U.S. patent application number 15/075750 was filed with the patent office on 2016-07-14 for alkoxylation methods.
The applicant listed for this patent is Nektar Therapeutics. Invention is credited to John R. Handley, Antoni Kozlowski, Greg Lavaty, Samuel P. McManus, Anthony G. Schaefer, David Swallow, Sachin Tipnis.
Application Number | 20160200867 15/075750 |
Document ID | / |
Family ID | 43639124 |
Filed Date | 2016-07-14 |
United States Patent
Application |
20160200867 |
Kind Code |
A1 |
Kozlowski; Antoni ; et
al. |
July 14, 2016 |
ALKOXYLATION METHODS
Abstract
Among other aspects, provided herein is a mixed-acid salt of a
water-soluble polymer-drug conjugate, along with related methods of
making and using the same. The mixed-salt acid salt is stably
formed, and appears to be more resistant to hydrolytic degradation
than the corresponding predominantly pure acid salt or free base
forms of the polymer-drug conjugate. The mixed acid salt is
reproducibly prepared and recovered, and provides surprising
advantages over non-mixed acid salt forms of the water-soluble
polymer drug conjugate.
Inventors: |
Kozlowski; Antoni;
(Huntsville, AL) ; McManus; Samuel P.;
(Guntersville, AL) ; Tipnis; Sachin; (Houston,
TX) ; Lavaty; Greg; (Sugar Land, TX) ;
Swallow; David; (Spring, TX) ; Handley; John R.;
(Duluth, MN) ; Schaefer; Anthony G.; (Huntsville,
AL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nektar Therapeutics |
San Francisco |
CA |
US |
|
|
Family ID: |
43639124 |
Appl. No.: |
15/075750 |
Filed: |
March 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14284067 |
May 21, 2014 |
9320808 |
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15075750 |
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13510555 |
Jan 22, 2013 |
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PCT/US2010/057289 |
Nov 18, 2010 |
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14284067 |
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61290072 |
Dec 24, 2009 |
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61262463 |
Nov 18, 2009 |
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Current U.S.
Class: |
548/520 ;
568/620; 568/623 |
Current CPC
Class: |
C07C 51/412 20130101;
C08G 65/329 20130101; A61P 11/00 20180101; A61K 31/4745 20130101;
A61P 15/00 20180101; C08G 65/331 20130101; C07D 491/22 20130101;
A61P 1/04 20180101; A61P 35/00 20180101; C07C 53/18 20130101; A61K
47/60 20170801; C08G 65/33396 20130101; C08G 65/48 20130101; C08L
2203/02 20130101 |
International
Class: |
C08G 65/331 20060101
C08G065/331; C08G 65/333 20060101 C08G065/333 |
Claims
1-50. (canceled)
51. A method comprising the step of alkoxylating in a suitable
solvent a previously isolated alkoxylatable oligomer to form an
alkoxylated polymeric material, wherein the previously isolated
alkoxylatable oligomer has a known and defined weight-average
molecular weight of greater than 300 Daltons.
52. The method of claim 51, wherein the previously isolated
alkoxylatable oligomer has a known and defined weight-average
molecular weight of greater than 500 Daltons.
53. The method of claim 51, wherein both the previously isolated
alkoxylatable oligomer and the alkoxylated polymeric product are
soluble in the suitable solvent.
54. The method of claim 51, wherein the previously isolated
alkoxylatable oligomer is prepared by (a) alkoxylating a precursor
molecule having a molecular weight of less than 300 Daltons to form
a reaction mixture comprising an alkoxylatable oligomer, and (b)
isolating the alkoxylatable oligomer from the reaction mixture.
55. The method of claim 51 where the alkoxylation utilizes ethylene
oxide as an alkoxylation agent.
56. The method of claim 54, wherein the precursor molecule is
selected from the group consisting of glycerol, diglycerol,
triglycerol, hexaglycerol, mannitol, sorbitol, pentaerythritol,
dipentaerthitol, and tripentaerythritol.
57. The method of claim 51, wherein both the previously isolated
alkoxylatable oligomer and the alkoxylated polymeric product each
has from one to eight primary hydroxyl groups.
58. The method of claim 57, wherein each of the one to eight
primary hydroxyl groups is the result of an alkoxylation
reaction.
59. The method of claim 57, wherein the neither the previously
isolated alkoxylatable oligomer and the alkoxylated polymeric
product has a hydroxyl group of the precursor molecule.
60. The method of claim 51, wherein both the previously isolated
alkoxylatable oligomer and the alkoxylated polymeric product each
has a branched structure.
61. The method of claim 60, wherein the branched structure is a 4-
to 8-arm branched structure.
62. The method of claim 61, wherein the branched structure is a
4-arm branched structure.
63. The method of claim 61, wherein the branched structure is a
5-arm branched structure.
64. The method of claim 61, wherein the branched structure is a
6-arm branched structure.
65. The method of claim 61, wherein the branched structure is an
8-arm branched structure.
66. The method of claim 51, wherein the previously isolated
alkoxylatable oligomer has the following structure: ##STR00032##
wherein the average value of n within the structure is from 2 to
50.
67. The method of claim 51, wherein the alkoxylated polymeric
material has the following structure: ##STR00033## wherein the
average value of all the instances of n within the structure is
from 10 to 1000.
68. The method of claim 51, wherein the previously isolated
alkoxylatable oligomer has the following structure: ##STR00034##
wherein the average value of all instances of the value of n within
the structure is from 2 to 35.
69. The method of claim 51, wherein the alkoxylated polymeric
material has the following structure: ##STR00035## wherein the
average value of all instances of the value of n within the
structure is from 10 to 750.
70. The method of claim 51, wherein the previously isolated
alkoxylatable oligomer has the following structure: ##STR00036##
wherein the average value of all instances of the value of n within
the structure is 2 to 35.
71. The method of claim 51, wherein the alkoxylated polymeric
material has the following structure: ##STR00037## wherein the
average value of all instances of the value of n within the
structure is 10 to 600.
72. The method claim 67, wherein the average value of all instances
of the value of n within the structure is from 50 to 400.
73. The method claim 67, wherein the average value of all instances
of the value of n within the structure is from 50 to 300.
74. The method of claim 66, wherein all values of n are within
three standard deviations of each other.
75. The method of claim 66, wherein all values of n are within two
standard deviations of each other.
76. The method claim 66, wherein all values of n are within one
standard deviation of each other.
77. The method of claim 51, wherein the suitable solvent include
organic solvents selected from the group consisting of
tetrahydrofuran (THF), dimethylformamide (DMF), toluene, benzene,
xylenes, mesitylene, tetrachloroethylene, anisole, and mixtures of
the foregoing.
78. The method of claim 51, wherein the suitable solvent is
selected from the group consisting of toluene, xylene, mesitylene,
tetrahydrofuran (THF), and mixtures of foregoing.
79. The method of claim 51, wherein the suitable solvent is toluene
used in quantities that after ethoxylation the solvent consists
more than 25 wt % and less than 75 wt % of the reaction
mixture.
80. The method of claim 51, wherein the step of alkoxylating is
carried out under alkoxylating conditions, wherein the alkoxylation
conditions include the presence of a strong base.
81. The method of claim 80, wherein the strong base is selected
from the group consisting of one or more alkali metals.
82. The method of claim 80, wherein the strong base is selected
from the group consisting of metallic potassium, metallic sodium,
sodium-potassium alloys, and a hydroxide.
83. The method of claim 82, wherein the hydroxide is NaOH, KOH and
mixtures thereof.
84. The method of claim 82, wherein the strong base is a
sodium-potassium alloy.
85. The method of claim 80, wherein the strong base is an alkoxide
of a previously isolated alkoxylatable oligomer.
86. The method of claim 80, wherein the strong base is present in a
catalytic amount.
87. The method of claim 86, wherein the catalytic amount is from
0.001 to 10.0 weight percent strong base based upon the weight of
the total reaction mixture.
88. The method of claim 86, wherein the catalytic amount is from
0.01 to about 6.0 weight percent strong base based upon the weight
of the total reaction mixture.
89. The method of claim 51, wherein the step of alkoxylating is
carried out under alkoxylating conditions wherein the amount of
water present is less than 20 ppm.
90. The method of claim 89, wherein the step of alkoxylating is
carried out under alkoxylating conditions wherein the amount of
water present is less than 14 ppm.
91. The method of claim 90, wherein the alkoxylating step is
carried out under alkoxylating conditions wherein the amount of
water present is less than 8 ppm.
92. The method of claim 1, wherein the alkoxylating step is carried
out at a temperature between 80.degree. C. and 140.degree. C.
93. A composition comprising the alkoxylated polymeric product
prepared in accordance with the method claim 51.
94. The composition of claim 93, wherein the purity of the
alkoxylated polymeric product is greater than 92 wt % and the total
content of high molecular weight impurities and diols is less than
8 wt %.
95. The composition of claim 94, wherein the purity is greater than
98 wt % and the total content of high molecular weight impurities
and diols is less than 2 wt %.
96. The method of claim 51, further comprising the step of
modifying the alkoxylated polymeric material to bear a reactive
group to thereby form a water-soluble polymer reagent.
97. The method of claim 96, wherein the water-soluble polymer
reagent has the following structure: ##STR00038## wherein each n is
from about 40 to about 500.
98-100. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to each of U.S. Provisional Patent Application
Ser. No. 61/262,463, filed 18 Nov. 2009, and U.S. Provisional
Patent Application Ser. No. 61/290,072, filed 24 Dec. 2009, both of
which are incorporated herein by reference in their entireties.
FIELD
[0002] This disclosure relates generally to mixed acid salt
compositions of water-soluble polymer-drug conjugates,
pharmaceutical compositions thereof, and methods for preparing,
formulating, administering and using such mixed acid salt
compositions. This disclosure also relates generally to
alkoxylation methods for preparing alkoxylated polymeric materials
from a previously isolated alkoxylated oligomer, as well as to
compositions comprising the alkoxylated polymeric material, methods
for using the alkoxylated polymeric material, and the like.
BACKGROUND
[0003] Over the years, numerous methods have been proposed for
improving the stability and delivery of biologically active agents.
Challenges associated with the formulation and delivery of
pharmaceutical agents can include poor aqueous solubility of the
pharmaceutical agent, toxicity, low bioavailability, instability,
and rapid in-vivo degradation, to name just a few. Although many
approaches have been devised for improving the delivery of
pharmaceutical agents, no single approach is without its potential
drawbacks. For instance, commonly employed drug delivery approaches
aimed at solving or at least ameliorating one or more of these
problems include drug encapsulation, such as in a liposome, polymer
matrix, or unimolecular micelle, covalent attachment to a
water-soluble polymer such as polyethylene glycol, use of gene
targeting agents, formation of salts, and the like.
[0004] Covalent attachment of a water-soluble polymer can improve
the water-solubility of an active agent as well as alter its
pharmacological properties. Certain exemplary polymer conjugates
are described in U.S. Pat. No. 7,744,861, among others. In another
approach, an active agent having acidic or basic functionalities
can be reacted with a suitable base or acid and marketed in salt
form. Over half of all active molecules are marketed as salts
(Polymorphism in the Pharmaceutical Industry, Hilfiker, R., ed.,
Wiley-VCH, 2006). Challenges with salt forms include finding an
optimal salt, as well as controlling solid state behavior during
processing. Biopharmaceutical salts can be amorphous, crystalline,
and exist as hydrates, solvents, various polymorphs, etc.
Interestingly, rarely are salt forms, let alone mixed acid salt
forms, of polymer conjugates used in drug formulations.
[0005] Another challenge associated with preparing active agent
conjugates of water-soluble polymers waters is the ability to
prepare relatively pure water-soluble polymers in a consistent and
reproducible method. For example, poly(ethylene glycol) (PEG)
derivatives activated with reactive functional groups are useful
for coupling to active agents (such as small molecules and
proteins), thereby forming a conjugate between the PEG and the
active agent. When an active agent is conjugated to a polymer of
poly(ethylene glycol) or "PEG," the conjugated active agent is
conventionally referred to as having been "PEGylated."
[0006] When compared to the safety and efficacy of the active agent
in the unconjugated form, the conjugated version exhibits
different, and often clinically beneficial, properties. The
commercial success of PEGylated active agents such as PEGASYS.RTM.
PEGylated interferon alpha-2a (Hoffmann-La Roche, Nutley, N.J.),
PEG-INTRON.RTM. PEGylated interferon alpha-2b (Schering Corp.,
Kennilworth, N.J.), and NEULASTA.RTM. PEG-filgrastim (Amgen Inc.,
Thousand Oaks, Calif.) demonstrates the degree to which PEGylation
has the potential to improve one or more properties of an active
agent.
[0007] In preparing a conjugate, a polymeric reagent is typically
employed to allow for a relatively straightforward synthetic
approach for conjugate synthesis. By combining a composition
comprising a polymeric reagent with a composition comprising the
active agent, it is possible--under the appropriate reaction
conditions--to carry out a relatively convenient conjugate
synthesis.
[0008] The preparation of the polymeric reagent suitable to the
regulatory requirements for drug products, however, is often
challenging. Conventional polymerization approaches result in
relatively impure compositions and/or low yield. Although such
impurities and yields may not be problematic outside the
pharmaceutical field, safety and cost represent important concerns
in the context of medicines for human use. Thus, conventional
polymerization approaches are not suited for the synthesis of
polymeric reagents intended for the manufacture of pharmaceutical
conjugates.
[0009] In the case of multiarm polymers, there is a dearth of
available, desirable water soluble polymers that have well
controlled and well defined properties with the absence of
significant amounts of undesirable impurities. Thus one can readily
obtain, for example, a high molecular weight multiarm poly(ethylene
glycol) but drug conjugates manufactured from commercial polymers
may have significant amounts (i.e. >8%) of polymer-drug
conjugate having either very low or very high molecular weight
biologically active impurities. This extent of active impurities in
a drug composition may render such compositions unacceptable and
thus render approval of such drugs challenging if not
impossible.
SUMMARY OF THE INVENTION
[0010] In one or more embodiments of the invention, a composition
is provided, the composition comprising mixed salts of water
soluble polymer-active agent conjugates, wherein the active agent
in the conjugate has at least one amine or other basic
nitrogen-containing group, and further wherein the amine or other
basic nitrogen-containing group is either protonated or
unprotonated (i.e., as the free base), where any given protonated
amine or other basic nitrogen containing group is an acid addition
salt of either a strong inorganic acid or a strong organic acid
such as, for example, trifluoroacetic acid (TFA).
[0011] Examples of strong inorganic acids include hydrohalic acids
(e.g., hydrochloric acid, hydrofluoric, hydroiodic, and
hydrobromic), sulfuric acid, nitric acid, phosphoric acid, and
nitrous acid.
[0012] In one or more embodiments of the invention, the protonated
form comprises an addition salt of a hydrohalic acid.
[0013] In one or more embodiments of the invention, the protonated
form comprises an addition salt of hydrochloric acid.
[0014] Examples of strong organic acids include organic acids
having a pKa of less than about 2.00. Examples include
trichloroacetic acid, dichloroacetic acid, as well as mixed
haloacetic acids such as fluorodichloroacetic acid,
fluorochloroacetic acid, chlorodifluoroacetic acid and the
like.
[0015] In one or more embodiments of the invention, the water
soluble polymer is linear or multi-armed.
[0016] In one or more embodiments of the invention, the water
soluble polymer is a poly(alkylene glycol) such as poly(ethylene
glycol) or a copolymer or terpolymer thereof.
[0017] In one or more embodiments of the invention, the active
agent is selected from a small molecule drug, a peptide, and a
protein.
[0018] In one or more embodiments of the invention, the active
agent is a camptothecin.
[0019] In one or more embodiments of the invention, the composition
comprises a mixed salt of a water-soluble polymer-active agent
conjugate corresponding to structure (I):
##STR00001##
wherein n is an integer ranging from 20 to about 600 (specific
protonated amino nitrogen atoms and counterions not shown), and for
each amine group within each irinotecan, each amino group is either
protonated or unprotonated, where any given protonated amine group
is an acid salt form of an inorganic acid or an organic acid such
as trifluoroacetic acid.
[0020] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates) the mole percent of active agent amino groups (or other
basic nitrogen atoms) in the composition that are protonated as the
TFA salt is greater than each of the mole percent of active agent
amino groups in the composition that are protonated as an inorganic
acid salt and the mole percent of active agent amino groups in the
composition in free base form.
[0021] In yet an alternative embodiment, with respect to a
composition of conjugates (e.g., a composition of four-arm
conjugates) the mole percent of active agent amine groups (or other
basic nitrogen atoms) in the composition that are protonated as the
TFA salt is greater than the mole percent of active agent amine
groups in the composition that are in free base (i.e.,
unprotonated) form.
[0022] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates) at least 20 mole percent of active agent amine groups
in the composition are protonated as the TFA salt.
[0023] In one or more embodiments, with respect to a composition of
conjugates (e.g., a composition of four-arm conjugates) at least 25
mole percent of active agent amine groups in the composition are
protonated as the TFA salt.
[0024] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates), about 20-45 mole percent of active agent amino groups
in the composition are protonated as the TFA salt.
[0025] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates), about 24-38 mole percent of active agent amino groups
in the composition are protonated as the TFA salt.
[0026] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates), about 35-65 mole percent of active agent amino groups
in the composition are protonated as the TFA salt.
[0027] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates), about 30-65 mole percent of the active agent amino
groups in the composition are protonated as an inorganic acid salt
(such as the HCl salt).
[0028] In yet one or more additional embodiments of the invention,
with respect to a composition of conjugates (e.g., a composition of
four-arm conjugates), about 32-60 mole percent of the active agent
amino groups in the composition are protonated as an inorganic acid
salt (such as the HCl salt).
[0029] In yet one or more further embodiments of the invention,
with respect to a composition of conjugates (e.g., a composition of
four-arm conjugates), about 35-57 mole percent of the active agent
amino groups in the composition are protonated as an inorganic acid
salt (such as the HCl salt).
[0030] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates), about 25-40 mole percent of the active agent amino
groups in the composition are protonated as an inorganic acid salt
(such as the HCl salt), and about 5-35 mole percent of the active
agent amino groups in the composition are non-protonated (i.e., as
the free base).
[0031] In one or more embodiments of the invention, with respect to
a composition of conjugates (e.g., a composition of four-arm
conjugates), about 32-60 mole percent of the active agent amino
groups in the composition are protonated as an inorganic acid salt
(such as the HCl salt), and about 5-35 mole percent of the active
agent amino groups in the composition are non-protonated (i.e., as
the free base).
[0032] In one or more embodiments of the invention, a
trifluoroacetic acid/hydrochloric acid mixed salt of a conjugate is
provided, the conjugate having the following structure:
##STR00002##
wherein n is an integer ranging from about 20 to about 500
(including about 40 to about 500) (noting that in the above
structure, specific basic nitrogen atoms in protonated form and
corresponding anions are not shown). In one or more embodiments of
the invention, a portion of amino groups in conjugate encompassed
by the structure immediately above are non-protonated. Exemplary
molar ratios of protonated and non-protonated forms as provided
above and further herein apply to the foregoing conjugate.
[0033] In one or more embodiments of the invention, a method for
providing a mixed salt of a water-soluble polymer-active agent
conjugate is provided, comprising the steps of: (i) deprotecting an
inorganic acid salt of an amine-containing active agent in
protected form by treatment with trifluoroacetic acid (TFA) or
other organic acid deprotecting reagent to form a deprotected
active agent acid salt, (ii) coupling the deprotected active agent
acid salt of step (i) with a water-soluble polymer reagent in the
presence of a base (e.g., trimethyl amine, triethyl amine, and
dimethylamino-pyridine) to form a polymer-active agent conjugate,
and (iii) recovering the polymer-active agent conjugate, where the
recovered polymer-active agent conjugate is characterized by having
active agent amino groups therein individually present in a form
selected from the group consisting of free base form
(non-protonated), inorganic acid salt form, and TFA or other
organic acid salt form. In one or more embodiments of the
invention, the method further comprises determining the relative
molar amounts of inorganic acid and TFA in the deprotected acid
salt formed in step (i). In one or more embodiments of the
invention, the inorganic acid salt in step (i) is a hydrohalic acid
salt such as a hydrochloric acid salt. In one or more embodiments
of the invention, the amount of base in step (ii) ranges from
1.00-2.00 (moles TFA+moles acid). In one or more related
embodiments, the amount of base in step (ii) ranges from 1.00 to
1.50 (moles TFA+moles inorganic acid), where the parenthesis
indicates multiplication. In one or more related embodiments, the
amount of base in step (ii) ranges from 1.00 to 1.20 (moles
TFA+moles inorganic acid). In one particular embodiment, the number
of equivalents of base is 1.05 ((moles TFA+moles inorganic
acid).
[0034] In one or more embodiments of the invention, the
water-soluble polymer reagent is an activated polyethylene glycol
ester (i.e., a polyethylene glycol reagent having at least one
activated ester group). In one or more embodiments of the
invention, the water-soluble polymer reagent is a polyethylene
glycol reagent having three or more polymer arms.
[0035] In one or more embodiments of the invention, the active
agent amine groups in the polymer-active agent conjugate are
selected from the group consisting of secondary amine groups and
tertiary amine groups. In one or more embodiments of the invention,
the active agent amine groups are tertiary amino groups. In yet
another embodiment, the polymer-active agent conjugate comprises a
basic nitrogen atom that, as its corresponding conjugate acid, has
a pK.sub.a in a range of about 10-11.5.
[0036] In one or more embodiments of the invention the active agent
is selected from a small molecule, a peptide and a protein. In one
or more embodiments of the invention, the active agent is a
camptothecin. Illustrative camptothecin molecules are selected from
camptothecin, irinotecan, and 7-ethyl-10-hydroxy-camptothecin
(SN-38). Exemplary sites for covalent attachment to a water-soluble
polymer include the 7-, 10-, and 20-ring positions of the
camptothecin skeleton, among others.
[0037] In one or more embodiments of the invention, a
pharmaceutically acceptable composition is provided, the
pharmaceutically acceptable composition comprising (i) a mixed salt
according to any one or more of the embodiments described herein,
and (ii) lactate buffer, optionally in lyophilized form. In one or
more embodiments of the invention, the pharmaceutically acceptable
composition is a sterile composition. In one or more embodiments of
the invention, the pharmaceutically acceptable composition is
optionally provided in a container (e.g., a vial), optionally
containing the equivalent of a 100-mg dose of irinotecan in
unconjugated form.
[0038] In one or more embodiments of the invention, a method is
provided, the method comprising administering a
conjugate-containing composition described herein (where the active
agent is an anti-cancer agent) to an individual suffering from one
or more types of cancerous solid tumors, wherein the
conjugate-containing composition is optionally dissolved in a
solution of 5% w/w dextrose. In one or more embodiments of the
invention, administration is effected via intravenous infusion.
[0039] In one or more embodiments of the invention, a method for
preparing a mixed salt of a water-soluble polymer-active agent
conjugate is provided, the method comprising the steps of: (i)
deprotecting t-Boc glycine-irinotecan.HCl by treatment with
trifluoroacetic acid (TFA) to form deprotected glycine-irinotecan
HCl/TFA mixed salt, (ii) coupling the deprotected
glycine-irinotecan HCl/TFA mixed salt with
4-arm-pentaerythritolyl-polyethylene glycol-carboxymethyl
succinimide in the presence of a base under conditions effective to
form a conjugate, 4-arm-pentaerythritolyl-polyethylene
glycol-carboxymethyl glycine-irinotecan (also referred to as
pentaerythritolyl-4-arm-(PEG-1-methylene-2-oxo-vinylamino acetate
linked-Irinotecan) and (iii) recovering the conjugate from step
(ii), wherein the conjugate is a mixed salt comprising amine groups
in a combination of free base, HCl, and TFA salt form. In one or
more embodiments of the invention, the method further comprises
purifying the conjugate (e.g., comprising recrystallizing the
conjugate to form a recrystallized conjugate). In one or more
embodiments of the invention, a recrystallized product is provided,
the recrystallized product being a mixed acid salt comprising
active agent amino groups existing as a combination of free base,
HCl, and TFA salt forms.
