstituted aromatic or heteroaromatic groups or
alkyl moieties of part of an aliphatic ring connecting R.sub.5 and
R.sub.6;
[0061] R.sub.7 is a selected from the group consisting of hydrogen,
a cationic counterion, including but not limited to sodium,
potassium or ammonium, a metal, or a substituted or unsubstituted
C.sub.1-6 alkyl group; and
[0062] R.sub.8-12 are independently selected from the group
consisting of hydrogen, halogen, substituted or unsubstituted
C.sub.1-3 alkyl groups, or alkyl moieties of an aromatic or
aliphatic ring incorporating two of the R.sub.8-12 sites.
[0063] More preferably R.sub.5 and R.sub.6 are independently
selected from H or CH.sub.3 and R.sub.7 is selected from hydrogen
or a cationic counterion as defined above.
[0064] In the most preferred embodiment the allosteric hemoglobin
modifier compound is
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid (efaproxiral) (5). ##STR9##
[0065] As used herein the term "impurity" includes any substance
that does not belong in the allosteric hemoglobin modifier
composition, typically resulting from the synthesis of the
allosteric hemoglobin modifier or from products of its degradation.
The term impurity includes, but is not limited to polymeric
impurities, such as poly (ethyl methacrylate) and precursors to
this compound, which are referred to herein collectively as PEM, as
well as other related impurities resulting from the synthetic
process.
[0066] As used herein the term "polymeric impurity" refers to any
polymerized byproduct of the process employed to produce an
allosteric hemoglobin modifier compound. In one embodiment, the
polymeric impurity is selected from a compound having the following
structure: ##STR10##
[0067] wherein
[0068] R, R', R'' and R''' are independently selected from the
group consisting of a substituted or unsubstituted C.sub.1-12 alkyl
group and hydrogen; and
[0069] n is any number of units appropriate for a polymer of
repeating units.
[0070] A polymeric impurity produced as a result of the synthesis
of compound (5) as described herein is poly (ethyl methacrylate)
(PEM).
[0071] Synthesis of the allosteric hemoglobin modifiers of the
invention can result in polymeric byproducts of up to approximately
10% by weight depending upon reaction conditions. As noted above,
in the synthesis of efaproxiral sodium (5) via Process B, the
polymeric impurity PEM is typically formed in concentrations of
from approximately 0.5% (5000 ppm) to 9% (90,000 ppm) by
weight.
[0072] "Related impurities" refer to all nonpolymeric impurities
resulting from the synthetic process such as structural isomers or
decomposition products. Related impurities include but are not
limited to 3-monomethyl efaproxiral (3MMRS13), .alpha.-desmethyl
efaproxiral (DDMRS13), monomethyl a to COOH (DMRS13), 3,4-dimethyl
efaproxiral (3,4DMRS13), a ethyl efaproxiral, diacid (DA),
3,5-dimethyl aniline, amidophenol and the ethyl esters of the
related impurities having the structures described in Table 1.
Other related impurities would be obvious to one of skill in the
art. With respect to the related impurities, the purification
method described herein either meets or exceeds the acceptable
levels of impurities for a high dose parenteral drug set forth in
the International Conference on Harmonisation (ICH) Guidelines,
which are published in the Federal Register or by the EMEA. For
high dose drugs the limit for any unqualified impurity is NMT
0.05%. In a preferred embodiment of the invention, the related
impurities are collectively present in an amount not exceeding
about 0.75% (75000 ppm) by weight.
[0073] By "substantially free" of impurities it is meant a degree
of polymeric impurity not exceeding about 0.5% (5000 ppm) by
weight, more preferably a degree of polymeric impurity not
exceeding 0.1%, 0.07%, 0.05%, 0.025%, 0.02%, 0.015%, 0.01% (100
ppm), 0.009%, and most preferably an amount of polymeric impurity
not exceeding 0.008% (80 ppm), 0.007% or 0.006% (60 ppm) by weight
or less, including 0.005% (50 ppm), 0.004% (40 ppm), 0.003% (30
ppm) and 0.001% (10 ppm) and a degree of related impurities not
exceeding about 0.1% by weight, more preferably a degree of related
impurity not exceeding 0.09%, 0.08%, 0.07%, 0.06% or 0.05%.