[0040] In one or more embodiments of the invention, a method of
treating a mammal suffering from cancer is provided, the method
comprising administering a therapeutically effective amount of a
mixed salt of a water soluble polymer-camptothecin conjugate
comprising a camptothecin having amine or other basic nitrogen
containing groups in both free base and in protonated form, where
the each protonated form exists as an acid addition salt of either
a strong inorganic acid and trifluoroacetic acid. The mixed acid
salt is administered to the mammal effective to produce a slowing
or inhibition of solid tumor growth in the subject. In one or more
embodiments of the invention, the cancerous solid tumor is selected
from the group consisting of colorectal, ovarian, cervical, breast
and non-small cell lung.
[0041] In one or more embodiments of the invention, a mixed acid
salt of an active agent conjugate as described herein is provided,
wherein the active is an anti-cancer agent for the manufacture of a
medicament for treating cancer.
[0042] In another aspect, a method is provided, the method
comprising the step of alkoxylating in a suitable solvent a
previously isolated alkoxylatable oligomer to form an alkoxylated
polymeric product, wherein the previously isolated alkoxylatable
oligomer has a known and defined weight-average molecular weight of
greater than 300 Daltons (e.g., greater than 500 Daltons).
[0043] In one or more embodiments of the foregoing aspect of the
invention, a composition is provided, the composition comprising an
alkoxylated polymeric product prepared by a method comprising the
step of alkoxylating in a suitable solvent a previously isolated
alkoxylatable oligomer to form an alkoxylated polymeric product,
wherein the previously isolated alkoxylatable oligomer has a known
and defined weight-average molecular weight of greater than 300
Daltons (e.g., greater than 500 Daltons).
[0044] In one or more embodiments of the invention, a composition
is provided, the composition comprising an alkoxylated polymeric
product having a purity of greater than 92 wt % and the total
combined content of high molecular weight products and diols is
less than 8 wt % (e.g., less than 2 wt %), as determined by, for
example, gel filtration chromatography (GFC) analysis.
[0045] In one or more embodiments of the invention, the alkoxylated
polymer product has the following structure:
##STR00003##
wherein each n is an integer from 20 to 1000 (e.g., from 50 to
1000).
[0046] In one or more embodiments of the invention, a method is
provided, the method comprising the steps of (i) alkoxylating in a
suitable solvent a previously isolated alkoxylatable oligomer to
form an alkoxylated polymeric material, wherein the previously
isolated alkoxylatable oligomer has a known and defined
weight-average molecular weight of greater than 300 Daltons (e.g.,
greater than 500 Daltons), and (ii) optionally, further activating
the alkoxylated polymeric product to provide an activated
alkoxylated polymeric product that is useful as (among other
things) a polymeric reagent for preparing polymer-drug
conjugates.
[0047] In one or more embodiments of the invention, a method is
provided, the method comprising the step of activating an
alkoxylated polymeric product obtained from and/or contained within
a composition comprising an alkoxylated polymeric product having a
purity of greater than 90% to thereby form an activated alkoxylated
polymeric product that is useful as (among other things) a polymer
reagent for preparing polymer-drug conjugates.
[0048] In one or more embodiments of the invention, a method is
provided, the method comprising the step of conjugating an
activated alkoxylated polymeric product to an active agent, wherein
the activated alkoxylated polymeric product was prepared by a
method comprising the step of activating an alkoxylated polymeric
product obtained from and/or contained within a composition
comprising an alkoxylated polymeric product having a purity of
greater than 90% to thereby form an activated alkoxylated polymeric
product.
[0049] In one or more embodiments of the invention, a mixed salt of
a water-soluble polymer-active agent conjugate is provided, the
conjugate having been prepared by coupling (under conjugation
conditions) an amine-bearing active agent (e.g., a deprotected
glycine-irinotecan) to a polymer reagent (e.g., a 4-arm
pentaerythritolyl-poly(ethylene glycol)-carboxymethyl succinimide)
in the presence of a base to form a conjugate, wherein the
conjugate is in the form of a mixed salt conjugate (e.g., the
conjugate possesses nitrogen atoms, each one of which will either
be protonated or unprotonated, where any given protonated amino
group is an acid salt possessing one of two different anions), and
further wherein, optionally, the polymer reagent is prepared from
an alkoxylation product prepared as described herein.
[0050] Additional embodiments of the present method, compositions,
and the like will be apparent from the following description,
drawings, examples, and claims. As can be appreciated from the
foregoing and following description, each and every feature
described herein, and each and every combination of two or more of
such features, is included within the scope of the present
disclosure provided that the features included in such a
combination are not mutually inconsistent. In addition, any feature
or combination of features may be specifically excluded from any
embodiment of the present invention. Additional aspects and
advantages of the present invention are set forth in the following
description and claims, particularly when considered in conjunction
with the accompanying examples and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 is a graph illustrating the results of stress
stability studies on three different samples of
4-arm-PEG-Gly-Irino-20K, each having a different composition with
respect to relative amounts of trifluoroacetic acid and
hydrochloride salts, as well as free base. Samples tested included
>99% HCl salt (<1% free base, triangles), 94% total salt (6%
free base, squares), and 52% total salt (48% free base, circles).
The samples were stored at 25.degree. C. and 60% relative humidity;
the plot illustrates degradation of compound over time, as
described in detail in Example 3.
[0052] FIG. 2 is a graph illustrating the increase in free
irinotecan over time in samples of 4-arm-PEG-Gly-Irino-20K stored
at 40.degree. C. and 75% relative humidity, each having a different
composition with respect to relative amounts of trifluoroacetic
acid and hydrochloride salts, as well as free base. Samples tested
correspond to product containing >99% HCl salt (<1% free
base, squares) and product containing 86% total salts (14% free
base, diamonds), as described in Example 3.
[0053] FIG. 3 is a graph illustrating the increase over time in
small PEG species (PEG degradation products) in samples of
4-arm-PEG-Gly-Irinio-20K stored at 40.degree. C. and 75% relative
humidity, as described in detail in Example 3. Samples tested
correspond to product containing >99% HCl salt (<1% free
base, squares) and product containing 86% total salts (14% free
base, diamonds).
[0054] FIG. 4 is a compilation of overlays of chromatograms
exhibiting release of irinotecan via hydrolysis from mono-(DS-1),
di-(DS-2), tri-(DS-3) and tetra-irinotecan substituted (DS-4)
4-arm-PEG-Gly-Irino-20K as described in detail in Example 5.
[0055] FIG. 5 is a graph illustrating the results of hydrolysis of
various species of 4-arm-PEG-Gly-Irino-20K as described above in
aqueous buffer at pH 8.4 in the presence of porcine
carboxypeptidase B in comparison to hydrolysis kinetics modeling
data as described in Example 5. For the kinetics model, the
hydrolysis of all species was assumed to be 1.sup.st order
kinetics. The 1.sup.st order reaction rate constant for
disappearance of DS4 (0.36 hr.sup.-1) was used to generate all
curves.
[0056] FIG. 6 is a graph illustrating the hydrolysis of various
species of 4-arm-PEG-Gly-Irino-20K as described above in human
plasma in comparison to hydrolysis kinetics modeling data. Details
are provided in Example 5. For the kinetics model, the hydrolysis
of all species was assumed to be 1.sup.st order kinetics. The
1.sup.st order reaction rate constant for disappearance of DS 4
(0.26 hr.sup.-1) was used to generate all curves.
[0057] FIG. 7 is a chromatogram following gel filtration
chromatography of a material prepared a described in Example 8.
[0058] FIG. 8 is a chromatogram following gel filtration
chromatography of a material prepared a described in Example 9.
DETAILED DESCRIPTION
[0059] Various aspects of the invention now will be described more
fully hereinafter. Such aspects may, however, be embodied in many
different forms and should not be construed as limited to the
embodiments set forth herein; rather, these embodiments are
provided so that this disclosure will be thorough and complete, and
will fully convey the scope of the invention to those skilled in
the art.
[0060] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entireties. In the event of an inconsistency
between the teachings of this specification and the art
incorporated by reference, the meaning of the teachings in this
specification shall prevail.
[0061] It must be noted that, as used in this specification, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to a "polymer" includes a single polymer as well as two
or more of the same or different polymers, reference to a
"conjugate" refers to a single conjugate as well as two or more of
the same or different conjugates, reference to an "excipient"
includes a single excipient as well as two or more of the same or
different excipients, and the like.
[0062] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions described below.
[0063] A "functional group" is a group that may be used, under
normal conditions of organic synthesis, to form a covalent linkage
between the entity to which it is attached and another entity,
which typically bears a further functional group. The functional
group generally includes multiple bond(s) and/or heteroatom(s).
Preferred functional groups are described herein.
[0064] The term "reactive" refers to a functional group that reacts
readily or at a practical rate under conventional conditions of
organic synthesis. This is in contrast to those groups that either
do not react or require strong catalysts or impractical reaction
conditions in order to react (i.e., a "nonreactive" or "inert"
group).
[0065] A "protecting group" is a moiety that prevents or blocks
reaction of a particular chemically reactive functional group in a
molecule under certain reaction conditions. The protecting group
will vary depending upon the type of chemically reactive group
being protected as well as the reaction conditions to be employed
and the presence of additional reactive or protecting groups in the
molecule. Functional groups that may be protected include, by way
of example, carboxylic acid groups, amino groups, hydroxyl groups,
thiol groups, carbonyl groups and the like. Representative
protecting groups for carboxylic acids include esters (such as a
p-methoxybenzyl ester), amides and hydrazides; for amino groups,
carbamates (such as tert-butoxycarbonyl) and amides; for hydroxyl
groups, ethers and esters; for thiol groups, thioethers and
thioesters; for carbonyl groups, acetals and ketals; and the like.
Such protecting groups are well-known to those skilled in the art
and are described, for example, in T. W. Greene and G. M. Wuts,
Protecting Groups in Organic Synthesis, Third Edition, Wiley, New
York, 1999, and in P. J. Kocienski, Protecting Groups, Third Ed.,
Thieme Chemistry, 2003, and references cited therein.
[0066] A functional group in "protected form" refers to a
functional group bearing a protecting group. As used herein, the
term "functional group" or any synonym thereof is meant to
encompass protected forms thereof.
[0067] "PEG" or "poly(ethylene glycol)" as used herein, is meant to
encompass any water-soluble poly(ethylene oxide). Typically, PEGs
for use in the present invention will comprise one of the two
following structures: "--(CH.sub.2CH.sub.2O).sub.n--" or
"--(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2--," depending upon
whether or not the terminal oxygen(s) has been displaced, e.g.,
during a synthetic transformation. The variable (n) ranges from 3
to about 3000, and the terminal groups and architecture of the
overall PEG may vary.
[0068] A water-soluble polymer may bear one or more "end-capping
group," (in which case it can stated that the water-soluble polymer
is "end-capped." With regard to end-capping groups, exemplary
end-capping groups are generally carbon- and hydrogen-containing
groups, typically comprised of 1-20 carbon atoms and an oxygen atom
that is covalently bonded to the group. In this regard, the group
is typically alkoxy (e.g., methoxy, ethoxy and benzyloxy) and with
respect to the carbon-containing group can optionally be saturated
or unsaturated, as well as aryl, heteroaryl, cyclo, heterocyclo,
and substituted forms of any of the foregoing.
[0069] The end-capping group can also comprise a detectable label.
When the polymer has an end-capping group comprising a detectable
label, the amount or location of the polymer and/or the moiety
(e.g., active agent) to which the polymer is attached can be
determined by using a suitable detector. Such labels include,
without limitation, fluorescers, chemiluminescers, moieties used in
enzyme labeling, colorimetric (e.g., dyes), metal ions, radioactive
moieties, and the like.
[0070] "Water-soluble", in the context of a polymer of the
invention or a "water-soluble polymer segment" is any segment or
polymer that is at least 35% (by weight), preferably greater than
70% (by weight), and more preferably greater than 95% (by weight)
soluble in water at room temperature. Typically, a water-soluble
polymer or segment will transmit at least about 75%, more
preferably at least about 95% of light, transmitted by the same
solution after filtering.
[0071] The term "activated," when used in conjugation with a
particular functional group, refers to a reactive functional group
that reacts readily with an electrophile or nucleophile on another
molecule. This is in contrast to those groups that require strong
bases or highly impractical reaction conditions in order to react
(i.e., a "nonreactive" or "inert" group).
[0072] "Electrophile" refers to an ion or atom or a neutral or
ionic collection of atoms having an electrophilic center. i.e., a
center that is electron seeking or capable of reacting with a
nucleophile.
[0073] "Nucleophile" refers to an ion or atom or a neutral or ionic
collection of atoms having a nucleophilic center, i.e., a center
that is seeking an electrophilic center or capable of reacting with
an electrophile.
[0074] The terms "protected" or "protecting group" or "protective
group" refer to the presence of a moiety (i.e., the protecting
group) that prevents or blocks reaction of a particular chemically
reactive functional group in a molecule under certain reaction
conditions. The protecting group will vary depending upon the type
of chemically reactive group being protected as well as the
reaction conditions to be employed and the presence of additional
reactive or protecting groups in the molecule, if any. Protecting
groups known in the art can be found in Greene, T. W., et al.,
PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 3rd ed., John Wiley &
Sons, New York, N.Y. (1999).
[0075] "Molecular mass" in the context of a water-soluble polymer
such as PEG, refers to the weight average molecular weight of a
polymer, typically determined by size exclusion chromatography,
light scattering techniques, or intrinsic viscosity determination
in an organic solvent like 1,2,4-trichlorobenzene.
[0076] The terms "spacer" and "spacer moiety" are used herein to
refer to an atom or a collection of atoms optionally used to link
interconnecting moieties such as a terminus of a series of monomers
and an electrophile. The spacer moieties of the invention may be
hydrolytically stable or may include a physiologically hydrolyzable
or enzymatically degradable linkage.
[0077] A "hydrolyzable" bond is a relatively labile bond that
reacts with water (i.e., is hydrolyzed) under physiological
conditions. The tendency of a bond to hydrolyze in water will
depend not only on the general type of linkage connecting two
central atoms but also on the substituents attached to these
central atoms. Illustrative hydrolytically unstable linkages
include carboxylate ester, phosphate ester, anhydrides, acetals,
ketals, acyloxyalkyl ether, imines, orthoesters, peptides and
oligonucleotides.
[0078] An "enzymatically degradable linkage" means a linkage that
is subject to degradation by one or more enzymes.
[0079] A "hydrolytically stable" linkage or bond refers to a
chemical bond that is substantially stable in water, that is to
say, does not undergo hydrolysis under physiological conditions to
any appreciable extent over an extended period of time. Examples of
hydrolytically stable linkages include but are not limited to the
following: carbon-carbon bonds (e.g., in aliphatic chains), ethers,
amides, urethanes, and the like. Generally, a hydrolytically stable
linkage is one that exhibits a rate of hydrolysis of less than
about 1-2% per day under physiological conditions. Hydrolysis rates
of representative chemical bonds can be found in most standard
chemistry textbooks.
[0080] "Multi-armed" in reference to the geometry or overall
structure of a polymer refers to polymer having 3 or more
polymer-containing "arms" connected to a "core" molecule or
structure. Thus, a multi-armed polymer may possess 3 polymer arms,
4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8
polymer arms or more, depending upon its configuration and core
structure. One particular type of multi-armed polymer is a highly
branched polymer referred to as a dendritic polymer or
hyperbranched polymer having an initiator core of at least 3
branches, an interior branching multiplicity or 2 or greater, a
generation of 2 or greater, and at least 25 surface groups within a
single dendrimer molecule. For the purposes herein, a dendrimer is
considered to possess a structure distinct from that of a
multi-armed polymer. That is to say, a multi-armed polymer as
referred to herein explicitly excludes dendrimers. Additionally, a
multi-armed polymer as provided herein possesses a non-crosslinked
core.
[0081] A "dendrimer" or "hyperbranched polymer" is a globular, size
monodisperse polymer in which all bonds emerge radially from a
central focal point or core with a regular branching pattern and
with repeat units that each contribute a branch point. Dendrimers
are typically although not necessarily formed using a nano-scale,
multistep fabrication process. Each step results in a new
"generation" that has two or more times the complexity of the
previous generation. Dendrimers exhibit certain dendritic state
properties such as core encapsulation, making them unique from
other types of polymers.
[0082] "Branch point" refers to a bifurcation point comprising one
or more atoms at which a polymer splits or branches from a linear
structure into one or more additional polymer arms. A multi-arm
polymer may have one branch point or multiple branch points, so
long as the branches are not regular repeats resulting in a
dendrimer.
[0083] "Substantially" or "essentially" means nearly totally or
completely, for instance, 95% or greater of some given
quantity.
[0084] "Alkyl" refers to a hydrocarbon chain ranging from about 1
to 20 atoms in length. Such hydrocarbon chains are preferably but
not necessarily saturated and may be branched or straight chain.
Exemplary alkyl groups include methyl, ethyl, isopropyl, n-butyl,
n-pentyl, 2-methyl-1-butyl, 3-pentyl, 3-methyl-3-pentyl, and the
like.
[0085] "Lower alkyl" refers to an alkyl group containing from 1 to
6 carbon atoms, and may be straight chain or branched, as
exemplified by methyl, ethyl, n-butyl, i-butyl and t-butyl.
[0086] "Cycloalkyl" refers to a saturated cyclic hydrocarbon chain,
including bridged, fused, or spiro cyclic compounds, preferably
made up of 3 to about 12 carbon atoms, more preferably 3 to about
8.
[0087] "Non-interfering substituents" are those groups that, when
present in a molecule, are typically non-reactive with other
functional groups contained within the molecule.
[0088] The term "substituted" as in, for example, "substituted
alkyl," refers to a moiety (e.g., an alkyl group) substituted with
one or more non-interfering substituents, such as, but not limited
to: C.sub.3-C.sub.8 cycloalkyl, e.g., cyclopropyl, cyclobutyl, and
the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano;
alkoxy, lower phenyl; substituted phenyl; and the like. For
substitutions on a phenyl ring, the substituents may be in any
orientation (i.e., ortho, meta or para).
[0089] "Alkoxy" refers to an --O--R group, wherein R is alkyl or
substituted alkyl, preferably C.sub.1-C.sub.20 alkyl (e.g.,
methoxy, ethoxy, propyloxy, etc.), preferably C.sub.1-C.sub.7.
[0090] As used herein. "alkenyl" refers to branched and unbranched
hydrocarbon groups of 1 to 15 atoms in length, containing at least
one double bond, such as ethenyl (vinyl), 2-propen-1-yl (allyl),
isopropenyl, 3-buten-1-yl, and the like.
[0091] The term "alkynyl" as used herein refers to branched and
unbranched hydrocarbon groups of 2 to 15 atoms in length,
containing at least one triple bond, such as ethynyl, 1-propynyl,
3-butyn-1-yl, 1-octyn-1-yl, and so forth.
[0092] The term "aryl" means an aromatic group having up to 14
carbon atoms. Aryl groups include phenyl, naphthyl, biphenyl,
phenanthrecenyl, naphthacenyl, and the like.
[0093] "Substituted phenyl" and "substituted aryl" denote a phenyl
group and aryl group, respectively, substituted with one, two,
three, four, or five (e.g., 1-2, 1-3, 1-4, or 1-5 substituents)
chosen from halo (F, Cl, Br, I), hydroxyl, cyano, nitro, alkyl
(e.g., C.sub.1-6alkyl), alkoxy (e.g., C.sub.1-C.sub.6 alkoxy),
benzyloxy, carboxy, aryl, and so forth.
[0094] An inorganic acid is an acid that is absent carbon atoms.
Examples include hydrohalic acids, nitric acid, sulfuric acid,
phosphoric acid and the like.
[0095] "Hydrohalic acid" means a hydrogen halide such as
hydrofluoric acid (HF), hydrochloric acid (HCl), hydrobromic acid
(HBr), and hydroiodic acid (HI).
[0096] "Organic acid" means any organic compound (i.e., having at
least one carbon atom) possessing one or more carboxy groups
(--COOH). Some specific examples include formic acid, lactic acid,
benzoic acid, acetic acid, trifluoroacetic acid, dichloroacetic
acid, trichloroacetic acid, mixed chlorofluoroacetic acids, citric
acid, oxalic acid, and the like.
[0097] "Active agent" as used herein includes any agent, drug,
compound, and the like which provides some pharmacologic, often
beneficial, effect that can be demonstrated in-vivo or in vitro. As
used herein, these terms further include any physiologically or
pharmacologically active substance that produces a localized or
systemic effect in a patient. As used herein, especially in
reference to synthetic approaches described herein, a "active
agent" is meant to encompass derivatized or linker modified
versions thereof, such that upon administration in vivo, the parent
"bioactive" molecule is released.
[0098] "Pharmaceutically acceptable excipient" and
"pharmaceutically acceptable carrier" refer to an excipient that
can be included in a composition comprising an active agent and
that causes no significant adverse toxicological effects to the
patient.
[0099] "Pharmacologically effective amount," "physiologically
effective amount," and "therapeutically effective amount" are used
interchangeably herein to mean the amount of an active agent
present in a pharmaceutical preparation that is needed to provide a
desired level of active agent and/or conjugate in the bloodstream
or in a target tissue or site in the body. The precise amount will
depend upon numerous factors, e.g., the particular active agent,
the components and physical characteristics of the pharmaceutical
preparation, intended patient population, and patient
considerations, and can readily be determined by one skilled in the
art, based upon the information provided herein and available in
the relevant literature.
[0100] "Multi-functional" in the context of a polymer means a
polymer having 3 or more functional groups, where the functional
groups may be the same or different, and are typically present on
the polymer termini. Multi-functional polymers will typically
contain from about 3-100 functional groups, or from 3-50 functional
groups, or from 3-25 functional groups, or from 3-15 functional
groups, or from 3 to 10 functional groups, i.e., contains 3, 4, 5,
6, 7, 8, 9 or 10 functional groups.
[0101] "Difunctional" and "bifunctional" are used interchangeably
herein and mean an entity such as a polymer having two functional
groups contained therein, typically at the polymer termini. When
the functional groups are the same, the entity is said to be
homodifunctional or homobifunctional. When the functional groups
are different, the entity is said to be heterodifunctional or
heterobifunctional.
[0102] A basic or acidic reactant described herein includes
neutral, charged, and any corresponding salt forms thereof.
[0103] The terms "subject," "individual" and "patient" are used
interchangeably herein and refer to a vertebrate, preferably a
mammal. Mammals include, but are not limited to, murines, rodents,
simians, humans, farm animals, sport animals and pets. Such
subjects are typically suffering from or prone to a condition that
can be prevented or treated by administration of a water-soluble
polymer-active agent conjugate as described herein.
[0104] The term "about," particularly in reference to a given
quantity, is meant to encompass deviations of plus or minus five
percent.
[0105] "Treatment" and "treating" of a particular condition
include: (1) preventing such a condition, i.e., causing the
condition not to develop, or to occur with less intensity or to a
lesser degree in a subject that may be exposed to or predisposed to
the condition but does not yet experience or display the condition,
and (2) inhibiting the condition, i.e., arresting the development
or reversing the condition.
[0106] "Optional" or "optionally" means that the subsequently
described circumstance may but need not necessarily, so that the
description includes instances where the circumstance occurs and
instances where it does not.
[0107] A "small molecule" is an organic, inorganic, or
organometallic compound typically having a molecular weight of less
than about 1000, preferably less than about 800 daltons. Small
molecules as referred to herein encompass oligopeptides and other
biomolecules having a molecular weight of less than about 1000.