[0074] As would be known to one of skill in the art a composition
which is 97% substantially free of impurities would also be
considered to be 97% substantially pure, etc.
[0075] As used herein the term "catalyst" refers to a substance
that initiates or accelerates the rate of a chemical reaction
without being consumed in the reaction. Typically, a catalyst
lowers the activation energy for a chemical reaction by providing
an alternate pathway for the reaction. Catalysts that promote the
formation of polymerized byproducts include, but are not limited to
metals such as copper, iron, nickel, palladium and rhodium. The
metals that promote the formation of polymeric byproducts are
referred to herein as "catalytic metals."
[0076] As used herein the term "extraction" or "extracting" refers
to a liquid-liquid partition or the process of transferring a
dissolved substance from one liquid phase to another (immiscible or
partially miscible) liquid phase in contact with it.
[0077] As used herein the term "recrystallization" or
"recrystallizing" is a standard term which refers to a means for
purifying materials by precipitation from a solvent(s).
[0078] Note, that throughout this application various citations are
provided. Each citation is specifically incorporated herein in its
entirety by reference.
[0079] The present invention includes novel compositions of
allosteric hemoglobin modifier compounds that are substantially
free of impurities, particularly polymeric impurities. The
compositions of allosteric hemoglobin modifier compounds included
within the scope of this invention are illustrated by the general
structure set forth above. As noted above, an impurity includes any
substance that is not the desired allosteric hemoglobin modifier
composition. Typically, the impurities present in the compositions
are a result of the process employed to produce the allosteric
hemoglobin modifier compound. For example, the polymeric impurities
are produced by the polymerization of one of the starting materials
used to synthesize these compounds via the method exemplified by
Process B. In a preferred embodiment of the invention the
allosteric hemoglobin modifier composition is the sodium salt of
efaproxiral (5) containing less than 100 ppm (0.01%) of polymeric
impurities, specifically the polymeric impurity PEM and less than
1000 ppm (0.1%) of any related impurity. In a most preferred
embodiment, the allosteric hemoglobin modifier composition contains
less than 80 ppm of polymeric impurities and less than 500 ppm of
any related impurity.
[0080] The present invention includes methods for the preparation
of compositions of allosteric hemoglobin modifier compounds that
are substantially free of impurities. By "substantially free" of
impurities it is meant a degree of polymeric impurity not exceeding
about 0.5% (5000 ppm) by weight, more preferably a degree of
polymeric impurity not exceeding 0.1%, 0.07%, 0.05%, 0.025%, 0.02%,
0.015%, 0.01% (100 ppm), 0.009%, and most preferably an amount of
polymeric impurity not exceeding 0.008% (80 ppm), 0.007% or 0.006%
(60 ppm) by weight or less, including 0.005% (50 ppm), 0.004% (40
ppm), 0.003% (30 ppm), and 0.001% (10 ppm) and a degree of related
impurities not exceeding about 0.1% by weight, more preferably a
degree of related impurity not exceeding 0.09%, 0.08%, 0.07%, 0.06%
or 0.05%. In this embodiment of the invention the method for
preparing compositions of allosteric hemoglobin modifier compounds
is comprised of the steps of a) coupling a substituted aniline with
4-hydroxyphenylacetic acid to yield the corresponding substituted
phenol; b) adding the product of step (a) to an alkyl ester halide
to yield a substituted ester; and c) saponifying the substituted
ester to provide the salt of the acid, wherein all steps are
performed in a reaction vessel that does not contain metals that
promote the formation of polymeric byproducts, referred to herein
as catalytic metals. Examples of catalytic metals include, but are
not limited to copper, iron, nickel, palladium and rhodium.
Stainless steel (SS) is generally comprised of predominantly
nickel, chromium and molybdenum. Acceptable materials for reaction
vessels include, but are not limited to glass lined stainless
steel, passivated stainless steel, Hastelloy.RTM. or similar
alloys. The Hastelloy 276.RTM. alloy is comprised of predominantly
nickel, chromium and molybdenum.