[0108] A "peptide" is a molecule composed of from about 13 to 50 or
so amino acids. An oligopeptide typically contains from about 2 to
12 amino acids.
[0109] Unless explicitly stated to the contrary, the terms "partial
mixed salt" and "mixed salt" as used herein are used
interchangeably, and, in the case of a polymer conjugate (and
corresponding compositions comprising a plurality of such polymer
conjugates), refer to a conjugates and compositions comprising one
or more basic amino (or other basic nitrogen containing) groups,
where (i) any given one of the basic amino groups in the conjugate
or conjugate composition is either non-protonated or protonated and
(ii) with respect to any given protonated basic amino group, the
protonted basic amino group will have one of two different
counterions. (The term "partial mixed salt" refers to the feature
where not all amino groups in the compound or composition are
protonated--hence the composition being a "partial" salt, while
"mixed" refers to the feature of multiple counterions). A mixed
salt as provided herein encompasses hydrates, solvates, amorphous
forms, crystalline forms, polymorphs, isomers, and the like.
[0110] An amine (or other basic nitrogen) group that is in "free
base" form is one where the amine group, i.e., a primary,
secondary, or tertiary amine, possesses a free electron pair. The
amine is neutral, i.e., is uncharged.
[0111] An amine group that is in "protonated form" exists as a
protonated amine, so that the amino group is positively charged. As
used herein, an amine group that is protonated can also be in the
form of an acid addition salt resulting from reaction of the amine
with an acid such as an inorganic acid or an organic acid.
[0112] The "mole percent" of an active agent's amino groups refers
to the fraction or percentage of amino groups in an active agent
molecule contained in a polymer conjugate that are in one
particular form or another, where the total mole percent of amino
groups in the conjugate is 100 percent.
[0113] As used herein, "psi" means pounds per square inch.
Overview: Mixed Salts Conjugates, Alkoxylation Methods, and
Compositions of Conjugates (and Mixed Salt Forms Thereof) Prepared
From Polymer Reagents Prepared from Polymeric Products Using the
Alkoxylation Methods
[0114] Mixed Salts:
[0115] As previously indicated, in one or more aspects of the
invention, a water-soluble polymer and active agent conjugate is
provided, wherein the conjugate is in the form of a mixed salt.
Such conjugates represent novel solid state forms and are based at
least in part on the discovery that, in spite of treatment with
base in their formation, conjugates precipitate as mixed salts.
Moreover, it has been discovered that conjugates can reliably and
reproducibly be produced as a mixed salts--where any given basic
nitrogen atom within the conjugate (and within the active agent
component of the conjugate) is present in one of a variety of
forms. Specifically, the conjugates provided herein possess active
agent basic nitrogen atoms, e.g., amino groups, each one of which
will either be protonated or unprotonated, where any given
protonated amino group is an acid salt possessing one of two
different anions. Moreover, it has been discovered that the mixed
salt form of the conjugate has several unexpected and advantageous
characteristics (i.e., greater stability against degradation of the
polymer backbone, greater hydrolytic stability, etc.,) when
compared to the corresponding free base or single acid salt forms
of the conjugate.
[0116] Alkoxylation Methods:
[0117] As also previously indicated, in one or more aspects of the
invention, a method is provided, the method comprising the step of
alkoxylating in a suitable solvent a previously isolated
alkoxylatable oligomer to form an alkoxylated polymeric product,
wherein the previously isolated alkoxylatable oligomer has a known
and defined weight-average molecular weight of greater than 300
Daltons (e.g., greater than 500 Daltons). Among other advantages,
the alkoxylation methods provided herein result in polymeric
products that are superior (e.g., in terms of consistency and
purity) than polymeric products prepared by previously known
methods. In one or more embodiments, a polymer formed by the
present alkoxylation methods may advantageously be used to prepare
a mixed acid salt as described herein.
[0118] Compositions of Conjugates (and Mixed Salt Forms Thereof)
Prepared from Polymer Reagents Prepared from Polymeric Products
Using the Alkoxylation Methods:
[0119] As also previously indicated, in one or more embodiments of
the invention, a mixed salt of a water-soluble polymer-active agent
conjugate is provided, wherein the conjugate is prepared by
coupling (under conjugation conditions) an amine-bearing active
agent (e.g., a deprotected glycine-irinotecan) to a polymer reagent
(e.g., 4-arm pentaerythritolyl-poly(ethylene glycol)-carboxymethyl
succinimide) in the presence of a base to form a conjugate, wherein
the conjugate is a mixed salt conjugate (e.g., the conjugate
possesses nitrogen atoms, each one of which will either be
protonated or unprotonated, where any given protonated amino group
is an acid salt possessing one of two different anions), and
further wherein, optionally, the polymer reagent is prepared from a
alkoxylation product prepared as described herein.
Conjugates--The Polymer Generally
[0120] Water-soluble polymer-active agent conjugates (regardless of
the specific form taken, e.g., a base form, salt form, mixed salt,
and so forth) include a water-soluble polymer. Typically, in order
to form a conjugate, a water-soluble polymer--in the form of a
polymer reagent--coupled (under conjugation conditions) to an
active agent at an electrophile or nucleophile contained within the
active agent. For example, a water-soluble polymer (again, in the
form of a polymer reagent bearing, e.g., an activated ester) can be
coupled to an active agent possessing one or more basic amine
groups (or other basic nitrogen atoms), i.e., an amine having a pK
from about 7.5 to about 11.5 (determined after conjugation).
[0121] The water-soluble polymer component of the conjugate is
typically a water-soluble and non-peptidic polymer. Representative
polymers include poly(alkylene glycol), poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharide),
poly(.alpha.-hydroxy acid), poly(acrylic acid), poly(vinyl
alcohol), polyphosphazene, polyoxazoline,
poly(N-acryloylmopholine), or copolymers or terpolymers thereof.
One particular water-soluble polymer is polyethylene glycol or PEG
comprising the repeat unit (CH.sub.2CH.sub.2O).sub.n--, where n
ranges from about 3 to about 2700 or even greater, or preferably
from about 25 to about 1300. Typically, the weight average
molecular weight of the water-soluble polymer in the partial mixed
acid salt ranges from about 100 daltons to about 150,000 daltons.
Illustrative overall molecular weights for the conjugate may range
from about 800 to about 80,000 daltons, or from about 900 to about
70,000 daltons. Additional representative molecular weight ranges
are from about 1,000 to about 40,000 daltons, or from about 5,000
to about 30,000 daltons, or from about 7500 daltons to about 25,000
daltons, or even from about 20,000 to about 80,000 daltons for
higher molecular weight embodiments of the instant partial mixed
salts.
[0122] The water-soluble polymer can be in any of a number of
geometries or forms, including linear, branched, forked. In
exemplary embodiments, the polymer is often linear or multi-armed.
Water-soluble polymers can be obtained commercially as simply the
water-soluble polymer. In addition, water-soluble polymers can be
conveniently obtained in an activated form as a polymer reagent
(which optionally may be coupled to an active agent without further
modification or activation). Descriptions of water-soluble polymers
and polymer reagents can be found in Nektar Advanced PEGylation
Catalog, 2005-2006, "Polyethylene Glycol and Derivatives for
Advanced PEGylation" and are available for purchase from NOF
Corporation and JenKem Technology USA, among others.
[0123] An exemplary branched polymer having two polymer arms in a
branched pattern is the following, often referred to as PEG-2 or
mPEG-2:
##STR00004##
wherein indicates the location for additional atoms to form any of
functional groups suitable for reaction with an electrophile or
nucleophile contained within an active agent. Exemplary functional
groups include NHS ester, aldehyde, and so forth.
[0124] For polymer structures described herein that contain the
variable, "n," such variable corresponds to an integer and
represents the number of monomer subunits within the repeating
monomeric structure of the polymer.
[0125] On exemplary architecture for use in preparing the
conjugates are multi-arm water-soluble polymer reagents having for
example 3, 4, 5, 6 or 8 polymer arms, each optimally bearing a
functional group. A multi-arm polymer reagent may possess any of a
number of cores (e.g., a polyol core) from which the polymer arms
emanate. Exemplary polyol cores include glycerol, glycerol dimer
(3,3'-oxydipropane-1,2-diol) trimethylolpropane, sugars (such as
sorbitol or pentaerythritol, pentaerythritol dimer), and glycerol
oligomers, such as hexaglycerol or
3-(2-hydroxy-3-(2-hydroxyethoxy)propoxy)propane-1,2-diol, and other
glycerol condensation products. Exemplary, the cores and the
polymer arms emanating therefrom can be of the following
formulae:
##STR00005##
[0126] In an exemplified embodiment, the water soluble polymer is a
4-arm polymer as shown above, where n may range from about 20 to
about 500, or from about 40 to about 500.
[0127] In the multi-arm embodiments described herein, each polymer
arm typically has a molecular weight corresponding to one of the
following: 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500,
2000, 3000, 4000, 5000, 6000, 7000, 7500, 8000, 9000, 10000,
12,000, 15000, 17,500, 18,000, 19,000, 20,000 Daltons or greater.
Overall molecular weights for the multi-armed polymer
configurations described herein (that is to say, the molecular
weight of the multi-armed polymer as a whole) generally correspond
to one of the following: 800, 1000, 1200, 1600, 2000, 2400, 2800,
3200, 3600, 4000, 5000, 6000, 8000, 10,000, 12,000, 15,000, 16,000,
20,000, 24,000, 25,000, 28,000, 30,000, 32,000, 36,000, 40,000,
45,000, 48,000, 50,000, 60,000, 80,000 or 100,000 or greater.
[0128] The water-soluble polymer, e.g., PEG, may be covalently
linked to the active agent via an intervening linker. The linker
may contain any number of atoms. Generally speaking, the linker has
an atom length satisfying one or more of the following ranges: from
about 1 atom to about 50 atoms; from about 1 atom to about 25
atoms; from about 3 atoms to about 12 atoms; from about 6 atoms to
about 12 atoms; and from about 8 atoms to about 12 atoms. When
considering atom chain length, only atoms contributing to the
overall distance are considered. For example, a linker having the
structure,
--CH.sub.2--C(O)--NH--CH.sub.2CH.sub.2O--CH.sub.2CH.sub.2O--C(O)--O--
is considered to have a chain length of 11 atoms, since
substituents are not considered to contribute significantly to the
length of the linker. Illustrative linkers include bifunctional
compounds such as amino acids (e.g., alanine, glycine, isoleucine,
leucine, phenlalanine, methionine, serine, cysteine, sarcosine,
valine, lysine, and the like). The amino acid may be a
naturally-occurring amino acid or a non-naturally occurring amino
acid. Suitable linkers also include oligopeptides.
[0129] The multi-arm structures above are drawn primarily to
illustrate the polymer core having PEG chains attached thereto, and
although not drawn explicitly, depending upon the nature of the
active agent and attachment chemistry employed, the final structure
may optionally include an additional ethylene group,
--CH.sub.2CH.sub.2--, attached to the oxygen atoms at the terminus
of each polymer arm, and/or may optionally contain any of a number
of intervening linker atoms to facilitate covalent attachment to an
active agent. In a particular embodiment, each of the PEG arms
illustrated above further comprises a carboxy methyl group,
--CH.sub.2--C(O)O--, covalently attached to the terminal oxygen
atom.
New Alkoxylation Method for Improved Polymer Compositions
[0130] As indicated previously, water-soluble polymers that have
utility in (for example) preparing conjugates with active agents
(as well as salt and mixed salt forms thereof) can be obtained
commercially. As further described herein, however, methods for
preparing water-soluble polymers--which methods distinguish over
previously described methods for preparing water-soluble
polymers--are provided that are particularly suited for preparing
conjugates with active agents (as well as salt and mixed salt forms
thereof).
[0131] In this regard, a method is provided, the method comprising
the step of alkoxylating in a suitable solvent a previously
isolated alkoxylatable oligomer to form an alkoxylated polymeric
product, wherein the previously isolated alkoxylatable oligomer has
a known and defined weight-average molecular weight of greater than
300 Daltons (e.g., greater than 500 Daltons).
The Alkoxylating Step in the New Alkoxylation Method
[0132] The alkoxylating step is carried out using alkoxylation
conditions, such that the sequential addition of monomers is
effected through repeated reactions of an oxirane compound. When
the alkoxylatable oligomer initially has one or more hydroxyl
functional groups, one or more of these hydroxyl groups in the
alkoxylatable oligomer will be converted into a reactive alkoxide
by reaction with a strong base. Then, an oxirane compound reacts
with an alkoxylatable functional group (e.g., a reactive alkoxide),
thereby not only adding to the reactive alkoxide, but doing so in a
way that also terminates in another reactive alkoxide. Thereafter,
repeated reactions of an oxirane compound at the reactive alkoxide
terminus of the previously added and reacted oxirane compound
effectively produces a polymer chain.
[0133] Although each of the one or more alkoxylatable functional
groups is preferably hydroxyl, other groups such as amines, thiols
and the hydroxyl group of a carboxylic acid can serve as an
acceptable alkoxylatable functional group. Also, because of the
acidity of the hydrogens of the alpha carbon atoms in aldehydes,
ketones, nitriles and amides, addition at the alpha carbon atoms of
these groups can serve as an acceptable alkoxylatable functional
group.
[0134] The oxirane compound contains an oxirane group and has the
following formula:
##STR00006##
wherein (with respect to this structure):
[0135] R.sup.1 is selected from the group consisting of H and alkyl
(preferably lower alkyl when alkyl);
[0136] R.sup.2 is selected from the group consisting of H and alkyl
(preferably lower alkyl when alkyl);
[0137] R.sup.3 is selected from the group consisting of H and alkyl
(preferably lower alkyl when alkyl); and
[0138] R.sup.4 is selected from the group consisting of H and alkyl
(preferably lower alkyl when alkyl).
[0139] With respect to the above oxirane compound formula, it is
particularly preferred that each of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 is H, and it is preferred that only one of R.sup.1,
R.sup.2, R.sup.3 and R.sup.4 is alkyl (e.g., methyl and ethyl) and
the remaining substituents are H. Exemplary oxirane compounds are
ethylene oxide, propylene oxide and 1,2-butylene oxide. The amount
of oxirane compound added to result in optimal alkoxylation
conditions depends upon a number of factors, including the amount
of starting alkoxylatable oligomer, the desired size of the
resulting alkoxylated polymeric material and the number of
alkoxylatable functional groups on the alkoxylatable oligomer.
Thus, when a larger alkoxylated polymeric material is desired,
relatively more oxirane compound is present in the alkoxylation
conditions. Similarly, if (Oa) represents the amount of oxirane
compound needed to achieve a given size of polymer "growth" on a
single alkoxylatable functional group, then an alkoxylatable
oligomer bearing two alkoxylatable functional groups requires
2x(Oa), an alkoxylatable oligomer bearing three alkoxylatable
functional groups requires 3x(Oa), an alkoxylatable oligomer
bearing four alkoxylatable functional groups requires 4x(Oa) and so
on. In all cases, one of ordinary skill in the art can determine an
appropriate amount of oxirane compound required for alkoxylation
conditions by taking into account the desired molecular weight of
alkoxylated polymeric material and following routine
experimentation.
[0140] The alkoxylation conditions include the presence of a strong
base. The purpose of the strong base is to deprotonate each acidic
hydrogen (e.g., the hydrogen of a hydroxyl group) present in the
alkoxylatable oligomer and form an alkoxide ionic species (or an
ionic species for non-hydroxyl alkoxylatable functional groups).
Preferred strong bases for use as part of the alkoxylation
conditions are: alkali metals, such as metallic potassium, metallic
sodium, and alkali metals mixtures such as sodium-potassium alloys;
hydroxides, such as NaOH and KOH; and alkoxides (e.g., present
following addition of an oxirane compound). Other strong bases can
be used and can be identified by one of ordinary skill in the art.
For example a given base can be used as a strong base herein if the
strong base can form an alkoxide ionic species (or an ionic species
for non-hydroxyl alkoxylatable functional groups) and also provide
a cation that does not encumber the alkoxide ionic species so as to
hinder (or effectively hinder through an impractically slow)
reaction of the alkoxide ionic species with the oxirane molecule.
The strong base is present in a generally small and calculated
amount, which amount can fall into one or more of the following
ranges: from 0.001 to 10.0 weight percent based upon the weight of
the total reaction mixture; and from 0.01 to about 6.0 weight
percent based upon the weight of the total reaction mixture.
[0141] The alkoxylation conditions include a temperature suitable
for alkoxylation to occur. Exemplary temperatures that may be
suitable for alkoxylation to occur include those falling into one
or more of the following ranges: from 10.degree. C. to 260.degree.
C.; from 20.degree. C. to 240.degree. C.; from 30.degree. C. to
220.degree. C.; from 40.degree. C. to 200.degree. C.; from
50.degree. C. to 200.degree. C.; from 80.degree. C. to 140.degree.
C.; and from 100.degree. C. to 120.degree. C.
[0142] The alkoxylation conditions include a pressure suitable for
alkoxylation to occur. Exemplary pressures that may be suitable for
alkoxylation to occur include those falling into one or more of the
following ranges: from 10 psi to 1000 psi; from 15 psi to 500 psi;
from 20 psi to 250 psi; from 25 psi to 100 psi. In addition, the
alkoxylation pressure can be about atmospheric pressure at sea
level (e.g., 14.696 pounds per square inch+/-10%).
[0143] In some instances, the alkoxylation conditions include
addition of the oxirane compound in liquid form. In some instances,
the alkoxylation conditions include addition of the oxirane
compound in vapor form.
[0144] The alkoxylation conditions can include the use of a
suitable solvent. Optimally, the system in which the alkoxylation
conditions occur will not include any component (including any
solvent) that can be deprotonated (or remains substantially
protonated under the conditions of pH, temperature, and so forth
under which the alkoxylation conditions will occur). Suitable
solvents for alkoxylation include organic solvents selected from
the group consisting of, tetrahydrofuran (THF), dimethylformamide
(DMF), toluene, benzene, xylenes, mesitylene, tetrachloroethylene,
anisole, dimethylacetamide, and mixtures of the foregoing. Less
ideal solvents (but nonetheless still contemplated) for use as part
of the alkoxylation conditions are acetonitrile, phenylacetonitrile
and ethyl acetate; in some instances, the alkoxylation conditions
will not include as a solvent any of acetonitrile,
phenylacetonitrile and ethyl acetate.
[0145] In one or more embodiments of the invention, when the
alkoxylation conditions are conducted in the liquid phase, the
alkoxylation conditions are conducted such that both the
alkoxylatable oligomer and the desired alkoxylated polymeric
material formed from alkoxylating the alkoxylatable oligomer not
only have similar solubilities (and, preferably, substantially the
same solubility) in the suitable solvent used, but are also both
substantially soluble in the suitable solvent. For example, in one
or more embodiments, the alkoxylatable oligomer will be
substantially soluble in the solvent used in the alkoxylation
conditions and the resulting alkoxylated polymeric material also
will be substantially soluble in the alkoxylation conditions.
[0146] In one or more embodiments, this substantially same
solubility of the alkoxylated oligomer and the alkoxylated
polymeric material in a suitable solvent stands in contrast to the
solubility of a precursor molecule (used, for example, in the
preparation of the previously isolated alkoxylated oligomer) in the
suitable solvent, wherein the precursor molecule can have a lower
(and even substantially lower) solubility in the suitable solvent
than the alkoxylated oligomer and/or the alkoxylated polymeric
material. By way of example only, the alkoxylated oligomer and the
alkoxylated polymeric material will both have a pentaerythritol
core and will both be substantially soluble in toluene, but
pentaerythritol itself has limited solubility in toluene.
[0147] It is particularly preferred that the solvent employed in
the alkoxylation conditions is toluene. The amount of toluene used
for the reaction is greater than 25 wt % and less than 75 wt % of
the reaction mixture, based on the weight of reaction mixture after
complete addition of the oxirane compound. One of ordinary skill in
the art can calculate the starting amount of the solvent by taking
into account the desired molecular weight of the polymer, the
number of sites for which alkoxylation will take place, the weight
of the alkoxylatable oligomer used, and so forth.
[0148] It is preferred that the amount of the toluene is measured
so that the amount is sufficient for the alkoxylation conditions
providing the desired alkoxylated polymeric material.
[0149] In addition, it is particularly preferred that the
alkoxylation conditions have substantially no water present. Thus,
it is preferred that the alkoxylation conditions have a water
content of less than 100 ppm, more preferably 50 ppm, still more
preferably 20 ppm, much more preferably less than 14 ppm, and even
still more preferably less than 8 ppm.
[0150] The alkoxylation conditions take place in a suitable
reaction vessel, typically a stainless steel reactor vessel.
[0151] In one or more embodiments, the alkoxylatable oligomer
and/or precursor molecule lacks an isocyanate group attached to a
carbon bearing an alpha hydrogen is acceptable. In one or more
embodiments, the previously prepared alkoxylatable oligomer and/or
precursor molecule lacks an isocyanate group.
The Alkoxylatable Oligomer in the New Alkoxylation Method
[0152] The alkoxylatable oligomer used in the new alkoxylation
method must have at least one alkoxylatable functional group. The
alkoxylatable oligomer, however, can have one, two, three, four,
five, six, seven, eight or more alkoxylatable functional groups,
with a preference for an alkoxylatable oligomer having from one to
six alkoxylatable functional groups.
[0153] As stated previously, each alkoxylatable functional group
within the alkoxylatable oligomer can be independently selected
from the group consisting of hydroxyl, carboxylic acid, amine,
thiol, aldehyde, ketone, and nitrile. In those instances where
there is more than one alkoxylatable functional group within the
alkoxylatable oligomer, it is typical that each alkoxylatable
functional group is the same (e.g., each alkoxylatable functional
group within the alkoxylatable oligomer is hydroxyl), although
instances of different alkoxylatable functional groups within the
same alkoxylatable oligomer are contemplated as well. When the
alkoxylatable functional group is hydroxyl, it is preferred that
the hydroxyl is a primary hydroxyl.
[0154] The alkoxylatable oligomer can take any of a number of
possible geometries.
[0155] For example, the alkoxylatable oligomer can be linear. In
one example of a linear alkoxylatable oligomer, one terminus of the
linear alkoxylatable oligomer is a relatively inert functional
group (e.g., an end-capping group) and the other terminus is an
alkoxylatable functional group (e.g., hydroxyl). An exemplary
alkoxylatable oligomer of this structure is methoxy-PEG-OH, or mPEG
in brief, in which one terminus is the relatively inert methoxy
group, while the other terminus is a hydroxyl group. The structure
of mPEG is given below.
CH.sub.3O--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.-
2--OH
(wherein, for the immediately preceding structure only, n is an
integer from 13 to 100).
[0156] Another example of a linear geometry for which the
alkoxylatable oligomer can take is a linear organic polymer bearing
alkoxylatable functional groups (either the same or different) at
each terminus. An exemplary alkoxylatable oligomer of this
structure is alpha-, omega-dihydroxylpoly(ethylene glycol), or
HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH
(wherein, for the immediately preceding structure only, n is an
integer from 13 to 100), which can be represented in brief form as
HO-PEG-OH where it is understood that the -PEG-symbol represents
the following structural unit:
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--
(wherein, for the immediately preceding structure only, n is an
integer from 13 to 100),
[0157] Another geometry for which the alkoxylatable oligomer may
have is a "multi-armed" or branched structure. With respect to such
branched structures, one or more atoms in the alkoxylatable
oligomer serves as a "branching point atom," through which two,
three, four or more (but typically two, three or four) distinct
sets of repeating monomers or "arms" are connected (either directly
or through one or more atoms). At a minimum, a "multi-arm"
structure as used herein has three or more distinct arms, but can
have as many as four, five, six, seven, eight, nine, or more arms,
with 4- to 8-arm multi-arm structures preferred (such as a 4-arm
structure, a 5-arm structure, a 6-arm structure, and an 8-arm
structure).