[0081] Example 1 describes the synthesis of the sodium salt of
efaproxiral (efaproxiral-Na) (5) according to the method of this
invention using either a Hastelloy or SS reaction vessel. The
synthesis of efaproxiral-Na (5) can be performed in any reaction
vessel that does not promote the formation of the polymeric
impurity PEM, including but not limited to an SS (316) reactor, a
Hastelloy 276.RTM. reactor or a glass-lined SS reactor. In a
preferred embodiment, the synthesis of (5) is performed in a
Hastelloy 276.RTM., SS or glass-lined SS reactor. The product
prepared by the method described in Example 1 contained less than
3% by weight of the poly (ethyl methacrylate) (PEM) impurity. The
use of a Monel.RTM. reaction vessel, on the other hand, resulted in
a product that contained about 9% by weight PEM. Monel.RTM. is an
alloy comprised predominantly of copper and nickel. Thus, the
change in reaction vessel significantly reduced the amount of PEM
formed during the reaction.
[0082] The present invention also includes improved methods for
purifying crude synthetic compositions of allosteric hemoglobin
modifier compositions containing impurities, particularly polymeric
impurities. In this embodiment of the invention, the method for
preparing compositions of allosteric hemoglobin modifier compounds
is comprised of the steps of a) coupling a substituted aniline with
4-hydroxyphenylacetic acid to yield the corresponding substituted
phenol; b) adding the product of step (a) to an alkyl ester halide
to yield a substituted alkyl ester; c) saponifying the substituted
alkyl ester to provide the salt of the acid; and d) purifying the
product obtained from step c) to obtain a product which is
substantially free of impurities, particularly polymeric
impurities, wherein all steps are performed in a reaction vessel
that does not contain metals that promote the formation of
polymeric byproducts. The improved methods for purifying these
compositions include, but are not limited to extracting the crude
compositions with any water immiscible or partially immiscible
solvent in which the polymeric impurity is soluble, including but
not limited to methyl isobutyl ketone (MIBK), isopropyl acetate,
ethyl acetate, methyl ethyl ketone, chlorinated solvents selected
from the group including, but not limited to chloroform and
methylene chloride and recrystallizing the crude compositions with
solvents including, but not limited to ethanol, acetone and
mixtures of acetone/ethanol, acetone/methanol and
ethanol/methanol/acetone/water. In a preferred embodiment, a
solution of the recrystallized product is further purified by
filtering through a polymeric filter, wherein said polymeric filter
is selected from the group including, but not limited to
poly(vinylidene difluoride) (PVDF) or a cellulose ester filter.
[0083] The improved methods of this invention for purifying crude
synthetic compositions of allosteric hemoglobin modifier compounds
are outlined in FIG. 3, using the synthesis of efaproxiral-Na (5)
for purposes of illustration. Compared to prior art methods, the
improved method of the invention includes addition of a
purification step via extraction, a recrystallization step, a
filtration step and synthesis in reaction vessels that do not
contain metals that promote the formation of polymeric byproducts.
Modifications were made in particular to control the levels of
polymeric impurities, such as poly (ethyl methacrylate) or PEM. In
one embodiment of the invention, the crude synthetic product
(allosteric hemoglobin modifier compound) is extracted with a
solvent such as methyl isobutyl ketone (MIBK), to remove
impurities, specifically the polymeric impurity, prior to
saponification. Examples of solvents that can be used to extract
the crude product include any water immiscible or partially
immiscible solvent in which the polymeric impurity is soluble
including, but not limited to MIBK, isopropyl acetate, ethyl
acetate, methyl ethyl ketone and chlorinated solvents selected from
the group including, but not limited to chloroform and methylene
chloride. The crude product is extracted at least one time, however
in other embodiments the crude product may be extracted two or more
times depending upon the impurities being removed. Exact extraction
protocols can be determined without difficulty by one skilled in
the art.
[0084] Example 2 describes the purification of efaproxiral-Na (5)
prepared according to the method of Example 1 by extraction with
methyl isobutyl ketone (MIBK).
[0085] Example 3 describes the purification of efaproxiral sodium
(5) prepared according to the methods of Examples 1 and 2 by
recrystallization with acetone/ethanol. The crude synthetic
product, or the extracted crude synthetic product, is
recrystallized using a solvent system such as ethanol, acetone and
mixtures of acetone/ethanol, acetone/methanol and
ethanol/methanol/acetone/water to reduce the amount of impurities,
specifically polymeric impurities. Examples of solvent(s) that can
be used to recrystallize these products include, but are not
limited to ethanol, acetone and mixtures of acetone/ethanol,
acetone/methanol and ethanol/methanol/acetone/water. The purified
product contained less than 100 ppm of PEM.
[0086] In the most preferred embodiment of the invention, the
allosteric hemoglobin modifier compounds are synthesized in
reaction vessels that do not contain metals that promote the
formation of polymeric byproducts and the crude synthetic products
produced are then purified by extraction with methyl isobutyl
ketone (MIBK) followed by an ethanol/acetone recrystallization.
[0087] Example 4 describes a method developed to detect and
quantify trace amounts of impurities in compositions of allosteric
hemoglobin compounds, specifically polymeric impurities and more
specifically the polymeric impurity PEM using a GC/MS method in
which the sample is pyrolyzed prior to introduction onto the GC
column. (Matheson et al. (May 1997) American Laboratory, pp
24C-24F; Irwin (1982) in Analytical Pyrolysis A Comprehensive
Guide, Marcel Dekker, Inc.). The sample flow within the pyrolysis
gas chromatography mass spectrometry (PY/GC/MS) instrument is
outlined in Scheme 2 using the impurity PEM for purposes of
illustration. The mass spectrometry data are collected using single
ion monitoring (SIM) (Hites (1997) in Handbook of Instrumental
Techniques for Analytical Chemistry, Settles, F. Ed., Prentice-Hall
Inc. p. 620) to improve the signal to noise ratio, and selectively
monitors a particular mass fragment arising from the ethyl
methacrylate monomer. Using this method, levels of PEM as low as 10
ppm can reliably be quantified. ##STR11##
[0088] An isotopic internal standard (deuterated poly (ethyl-d5
methacrylate) (PEM-d5)) is added to the sample prior to analysis.
(PEM-d5-M.sub.w 12,555, M.sub.n 12260, PI 1.02. It is believed that
this is the first report of the use of an internal standard,
particularly an isotopic internal standard prior to analysis by
PY/GC/MS for a trace level analysis of polymers in drugs.
Currently, no internal standard is used in the analysis of samples
by this method. The internal standard is added in approximately the
same expected concentration as the endogenous PEM. The use of an
internal standard, particularly an isotopic internal standard
greatly improves the precision and accuracy in the analytical
method making quantitative measurements in the 10-100 ppm range
possible. The increase in precision and accuracy is due to the fact
that virtually the same compound, differing only in isotopic
content and hence in molecular weight, is being subjected to the
same pyrolysis conditions as the compound being analyzed. As noted
above, the internal standard used in Example 4 was an isotope of
hydrogen, namely PEM-d5, however isotopes of atoms other than
hydrogen can be used including, but not limited to isotopes of
carbon, oxygen and nitrogen. Examples of such isotopes include, but
are not limited to deuterium (D), carbon 13 (.sup.13C), oxygen 18
(.sup.18O) and nitrogen (.sup.15N). Scheme 3 depicts the monomers
resulting from pyrolysis of PEM and PEM-d5 and Scheme 4 depicts the
actual ions that are detected in the mass spectrometer from PEM and
PEM-d5. ##STR12## ##STR13##
[0089] FIG. 4 depicts a total ion chromatograph (TIC) for a sample
of efaproxiral, showing all of the ions resulting from pyrolysis
and ionization of the drug. With reference to FIG. 4, it can be
seen that the sample contains low levels of PEM (less than 50 ppm)
and PEM-d5 (internal standard). FIG. 5 depicts an expansion of the
TIC of FIG. 4 in the region of 4.7 to 5.7 minutes showing the
retention time of ions resulting from chromatographically separated
PEM-d.sub.5 (main ion: m/e 104; 5.25 minutes) and PEM (main ion:
m/e 99; 5.38 minutes). FIGS. 6A and 6B depict the chromatogram
(FIG. 6A) and the mass spectra (SIM) (FIG. 6B) of PEM-d.sub.5 using
SIM mode analysis. FIGS. 7A and 7B depict the chromatogram (FIG.
7A) and the mass spectra (SIM) (FIG. 7B) of PEM using SIM mode
analysis.