[0158] Exemplary multi-arm structures for the alkoxylatable
oligomer are provided below:
##STR00007##
wherein (for the immediately preceding structure only) the average
value of n is from 1 to 50, e.g., from 10 to 50, (or otherwise
defined such that the molecular weight of the structure is from 300
Daltons to 9,000 Daltons (e.g., from about 500 Daltons to 5,000
Daltons);
##STR00008##
wherein (for the immediately preceding structure only) the average
value of n is from 2 to 50, e.g., from 10 to 50 (or otherwise
defined such that the molecular weight of the structure is from 300
Daltons to 9,000 Daltons (e.g., from about 500 Daltons to 5,000
Daltons);
##STR00009##
wherein (for the immediately preceding structure only) the average
value of n is from 2 to 35, e.g., from 8 to about 40 (or otherwise
defined such that the molecular weight of the structure is from 750
Daltons to 9,500 Daltons (e.g., from 500 Daltons to 5,000 Daltons);
and
##STR00010##
wherein (for the immediately preceding structure only) the average
value of n is 2 to 35. e.g., from 5 to 35, (or otherwise defined
such that the molecular weight of the structure is from 1,000
Daltons to 13,000 Daltons (e.g., from 500 Daltons to 5,000
Daltons).
[0159] For each of the four immediately preceding structures, it is
preferred that the value of n, in each instance, is substantially
the same. Thus, it is preferred that when all values of n are
considered for a given alkoxylatable oligomer, all values of n for
that alkoxylatable oligomer are within three standard deviations,
more preferably within two standard deviations, and still more
preferably within one standard deviation.
[0160] In terms of the molecular weight of the alkoxylatable
oligomer, the alkoxylatable oligomer will have a known and defined
weight-average molecular weight. For use herein, a weight-average
molecular weight can only be known and defined for an alkoxylatable
oligomer when the alkoxylatable oligomer is isolated from the
synthetic milieu from which it was generated. Exemplary
weight-average molecular weights for the alkoxylatable oligomer
will fall into one or more of the following ranges: greater than
300 Daltons; greater than 500 Daltons; from 300 Daltons to 15,000
Daltons; from 500 Daltons to 5,000 Daltons; from 300 Daltons to
10,000 Daltons; from 500 Daltons to 4,000 Daltons; from 300 Daltons
to 5,000 Daltons; from 500 Daltons to 3,000 Daltons; from 300
Daltons to 2,000 Daltons; from 500 Daltons to 2,000 Daltons; from
300 Daltons to 1,000 Daltons; from 500 Daltons to 1,000 Daltons;
from 1,000 Daltons to 10,000 Daltons; from 1,000 Daltons to 5,000
Daltons; from 1,000 Daltons to 4,000 Daltons; from 1,000 Daltons to
3,000 Daltons; from 1,000 Daltons to 2,000 Daltons; from 1,500
Daltons to 15,000 Daltons; from 1,500 Daltons to 5,000 Daltons;
from 1,500 Daltons to 10,000 Daltons; from 1,500 Daltons to 4,000
Daltons; from 1,500 Daltons to 3,000 Daltons; from 1.500 Daltons to
2,000 Daltons; from 2,000 Daltons to 5,000 Daltons; from 2,000
Daltons to 4,000 Daltons; and from 2,000 Daltons to 3,000
Daltons.
[0161] For purposes of the present invention, the alkoxylatable
oligomer is preferably previously isolated. By previously isolated
is meant the alkoxylatable oligomer exists outside and separate
from the synthetic milieu from which it was generated (most
typically outside of the alkoxylating conditions used to prepare
the alkoxylatable oligomer) and can optionally be stored for a
relatively long period of time or optionally stored over a shorter
time without substantially changing for subsequent use. Thus, an
alkoxylatable oligomer is previously isolated if, for example, it
is housed in an inert environment. In this regard, a previously
isolated alkoxylated oligomer can be housed in a container
substantially lacking (e.g., less than 0.1 wt %) an oxirane
compound. Also, a previously isolated alkoxylatable oligomer does
not change its molecular weight more than 10% over the course of 15
days. Thus, in one or more embodiments of the invention, the
concept of "previously isolated" stands in contrast to (for
example) a situation where an ongoing and uninterrupted
alkoxylation reaction is allowed to proceed from precursor
molecule, into a structure that corresponds an alkoxylatable
oligomer, to a structure that corresponds to an alkoxylated
polymeric material; the concept of "previously isolated" requires
that the alkoxylatable oligomer exists apart from the conditions
from which it formed. Pursuant to the present invention, however,
the previously isolated alkoxylatable oligomer will be subjected to
an alkoxylation step once it is added to, as a separate step,
alkoxylation conditions.
Sources of the Alkoxylatable Oligomer in the New Alkoxylation
Method
[0162] The alkoxylatable oligomer can be obtained via synthetic
means. In this regard, the alkoxylatable oligomer is prepared by
(a) alkoxylating a precursor molecule having a molecular weight of
less than 300 Daltons (e.g., less than 500 Daltons) to form a
reaction mixture comprising an alkoxylatable oligomer or
prepolymer, and (b) isolating the alkoxylatable oligomer from the
reaction mixture. The step of alkoxylating the precursor molecule
largely follows the conditions and requirements of the alkoxylating
step previously discussed. The step of isolating the alkoxylatable
oligomer can be carried out using any art known step, but can
include allowing all oxirane compound to be consumed in the
reaction, actively performing a quenching step, separating the
final reaction mixture through art-known approaches (including, for
example, distilling off all volatile materials, removing solid
reaction by-product by filtration or washing and applying
chromatographic means).
[0163] In addition, the alkoxylatable oligomer can be obtained from
commercial sources. Exemplary commercial sources include NOF
Corporation (Tokyo Japan) which provides alkoxylatable oligomers
under the names SUNBRIGHT DKH.RTM. poly(ethylene glycol),
SUNBRIGHT.RTM. GL glycerine, tri-poly(ethylene glycol) ether,
SUNBRIGHT PTE.RTM. pentaerythritol, tetra-poly(ethylene glycol)
ether, SUNBRIGHT.RTM. DG di-glycerine, tetra-poly(ethylene glycol)
ether, and SUNBRIGHT HGEO.RTM. hexa-glycerine, octa-poly(ethylene
glycol) ether. Preferred alkoxylatable oligomers include those
having the structures of SUNBRIGHT PTE.RTM.-2000 pentaerythritol,
tetra-poly(ethylene glycol) ether (which has a weight-average
molecular weight of about 2,000 Daltons) and SUNBRIGHT.RTM. DG-2000
di-glycerine, tetra-poly(ethylene glycol) ether (which has a
weight-average molecular weight of about 2,000 Daltons).
[0164] Precursor molecules can be any small molecule (e.g., a
molecular weight less than the weight-average molecular weight of
the alkoxylatable oligomer) having one or more alkoxylatable
functional groups.
[0165] Exemplary precursor molecules include polyols, which are
small molecules (typically of a molecular weight of less than 300
Daltons, e.g., less than 500 Daltons) having a plurality of
available hydroxyl groups. Depending on the desired number of
polymer arms in the alkoxylatable oligomer or prepolymer, the
polyol serving as the precursor molecule will typically comprise 3
to about 25 hydroxyl groups, preferably about 3 to about 22
hydroxyl groups, most preferably about 4 to about 12 hydroxyl
groups. Preferred polyols include glycerol oligomers or polymers
such as hexaglycerol, pentaerythritol and oligomers or polymers
thereof (e.g., dipentaerythritol, tripentaerythritol,
tetrapentaerythritol, and ethoxylated forms of pentaerythritol),
and sugar-derived alcohols such as sorbitol, arabanitol, and
mannitol. Also, many commercially available polyols, such as
various isomers of inositol (i.e.
1,2,3,4,5,6-hexahydroxycyclohexane),
2,2-bis(hydroxymethyl)-1-butanol,
(2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS),
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol,
{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}acetic acid
(Tricine),
2-[(3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}propyl)amino]-2-(hydr-
oxymethyl)-1,3-propanediol,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid
(TES),
4-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-butanesulfonic
acid, and
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol
hydrochloride can serve as an acceptable precursor molecule. In
those cases in which the precursor molecule has an ionizable group
or groups that will interfere with the alkoxylation step, those
ionizable groups must be protected or modified prior to carrying
out the alkoxylation step.
[0166] Exemplary preferred precursor molecules include those
precursor molecules selected from the group consisting of glycerol,
diglycerol triglycerol, hexaglycerol, mannitol, sorbitol,
pentaerythritol, dipentaerthitol, and tripentaerythritol.
[0167] In one or more embodiments of the invention, it is preferred
that neither the previously isolated alkoxylatable oligomer nor the
alkoxylated polymeric product has an alkoxylatable functional group
(e.g., hydroxyl group) of the precursor molecule.
The Alkoxylated Polymeric Materials Generated by the New
Alkoxylation Method
[0168] The alkoxylated polymeric material prepared under the
methods described herein will have a basic architecture
corresponding to the structure of the alkoxylatable oligomer (i.e.,
a linear alkoxylatable oligomer results in a linear alkoxylated
polymericmaterial, a four-armed alkoxylatable oligomer results in a
four-armed alkoxylated polymer material, so forth). As a
consequence, the alkoxylated polymeric material will take any of a
number of possible geometries, including linear, branched and
multi-armed.
[0169] With respect to branched structures, a branched alkoxylated
polymeric material will have three or more distinct arms, but can
have as many as four, five, six, seven, eight, nine, or more arms,
with 4- to 8-arm branched structures preferred (such as a 4-arm
branched structure, 5-arm branched structure, 6-arm branched
structure, and 8-arm branched structure).
[0170] Exemplary branched structures for the alkoxylated polymeric
material are provided below:
##STR00011##
wherein (for the immediately preceding structure only) the average
value of n satisfies one or more of the following ranges: from 10
to 1,000; from 10 to 500; from 10 to 250; from 50 to 1000; from 50
to 250; and from 50 to 120 (or otherwise defined such that the
molecular weight of the structure is from 2,000 Daltons to 180,000
Daltons, e.g., from 2,000 Daltons to 120,000 Daltons);
##STR00012##
wherein (for the immediately preceding structure only) the average
value of n satisfies one or more of the following ranges: from 10
to 1,000; from 10 to 500; from 10 to 250; from 50 to 1,000; from 50
to 250; and from 50 to 120 (or otherwise defined such that the
molecular weight of the structure is from 2,000 Daltons to 180,000
Daltons, e.g., from 2,000 Daltons to 120,000 Daltons);
##STR00013##
wherein (for the immediately preceding structure only) the average
value of n is satisfies one or more of the following ranges: from
10 to 750; from 40 to 750; from 50 to 250; and from 50 to 120 (or
otherwise defined such that the molecular weight of the structure
is from 3,000 Daltons to 200,000 Daltons, e.g., from 12,000 Daltons
to 200,000 Daltons); and
##STR00014##
wherein (for the immediately preceding structure only) the average
value of n is satisfies one or more of the following ranges: from
10 to 600 and from 35 to 600 (or otherwise defined such that the
molecular weight of the structure is from 4,000 Daltons to 215,000
Daltons, e.g., from 12,000 Daltons to 215,000 Daltons).
[0171] For each of the four immediately provided structures, it is
preferred that the value of n, in each instance, is substantially
the same. Thus, it is preferred that when all values of n are
considered for a given alkoxylated polymeric material, all values
of n for that alkoxylated polymeric material alkoxylatable oligomer
or prepolymer are within three standard deviations, more preferably
within two standard deviations, and still more preferably within
one standard deviation.
[0172] In terms of the molecular weight of the alkoxylated
polymeric material, the alkoxylated polymeric material will have a
known and defined number-average molecular weight. For use herein,
a number-average molecular weight can only be known and defined for
material that is isolated from the synthetic milieu from which it
was generated.
[0173] The total molecular weight of the alkoxylated polymeric
product can be a molecular weight suited for the intended purpose.
An acceptable molecular weight for any given purpose can be
determined through trial and error via routine experimentation.
[0174] Exemplary molecular weights for the alkoxylated polymeric
product, will have a number-average molecular weight falling within
one or more of the following ranges: from 2,000 Daltons to 215,000
Daltons; from 5,000 Daltons to 215,000 Daltons; from 5,000 Daltons
to 150,000 Daltons; from 5,000 Daltons to 100,000 Daltons; from
5,000 Daltons to 80,000 Daltons; from 6,000 Daltons to 80,000
Daltons; from 7,500 Daltons to 80,000 Daltons; from 9,000 Daltons
to 80,000 Daltons; from 10,000 Daltons to 80,000 Daltons; from
12,000 Daltons to 80,000 Daltons; from 15,000 Daltons to 80,000
Daltons; from 20,000 Daltons to 80,000 Daltons; from 25,000 Daltons
to 80,000 Daltons; from 30,000 Daltons to 80,000 Daltons; from
40,000 Daltons to 80,000 Daltons; from 6,000 Daltons to 60,000
Daltons; from 7.500 Daltons to 60,000 Daltons; from 9,000 Daltons
to 60,000 Daltons; from 10,000 Daltons to 60,000 Daltons; from
12,000 Daltons to 60,000 Daltons; from 15,000 Daltons to 60,000
Daltons; from 20,000 Daltons to 60,000 Daltons; from 25,000 Daltons
to 60,000 Daltons; from 30,000 Daltons to 60,000; from 6,000
Daltons to 40,000 Daltons; from 9,000 Daltons to 40,000 Daltons;
from 10,000 Daltons to 40,000 Daltons; from 15,000 Daltons to
40,000 Daltons; from 19,000 Daltons to 40,000 Daltons; from 15,000
Daltons to 25,000 Daltons; and from 18,000 Daltons to 22,000
Daltons.
[0175] For any given alkoxylated polymeric material, an optional
step can be carried out so as to further transform the alkoxylated
polymeric material so that it bears a specific reactive group to
form a polymeric reagent. Thus, using techniques well known in the
art, the alkoxylated polymeric material can be functionalized to
include a reactive group (e.g., carboxylic acid, active ester,
amine, thiol, maleimide, aldehyde, ketone, and so forth).
[0176] In carrying out an optional step to further transform the
alkoxylated polymeric product so that it bears a specific reactive
group, such an optional step is carried out in a suitable solvent.
One of ordinary skill in the art can determine whether any specific
solvent is appropriate for any given reaction step. Often, however,
the solvent is preferably a nonpolar solvent or a polar solvent.
Nonlimiting examples of nonpolar solvents include benzene, xylenes
and toluene. Exemplary polar solvents include, but are not limited
to, dioxane, tetrahydrofuran (THF), i-butyl alcohol, DMSO (dimethyl
sulfoxide), HMPA (hexamethylphosphoramide), DMF
(dimethylformamide), DMA (dimethylacetamide), and NMP
(N-methylpyrrolidinone).
Further Compositions of the Alkoxylated Polymeric Material
[0177] Another aspect of the invention provided herein are
compositions comprising the alkoxylated polymeric material, which
include not only any compositions comprising the alkoxylated
polymeric material, but also compositions in which the alkoxylated
polymeric material is further transformed into, for example, a
polymer reagent, as well as compositions of conjugates formed from
coupling such polymer reagents with an active agent. Among other
things, a benefit of the method described herein is the ability to
achieve high purity alkoxylated polymeric material-containing
compositions. The compositions can be characterized as having:
substantially low content of both high molecular weight impurities
(e.g., polymer-containing species having a molecular weight greater
than the molecular weight of the desired alkoxylated polymeric
material) and low content of low molecular weight diol impurities
(i.e., HO-PEG-OH), either impurity type (and preferably both
impurity types) totaling less than 8 wt %, and more preferably less
than 2 wt %. In addition or alternatively, the compositions can
also be characterized as having a purity of alkoxylated polymeric
material (as well as compositions comprising polymer reagents
formed from the alkoxylated polymeric material, and compositions of
conjugates formed from conjugating such polymer reagents and an
active agent) of greater than 92 wt %, of greater than 93 wt %, or
greater than 94 wt %, of greater than 95 wt %, preferably of
greater than 96 wt %, and more preferably greater than 97 wt %. Gel
permeation chromatography (GPC) and gel filtration chromatography
(GFC) can be used to characterize the alkoxylated polymeric
material. Those chromatographic methods allow separation of the
composition to its components according to molecular weight. The
exemplary GFC traces of products described in the Example 8 and
Example 9 are provided as FIG. 7 and FIG. 8.
Exemplary Uses of the Alkoxylated Polymeric Materials and
Compositions Formed Therefrom
[0178] The alkoxylated polymeric material provided herein as well
as those alkoxylated polymeric products that have been further
modified to bear a specific reactive group (hereinafter referred to
as a "polymer reagent") are useful for conjugation to, for example,
active agents. Preferred groups of the biologically active agents
suited for reaction with the polymeric reagents described herein
are electrophilic and nucleophilic groups. Exemplary groups include
primary amines, carboxylic acids, alcohols, thiols, hydrazines and
hydrazides. Such groups suited to react with the polymeric reagents
described herein are known to those of ordinary skill in the art.
Thus, the invention provides a method for making a conjugate
comprising the step of contacting, under conjugation conditions, an
active agent with a polymeric reagent described herein.
[0179] Suitable conjugation conditions are those conditions of
time, temperature, pH, reagent concentration, reagent functional
group(s), available functional groups on the active agent, solvent,
and the like sufficient to effect conjugation between a polymeric
reagent and an active agent. As is known in the art, the specific
conditions depend upon, among other things, the active agent, the
type of conjugation desired, the presence of other materials in the
reaction mixture, and so forth. Sufficient conditions for effecting
conjugation in any particular case can be determined by one of
ordinary skill in the art upon a reading of the disclosure herein,
reference to the relevant literature, and/or through routine
experimentation.
[0180] For example, when the polymeric reagent contains an
N-hydroxysuccinimide active ester (e.g., succinimidyl succinate,
succinimidyl propionate, and succinimidyl butanoate), and the
active agent contains an amine group, conjugation can be effected
at a pH of from about 7.5 to about 9.5 at room temperature. In
addition, when the polymer reagent contains a vinylsulfone reactive
group or a maleimide group and the pharmacologically active agent
contains a sulfhydryl group, conjugation can be effected at a pH of
from about 7 to about 8.5 at room temperature. Moreover, when the
reactive group associated with the polymer reagent is an aldehyde
or ketone and the pharmacologically active agent contains a primary
amine, conjugation can be effected by reductive amination wherein
the primary amine of the pharmacologically active agent reacts with
the aldehyde or ketone of the polymer. Taking place at pH's of from
about 6 to about 9.5, reductive amination initially results in a
conjugate wherein the pharmacologically active agent and polymer
are linked via an imine bond. Subsequent treatment of the imine
bond-containing conjugate with a suitable reducing agent such as
NaCNBH.sub.3 reduces the imine to a secondary amine. For additional
information concerning these and other conjugation reactions,
reference is made to Hermanson "Bioconjugate Techniques," Academic
Press, 1996.
[0181] Exemplary conjugation conditions include carrying out the
conjugation reaction at a pH of from about 4 to about 10, and at,
for example, a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5,
8.0, 8.5, 9.0, 9.5, or 10.0. The reaction is allowed to proceed
from about 5 minutes to about 72 hours, preferably from about 30
minutes to about 48 hours, and more preferably from about 4 hours
to about 24 hours. The temperature under which conjugation can take
place is typically, although not necessarily, in the range of from
about 0.degree. C. to about 40.degree. C., and is often at room
temperature or less. The conjugation reactions are often carried
out using a phosphate buffer solution, sodium acetate, or similar
system.
[0182] With respect to reagent concentration, an excess of the
polymer reagent is typically combined with the active agent. In
some cases, however, it is preferred to have stoichiometic amounts
of reactive groups on the polymer reagent to the reactive groups of
the active agent. Thus, for example, one mole of a polymer reagent
bearing four reactive groups is combined with four moles of active
agent. Exemplary ratios of reactive groups of polymer reagent to
active agent include molar ratios of about 1:1 (reactive group of
polymer reagent:active agent), 1:0.1, 1:0.5, 1:1.5, 1:2, 1:3, 1:4,
1:5, 1:6, 1:8, or 1:10. The conjugation reaction is allowed to
proceed until substantially no further conjugation occurs, which
can generally be determined by monitoring the progress of the
reaction over time.
[0183] Progress of the reaction can be monitored by withdrawing
aliquots from the reaction mixture at various time points and
analyzing the reaction mixture by chromatographic methods. SDS-PAGE
or MALDI-TOF mass spectrometry, NMR, IR, or any other suitable
analytical method. Once a plateau is reached with respect to the
amount of conjugate formed or the amount of unconjugated polymer
reagent remaining, the reaction is assumed to be complete.
Typically, the conjugation reaction takes anywhere from minutes to
several hours (e.g., from 5 minutes to 24 hours or more). The
resulting product mixture is preferably, but not necessarily
purified, to separate out excess active agent, strong base,
condensing agents and reaction by-products and solvents. The
resulting conjugates can then be further characterized using
analytical methods such as chromatographic methods, spectroscopic
methods, MALDI, capillary electrophoresis, and/or gel
electrophoresis. The polymer-active agent conjugates can be
purified to obtain/isolate different conjugated species.
[0184] With respect to an active agent, the alkoxylated polymeric
material and a polymer reagent prepared from the alkoxylated
polymeric material can be combined under suitable conjugation
conditions to result in a conjugate. In this regard, exemplary
active agents can be an active agent selected from the group
consisting of a small molecule drug, an oligopeptide, a peptide,
and a protein. The active agent for use herein can include but are
not limited to the following: adriamycin, .gamma.-aminobutyric acid
(GABA), amiodarone, amitryptyline, azithromycin, benzphetamine,
bromopheniramine, cabinoxamine, calcitonin chlorambucil,
chloroprocaine, chloroquine, chlorpheniramine, chlorpromazine,
cinnarizine, clarthromycin, clomiphene, cyclobenzaprine,
cyclopentolate, cyclophosphamide, dacarbazine, daunomycin,
demeclocycline, dibucaine, dicyclomine, diethylproprion, diltiazem,
dimenhydrinate, diphenhydramine, disopyramide, doxepin,
doxycycline, doxylamine, dypyridame, EDTA, erythromycin,
flurazepam, gentian violet, hydroxychloroquine, imipramine,
insulin, irinotecan, levomethadyl, lidocaine, loxarine,
mechlorethamine, melphalan, methadone, methotimeperazine,
methotrexate, metoclopramide, minocycline, naftifine, nicardipine,
nizatidine, orphenadrine, oxybutin, oxytetracycline,
phenoxybenzamine, phentolamine, procainamide, procaine, promazine,
promethazine, proparacaine, propoxycaine, propoxyphene, ranitidine,
tamoxifen, terbinafine, tetracaine, tetracycline, tranadol,
triflupromazine, trimeprazine, trimethylbenzamide, trimipramine,
trlpelennamine, troleandomycin, tyramine, uracil mustard,
verapamil, and vasopressin.
[0185] Further exemplary active agents include those selected from
the group consisting of acravistine, amoxapine, astemizole,
atropine, azithromycin, benzapril, benztropine, beperiden,
bupracaine, buprenorphine, buspirone, butorphanol, caffeine,
camptothecin and molecules belonging to the camptothecin family,
ceftriaxone, chlorpromazine, ciprofloxacin, cladarabine,
clemastine, clindamycin, clofazamine, clozapine, cocaine, codeine,
cyproheptadine, desipramine, dihydroergotamine, diphenidol,
diphenoxylate, dipyridamole, docetaxel, doxapram, ergotamine,
famciclovir, fentanyl, flavoxate, fludarabine, fluphenazine,
fluvastin, ganciclovir, granisteron, guanethidine, haloperidol,
homatropine, hydrocodone, hydromorphone, hydroxyzine, hyoscyamine,
imipramine, itraconazole, keterolac, ketoconazole, levocarbustine,
levorphone, lincomvcin, lomefloxacin, loperamide, losartan,
loxapine, mazindol, meclizine, meperidine, mepivacaine,
mesoridazine, methdilazine, methenamine, methimazole,
methotrimeperazine, methysergide, metronidazole, minoxidil,
mitomycin c, molindone, morphine, nafzodone, nalbuphine, naldixic
acid, nalmefene, naloxone, naltrexone, naphazoline, nedocromil,
nicotine, norfloxacin, ofloxacin, ondansteron, oxycodone,
oxymorphone, paclitaxel, pentazocine, pentoxyfylline, perphenazine,
physostigmine, pilocarpine, pimozide, pramoxine, prazosin,
prochlorperazine, promazine, promethazine, quinidine, quinine,
rauwolfia alkaloids, riboflavin, rifabutin, risperidone,
rocuronium, scopalamine, sufentanil, tacrine, terazosin,
terconazole, terfenadine, thiordazine, thiothixene, ticlodipine,
timolol, tolazamide, tolmetin, trazodone, triethylperazine,
trifluopromazine, trihexylphenidyl, trimeprazine, trimipramine,
tubocurarine, vecuronium, vidarabine, vinblastine, vincristine and
vinorelbine.
[0186] Still further exemplary active agents include those selected
from the group consisting of acetazolamide, acravistine, acyclovir,
adenosine phosphate, allopurinal, alprazolam, amoxapine, amrinone,
apraclonidine, azatadine, aztreonam, bisacodyl, bleomycin,
bromopheniramine, buspirone, butoconazole, camptothecin and
molecules within the camptothecin family, carbinoxamine,
cefamandole, cefazole, cefixime, cefmetazole, cefonicid,
cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone,
cephapirin, chloroquine, chlorpheniramine, cimetidine, cladarabine,
clotrimazole, cloxacillin, didanosine, dipyridamole, doxazosin,
doxylamine, econazole, enoxacin, estazolam, ethionamide,
famciclovir, famotidine, fluconazole, fludarabine, folic acid,
ganciclovir, hydroxychloroquine, iodoquinol, isoniazid,
itraconazole, ketoconazole, lamotrigine, lansoprazole, lorcetadine,
losartan, mebendazole, mercaptopurine, methotrexate, metronidazole,
miconazole, midazolam, minoxidil, nafzodone, naldixic acid, niacin,
nicotine, nizatidine, omeperazole, oxaprozin, oxiconazole,
papaverine, pentostatin, phenazopyridine, pilocarpine, piroxicam,
prazosin, primaquine, pyrazinamide, pyrimethamine, pyroxidine,
quinidine, quinine, ribaverin, rifampin, sulfadiazine,
sulfamethizole, sulfamethoxazole, sulfasalazine, sulfasoxazole,
terazosin, thiabendazole, thiamine, thioguanine, timolol,
trazodone, triampterene, triazolam, trimethadione, trimethoprim,
trimetrexate, triplenamine, tropicamide, and vidarabine.
[0187] Still further exemplary active agents include those
belonging to the camptothecin family of molecules. For example, the
active agent can possess the general structure:
##STR00015##
wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are each
independently selected from the group consisting of: hydrogen;
halo; acyl; alkyl (e.g., C1-C6 alkyl); substituted alkyl; alkoxy
(e.g., C1-C6 alkoxy); substituted alkoxy; alkenyl; alkynyl;
cycloalkyl; hydroxyl; cyano; nitro; azido; amido; hydrazine; amino;
substituted amino (e.g., monoalkylamino and dialkylamino);
hydroxcarbonyl; alkoxycarbonyl; alkylcarbonyloxy;
alkylcarbonylamino; carbamoyloxy; arylsulfonyloxy;
alkylsulfonyloxy; --C(R.sub.7)=N--(O).sub.i--R.sub.8 wherein
R.sub.7 is H, alkyl, alkenyl, cycloalkyl, or aryl, i is 0 or 1, and
R.sub.8 is H, alkyl, alkenyl, cycloalkyl, or heterocycle; and
R.sub.9C(O)O-- wherein R.sub.9 is halogen, amino, substituted
amino, heterocycle, substituted heterocycle, or
R.sub.10--O--(CH.sub.2).sub.m-- where m is an integer of 1-10 and
R.sub.10 is alkyl, phenyl, substituted phenyl, cycloalkyl,
substituted cycloalkyl, heterocycle, or substituted heterocycle; or
R.sub.2 together with R.sub.3 or R.sub.3 together with R form
substituted or unsubstituted methylenedioxy, ethylenedioxy, or
ethyleneoxy; R.sub.6 is H or OR', wherein R' is alkyl, alkenyl,
cycloalkyl, haloalkyl, or hydroxyalkyl. Although not shown, analogs
having a hydroxyl group corresponding to a position other than the
20-position (e.g., 10-, or 11-position, and so forth) in the
immediately preceding structure are encompassed within possible
active agents.
[0188] An exemplary active agent is irinotecan.
##STR00016##
[0189] Another exemplary active agent is
7-ethyl-10-hydroxy-camptothecin (SN-38), the structure of which is
shown below.
##STR00017##
[0190] Yet other exemplary class of active agents include those
belonging to the taxane family of molecules. An exemplary active
agent from this class of molecules is docetaxel where the H of the
hydroxy at the 2' hydroxyl group is involved in forming the
preferred multi-armed polymer conjugate:
##STR00018##
[0191] The polymer reagents described herein can be attached,
either covalently or noncovalently, to a number of entities
including films, chemical separation and purification surfaces,
solid supports, metal surfaces such as gold, titanium, tantalum,
niobium, aluminum, steel, and their oxides, silicon oxide,
macromolecules (e.g., proteins, polypeptides, and so forth), and
small molecules. Additionally, the polymer reagents can also be
used in biochemical sensors, bioelectronic switches, and gates. The
polymer reagents can also be employed as carriers for peptide
synthesis, for the preparation of polymer-coated surfaces and
polymer grafts, to prepare polymer-ligand conjugates for affinity
partitioning, to prepare cross-linked or non-cross-linked
hydrogels, and to prepare polymer-cofactor adducts for
bioreactors.
[0192] Optionally, the conjugate can be provided as a
pharmaceutical composition for veterinary and for human medical
use. Such a pharmaceutical compositions is prepared by combining
the conjugate with one or more pharmaceutically acceptable
excipients, and optionally any other therapeutic ingredients.
[0193] Exemplary pharmaceutically acceptable excipients, without
limitation, those selected from the group consisting of
carbohydrates, inorganic salts, antimicrobial agents, antioxidants,
surfactants, buffers, acids, bases, and combinations thereof.
[0194] A carbohydrate such as a sugar, a derivatized sugar such as
an alditol, aldonic acid, an esterified sugar, and/or a sugar
polymer may be present as an excipient. Specific carbohydrate
excipients include, for example: monosaccharides, such as fructose,
maltose, galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol
(glucitol), pyranosyl sorbitol, myoinositol, and the like.
[0195] The excipient can also include an inorganic salt or buffer
such as citric acid, sodium chloride, potassium chloride, sodium
sulfate, potassium nitrate, sodium phosphate monobasic, sodium
phosphate dibasic, and combinations thereof.
[0196] The composition can also include an antimicrobial agent for
preventing or deterring microbial growth. Nonlimiting examples of
antimicrobial agents suitable for one or more embodiments of the
present invention include benzalkonium chloride, benzethonium
chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol,
phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and
combinations thereof.
[0197] An antioxidant can be present in the composition as well.
Antioxidants are used to prevent oxidation, thereby preventing the
deterioration of the conjugate or other components of the
preparation. Suitable antioxidants for use in one or more
embodiments of the present invention include, for example, ascorbyl
palmitate, butylated hydroxyanisole, butylated hydroxytoluene,
hypophosphorous acid, monothioglycerol, propyl gallate, sodium
bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite,
and combinations thereof.
[0198] A surfactant can be present as an excipient. Exemplary
surfactants include: polysorbates, such as "Tween 20" and "Tween
80," and pluronics such as F68 and F88 (both of which are available
from BASF, Mount Olive, N.J.); sorbitan esters; lipids, such as
phospholipids such as lecithin and other phosphatidylcholines,
phosphatidylethanolamines (although preferably not in liposomal
form), fatty acids and fatty esters; steroids, such as cholesterol;
and chelating agents, such as EDTA, zinc and other such suitable
cations.
[0199] Acids or bases can be present as an excipient in the
composition. Nonlimiting examples of acids that can be used include
those acids selected from the group consisting of hydrochloric
acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic
acid, formic acid, trichloroacetic acid, nitric acid, perchloric
acid, phosphoric acid, sulfuric acid, fumaric acid, and
combinations thereof. Examples of suitable bases include, without
limitation, bases selected from the group consisting of sodium
hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,
ammonium acetate, potassium acetate, sodium phosphate, potassium
phosphate, sodium citrate, sodium formate, sodium sulfate,
potassium sulfate, potassium fumerate, and combinations
thereof.
[0200] The amount of the conjugate (i.e., the conjugate formed
between the active agent and the polymeric reagent) in the
composition will vary depending on a number of actors, but will
optimally be a therapeutically effective dose when the composition
is stored in a unit dose container (e.g., a vial). In addition, the
pharmaceutical preparation can be housed in a syringe. A
therapeutically effective dose can be determined experimentally by
repeated administration of increasing amounts of the conjugate in
order to determine which amount produces a clinically desired
endpoint.
[0201] The amount of any individual excipient in the composition
will vary depending on the activity of the excipient and particular
needs of the composition. Typically, the optimal amount of any
individual excipient is determined through routine experimentation,
i.e., by preparing compositions containing varying amounts of the
excipient (ranging from low to high), examining the stability and
other parameters, and then determining the range at which optimal
performance is attained with no significant adverse effects.
[0202] Generally, however, the excipient will be present in the
composition in an amount of about 1% to about 99% by weight,
preferably from about 5% to about 98% by weight, more preferably
from about 15 to about 95% by weight of the excipient, with
concentrations less than 30% by weight most preferred.
[0203] These foregoing pharmaceutical excipients along with other
excipients are described in "Remington: The Science & Practice
of Pharmacy", 19.sup.th ed., Williams & Williams, (1995), the
"Physician's Desk Reference", 52.sup.nd ed., Medical Economics,
Montvale, N.J. (1998), and Kibbe, A. H., Handbook of Pharmaceutical
Excipients, 3.sup.rd Edition, American Pharmaceutical Association,
Washington, D.C., 2000.
[0204] The pharmaceutically acceptable compositions encompass all
types of formulations and in particular those that are suited for
injection, e.g., powders or lyophilates that can be reconstituted
as well as liquids. Examples of suitable diluents for
reconstituting solid compositions prior to injection include
bacteriostatic water for injection, dextrose 5% in water,
phosphate-buffered saline, Ringer's solution, saline, sterile
water, deionized water, and combinations thereof. With respect to
liquid pharmaceutical compositions, solutions and suspensions are
envisioned.
[0205] The compositions of one or more embodiments of the present
invention are typically, although not necessarily, administered via
injection and are therefore generally liquid solutions or
suspensions immediately prior to administration. The pharmaceutical
preparation can also take other forms such as syrups, creams,
ointments, tablets, powders, and the like. Other modes of
administration are also included, such as pulmonary, rectal,
transdermal, transmucosal, oral, intrathecal, subcutaneous,
intra-arterial, and so forth.
[0206] The invention also provides a method for administering a
conjugate as provided herein to a patient suffering from a
condition that is responsive to treatment with conjugate. The
method comprises administering to a patient, generally via
injection, a therapeutically effective amount of the conjugate
(preferably provided as part of a pharmaceutical composition). As
previously described, the conjugates can be administered injected
parenterally by intravenous injection. Suitable formulation types
for parenteral administration include ready-for-injection
solutions, dry powders for combination with a solvent prior to use,
suspensions ready for injection, dry insoluble compositions for
combination with a vehicle prior to use, and emulsions and liquid
concentrates for dilution prior to administration, among
others.
[0207] The method of administering may be used to treat any
condition that can be remedied or prevented by administration of
the conjugate. Those of ordinary skill in the art appreciate which
conditions a specific conjugate can effectively treat.
Advantageously, the conjugate can be administered to the patient
prior to, simultaneously with, or after administration of another
active agent.
[0208] The actual dose to be administered will vary depending upon
the age, weight, and general condition of the subject as well as
the severity of the condition being treated, the judgment of the
health care professional, and conjugate being administered.
Therapeutically effective amounts are known to those skilled in the
art and/or are described in the pertinent reference texts and
literature. Generally, a therapeutically effective amount will
range from about 0.001 mg to 100 mg, preferably in doses from 0.01
mg/day to 75 mg/day, and more preferably in doses from 0.10 mg/day
to 50 mg/day. A given dose can be periodically administered up
until, for example, related symptoms lessen and/or are eliminated
entirely.
[0209] The unit dosage of any given conjugate (again, preferably
provided as part of a pharmaceutical preparation) can be
administered in a variety of dosing schedules depending on the
judgment of the clinician, needs of the patient, and so forth. The
specific dosing schedule will be known by those of ordinary skill
in the art or can be determined experimentally using routine
methods. Exemplary dosing schedules include, without limitation,
administration once daily, three times weekly, twice weekly, once
weekly, twice monthly, once monthly, and any combination thereof.
Once the clinical endpoint has been achieved, dosing of the
composition is halted.
[0210] One advantage of administering certain conjugates described
herein is that individual water-soluble polymer portions can be
cleaved when a hydrolytically degradeable linkage is included
between the residue of the active agent moiety and water-soluble
polymer. Such a result is advantageous when clearance from the body
is potentially a problem because of the polymer size. Optimally,
cleavage of each water-soluble polymer portion is facilitated
through the use of physiologically cleavable and/or enzymatically
degradable linkages such as amide, carbonate or ester-containing
linkages. In this way, clearance of the conjugate (via cleavage of
individual water-soluble polymer portions) can be modulated by
selecting the polymer molecular size and the type functional group
that would provide the desired clearance properties. One of
ordinary skill in the art can determine the proper molecular size
of the polymer as well as the cleavable functional group. For
example, one of ordinary skill in the art, using routine
experimentation, can determine a proper molecular size and
cleavable functional group by first preparing a variety of polymer
derivatives with different polymer weights and cleavable functional
groups, and then obtaining the clearance profile (e.g., through
periodic blood or urine sampling) by administering the polymer
derivative to a patient and taking periodic blood and/or urine
sampling. Once a series of clearance profiles have been obtained
for each tested conjugate, a suitable conjugate can be
identified.
Mixed Salts--Considerations Concerning the Active Agent, "D"
[0211] As indicated previously, water-soluble polymer conjugates
and compositions containing these conjugates may be provided as
mixed salts. In the mixed salt conjugate and composition context,
the active agent is a small molecule drug, an oligopeptide, a
peptide, or a protein, that, when conjugated to the water-soluble
polymer, contains at least one basic nitrogen atom such as an amine
group (e.g., an amine or other basic nitrogen containing group that
is not conjugated to the water-soluble polymer). In the mixed salt,
the basic nitrogen atoms are each individually either protonated or
unprotonated, where the protonated nitrogen atoms exist as acid
salts of two different anions.
[0212] Active agents containing at least one amine group or basic
nitrogen atom suitable for providing a mixed acid salt as described
herein include but are not limited to the following: adriamycin,
y-aminobutyric acid (GABA), amiodarone, amitryptyline,
azithromycin, benzphetamine, bromopheniramine, cabinoxamine,
calcitonin chlorambucil, chloroprocaine, chloroquine,
chlorpheniramine, chlorpromazine, cinnarizine, clarthromycin,
clomiphene, cyclobenzaprine, cyclopentolate, cyclophosphamide,
dacarbazine, daunomycin, demeclocycline, dibucaine, dicyclomine,
diethylproprion, diltiazem, dimenhydrinate, diphenhydramine,
disopyramide, doxepin, doxycycline, doxylamine, dypyridame, EDTA,
erythromycin, flurazepam, gentian violet, hydroxychloroquine,
imipramine, insulin, irinotecan, levomethadyl, lidocaine, loxarine,
mechlorethamine, melphalan, methadone, methotimeperazine,
methotrexate, metoclopramide, minocycline, naftifine, nicardipine,
nizatidine, orphenadrine, oxybutin, oxytetracycline,
phenoxybenzamine, phentolamine, procainamide, procaine, promazine,
promethazine, proparacaine, propoxycaine, propoxyphene, ranitidine,
tamoxifen, terbinafine, tetracaine, tetracycline, tranadol,
triflupromazine, trimeprazine, trimethylbenzamide, trimipramine,
trlpelennamine, troleandomycin, tyramine, uracil mustard,
verapamil, and vasopressin.
[0213] Additional active agents include those comprising one or
more nitrogen-containing heterocycles such as acravistine,
amoxapine, astemizole, atropine, azithromycin, benzapril,
benztropine, beperiden, bupracaine, buprenorphine, buspirone,
butorphanol, caffeine, camptothecin and molecules belonging to the
camptothecin family, ceftriaxone, chlorpromazine, ciprofloxacin,
cladarabine, clemastine, clindamycin, clofazamine, clozapine,
cocaine, codeine, cyproheptadine, desipramine, dihydroergotamine,
diphenidol, diphenoxylate, dipyridamole, doxapram, ergotamine,
famciclovir, fentanyl, flavoxate, fludarabine, fluphenazine,
fluvastin, ganciclovir, granisteron, guanethidine, haloperidol,
homatropine, hydrocodone, hydromorphone, hydroxyzine, hyoscyamine,
imipramine, itraconazole, keterolac, ketoconazole, levocarbustine,
levorphone, lincomycin, lomefloxacin, loperamide, losartan,
loxapine, mazindol, meclizine, meperidine, mepivacaine,
mesoridazine, methdilazine, methenamine, methimazole,
methotrimeperazine, methysergide, metronidazole, minoxidil,
mitomycin c, molindone, morphine, nafzodone, nalbuphine, naldixic
acid, nalmefene, naloxone, naltrexone, naphazoline, nedocromil,
nicotine, norfloxacin, ofloxacin, ondansteron, oxycodone,
oxymorphone, pentazocine, pentoxyfylline, perphenazine,
physostigmine, pilocarpine, pimozide, pramoxine, prazosin,
prochlorperazine, promazine, promethazine, quinidine, quinine,
rauwolfia alkaloids, riboflavin, rifabutin, risperidone,
rocuronium, scopalamine, sufentanil, tacrine, terazosin,
terconazole, terfenadine, thiordazine, thiothixene, ticlodipine,
timolol, tolazamide, tolmetin, trazodone, triethylperazine,
trifluopromazine, trihexylphenidyl, trimeprazine, trimipramine,
tubocurarine, vecuronium, vidarabine, vinblastine, vincristine and
vinorelbine.
[0214] Additional active agents include those comprising an
aromatic ring nitrogen such as acetazolamide, acravistine,
acyclovir, adenosine phosphate, allopurinal, alprazolam, amoxapine,
amrinone, apraclonidine, azatadine, aztreonam, bisacodyl,
bleomycin, bromopheniramine, buspirone, butoconazole, camptothecin
and molecules within the camptothecin family, carbinoxamine,
cefamandole, cefazole, cefixime, cefmetazole, cefonicid,
cefoperazone, cefotaxime, cefotetan, cefpodoxime, ceftriaxone,
cephapirin, chloroquine, chlorpheniramine, cimetidine, cladarabine,
clotrimazole, cloxacillin, didanosine, dipyridamole, doxazosin,
doxylamine, econazole, enoxacin, estazolam, ethionamide,
famciclovir, famotidine, fluconazole, fludarabine, folic acid,
ganciclovir, hydroxychloroquine, iodoquinol, isoniazid,
itraconazole, ketoconazole, lamotrigine, lansoprazole, lorcetadine,
losartan, mebendazole, mercaptopurine, methotrexate, metronidazole,
miconazole, midazolam, minoxidil, nafzodone, naldixic acid, niacin,
nicotine, nizatidine, omeperazole, oxaprozin, oxiconazole,
papaverine, pentostatin, phenazopyridine, pilocarpine, piroxicam,
prazosin, primaquine, pyrazinamide, pyrimethamine, pyroxidine,
quinidine, quinine, ribaverin, rifampin, sulfadiazine,
sulfamethizole, sulfamethoxazole, sulfasalazine, sulfasoxazole,
terazosin, thiabendazole, thiamine, thioguanine, timolol,
trazodone, triampterene, triazolam, trimethadione, trimethoprim,
trimetrexate, triplenamine, tropicamide, and vidarabine.
[0215] A preferred active agent is one belonging to the
camptothecin family of molecules. For example, the active agent may
possess the general structure:
##STR00019##
wherein R.sub.1-R.sub.5 are each independently selected from the
group consisting of hydrogen; halo; acyl; alkyl (e.g., C1-C6
alkyl); substituted alkyl; alkoxy (e.g., C1-C6 alkoxy); substituted
alkoxy; alkenyl; alkynyl; cycloalkyl; hydroxyl; cyano; nitro;
azido; amido; hydrazine; amino; substituted amino (e.g.,
monoalkylamino and dialkylamino); hydroxcarbonyl; alkoxycarbonyl;
alkylcarbonyloxy; alkylcarbonylamino; carbamoyloxy;
arylsulfonyloxy; alkylsulfonyloxy;
--C(R.sub.7).dbd.N--(O).sub.i--R.sub.8 wherein R.sub.7 is H, alkyl,
alkenyl, cycloalkyl, or aryl, i is 0 or 1, and R.sub.8 is H, alkyl,
alkenyl, cycloalkyl, or heterocycle; and R.sub.9C(O)O-- wherein
R.sub.9 is halogen, amino, substituted amino, heterocycle,
substituted heterocycle, or R.sub.10--O--(CH.sub.2).sub.m-- where m
is an integer of 1-10 and R.sub.10 is alkyl, phenyl, substituted
phenyl, cycloalkyl, substituted cycloalkyl, heterocycle, or
substituted heterocycle; or R.sub.2 together with R.sub.3 or
R.sub.3 together with Re form substituted or unsubstituted
methylenedioxy, ethylenedioxy, or ethyleneoxy; R.sub.6 is H or OR',
wherein R' is alkyl, alkenyl, cycloalkyl, haloalkyl, or
hydroxyalkyl.
[0216] In reference to the foregoing structure, although not shown,
analogs having a hydroxyl group at other than the 20-position
(e.g., 10-, or 11-position, etc.) are similarly preferred.
[0217] In one particular embodiment, the active agent is irinotecan
(structure shown below).
##STR00020##
[0218] In yet another particular embodiment, the active agent is
7-ethyl-10-hydroxy-camptothecin (SN-38), a metabolite of
irinotecan, whose structure is shown below.
##STR00021##
Mixed Salts--Considerations Concerning the Conjugates
[0219] Illustrative mixed salt conjugates of a water-soluble
polymer and an active agent may possess any of a number of
structural features as described above. That is to say, the
conjugate may possess a linear structure, i.e., having one or two
active agent molecules covalently attached to a linear
water-soluble polymer, typically at each terminus of the linear
water-soluble polymer. Alternatively, the conjugate may possess a
forked, branched or multi-armed structure.
[0220] One exemplary multi-armed polymer conjugate corresponds to
the following generalized structure: R(-Q-POLY.sub.1-X-D).sub.q,
wherein R is an organic radical possessing from about 3 to about
150 carbon atoms, Q is a linker (preferably hydrolytically stable
and may be --O--, --S--, --NH--C(O)-- and --C(O)--NH--, POLY.sub.1
is a water-soluble, non-peptidic polymer, X is spacer that
comprises a hydrolyzable linkage, D is an active agent moiety, and
q ranges from 3 to 25 (e.g., 3 to 10, such as any of 3, 4, 5.6, 7,
8, 9 and 10).