[0090] In order to quantify the amount of ethyl methacrylate (EM)
(and ultimately the amount of PEM) a standard curve is established
for a range of PEM concentrations corresponding to the acceptable
range of PEM in the efaproxiral-Na sample. Each sample is then
analyzed in triplicate and the concentration of PEM is determined
using the linear calibration curve prepared based on the results of
PY/GC/MS of the deuterated PEM. Example 5 describes the preparation
of a typical standard calibration curve for the quantification of
PEM by PY/GC/MS. FIG. 8 depicts the standard calibration curve for
the PY/GC/MS method described in Example 5.
[0091] The invention is further illustrated by the following
non-limited examples. All scientific and technical terms have the
meanings as understood by one with ordinary sldll in the art. The
specific examples that follow illustrate the methods in which the
compositions of the invention may be prepared and are not to be
construed as limiting the invention in sphere or scope. The methods
may be adapted to variation in order to produce compositions
embraced by this invention but not specifically disclosed. Further,
variations of the methods to the produce, the same compositions in
somewhat different fashion will be evident to one skilled in the
art.
[0092] Example 6 describes the purification of efaproxiral sodium
(5) prepared according to the methods of Examples 1, 2 and 3 by
filtration of the recrystallized product obtained from Example 3
through the following three filters: a polytetrafluoroethylene
(PTFE) filter, a cellulose ester filter and a poly(vinylidene
difluoride) (PVDF) filter. The results are set forth in Table 3.
With reference to Table 3, it can be seen that PVDF filtration
reduced the levels of PEM from about 55 ppm to about 9 ppm.
EXAMPLES
[0093] Materials. The following reactions were carried out in
either Hastelloy 276 .RTM., SS (316) or glass-lined SS reactors.
The Gas Chromatography/Mass Spectrometry was performed using a
Hewlett Packard 5890 or 6890 Gas Chromatograph, interfaced with a
5971, 5972 or 5973 Mass Spectrometer; equipped with cryogenic
cooling option, GC/MS ChemStation software, version A.03.00 or
greater. Gas chromatography column, DB-5 Column 15 m.times.0.25
mm.times.0.25 .mu.m, VWR.
Example 1
Preparation of 2-[4-((((3
5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic
acid
[0094] FIG. 3 illustrates a general five step reaction scheme for
the preparation of
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid which is described in detail below.
Synthesis of Amidophenol (3)
[0095] With reference to FIG. 3, 4-hydroxyphenylacetic acid (2001
kg) (2) was added to xylene (760 L) with stirring. To this mixture,
3,5-xylidine (3,5-dimethyl aniline) (178 L) (1) was added. The
reaction mixture was heated to reflux and water was removed
azeotropically as the reaction proceeded. Upon completion, the
reaction mixture was distilled to provide amidophenol (3), which
solidified upon cooling. To recrystallize, ethanol (1180 L) and
methyl isobutyl ketone (MIBK) (56 L) were added to the solid and
the mixture was refluxed until dissolution. Upon dissolution water
was added (70.degree. C., 490 L) and mixture was stirred and cooled
slowly over 6 hours to about 0.degree. C. The mixture was then
stirred for at least one hour at this temperature. The mixture was
then filtered, and the solid washed with 1:2 ethanol/water at
5.degree. C., followed by a wash with xylene (452 L at 5.degree.
C.).
Synthesis of Efaproxiral Ethyl Ester (4)
[0096] Methyl isobutyl ketone (MIBK) (827 L) was added to the
crystallized amidophenol (3) and the mixture was refluxed to
azeotropically remove water. The reaction mixture was then cooled
to below 70.degree. C., and absolute ethanol (731 L) was added,
followed by anhydrous potassium carbonate (668 kg) and ethyl
2-bromoisobutyrate (366 L). The reaction mixture was refluxed for
at least 7 hours, then cooled to below 0.degree. C. The mixture was
filtered, and the solids were washed with MIBK such that the total
volume of the wash plus the filtrate was 1208 L. The mixture was
the distilled to remove the ethanol and the volume was adjusted
with MIBK to about 2163 L. The MIBK mixture was extracted with
dilute aqueous base (32 kg sodium bicarbonate in 604 L of water),
followed aqueous acid (63 L in 572 liters of water, and water
(3.times.700 L each). The mixture was then distilled to remove MIBK
and cooled to about 35.degree. C. Heptane (about 572 L) was added
and the solution was stirred while additional heptane
(approximately 1145 L) was slowly added over the course of one
hour. The mixture was then cooled to about 12.degree. C., stirred
for at least 2 hours and then filtered. The solid, efaproxiral
ethyl ester (4) was washed with heptane (318 L).