[0221] Another exemplary multi-armed polymer conjugate corresponds
to the following generalized structure:
R(-Q-POLY.sub.1-CH.sub.2C(O)--NH--CH.sub.2--C(O)--O-D).sub.q,
wherein: R is an organic radical possessing from 3 to 150 carbon
atoms; Q is a linker, wherein R, when taken together with Q to form
R(-Q-).sub.q, is a residue of a polyol or a polythiol after removal
of "q" hydroxyl or thiol protons, respectively, to form a point of
attachment for POLY.sub.1; POLY.sub.1, is a water-soluble polymer
selected from the group consisting of poly(alkylene glycol),
poly(olefinic alcohol), poly(vinylpyrrolidone),
poly(hydroxylalkyl-methacrylamide),
poly(hydroxyalkyl-methacrylate), poly(.alpha.-hydroxy acid),
poly(acrylic acid), poly(vinyl alcohol), polyphosphazene,
polyoxazoline, poly(N-acryloylmorpholine), and copolymers or
terpolymers thereof, D is a camptothecin attached at its 10-, 11-
or 20-ring position; and q has a value from 3 to 50 (e.g., 3 to 10,
such as any of 3, 4, 5, 6, 7, 8, 9 and 10).
[0222] One illustrative multi-armed polymer conjugate structure
corresponds to the following structure:
##STR00022##
[0223] The foregoing structure is referred to herein in shorthand
fashion as "4-arm-PEG-Gly-Irino"
(4-arm-pentaerythritolyl-PEG-carboxymethylglycine irinotecan); a
more complete name corresponds to
"pentaerythritolyl-4-arm-(PEG-1-methylene-2-oxo-vinylamino acetate
linked-irinotecan)." Basic amino and/or nitrogen groups in the
active agent portion of the conjugate are shown above in only
neutral form, with the understanding that the conjugate possesses
the features of a partial mixed salt as described in detail herein.
As can be seen from the structure above, the carboxymethyl modified
4-arm pentaerythritolyl PEG reagent possesses a glycine linker
intervening between the polymer portion and the active agent,
irinotecan.
[0224] In certain instances, due to incomplete conversions, less
than 100% yields, and other unavoidable complications routinely
encountered during chemical syntheses, in particular of multi-arm
polyethylene glycol-based materials, exemplary compositions
comprising "4-arm-PEG-Gly-Irino" can be characterized as
compositions comprising four-arm conjugates, wherein at least 90%
of the four-arm conjugates in the composition: [0225] (i) have a
structure encompassed by the formula,
[0225]
C--[CH.sub.2--O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2--C(O)-Term].-
sub.4, [0226] wherein [0227] n, in each instance, is an integer
having a value from 5 to 150 (e.g., about 113), and [0228] Term, in
each instance, is selected from the group consisting of --OH,
--OCH.sub.3,
[0228] ##STR00023## [0229] and --NH--CH.sub.2--C(O)--O--Irino
("GLY-irino"), wherein Irino is a residue of irinotecan; and [0230]
(ii) for each Term in the at least 90% of the four-arm conjugates
in the composition, at least 90% thereof are
--NH--CH.sub.2--C(O)--O--Irino.
[0231] Typically, although not necessarily, the number of polymer
arms will correspond to the number of active agent molecules
covalently attached to the water-soluble polymer core. That is to
say, in the case of a polymer reagent having a certain number of
polymer arms (e.g., corresponding to the variable "q"), each having
a reactive functional group (e.g., carboxy, activated ester such as
succinimidyl ester, benzotriazolyl carbonate, and so forth) at its
terminus, the optimized number of active agents (such as
irinotecan) that can be covalently attached thereto in the
corresponding conjugate is most desirably "q." That is to say, the
optimized conjugate is considered to have a drug loading value of
1.00(q) (or 100%). In a preferred embodiment, the multi-armed
polymer conjugate is characterized by a degree of drug loading of
0.90(q) (or 90%) or greater. Preferred drug loadings satisfy one or
more of the following: 0.92(q) or greater; 0.93(q) or greater;
0.94(q) or greater; 0.95(q) or greater; 0.96(q) or greater; 0.97(q)
or greater; 0.98(q) or greater; and 0.99(q) or greater. Most
preferably, the drug loading for a multi-armed polymer conjugate is
one hundred percent. A composition comprising a multi-arm water
soluble polymer conjugate mixed acid salt may comprise a mixture of
molecular conjugates having one active agent attached to the
polymer core, having two active agent molecules attached to the
polymer core, having three active agents attached to the polymer
core, and so on, up to and including a conjugate having "q" active
agents attached to the polymer core. The resulting composition will
possess an overall drug loading value, averaged over the conjugate
species contained in the composition. Ideally, the composition will
comprise a majority, e.g., greater than 50%, but more preferably
greater than 60%, still more preferably greater than 70%, still yet
more preferably greater than 80%, and most preferably greater than
90%) of drug fully loaded polymer conjugates (i.e., having "q"
active agent molecules for "q" arms, a single active agent molecule
for each arm).
[0232] As an illustration, in an instance in which the multi-armed
polymer conjugate contains four polymer arms, the idealized value
of the number of covalently attached drug molecules per multi-armed
polymer is four, and--with respect to describing the average in the
context of a composition of such conjugates--there will be a value
(i.e., percentage) of drug molecules loaded onto multi-armed
polymer ranging from about 90% to about 100% of the idealized
value. That is to say, the average number of drug molecules
covalently attached to a given four-armed polymer (as part of a
four-armed polymer composition) is typically 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, and 100% of the fully loaded value.
This corresponds to an average number of D per multi-arm polymer
conjugate ranging from about 3.60 to 4.0.
[0233] In yet another embodiment, for a multi-armed polymer
conjugate composition, e.g., where the number of polymer arms
ranges from about 3 to about 8. e.g., greater than 50%, but more
preferably greater than 60%, still more preferably greater than
70%, still yet more preferably greater than 80%, and most
preferably greater than 90%) of species present in the composition
are those having either an idealized number of drug molecules
attached to the polymer core ("q") or those having a combination of
("q") and ("q-1") drug molecules attached to the polymer core.
[0234] In certain instances, a multi-armed polymer conjugate such
as described herein is prepared, where the resulting conjugate
exhibits a high degree of substitution or drug loading in the
context of the ranges provided above. Illustrative conjugates thus
prepared will generally have a drug loading value of at least 9(0%,
and may typically possess drug loading values of greater than 91%,
or greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and in
some cases, at 100% of the fully loaded value. In particular,
multi-armed polymer conjugates prepared from multi-arm polymeric
starting materials that are prepared, e.g., in accordance with the
alkoxylation methodology provided herein, may exhibit higher drug
substitution values, due, at least in part, to the purity of the
polymeric starting material. As an example, 4-arm PEG-CM-SCM (e.g.,
having a molecular weight greater than about 10 kilodaltons)
prepared from 4-arm PEG-OH prepared according to the alkoxylation
method provided herein, may possess, on average, a higher level of
purity with respect to the particular polymer species present in
the 4-arm-PEG-CM-SCM reactant material than obtained with other
commercially available 4-arm PEG-OH starting materials (e.g.,
having fewer low molecular weight polymer impurities). The level of
purity of a multi-arm PEG starting material, especially those of
higher molecular weight, can contribute to the purity of the final
conjugate product in the event that non-desired polymer materials
present in the polymeric starting material are "carried along" in
subsequent transformation steps. In particular, in employing
synthetic methodologies having high yield reaction steps, e.g.,
carboxymethylation, coupling to an active agent such as deprotected
glycine-irinotecan, utilization of a polymeric starting material
having a relatively high amount of polymeric impurities, can impact
the purity and drug loading values of the resulting conjugate
species, in certain cases by several percent. Moreover, the
presence of even a small percentage of low molecular weight polymer
conjugate species in the final mixed salt conjugate can lead to
reduced bioavailability, since the small molecular weight
conjugates will clear more rapidly. Polymer conjugates prepared
from starting materials prepared using the alkoxylation method
described herein may therefore exhibit higher bioavailabilities
than polymer conjugates prepared from commercially available
multi-arm starting materials containing up to, e.g., 20% (e.g., 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or
20%), low molecular weight or other polymer impurities.
[0235] In accordance with the foregoing, the partial mixed salt
(and compositions containing the same) may comprise any one or more
of the following structures, in addition to the fully drug loaded
structure (i.e., having a glycine-modified irinotecan molecule
covalently attached to each of the four polymer arms):
##STR00024## ##STR00025##
For a given polymer arm terminus shown above having a carboxylic
acid (and therefore not covalently attached to drug, e.g.,
irinotecan), other possible termini extending from the 4-arm-PEG-CM
(--CH.sub.2C(O)--) arm include --OH, --OCH.sub.3,
##STR00026##
[0236] The multi-arm polymer conjugate compositions provided herein
are intended to encompass any and all stereoisomeric forms of the
conjugates comprised in such compositions. In a particular
embodiment of the conjugate, the stereochemistry at C-20 of
irinotecan, when in conjugated form such as in compositions of
4-arm-PEG-Gly-Irino, remains intact, i.e., C-20 retains its
(S)-configuration when in its conjugated form. See, e.g., Example
4.
[0237] Yet another preferred multi-armed structure is a
carboxymethyl modified 4-arm pentaerythritolyl PEG having a glycine
linker intervening between the polymer portion in each arm and the
active agent (polymer portion and linker shown above), where the
active agent is 7-ethyl-10-hydroxy-camptothecin. Again, included
herein are embodiments in which the multi-arm polymer is (i) fully
loaded, as well as having (ii) three
7-ethyl-10-hydroxy-camptothecin molecules covalently attached
thereto, (iii) two 7-ethyl-10-hydroxy-camptothecin molecules
covalently attached thereto, and (iv) one
7-ethyl-10-hydroxy-camptothecin molecule covalently attached to the
four-arm polymer core.
[0238] Yet another representative multi-armed conjugate structure
is a carboxymethyl modified 4-arm glycerol dimer
(3,3'-oxydipropane-1,2-diol) PEG having
7-ethyl-10-hydroxy-camptothecin (SN-38) molecules covalently
attached to the polymer core. Embodiments in which the multi-armed
polymer core is fully loaded with drug (i.e., having four
7-ethyl-10-hydroxy-camptothecin molecules covalently attached
thereo), or is less than fully loaded (i.e., having one, two, or
three 7-ethyl-10-hydroxy-camptothecin molecules covalently attached
thereto) are included herein. The conjugate having drug (i.e.,
7-ethyl-10-hydroxy-camptothecin) covalently attached to each
polymer arm is shown below.
##STR00027##
[0239] In yet another illustrative embodiment, the conjugate is a
multi-armed structure comprising a carboxymethyl modified 4-arm
glycerol dimer (3,3'-oxydipropane-1,2-diol) PEG having irinotecan
molecules covalently attached to the polymer core. Embodiments in
which the multi-armed polymer core is fully loaded with drug (i.e.,
having four irinotecan molecules covalently attached thereo), or is
less than fully loaded (i.e., having one, two, or three irinotecan
molecules covalently attached thereto) are included herein.
Parameters of the Mixed Salts
[0240] The subject compositions can be, among other things, partial
mixed acid salts. That is to say, mixed salt conjugates are
provided in a composition such that basic nitrogen atoms in the
conjugate (as well as in the bulk composition) may individually be
present in either protonated or non-protonated forms with the
protonated nitrogen atoms (referred to as acid salts) having one of
two different counter anions. One anion corresponds to the
conjugate base of a strong inorganic acid such as a hydrohalic
acid, sulfuric acid, nitric acid, phosphoric acid, nitrous acid,
and the like; the other anion corresponds to the conjugate base of
a strong organic acid such as trifluoroacetate. The subject mixed
acid salt compositions are stably and reproducibly prepared.
[0241] A mixed acid salt as provided herein is characterized in
terms of its bulk or macro properties. That is to say, basic
nitrogen atoms (i.e., amino groups) in the conjugate exist
individually in either neutral (non-protonated) or protonated form,
the protonated forms associated with one of two different possible
counterions. While the present compositions are characterized based
on bulk properties, different individual molecular species are
contained within the bulk composition. Taking the exemplary 4-arm
polymer conjugate described in Example 1, 4-arm-PEG-Gly-Irino-20K,
the mixed acid salt product contains any of a number of individual
molecular species. One molecular species is one in which each
polymer arm contains an irinotecan molecule that is in neutral
form, i.e., its amino group is unprotonated. See structure I below.
Another molecular species is one in which each polymer arm contains
an irinotecan molecule in protonated form. See Structure IV below.
An additional molecular species is one in which three of the
polymer arms contain an irinotecan molecule that is in protonated
form, and one polymer arm contains an irinotecan molecule in
neutral form (structure II). In another molecular species, two of
the four polymer arms contain an irinotecan molecule in neutral
form (i.e., its amino group is unprotonated), and two of the four
polymer arms contain an irinotecan molecule that is in protonated
form (structure II). Within all of the molecular species described
above with the exception of the first "all neutral" form,
sub-species of molecules are possible containing different
combinations of counterions. The schematic below illustrates
various possible combinations; the table that follows indicates
possible combinations of protonated acid salts corresponding to
each structure.
##STR00028##
TABLE-US-00001 III IV I II P, P, P, P, P, P, P No P P, P
combinations combinations combinations all unprotonated, TFA, TFA
TFA, TFA, TFA Cl, Cl, Cl, Cl i.e., the same TFA, Cl Cl, Cl, Cl TFA,
TFA, Cl, Cl TFA, Cl, Cl TFA, TFA Cl, TFA, TFA Cl, TFA, TFA, TFA Cl,
Cl, TFA, TFA TFA, Cl, Cl, Cl
[0242] As demonstrated in Example 1 and in Example 6, certain
exemplary polymer prodrug conjugates are obtained as mixed acid
salts of both hydrochloric acid and trifluoroacetic acid. In
Example 1, hydrochloric acid is introduced by the use of an acid
salt form of the active agent molecule to form the resulting
polymer conjugate, while the trifluoroacetic acid is introduced to
the reaction mixture in a deprotection step (although any strong
acid may be used). Following covalent attachment of the active
agent (or modified active agent as illustrated in Example 1) to the
water soluble polymer reagent, and treatment with base, even in
instances in which additional purification steps are carried out,
the resulting conjugate is unexpectedly and reproducibly obtained
as a partial mixed acid salt having surprising and beneficial
properties, to be described in greater detail below. Even after
repeated purifications, it has been discovered there is a
persistent and repeatable association of the exemplary strong
inorganic acid, hydrochloric acid, and trifluoroacetic acid in the
resulting conjugate. See, e.g., Example 2, Table 1 and Example 6,
Table 2.
[0243] The mixed acid salt conjugates described herein preferably
contain fairly well-defined proportions and ranges of each
component (i.e., free base, inorganic acid salt, organic acid
salt). The characteristics of the mixed acid salt product, may of
course, vary depending upon changes to the synthesis conditions
employed. In looking at the compositions prepared in accordance
with the method described in Example 1, the polymer conjugate mixed
acid salt is consistently recovered as having the greatest relative
molar amount of basic nitrogen atoms in protonated form in
comparison to free base (or unprotonated) nitrogens (calculated
with respect to basic nitrogen atoms in the active agent). Thus, if
all basic nitrogens in the active agent portion of the conjugate
are unprotonated, the corresponding molar percent would be 100. In
one embodiment, the partial mixed salt composition is characterized
as having the greatest relative molar amount of TFA salt (in
comparison to hydrochloride salt and free base). In yet another
particular embodiment, the partial mixed salt composition is
characterized as typically comprising a lesser relative molar
amount of hydrohalic salt (in comparison to TFA salt), and even
less of unprotonated (free base) nitrogens. In one embodiment, the
partial mixed salt composition comprises approximately 30-75 mole
percent TFA salt, approximately 15-45 mole percent hydrohalic acid
salt, and 2-55 mole percent free base. These relative amounts may
of course vary with variations in process conditions for making the
mixed acid salt. For example, in yet another embodiment, the mole
percentage of trifluoroacetic acid salt ranges from about 45 to 70,
the mole percentage of hydrochloric acid salt ranges from about 20
to 38, and the mole percentage of free base ranges from about 10 to
35. Generally, for the earlier batches of conjugates prepared,
active agent basic nitrogen (e.g., amino) groups within the
conjugate are present in the highest molar percentage as the
trifluoroacetic acid salt, in the second highest molar percentage
as the hydrochloric acid salt, and in the third or least highest
molar percentage as the free base. In certain embodiments, the mole
percentages of hydrochloride salt and free base in the conjugate
are about the same. Taking the average relative molar amounts of
trifluoroacetic acid salt, hydrochloride salt, and free base in the
conjugate over lots tested, on average, the product contained about
50 mole percent trifluoroacetic acid salt, about 30 mole percent
hydrochloric acid salt, and about 20 mole percent free base.
[0244] Turning now to Example 6, it can be seen that mixed acid
salt conjugates have been prepared, where the relative molar
amounts of each of TFA salt, hydrodrochloride salt, and
unprotonated material among the four different lots exhibit a high
level of consistency. Similar to the results in Example 1, the
polymer conjugate mixed acid salt is consistently recovered as
having the greatest relative molar amount of basic nitrogen atoms
in protonated form in comparison to free base (or unprotonated)
nitrogens (calculated with respect to basic nitrogen atoms in the
active agent). In the lots summarized in Table 2, the partial mixed
salt compositions having the greatest relative molar amount of HCl
salt in comparison to TFA salt and free base. In yet another
particular embodiment, the partial mixed salt composition may be
characterized as typically comprising a lesser relative molar
amount of TFA salt in comparison to the HCl salt, and even less of
unprotonated (free base) nitrogens. In one embodiment, the partial
mixed salt composition will comprises at least about 20 mole
percent TFA, or at least about 25 mole percent TFA. Exemplary
ranges of TFA salt within the mixed salt composition may range from
about 20-45 mole percent, or from about 24-38 mole percent, or even
from about 35 to 65 mole percent. With respect to hydrochloride
salt, the composition may, in certain embodiments, possess from
about 30 to 65 mole percent hydrochloride, or from about 32 to 60
mole percent hydrochloride, or preferably, from about 35 to 57 mole
percent hydrochloride.
[0245] The mixed acid salt conjugates described herein were
generally found to possess greater stability than either the pure
HCl salt or the free base forms of the conjugate. See, e.g.,
Example 3 and FIG. 1, illustrating the results of stress stability
tests on compositions containing varied amounts of salt and free
base forms of an exemplary conjugate, 4-arm-PEG-GLY-IRT. A positive
correlation was observed between increased stability towards
hydrolysis and increased molar percentage of salt in the final
conjugate product. Based upon the slopes of the graphs, it can be
determined that as free base content increases, product stability
decreases. A correlation between decrease in product and increase
in irinotecan over time was observed, thereby leading to a
determination that the mode of decomposition observed under the
conditions employed was ester bond hydrolysis.
[0246] FIG. 2 further illustrates that stability (or resistance)
against hydrolytic degradation is greater for conjugates possessing
a greater degree of protonated amine groups (i.e., acid salt). For
instance, it was observed that conjugate product containing 14
molar percent or more free base was notably less stable towards
hydrolysis than the corresponding acid salt-rich product.
[0247] Additionally, as illustrated in FIG. 3, product rich in the
hydrochloride salt appears to be more susceptible to cleavage of
the water-soluble polymer backbone than the mixed salt form
containing a measurable amount of free base. Indeed, decomposition
of the mixed salt conjugate appears to be attributable primarily to
hydrolytic release of drug rather than cleavage of the polymer
backbone. Such backbone decomposition appears, however, to be
relevant only under accelerated stress conditions.
[0248] Since the two modes of decomposition observed seem to show
opposite trends with respect to stability or resistance to
degradation versus salt/free base content, this may (but does not
necessarily) indicate a preferred region of salt composition that
possesses a greater overall stability than either extremes of full
salt or full free base. Moreover, based upon preliminary studies,
the mixed salt appears to possess somewhat greater stability than
either the free base or hydrochloride salt form, thus indicating
its unexpected superiority over any of the more traditional pure
base or single salt forms thereof.
[0249] Further, mixed salt forms of the conjugate are prepared in
high lot-to-lot consistency--that is to say, having relatively
consistent molar ratios of trifluoroacetate, halide (or other
suitable inorganic acid anion) and free base in the final conjugate
product. As can be seen in Table 1 of Example 2, roughly 50 mole
percent of drug basic nitrogen groups are associated with
trifluoroacetic acid. This mole percentage is fairly consistently
observed from lot-to-lot. Similarly, roughly 30 mole percent of
conjugate drug amino (or other basic nitrogen) groups are fairly
consistently associated with hydrochloric acid, i.e., provided as
the HCl salt. It follows that the free base form of drug amino (or
other basic nitrogen) groups in the conjugate are also stably and
reproducibly prepared. Turning to the results provided in Example
6, based upon a slightly revised manufacturing method, it can be
seen that despite differences in the actual relative molar amounts
of protonated and unprotonated species, and within the protonated
species, TFA versus hydrochloride salt, mixed acid salts were
reproducibly prepared.
[0250] These collective results indicate the unexpected advantages
of a partial mixed salt of a water-soluble polymer-active agent
conjugate (in one embodiment, 4-arm-PEG-Gly-Irino-20K) over free
base alone or either salt in the absence of the other. The mixed
salt appears to have greater stability than either the free base or
hydrochloride salt, thus indicating its seeming advantages over
either of the more customary pure base or pure salt forms
thereof.
Mixed Salts Conjugates--Methods for Forming
[0251] A mixed acid salt of a water soluble polymer conjugate can
be readily prepared from commercially available starting materials
in view of the guidance presented herein, coupled with what is
known in the art. As described above, the mixed salt polymer-active
agent conjugate comprises a water-soluble polymer covalently
attached to one or more active agent molecules each possessing one
or more basic nitrogen atoms, such as an amino group, when in
conjugated form. Amine groups in the resulting conjugate may be
primary, secondary, or tertiary amino groups.
[0252] Linear, branched, and multi-arm water-soluble polymer
reagents are available from a number of commercial sources as
described above. Alternatively, PEG reagents such as a multi-armed
reactive PEG polymer may be synthetically prepared as described
herein.
[0253] The partial mixed acid salt can be formed using known
chemical coupling techniques for covalent attachment of activated
polymers, such as an activated PEG, to a biologically active agent
(See, for example, POLY(ETHYLENE GLYCOL) CHEMISTRY AND BIOLOGICAL
APPLICATIONS, American Chemical Society, Washington, D.C. (1997);
and U.S. Patent Publication Nos. 2009/0074704 and 2006/0239960).
Selection of suitable functional groups, linkers, protecting
groups, and the like to achieve a mixed acid salt in accordance
with the invention, will depend, in part, on the functional groups
on the active agent and on the polymer starting material and will
be apparent to one skilled in the art, based upon the content of
the present disclosure. In view of certain features of the partial
mixed acid salt, the method comprises provision of an amine- (or
other basic nitrogen)-containing active agent in the form of an
inorganic acid addition salt, and a trifluoroacetic acid treatment
step. Alternatively, the conjugate product or an intermediate in
the synthetic pathway can be reacted with an inorganic acid to form
an inorganic acid addition salt at a later stage in the process, to
thereby introduce a second counterion (in addition to
trifluoroacetate) into the reaction. Reference to an "active agent"
in the context of the synthetic method is meant to encompass an
active agent optionally modified to possess a linker covalently
attached thereto, to facilitate attachment to the water-soluble
polymer.
[0254] Generally, the method comprises the steps of (i)
deprotecting an inorganic acid salt of an amine- (or other basic
nitrogen)-containing active agent in protected form by treatment
with trifluoroacetic acid (TFA) to form a deprotected mixed acid
salt, (ii) coupling the deprotected inorganic acid salt of step (i)
with a water-soluble polymer reagent in the presence of a base to
form a polymer-active agent conjugate, and (iii) recovering the
polymer active agent conjugate. The resulting polymer-active agent
conjugate composition is characterized by having the one or more
amino (or other basic nitrogen-containing) groups present in a
combination of free base, acid salt, and TFA salt form. The product
therefore comprises both inorganic acid salt and trifluoroacetate
salt, as well as a proportion of basic groups in the conjugate that
are in unprotonated or free base form. Thus, the combined molar
amounts of inorganic acid salt and trifluoroacetic acid salt are
less than the total number of basic amino or other nitrogens
contained in the conjugate product.
[0255] In turning now to one of the preferred classes of active
agents, the camptothecins, since the 20-hydroxyl group of compounds
within the camptothecin family is sterically hindered, a single
step conjugation reaction is difficult to accomplish in significant
yields. As a result, a preferred method is to react the 20-hydroxyl
group of the bioactive starting material, e.g., irinotecan
hydrochloride, with a short linker or spacer moiety carrying a
functional group suitable for reaction with a water-soluble
polymer. Such an approach is applicable to many small molecules,
particularly those having a site of covalent attachment that is
inaccessible to an incoming reactive polymer. Preferred linkers for
reaction with a hydroxyl group to form an ester linkage include
t-BOC-glycine or other amino acids such as alanine, glycine,
isoleucine, leucine, phenylalanine, and valine having a protected
amino group and an available carboxylic acid group (See Zalipsky et
al., "Attachment of Drugs to Polyethylene Glycols", Eur. Polym. J.,
Vol. 19, No. 12, pp. 1177-1183 (1983)). Other spacer or linker
moieties having an available carboxylic acid group or other
functional group reactive with a hydroxyl group and having a
protected amino group can also be used in lieu of the amino acids
described above.
[0256] Typical labile protecting groups include t-BOC and FMOC
(9-flourenylmethloxycarbonyl). t-BOC is stable at room temperature
and easily removed with dilute solutions of trifluoroacetic acid
and dichloromethane. FMOC is a base labile protecting group that is
easily removed by concentrated solutions of amines (usually 20-55%
piperidine in N-methylpyrrolidone).
[0257] In the instant example, the carboxyl group of N-protected
glycine reacts with the 20-hydroxyl group of irinotecan
hydrochloride (or other suitable camptothecin, such as
7-ethyl-10-hydroxy-camptothecin, or any other active agent) in the
presence of a coupling agent (e.g., dicyclohexylcarbodiimide (DCC))
and a base catalyst (e.g., dimethylaminopyridine (DMAP) or other
suitable base) to provide N-protected linker modified active agent,
e.g., t-Boc-glycine-irinotecan hydrochloride. Although
hydrochloride is exemplified, other inorganic acid salts may be
used. Preferably, each reaction step is conducted under an inert
atmosphere.
[0258] In a subsequent step, the amino protecting group, t-BOC
(N-tert-butoxycarbonyl), is removed by treatment with
trifluoroacetic acid (TFA) under suitable reaction conditions. It
is in this step that trifluoroacetic acid is typically introduced
into the reaction mixture. The product is linker modified active
agent, e.g., 20-glycine-irinotecan TFA/HCl. Illustrative reaction
conditions are described in Example 1, and may be further optimized
by routine optimization by one of skill in the art. Optionally, the
molar amounts of inorganic acid and trifluoroacetic acid in the
decoupled product are determined by a suitable analytical method
such as HPLC or ion chromatography, to allow greater precision and
product consistency in the coupling step.
[0259] Deprotected active agent (optionally linker modified), e.g.,
20-glycine-irinotecan TFA/HCl, is then coupled to a desired polymer
reagent, e.g., 4-arm pentaerythritolyl-PEG-succinimide (or any
other similarly activated ester counterpart) in the presence of a
coupling agent (e.g., hydroxybenzyltriazole (HOBT)) and a base
(e.g., DMAP, trimethyl amine, triethyl amine, etc.), to form the
desired conjugate. In one embodiment of the method, the amount of
base added in the conjugation step is in a range of approximately
1.0 to 2.0 times, or from about 1.0 to 1.5 times, or from about 1.0
to 1.05 times, the sum of the moles of TFA and the moles of
inorganic acid determined for the starting material, in this case,
20-glycine-irinotecan TFA/HCl. By virtue of adjusting the amount of
base to the acid salt content of the 20-glycine-irinotecan TFA/HCl,
a relatively consistent ratio of TFA, inorganic acid (e.g., HCl),
and base is maintained in the coupling step, to thereby form a
partial mixed acid salt conjugate having a consistently narrow
range of TFA and inorganic acid contents. Preferably, the resulting
partial mixed acid salt is reproducibly prepared such that the
relative molar amounts of inorganic addition salt, trifluoroacetic
acid salt, and free base in the conjugate composition vary by no
more than about 25%, and even more preferably by no more than about
15%, from batch to batch. For the purposes of making such
determination, the foregoing measure of consistency is determined
over at least five batches (e.g., from 5 to 7), where failed
batches that are clearly outliers are excluded from the
calculation.
[0260] Although the conjugation step is conducted in the presence
of excess base, it is surprising to discover that the resulting
conjugate is stably formed as a partial mixed acid salt, i.e., such
that a significant amount of basic amino or other nitrogen
containing groups in the conjugate are protonated rather than being
in free base form. Reaction yields for the coupling reaction are
typically high, greater than about 90% (e.g., about 95% on
average).
[0261] The partial mixed acid salt conjugate is recovered, e.g., by
precipitation with ether (e.g., methyl tert-butyl ether, diethyl
ether) or other suitable solvent. The product may be further
purified by any suitable method. Methods of purification and
isolation include precipitation followed by filtration and drying,
as well as chromatography. Suitable chromatographic methods include
gel filtration chromatography, ion exchange chromatography, and
Biotage Flash chromatography. One preferred method of purification
is recrystallization. For example, the partial mixed acid salt is
dissolved in a suitable single or mixed solvent system (e.g.,
isopropanol/methanol), and then allowed to crystallize.
Recrystallization may be conducted multiple times, and the crystals
may also be washed with a suitable solvent in which they are
insoluble or only slightly soluble (e.g., methyl tert-butyl ether
or methyl-tert-butyl ether/methanol). The purified product may
optionally be further air or vacuum dried. Even upon repeated
purification, the product is typically recovered as a mixed acid
salt rather than as the free base. Even upon additional treatment
with base, the conjugate remained in the form of a partial mixed
acid salt having the features described herein.
[0262] The resulting conjugate is a partial mixed salt, i.e., where
certain of the basic nitrogen atoms are in neutral or free base
form and other basic nitrogen atoms, e.g., amino groups, are
protonated. The protonated amine groups are in the form of acid
salts with differing anions, one anion corresponding to the
conjugate base of an inorganic acid, the other anion being
trifluoroacetate (or the conjugate base of an organic acid as
previously described). As used herein, a partial mixed salt refers
to the bulk product rather than necessarily referring to individual
molecular species contained within the bulk product. Thus,
depending upon the particular conjugate structure, individual
molecular species contained within the mixed salt may contain amine
groups that are in free base and in protonated form as described
above. Alternatively, a mixed salt may contain a mixture of
molecular species (e.g., having all amine groups in free base form,
having all amine groups in protonated form, either as the salt of
an inorganic acid, the salt of trifluoroacetic acid or other
suitable organic acid, or a mixture of both, various combinations
of the foregoing, etc.), such that the features of the bulk product
are as described herein. In the event that the conjugate is a
polymer conjugate comprising only one active agent amine group, the
mixed salt must necessarily be such that the bulk product is a
mixture of molecular species to arrive at a mixed salt as described
generally herein.
[0263] Preferably, the mixed acid salt product is stored under
conditions suitable for protecting the product from exposure to any
one or more of oxygen, moisture, and light. Any of a number of
storage conditions or packaging protocols can be employed to
suitably protect the acid salt product during storage. In one
embodiment, the product is packaged under an inert atmosphere
(e.g., argon or nitrogen) by placement in one or more polyethylene
bags, and placed in an aluminum lined polyester heat sealable
bag.
[0264] Representative mole percents of TFA salt, hydrochloric acid
salt, and free base determined over a number of lots of
4-arm-PEG-Gly-Irino are summarized in Table 1 (Example 2) and Table
2 (Example 6). As can be seen, unexpectedly, even following
treatment with base and repeated purification, the conjugate
product is isolated not as a single non-protonated conjugate
species, but rather as a mixed acid salt.
Mixed Salts--Pharmaceutical Compositions Containing Mixed Salt
Conjugates
[0265] The partial mixed acid salt conjugates may be in the form of
a pharmaceutical formulation or composition for either veterinary
or human medical use. An illustrative formulation will typically
comprise a partial mixed acid salt conjugate in combination with
one or more pharmaceutically acceptable carriers, and optionally
any other therapeutic ingredients, stabilizers, or the like. The
carrier(s) must be pharmaceutically acceptable in the sense of
being compatible with the other ingredients of the formulation and
not unduly deleterious to the recipient/patient. The partial mixed
acid salt conjugate is optionally contained in bulk or in unit dose
form in a container or receptacle which includes packaging that
protects the product from exposure to moisture and oxygen.
[0266] The pharmaceutical composition may include polymeric
excipients/additives or carriers, e.g., polyvinylpyrrolidones,
derivatized celluloses such as hydroxymethylcellulose,
hydroxyethylcellulose, and hydroxypropylmethylcellulose, Ficolls (a
polymeric sugar), hydroxyethylstarch (HES), dextrates (e.g.,
cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin and
sulfobutylether-.beta.-cyclodextrin), polyethylene glycols, and
pectin. The compositions may further include diluents, buffers,
binders, disintegrants, thickeners, lubricants, preservatives
(including antioxidants), flavoring agents, taste-masking agents,
inorganic salts (e.g., sodium chloride), antimicrobial agents
(e.g., benzalkonium chloride), sweeteners, antistatic agents,
surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80",
and pluronics such as F68 and F88, available from BASF), sorbitan
esters, lipids (e.g., phospholipids such as lecithin and other
phosphatidylcholines, phosphatidylethanolamines, fatty acids and
fatty esters, steroids (e.g., cholesterol)), and chelating agents
(e.g., EDTA, zinc and other such suitable cations). Other
pharmaceutical excipients and/or additives suitable for use in the
compositions according to the invention are listed in "Remington:
The Science & Practice of Pharmacy", 19.sup.th ed., Williams
& Williams, (1995), and in the "Physician's Desk Reference",
52.sup.nd ed., Medical Economics, Montvale, N.J. (1998), and in
"Handbook of Pharmaceutical Excipients". Third Ed., Ed. A. H.
Kibbe, Pharmaceutical Press, 2000.
[0267] The mixed acid salt may be formulated in a composition
suitable for oral, rectal, topical, nasal, ophthalmic, or
parenteral (including intraperitoneal, intravenous, subcutaneous,
or intramuscular injection) administration. The mixed acid salt
composition may conveniently be presented in unit dosage form and
may be prepared by any of the methods well known in the art of
pharmacy. All methods include the step of bringing the mixed acid
salt into association with a carrier that constitutes one or more
accessory ingredients.
[0268] In one particular embodiment, the mixed acid salt, e.g.,
4-arm-PEG-Gly-Irino-20K, is provided in lyophilized form in a
sterile single use vial for use by injection. In one embodiment,
the amount of conjugate product contained in the single use vial is
the equivalent of a 100-mg dose of irinotecan. More particularly,
the lyophilized composition includes 4-arm-PEG-Gly-Irino-20K
combined with lactate buffer at pH 3.5. That is to say, the
lyophilized composition is prepared by combining
4-arm-PEG-Gly-Irino-20K, e.g., in an amount equivalent to a 100-mg
dose of irinotecan, with approximately 90 mg of lactic acid, and
the pH of the solution adjusted to 3.5 by addition of either acid
or base. The resulting solution is then lyophilized under sterile
conditions, and the resulting powder is stored at -20.degree. C.
prior to use. Prior to intravenous infusion, the lyophilized
composition is combined with a solution of dextrose, e.g., a 5%
(w/w) solution of dextrose.
[0269] The amount of mixed acid salt (i.e., active agent) in the
formulation will vary depending upon the specific active agent
employed, its activity, the molecular weight of the conjugate, and
other factors such as dosage form, target patient population, and
other considerations, and will generally be readily determined by
one skilled in the art. The amount of conjugate in the formulation
will be that amount necessary to deliver a therapeutically
effective amount of the compound, e.g., an alkaloid anticancer
agent such as irinotecan or SN-38, to a patient in need thereof to
achieve at least one of the therapeutic effects associated with the
compound, e.g., for treatment of cancer. In practice, this will
vary widely depending upon the particular conjugate, its activity,
the severity of the condition to be treated, the patient
population, the stability of the formulation, and the like.
Compositions will generally contain anywhere from about 1% by
weight to about 99% by weight conjugate, typically from about 2% to
about 95% by weight conjugate, and more typically from about 5% to
85% by weight conjugate, and will also depend upon the relative
amounts of excipients/additives contained in the composition. More
specifically, the composition will typically contain at least about
one of the following percentages of conjugate: 2%, 5%, 10%, 20%,
30%, 40%, 50%, 60%, or more by weight.
[0270] Compositions suitable for oral administration may be
provided as discrete units such as capsules, cachets, tablets,
lozenges, and the like, each containing a predetermined amount of
the conjugate as a powder or granules; or a suspension in an
aqueous liquor or non-aqueous liquid such as a syrup, an elixir, an
emulsion, a draught, and the like.
[0271] Formulations suitable for parenteral administration
conveniently comprise a sterile aqueous preparation of the mixed
acid salt conjugate, which can be formulated to be isotonic with
the blood of the recipient.
[0272] Nasal spray formulations comprise purified aqueous solutions
of the multi-armed polymer conjugate with preservative agents and
isotonic agents. Such formulations are preferably adjusted to a pH
and isotonic state compatible with the nasal mucous membranes.
[0273] Formulations for rectal administration may be presented as a
suppository with a suitable carrier such as cocoa butter, or
hydrogenated fats or hydrogenated fatty carboxylic acids.
[0274] Ophthalmic formulations are prepared by a similar method to
the nasal spray, except that the pH and isotonic factors are
preferably adjusted to match that of the eye.
[0275] Topical formulations comprise the multi-armed polymer
conjugate dissolved or suspended in one or more media such as
mineral oil, petroleum, polyhydroxy alcohols or other bases used
for topical formulations. The addition of other accessory
ingredients as noted above may be desirable.
[0276] Pharmaceutical formulations are also provided which are
suitable for administration as an aerosol. e.g., by inhalation.
These formulations comprise a solution or suspension of the desired
multi-armed polymer conjugate or a salt thereof. The desired
formulation may be placed in a small chamber and nebulized.
Nebulization may be accomplished by compressed air or by ultrasonic
energy to form a plurality of liquid droplets or solid particles
comprising the conjugates or salts thereof.
Mixed Salts--Methods of Using Mixed Salt Conjugates
[0277] The mixed acid salts described herein can be used to treat
or prevent any condition responsive to the unmodified active agent
in any animal, particularly in mammals, including humans. One
representative mixed acid salt,
4-arm-pentaerythritolyl-PEG-glycine-irinotecan, comprising the
anti-cancer agent, irinotecan, is particularly useful in treating
various types of cancer.
[0278] The partial mixed acid salts conjugates, in particular,
those where the small molecule drug is an anticancer agent such as
a camptothecin compound as described herein (e.g., irinotecan or
7-ethyl-10-hydroxy-camptothecin) or other oncolytic, are useful in
treating solid type tumors such as breast cancer, ovarian cancer,
colon cancer, gastric cancer, malignant melanoma, small cell lung
cancer, non-small cell lung cancer, thyroid cancers, kidney cancer,
cancer of the bile duct, brain cancer, cervical cancer, maxillary
sinus cancer, bladder cancer, esophageal cancer. Hodgkin's disease,
adrenocortical cancer, and the like. Additional cancers treatable
with the mixed acid salt include lymphomas, leukemias,
rhabdomyosarcoma, neuroblastoma, and the like. As stated above, the
mixed salt conjugates are particularly effective in targeting and
accumulating in solid tumors. The mixed salt conjugates are also
useful in the treatment of HIV and other viruses.
[0279] Representative conjugates such as
4-arm-pentaerythritolyl-PEG-glycine-irinotecan have also been shown
to be particularly advantageous when used to treat patients having
cancers shown to be refractory to treatment with one or more
anticancer agents.
[0280] Methods of treatment comprise administering to a mammal in
need thereof a therapeutically effective amount of a partial mixed
acid salt composition or formulation as described herein.
[0281] Additional methods include treatment of (i) metastatic
breast cancer that is resistant to anthracycline and/or taxane
based therapies, (ii) platinum-resistant ovarian cancer, (iii)
metastatic cervical cancer, and (iv) colorectal cancer in patients
with K-Ras mutated gene status by administering a partial mixed
acid salt composition.
[0282] In treating metastatic breast cancer, a mixed acid salt of a
conjugate such as 4-arm-pentaerythritolyl-PEG-glycine-irinotecan as
provided herein is administered to a patient with locally advanced
metastatic breast cancer at a therapeutically effective amount,
where the patient has had no more than two prior (unsuccessful)
treatments with anthracycline and/or taxane based
chemotherapeutics.
[0283] For treating platinum-resistant ovarian cancer, a
composition as provided herein is administered to a patient with
locally advanced or metastatic ovarian cancer at a therapeutically
effective amount, where the patient has shown tumor progression
during platinum-based therapy, with a progression-free interval of
less than six months.
[0284] In yet another approach, a mixed acid salt (e.g., such as
that in Example 1) is administered to a subject with locally
advanced colorectal cancer, where the colorectal tumor(s) has a
K-Ras oncogene mutation (K-Ras mutant types) such that the tumor
does not respond to EGFR-inhibitors, such as cetuximab. Subjects
are those having failed one prior 5-FU containing therapy, and are
also irinotecan naive.
[0285] A therapeutically effective dosage amount of any specific
mixed acid salt will vary from conjugate to conjugate, patient to
patient, and will depend upon factors such as the condition of the
patient, the activity of the particular active agent employed, the
type of cancer, and the route of delivery.
[0286] For camptothecin-type active agents such as irinotecan or
7-ethyl-10-hydroxy-camptothecin, dosages from about 0.5 to about
100 mg camptothecin/kg body weight, preferably from about 10.0 to
about 60 mg/kg, are preferred. When administered conjointly with
other pharmaceutically active agents, even less of the mixed acid
salt may be therapeutically effective. For administration of a
mixed acid salt of irinotecan, the dosage amount of irinotecan will
typically range from about 50 mg/m.sup.2 to about 350
mg/m.sup.2.
[0287] Methods of treatment also include administering a
therapeutically effective amount of a mixed acid salt composition
or formulation as described herein (e.g., where the active agent is
a camptothecin type molecule) in conjunction with a second
anticancer agent. Preferably, such camptothecin-based conjugates,
of course, in the form of a mixed acid salt, are administered in
combination with 5-fluorouracil and folinic acid as described in
U.S. Pat. No. 6,403,569.
[0288] The mixed acid salt compositions may be administered once or
several times a day, preferably once a day or less. The duration of
the treatment may be once per day for a period of from two to three
weeks and may continue for a period of months or even years. The
daily dose can be administered either by a single dose in the form
of an individual dosage unit or several smaller dosage units or by
multiple administration of subdivided dosages at certain
intervals.
[0289] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, that the foregoing description as well as the examples
that follow are intended to illustrate and not limit the scope of
the invention. Other aspects, advantages and modifications within
the scope of the invention will be apparent to those skilled in the
art to which the invention pertains.
EXPERIMENTAL
[0290] The practice of the invention will employ, unless otherwise
indicated, conventional techniques of organic synthesis and the
like, which are within the skill of the art. Such techniques are
fully described in the literature. Reagents and materials are
commercially available unless specifically stated to the contrary.
See, for example, M. B. Smith and J. March, March's Advanced
Organic Chemistry: Reactions Mechanisms and Structure, 6th Ed. (New
York: Wiley-Interscience, 2007), supra, and Comprehensive Organic
Functional Group Transformations 11, Volumes 1-7, Second Ed.: A
Comprehensive Review of the Synthetic Literature 1995-2003 (Organic
Chemistry Series), Eds. Katritsky, A. R., et al., Elsevier
Science.
[0291] In the following examples, efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.) but some experimental error and deviation should be accounted
for. Unless indicated otherwise, temperature is in degrees C. and
pressure is at or near atmospheric pressure at sea level.
[0292] The following examples illustrate certain aspects and
advantages of the present invention, however, the present invention
is in no way considered to be limited to the particular embodiments
described below.
ABBREVIATIONS
[0293] Ar argon
[0294] CM carboxymethyl or carboxymethylene (--CH.sub.2COOH)
[0295] DCC 1,3-dicyclohexylcarbodiimide
[0296] DCM dichloromethane
[0297] DMAP 4-(N,N-dimethylamino)pyridine
[0298] GLY glycine
[0299] HCl hydrochloric acid
[0300] RP-HPLC reverse-phase high performance liquid
chromatography
[0301] IPA isopropyl alcohol
[0302] IRT irinotecan
[0303] IPC ion pair chromatography
[0304] MeOH methanol
[0305] MTBE methyl tert-butyl ether
[0306] MW molecular weight
[0307] NMR nuclear magnetic resonance
[0308] PEG polyethylene glycol
[0309] RT room temperature
[0310] SCM succinimidylcarboxymethyl (--CH.sub.2--COO--N--
succinimidyl)
[0311] TEA triethylamine
[0312] TFA trifluoroacetic acid
[0313] THF tetrahydrofuran
Materials and Methods
[0314] Pentaerythritolyl-based 4-ARM-PEG.sub.20K-OH was obtained
from NOF Corporation (Japan). 4-ARM-PEG.sub.20K-OH possesses the
following structure (wherein each n is about 113):
C--(CH.sub.2O--(CH.sub.2CH.sub.2O).sub.NH).sub.4.
[0315] All .sup.1HNMR data was generated by a 300 or 400 MHz NMR
spectrometer manufactured by Bruker.
Example 1
Preparation 1: Preparation of
Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2 Oxo-Vinylamino Acetate
Linked-Irinotecan)-20K "4-Arm-PEG-Gly-Irino-20K Mixed Acid Salt
[0316] Reaction Scheme:
##STR00029## ##STR00030##
[0317] All solvents used in synthesis were anhydrous.
Step 1. Conjugation of t-Boc-Glycine to Irinotecan.HCl Salt
(>95% Yield)
[0318] Irinotecan.HCl.trihydrate (1 mole or 677 g) and DMF (10 L)
were charged into a distiller at 60.degree. C. Upon dissolution of
the irinotecan.HCl.trihydrate in DMF, full vacuum was slowly
applied in order to remove water from the irinotecan.HCl.trihydrate
by azeotropic distillation at 60.degree. C. Upon solids formation
from the residual DMF, heptane (up to 60 L) was charged into the
distiller to remove residual DMF at 40-50.degree. C. Upon removal
of heptane by visual inspection, the azeotropic distillation was
stopped and the solid (irinotecan.HCl) was allowed to cool to
17.+-.2.degree. C. For the coupling reaction, t-boc-glycine (1.2
mole), 4-DMAP (0.1 mole) dissolved in DCM (1 L), and DCM (19 L)
were charged into the distiller. Once the mixture was visually well
dispersed, melted DCC (1.5 mole) was added and reaction was allowed
to proceed. The reaction was carried out under an argon or nitrogen
blanket, with sufficient mixing and pot temperature at
17.+-.2.degree. C.