Synthesis of Efaproxiral Sodium (5)
[0097] Absolute ethanol (880 L) was first mixed with water (19 L),
followed by the addition of sodium hydroxide (36 kg). This mixture
was filtered, efaproxiral ethyl ester (4) was added and the
reaction mixture was refluxed for at least 3 hours. Sodium
hydroxide (10 N, 1 molar equivalent) was then added and reflux was
maintained for at least 5 hours after the last addition. The
mixture was then concentrated by distillation, and absolute ethanol
(1056 L) was added. The water content was less than 0.5%. The
reaction mixture was then cooled to about 40.degree. C., then
35.degree. C., and stirred for at least 2 hours. The mixture was
then concentrated under vacuum to about 1408 L, cooled to about
10.degree. C., and stirred for at least 5 hours. The mixture was
then filtered and the solid, which consisted primarily of the
sodium salt of 2-[4-((((3,5-dimethylphenyl)amino)
carbonyl)methyl)phenoxy]-2-methyl propionic acid (efaproxiral
sodium) (5), was washed with ethanol (282 L at 10.degree. C.).
Example 2
Purification of Efaproxiral Sodium (5) by Extraction with MIBK
[0098] Purified water (1658 L) was added to the product (5) (325
kg) obtained using the method described in Example 1. The mixture
was distilled under vacuum at a maximum temperature of 50.degree.
C. until about 423 L of solvent was removed. Another 423 L of
purified water was then added and the aqueous solution was
extracted with MIBK (390 L, below 30.degree. C.). The organic phase
was discarded, the aqueous phase was extracted again with MIBK (228
L, below 30.degree. C.) and the organic phase was discarded.
Example 3
Purification of Efaproxiral Sodium (5) by Recrystallization with
Acetone/Ethanol
[0099] The sodium salt of efaproxiral (5) synthesized as described
in Examples 1 and 2 in the aqueous solution was concentrated under
vacuum at a maximum temperature of 50.degree. C. to the maximum
extraction of solvent, after which absolute ethanol (406 L) was
added to provide a mixture having a water content of between 5 and
5.4%. The mixture was then cooled to about 47.degree. C., acetone
(975 L) was added and the mixture was stirred while maintaining the
temperature. After crystallization, the mixture was stirred for at
least one hour, after which an equal volume of acetone was added.
The mixture was then slowly cooled to a temperature of about
15.degree. C. and stirred for at least 5 hours. The crystals were
collected on a filter and washed with acetone (146 L).
Example 4
Quantitation of trace poly-ethyl methacrylate impurity in
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid
[0100] Described below is a method for measuring trace amounts of
PEM impurity in
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid using pyrolysis/gas chromatography/mass spectrometry
(PY/GC/MS). PY/GC/MS is useful when the analyte in question has a
large molecular weight and is either semi-volatile or nonvolatile.
As discussed in detail above, typically, the sample being analyzed
is heated in a controlled manner to create reproducible
pyrolytically-derived compound fragments, which are then analyzed
by normal GC or GC/MS. In the instant case, when a sample
containing trace amounts of PEM, is subjected to pyrolysis the
monomer ethyl methacrylate (EM) is generated, which is accurately
measured by GC or GC/MS. An internal standard, such as deuterated
PEM in methanol/methylene chloride, is introduced to provide the
required precision and accuracy in the analytical test. The
deuterated PEM is differentiated from the analyte PEM by its
greater mass units.
[0101] A 5 g sample of solid
2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid sodium salt (5) was dissolved in methanol and a
deuterated analog of PEM in methylene chloride was added to the
solution as an internal standard. Additional methylene chloride was
then added until a ratio of 40:60 methanol:methylene chloride was
attained. The methylene chloride was required to solubilize the PEM
released from the matrix. An aliquot of the sample was then placed
into a prepared quartz tube packed with glass wool. After the
solvent evaporated, the quartz tube was loaded into the pyrolysis
unit and analyzed by PY/GC/MS using known methods. The PEM
concentration in the sample was calculated using a linear
calibration curve prepared based on the results of PY/GC/MS of the
deuterated PEM as described in Example 5.
Example 5
Preparation of a Standard Calibration Curve for the Quantification
of PEM by PY/GC/MS
[0102] Three standard solutions were prepared and analyzed
according to the method described in Example 4. Three additional
standard solutions were also prepared in order to demonstrate that
acceptable linearity exceeds both ends of the calibration range
specified in the method. Table 2 shows the concentration of each
standard analyzed. Calibration levels corresponding to those
normally used in the method are indicated by an asterisk. The
standard solutions were then analyzed from the lowest to the
highest concentration and a concentration calibration curve was
constructed by plotting the Amount Ratio (X-axis) vs. the Response
Ratio (Y-axis) as follows.
[0103] The Amount Ratio (X-axis) is the concentration of PEM
divided by the concentration of PEM-d.sub.5 present in each
standard solution. All concentrations are expressed as mass of
compound sample analyzed, and are calculated by multiplying the
concentrations shown in Table 2 (ng/.mu.L) by the volume of
standard solution analyzed (.mu.L). In the instant case, the
resulting units are ng in sample analyzed. Implicit in the use of
the unit "ng/sample analyzed" is the assumption that the % transfer
of material to the column is the same for both the internal
standard and sample. The appropriateness of this assumption is
demonstrated in recovery experiments.
[0104] The "Response Ratio" (Y-axis) is calculated by dividing the
measured area of the PEM peak (m/z=99) by the measured area of the
PEM-d.sub.5 peak (m/z=104). A non-weighted least-squares linear
regression is performed on the paired data points (the
corresponding ratios for each standard) to determine the
calibration plot's line equation (slope and y-intercept). To
determine sample solution concentrations ("ng on-column"), the line
equation is solved for PEM concentration (i.e. y=mx+b is solved for
x) as shown in Equation 1: [ PEM ] = [ ( Area .times. .times. PEM
Area .times. .times. PEM .times. .times. d 5 ) - ( y .times.
.times. intercept ) ] slope .times. internal .times. .times.
standard .times. .times. concentration ( 1 ) ##EQU1##
[0105] wherein [0106] [PEM]=ng in sample analyzed concentration of
PEM in sample solution analyzed [0107] Area PEM=area of the m/z=99
(PEM) peak in the sample [0108] Area PEM d.sub.5=area of the
m/z=104 (PEM-d.sub.5) peak in the sample [0109] y
intercept=y-intercept of the calibration curve equation [0110]
slope=slope of the calibration curve equation and [0111] internal
standard concentration=concentration of PEM-d.sub.5 in the
sample.
[0112] To calculate concentration in the solid sample, the [PEM]
result derived from Equation 1 is multiplied by the volume of
sample solution analyzed (units of .mu.L) and divided by the
concentration of the solid sample in the sample solution (units of
mg/.mu.L). The result is a PEM concentration in ng PEM/mg
efaproxiral-Na. This number can also be expressed as ppm.
[0113] The calculation shown in Equation 1 is generally described
in general in: Hewlett-Packard MS ChemStation User's Guide for HP
G1034C MS Chemstation Software, Hewlett-Packard Company,
Publication number G1034-90043, First Edition, 2/93. The
calculation is automatically performed by the MS Chemstation
software for analyses involving internal standards. Using this
procedure, a calibration curve plot for the six standards analyzed
was constructed. The resulting calibration curve which is set forth
in FIG. 8, resulted in an R.sup.2 value of 0.994.
Example 6
Purification of Efaproxiral Sodium (5) by Filtration Through a
Poly(Vinylidene Difluoride) (PVDF) Filter
[0114] Formulation of Efaproxiral-Na
[0115] A sample of
2-[4,-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl
propionic acid sodium salt (efaproxiral-Na) prepared as described
in Examples 1, 2 and 3 was analyzed for PEM as described in Example
5. The results from that analysis demonstrated a PEM content of
50.3.+-.0.3 .mu.g PEM/g efaproxiral-Na (50 ppm). This RSR 13-Na
composition was then formulated for use as a drug product as
follows: To a 1 L volumetric flask was added sodium chloride (2.25
g), anhydrous monobasic sodium phosphate (135 mg) and dibasic
sodium phosphate, heptahydrate (7 mg), followed by approximately
800 mL of deionized water. The mixture was mixed until all of the
solids had dissolved. To this solution was added efaproxiral-Na
(21.3 g). The mixture was again mixed until all of the solids had
dissolved. The pH of the resulting solution was then adjusted to
approximately 7.9 using 0.1N HCl. Finally, the solution was diluted
to volume using deionized water. The resulting solution represents
a formulated efaproxiral drug product.
[0116] Analysis of the Formulated Efaproxiral-Na
[0117] To a 4 mL aliquot of the formulated efaproxiral drug product
was added 20 .mu.L of PEM-d.sub.5 internal standard solution in a
13.times.100 mm test tube. The mixture was vortexed to homogeneity
and frozen in a dry ice-isopropanol bath. The frozen sample was
then lyophilized to dryness overnight. To the resulting lyophilized
cake was added 400 .mu.L of methanol followed by votexing. To this
resulting mixture was added 600 .mu.L of methylene chloride
followed by vortexing. A representative sample of the prepared
mixture was then centrifuged on a table-top centrifuge for five
minutes. From the centrifuge tube, 5 .mu.L of supernatant solution
was transferred to a quartz tube for analysis by PY/GC/MS as
described above. The determined value for PEM in the formulated
drug product based on this analysis was 50.9.+-.5.0 .mu.g PEM/g
efaproxiral-Na.
[0118] Purification of Formulated Efaproxiral-Na by PVDF
Filtration
[0119] A sample of the formulated efaproxiral drug product (50 mL)
prepared as described above, was placed into a 50 mL glass syringe.
To the syringe was attached one of three 0.22 .mu.m, 25 mm
disposable syringe filters (3.9 cm.sup.2 filter area) (as set forth
in Table 3). The solution was then pushed through the selected
filter at a rate of approximately 8 mL/min. The entire 50 mL of
filtrate was collected in a clean glass container. A 4 mL
homogeneous aliquot of filtrate for each filter type was then
analyzed for PEM content as described above. The results of these
filtration experiments are set forth in the Table 3.
[0120] The foregoing description is considered to be illustrative
only of the principles of the invention. The words "comprise,"
"comprising," "include," "including," and "includes" when used in
this specification and in the following claims are intended to
specify the presence of one or more stated features, integers,
components, or steps, but they do not preclude the presence or
addition of one or more other features, integers, components,
steps, or groups thereof. Furthermore, since a number of
modifications and changes will readily occur to those skilled in
the art, it is not desired to limit the invention to the exact
composition and process shown or described above. Accordingly, all
suitable modifications and equivalents may be resorted to falling
within the scope of the invention as defined by the claims that
follow. TABLE-US-00001 TABLE 1 Related Impurities in RSR 13
Compositions Impurity Short Name Code Structure A
3-monomethylanilino efaproxiral 3MMRS13 ##STR14## B
.alpha.-desmethyl to COOH DDMRS13 ##STR15## C monomethyl .alpha. to
COOH DMRS13 ##STR16## D Diacid DA ##STR17## E 3,4-dimethyl
efaproxiral 3,4DMRS13 ##STR18## F .alpha.-ethyl-efaproxiral
.alpha.-ethyl-efaproxiral ##STR19## G 3,5-dimethylaniline 3,5-DMA
##STR20## H amidophenol amidophenol ##STR21## I efaproxiral ethyl
ester efaproxiral ethyl ester ##STR22##
[0121] TABLE-US-00002 TABLE 2 Calibration Standard Levels [PEM]
[PEM-d5] Standard Level (ng/.mu.L) (ng/.mu.L) 1 0.985 10.4 2 1.97*
10.4 3 3.94* 10.4 4 5.91 10.4 5 7.88* 10.4 6 9.85 10.4 *Calibration
levels corresponding to those typically used
[0122] TABLE-US-00003 TABLE 3 Filtration of Formulated Efaproxiral
Drug Product Filter Composition PEM (.mu.g/g efaproxiral-Na) No
Filter 55.6 PTFE 61.6 Cellulose Esters 15.6 PVDF 9.3
* * * * *