[0319] After a 2-4 hour reaction time, a sample was withdrawn to
measure residual irinotecan (IRT) peak area percent by
chromatography. Residual irinotecan was determined to be present in
an amount of no more than 5%. DCU formed during the coupling
reaction was removed by filtration, and washed with DCM. The
resulting filtrates containing crude t-boc-glycine-irinotecan.HCl
salt were combined and concentrated below 45.degree. C. under
vacuum to remove DCM. When approximately 75% of its initial volume
was removed by distillation, IPA was then added to the concentrate
to reach the initial volume, and the mixture further distilled
until the condensate volume reached about 25% of its initial
volume. The resulting clear solution was cooled to room
temperature, followed by its addition to heptane with mixing. The
mixture was mixed for an additional 0.5 to 1 hour, during which
time a precipitate formed. The precipitate was drained and filtered
to obtain a wet cake, and then washed with heptane (up to 6 L). The
wet cake was vacuum-dried to yield t-boc-glycine-irinotecan powder
for use in Step 2. Yield >95%.
Step 2. Deprotection of t-boc-glycine-Irinotecan
[0320] The t-boc-glycine-irinotecan (1 mole) from Step 1 was
dissolved in DCM with agitation to form a visually homogeneous
solution. To this solution was added TFA (15.8 mole) over a period
of 5 to 10 minutes and the resulting solution stirred for about 2
hours. Residual starting material was measured by RP-HPLC and
determined to be less than about 5%. Acetonitrile was then added to
the reaction solution to form a visually homogeneous solution at
RT. This solution was then added to MTBE (46.8 kg) being
sufficiently agitated at 35.degree. C. to promote crystallization.
Optionally to reduce MTBE use, DCM in the reaction solution was
replaced with acetonitrile by distillation at 15 to 40.degree. C.
After the solvent swap, the product-containing solution was added
into approximately 50% less volume of MTBE (23 kg) being
sufficiently agitated at the crystallization temperature
(35.degree. C.). Mixing was continued for a half to one hour. The
resulting solid was filtered and the cake washed with MTBE.
[0321] The wet cake was vacuum-dried to yield the
glycine-irinotecan salt powder for use in Step 3. Trifluoroacetate
and chloride content of the product was determined by ion
chromatography with a conductivity detector. (Yield >95%).
Step 3. PEGylation of Glycine-irinotecan using 4-arm-PEG-CM-SCM
[0322] The glycine-irinotecan.TFA/HCl salt powder from Step 2 was
added to a reaction vessel to which was added DCM (approx. 23 L).
The mixture was agitated for approximately 10 to 30 minutes to
allow the glycine-irinotecan-TFA/HCl salt to disperse in DCM.
Triethyl amine (approx. 1.05 moles (HCl+TFA) moles in
glycine-irinotecan TFA/HCl salt powder) was then added slowly, at a
rate which maintained the pot temperature at 24.degree. C. or
below. The resulting mixture was agitated for 10 to 30 minutes to
allow dissolution of the GLY-IRT (glycine-modified irinotecan) free
base.
[0323] Approximately 80% of the total quantity (6.4 kg) of 4-arm
PEG-SCM was added to the reaction vessel over a course of up to 30
minutes. After dissolution of the PEG reagent, reaction progress
was monitored by IPC. (In the event that the amount of
non-conjugated GLY-IRT was greater than 5% when the reaction
appeared to have reached a plateau, the remaining 20% of 4-arm PEG
SCM was then added to the reaction vessel, and the reaction
progress monitored until a constant value of unreacted GLY-IRT was
observed).
[0324] Crude product was precipitated by adding the reaction
solution into MTBE (113.6 L) agitated at room temperature over a
period of from 1-1.5 hours, followed by stirring. The resulting
mixture was transferred into a filter-drier with an agitator to
remove the mother liquor. The precipitate (crude product) was
partially vacuum-dried at approximately at 10 to 25.degree. C. with
minimum intermittent stirring.
[0325] Crude product was then placed into a reaction vessel, to
which was added IPA (72 L) and MeOH (8 L), followed by agitation
for up to 30 minutes. Heat was applied to achieve visually complete
dissolution (a clear solution) at 50.degree. C. pot temperature,
followed by agitation for 30 to 60 minutes. The solution was then
cooled to 37.degree. C., held there for several hours, followed by
cooling to 20.degree. C. The mixture was transferred into an
agitated filter dryer, and filtered to remove mother liquor to form
a cake on a filter. The cake was washed with 70% MTBE in IPA and
30% MeOH and partially vacuum-dried. This procedure was repeated
two additional times, with the exception that, prior to cooling,
the clear IPA/MeOH solution containing 4-arm PEG-Gly-IRT was
filtered using an in-line filter (1 um) at 50.degree. C. to remove
any potential particulates in the last (3rd) crystallization.
[0326] Three representative samples were taken from the washed wet
cake, and NHS levels were measured using NMR. The wet cake was
vacuum-dried.
[0327] The product ("API") was packaged into double bags sealed
under an inert atmosphere, and stored at -20.degree. C. without
exposure to light. Product yield was approximately 95/%.
Example 2
Characterization of "4-Arm-PEG-Gly-Irino-20K" Product as a Mixed
Salt
[0328] The product from Example 1 was analyzed by ion
chromatography (IC analysis). See Table 1 below for IC analytical
results for various product lots of 4-arm-PEG-Gly-Irino-20K.
TABLE-US-00002 TABLE 1 Mole Percent of Irinotecan bound to PEG TFA
HCl FREE LOT NO. SALT SALT BASE 010 59 36 5 (low) 020 64 (high) 30
6 030 27 (low) 24 4 9 (high) 040 53 26 21 050 54 26 20 060 57 28 15
070 53 33 14 080 53 27 20 090 44 19 36 100 33 41 26 Average of last
50 29 22 7 lots
[0329] Based upon the IC results provided in Table 1, it can be
seen that the product formed in Example 1, 4-arm-PEG-Gly-Irino-20K,
is a partial mixed salt of approximately 50 mole percent TFA salt,
30 mole percent HCl salt, and 20 mole percent free base, based upon
conjugated irinotecan molecules in the product. The mixture of
salts was observed even after repeated (1-3) recrystallizations of
the product. In the various product lots analyzed above, it can be
seen that about 35-65 mole percent of the irinotecan molecules in
the composition are protonated as the TFA salt, about 25-40 mole
percent of the irinotecan molecules in the composition are
protonated as the HCl salt, while the remaining 5-35 mole percent
of the irinotecan is non-protonated (i.e., as the free base).
[0330] The generalized structure of the product is shown below,
where the irinotecan moieties are shown in free base form, and in
association with HCl and TFA--as an indication of the mixed
salt.
##STR00031##
Example 3
Stress Stability Studies of 4-ARM-PEG-Gly-Irino-20K
[0331] Stability studies were conducted in an attempt to evaluate
the 4-arm-PEG-Gly-Irino-20K product composition. Compositions
containing varying amounts of protonated irinotecan, as well as
differing in the amount of TFA versus HCl salt were examined.
[0332] Stress Stability Studies
[0333] The product formed in Example 1, 4-arm-PEG-Gly-Irino-20K,
compound 5, (approximately 1-2 g) was weighed into PEG PE `whirl
top` bags and placed into another `whirl top` bag in order to
simulate the API packaging conditions. In one study (results shown
in FIG. 1), samples were placed in an environmental chamber at
25.degree. C./60% RH for 4 weeks. In another study, samples were
placed in an environmental chamber at 40.degree. C./75% RH for up
to several months (results shown in FIG. 2 and FIG. 3). Samples
were taken and analyzed on a periodic basis over the course of the
studies.
Results
[0334] The results of the studies are shown in FIG. 1, FIG. 2 and
FIG. 3. In FIG. 1, 4-arm-PEG-Gly-Irino-20K peak area percents for
samples stored at 25.degree. C. and 60% relative humidity are
plotted versus time. The data shown are for samples consisting of
>99% HCl salt (<1% free base, triangles), 94% total salt (6%
free base, squares), and 52% total salt (48% free base, circles).
The slopes of the graphs indicate that as free base content
increases, the stability of the product decreases. Under the stress
conditions employed (i.e., 25.degree. C. for up to 28 days), the
drop in 4-arm-PEG-Gly-Irino-20K peak area correlated well with the
increase in free irinotecan, indicating that the mode of
decomposition is primarily via hydrolysis of the ester bond to
release irinotecan. Based upon the results observed, it appears
that a greater amount of free base in the product leads to
decreased stability towards hydrolysis. Thus, product containing a
greater degree of protonated irinotecan appears to have a greater
stability against hydrolysis than product containing less
protonated irinotecan (based upon mole percent).
[0335] FIG. 2 and FIG. 3 show another set of data obtained from the
sample containing >99% HCl salt (<1% free base, squares) and
a sample consisting of 86% total salts (14% free base, diamonds)
that were stored at 40.degree. C. and 75% relative humidity. FIG. 2
shows the increase in free irinotecan over 3 months for both
samples. This data is consistent with the data from the previously
described study (summarized in FIG. 1), which shows that product
with a higher free base content is less stable with respect to
hydrolysis. FIG. 3 shows the increase in smaller PEG species for
the same samples over 3 months. The increase in smaller PEG species
is indicative of decomposition of the PEG backbone to provide
multiple PEG species. The data indicates that product corresponding
to the HCl salt is more prone to PEG backbone decomposition than
the mixed salt sample containing 14% free base. Thus, while not
intending to be bound by theory, it appears that that while the
partial mixed salt degrades primarily by hydrolytic release of
drug, the hydrochloride salt appears to degrade by a different
mechanism, i.e., degradation of the polymer backbone. Based upon
these preliminary results, the partial mixed salt product appears
to be preferred over the hydrochloride salt.
[0336] In summary, the two modes of decomposition observed exhibit
opposite trends with respect to salt/free base content.
Unexpectedly, these results suggest that there is a region of salt
composition that may possess an overall stability that is enhanced
over either of the traditional extremes of full salt and full free
base. The results further indicate the unforeseen advantages of a
partial mixed salt of 4-arm-PEG-Gly-Irino-20K over free base alone
or either salt in the absence of the other. The mixed salt was
shown to have greater stability than either the free base or
hydrochloride salt, thus indicating its superiority over either of
the more customary pure base or pure salt forms thereof.
Example 4
Chirality Study
[0337] The chirality of carbon-20 of irinotecan in
4-arm-PEG-Gly-Irino-20K was determined.
[0338] As detailed in documentation from the vendor, the irinotecan
hydrochloride starting material is optically active, with C-20 in
its (S)-configuration. The C-20 position in irinotecan bears a
tertiary alcohol, which is not readily ionizable, hence this site
is not expected to racemize except under extreme (strongly acidic)
conditions. To confirm the chirality at the C-20 in
4-arm-PEG-Gly-Irino-20K, a chiral HPLC method was used to analyze
irinotecan released from product via chemical hydrolysis.
[0339] Based upon the resulting chromatograms, no (R)-enantiomer
was detected for the 4-arm-PEG-Gly-Irino-20K samples. Following
hydrolysis, the irinotecan released from the conjugate was
confirmed to be the (S)-configuration.
Example 5
Hydrolysis Study
[0340] All PEGylated irinotecan species are considered as part of
4-arm-PEG-Gly-Irino-20K; each specie cleanly hydrolyzes to produce
irinotecan of >99% purity. Furthermore, the main, fully
drug-loaded DS4 species (drug covalently attached on each of the
four polymer arms) and the partially substituted species--DS3 (drug
covalently attached on three polymer arms). DS2 (drug covalently
attached on two of the polymer arms) and DS1 species (drug
covalently attached on a single polymer arm)--all hydrolyze at the
same rate to release free drug, irinotecan.
[0341] Experiments were performed to determine the fate of the
irinotecan-containing PEG species in 4-arm-PEG-Gly-Irino-20K under
transesterification (K.sub.2CO.sub.3 in CH.sub.3OH, 20.degree. C.)
and aqueous hydrolysis (pH 10, 20.degree. C.) conditions. The
transesterification reaction was >99% complete after 45 minutes.
The aqueous hydrolysis reaction was >99% complete within 24
hours. For both reaction types, control reactions using irinotecan
were performed under identical conditions and some artifact peaks
were observed. After adjustment for artifact peaks, in both cases,
the irinotecans produced had chromatographic purities of
>99%.
[0342] Based upon these results, it was concluded that essentially
all PEGylated species in 4-arm-PEG-Gly-Irino-20K release
irinotecan. Overlays of the HPLCs taken over time from the aqueous
hydrolysis reaction show the conversion of DS4 to DS3 to DS2 to DS1
to irinotecan. All of these species hydrolyze to release
irinotecan. See FIG. 4 demonstrating release of irinotecan via
hydrolysis from mono-, di-, tri- and tetra-substituted
4-arm-PEG-Gly-Irino-20K species.
[0343] Additional experiments were conducted to measure the rates
of hydrolysis for the major component of 4-arm-PEG-Gly-Irino-20K,
DS4, and its lesser substituted intermediates, DS3, DS2 and DS1 in
aqueous buffer (pH 8.4) in the presence of porcine carboxypeptidase
B and in human plasma. The hydrolysis in aqueous buffer (pH 8.4) in
the presence of porcine carboxypeptidase B was an attempt to
perform enzyme-based hydrolysis. The control experiment at pH 8.4
without the enzyme later showed that the hydrolysis was pH-driven,
and thus primarily a chemical hydrolysis. The data were,
nevertheless, valuable for comparison with the data obtained from
the hydrolysis performed in human plasma. These experiments showed
that the hydrolysis rates of the various components are not
significantly different and compare favorably with theoretical
predictions. Additional experiments measured the rates of
hydrolysis for the major components (DS4, DS3, DS2 and DS1) of
4-arm-PEG-Gly-Irino-20K in human plasma. These experiments also
show that the various components are hydrolyzed at the same rate
and compare favorably with theoretical predictions.
[0344] FIG. 5 and FIG. 6 present graphs which show the theoretical
hydrolysis rates versus experimental data for the chemical
hydrolysis (in the presence of enzyme) and plasma hydrolysis,
respectively. In both cases, the theoretical predictions are based
on identical rates for the hydrolysis of each species to produce
the next-lower homologue plus free irinotecan (i.e.,
DS4>DS3>DS2>DS1).
Example 6
Preparation 2: Preparation of
Pentaerythritolyl-4-Arm-(PEG-1-Methylene-2 Oxo-Vinylamino Acetate
Linked-Irinotecan)-20K "4-Arm-PEG-Gly-Irino-20K Mixed Acid Salt
Step 1. Synthesis of Boc-Glycine-irinotecan Hydrochloride (Gly-IRT
HCl)
Part 1: Drying of Irinotecan Hydrochloride Trihydrate
(IRT.HCl.3H.sub.2O)
[0345] IRT.HCl.3H.sub.2O (45.05 g, 66.52 mmol) was charged into a
reactor. Anhydrous N,N-dimethylformamide (DMF) (666 mL, 14.7 mL/g
of IRT.HCl.3H.sub.2O, DMF water content NMT 300 ppm) was charged to
the reactor. With slow agitation, the reactor was heated to
60.degree. C. (jacket temperature). After the irinotecan (IRT) was
fully dissolved (5-10 minutes), vacuum was slowly applied to reach
5-10 mbar and DMF was distilled off. When the volume of condensed
distillate (DMF) reached 85-90% of the initial DMF charge, the
vacuum was released. Heptane (1330 mL, 30.0 mL/g of
IRT.HCl.3H.sub.2O, water content NMT 50 ppm) was introduced into
the reactor and the jacket temperature was lowered to 50.degree. C.
Heptane was vacuum distilled (100-150 mbar) until the volume of the
distillate was about 90.degree. % of the initial charge of heptane.
Two more cycles of heptane distillation were carried out
(2.times.1330 mL heptane charge and distillation). A solvent phase
sample was taken from the reactor and was analyzed for DMF content
using GC to ensure a DMF content of less than 3% w/w. (In the event
the residual DMF was >3.0% w/w, a fourth azeotropic distillation
cycle would be performed). The resultant slurry was used for the
coupling reaction (Part 2).
Part 2: Coupling Reaction: Preparation of Boc-Gly-IRT.HCl
[0346] Dichloromethane (1330 mL, 29.5 mL DCM/g IRT.HCl.3H.sub.2O)
was charged into the reactor containing the slurry of dry IRT.HCl
(1.0 equiv) in residual heptanes (the approximate mass ratio of
residual heptanes to IRT.HCL was 3) which was being stirred. The
reaction contents were agitated for 15-30 minutes, and the batch
temperature was maintained at 17.degree. C. Boc-glycine (14.0 g,
79.91 mmol, 1.2 equiv) and DMAP (0.81 g, 6.63 mmol, 0.1 equiv) were
charged, as solids, into the reactor. A DCM solution of DCC (1.5
equiv in 40 mL of dichloromethane) was prepared and added into the
reactor over 15-30 min, and the resultant reaction mixture was
stirred at 17.degree. C. (batch temperature) for 2-3 hr. The
reaction was monitored by HPLC to ensure completion. A pre-made
quenching solution was charged into the reaction mixture to quench
any remaining DCC. Briefly, the pre-made quenching solution is a
pre-mixed solution of TFA and IPA in dichloromethane, prepared by
mixing TFA (1.53 mL, 0.034 mL/g IRT.HCl.3H.sub.2O) and IPA (3.05
mL, 0.068 mL/g IRT.HCl.3H.sub.2O) in DCM (15.3 mL, 0.34 mL/g
IRT.HCl.3H.sub.2O), and was added to the reactor V1 over 5-10
minutes when the conversion was at least 97%. The contents were
agitated for additional 30-60 min to allow quenching. The
DCU-containing reaction mixture was filtered through a 1 micron
filter into another reactor. The reaction filtrate was distilled to
1/3 its volume under vacuum at 35 C. Isopropyl alcohol (IPA) (490.5
mL, 10.9 mL/g IRT.HCl.3H.sub.2O) was added to the concentrated
mixture and the mixture was stirred for 30-60 min at 50.degree. C.
(jacket temperature). The resulting homogeneous solution was
concentrated by vacuum distillation to approximately 85% of the
initial IPA charge volume and the resultant concentrate was cooled
to 20.degree. C. (jacket temperature). The reaction mixture in IPA
was transferred over 60-80 min into heptane (1750 mL, 38.8 mL
heptane/g IRT.HCl.3H.sub.2O) at 20.degree. C. The resultant slurry
containing Boc-gly-IRT.HCl precipitate was stirred for an
additional 60-90 minutes and the product was collected by
filtration. The reaction flask was rinsed with heptane (2.times.490
mL, 20.0 mL Heptane/g IRT.HCl.3H.sub.2O) and the product cake was
washed with the rinse. The wet cake was dried at 20.degree. C. to
25.degree. C. under vacuum for a minimum of 12 hrs. Yield: 57.13 g
(110%, high due to residual solvents)
Step 2. Synthesis of Glycine-Irinotecan
Hydrochloride-Trifluoroacetate (Gly-IRT HCl-TFA) (Deprotection)
[0347] To an appropriately sized reactor was added dried
Boc-gly-IRT.HCl (41.32 g, 52.5 mmol, from step 1) under an inert
atmosphere. Anhydrous DCM (347 mL, 8.4 mL of DCM/g of
Boc-gly-IRT.HCl) was added to the reactor and the contents were
agitated at 17.degree. C. until complete dissolution (15-30 min
approximately). TFA (61.98 mL, 691.5 mmol, 1.5 mL/g of
Boc-gly-IRT.HCl) was added to the flask over 15-30 min and mixing
continued for 3.0 hours. The reaction was monitored for completion
by HPLC (limit: not less than 97%). The reaction was diluted with
acetonitrile (347 mL, 8.4 mL of ACN/g of Boc-gly-IRT.HCl). The
jacket temperature was set to 15.degree. C. and the reaction
mixture was concentrated under vacuum until the final residual pot
volume was approximately 85% of the initial acetonitrile charge
(295-305 mL approximately). The resulting acetonitrile solution was
added slowly to a reactor containing methyltert-butyl ether (MTBE,
1632 mL, 39.5 mL of MTBE/g of Boc-gly-IRT.HCl) over a period of
30-60 minutes. The precipitated product was gently mixed for 30
minutes and collected by filtration. The reactor was rinsed with
MTBE (410 mL) and the gly-IRT.HCl/TFA filter cake was washed with
the rinse. The product was dried under vacuum at 17.degree. C. for
a minimum of 12 hours. Yield: 42.1 g (102%).
Step 3. Synthesis of 4-armPEG20K-irinotecan
Hydrochloride-Trifuoroacetate
[0348] Gly-IRT HCl-TFA (10.0 g) was charged to a 250 mL reactor and
flushed with argon. The jacket temperature was set at 20.degree. C.
DCM (166 mL) and TEA (2.94 g) were added. The solution was mixed
for 10 minutes. An initial charge of 4-armPEG20K-SCM was added
(47.6 g) and the reaction mixture stirred for 30 minutes. A sample
was taken and analyzed by HPLC. The HPLC data showed 18% remaining
Gly-IRT. A second charge of 4-armPEG20K-SCM (10.7 g) was added to
the reaction mixture and the solution stirred for approximately 2
hours. A sample was withdrawn for HPLC analysis. The HPLC analysis
data showed 1.5% remaining Gly-IRT. The reaction solution was then
slowly added to MTBE (828 mL) to precipitate the product. The
precipitate was stirred for 30 minutes and collected via
filtration. The wet cake was washed with a mixture of 30%
Methanol/70% MTBE (830 mL). The product was then charged to a
reactor containing a mixture of 30% Methanol/70% MTBE (642 mL) and
the mixture was stirred at 20.degree. C. for 20 minutes. The
mixture was filtered and the wet cake was washed on the filter with
a mixture of 30% Methanol/70% MTBE (642 mL). The product was dried
under vacuum at 20.degree. C.
[0349] The dried product was charged to a reactor containing ethyl
acetate (642 mL). The mixture was heated to 35.degree. C. to
achieve complete dissolution. The warm solution was filtered if
necessary to remove undissolved particulates, and then cooled to
10.degree. C. with stirring. The precipitated
4-armPEG20K-glycine-irinotecan hydrochloride-trifluoroacetate
product was filtered and the wet cake was washed on the filter with
a mixture of 30% Methanol/70% MTBE (642 mL). The product was then
dried under vacuum at 20.degree. C. Yield: 54 g (approximately
85%).
[0350] Various lots prepared according to the process above were
analyzed by ion chromatography for salt composition.
TABLE-US-00003 TABLE 2 Mole Percent of Irinotecan bound to PEG LOT
TFA HCl FREE NO. SALT SALT BASE Lot 1 34 41 25 Lot 2 31 45 24 Lot 3
30 49 21 Lot 4 29 48 23
TABLE-US-00004 TABLE 3 Mean and Standard Deviations for Batches in
Table 2 MEAN SD 2SD 3SD 4SD TFA, mol % 31 2.4 4.8 7.2 9.6 Cl, mole
% 46 3.5 7.1 10.6 14.2 free base, mole % 23 1.0 1.9 2.9 3.9
[0351] As can be seen from the results in Table 2, batches prepared
as described show consistent ratios of TFA salt, hydrochloride salt
and free base. Based upon a review of the batch information, it
appears that a higher chloride content in the glycine-irinotecan
TFA/HCl intermediate leads to a higher the chloride content in the
final mixed salt conjugate product. By utilizing a starting
material such as irinotecan hydrochloride having a fairly constant
chloride content, a glycine-irinotecan TFA/HCl salt can be prepared
having a fairly constant chloride content.
[0352] Based upon a further review of batch information, it appears
that the higher the number of TEA equivalents utilized in step 3,
the lower the TFA and to a lesser extent, chloride, content in the
final mixed salt conjugate product. The measurement of chloride and
TFA content of the intermediate, i.e., gly-irinotecan TFA/HCl,
facilitated perhaps by greater dissolution of the intermediate
prior to analysis, by, for example, ion chromatography, may allow
for a more precise determination of stoichiometry, e.g., in the
amount of triethylamine added in the final reaction step.
[0353] Based upon the foregoing, preferred ranges of TFA in the
mixed acid salt conjugate are from about 20 to about 45 mole
percent, preferably from about 22 to 40 mole percent, or from about
24 to 38 mole percent. With respect to hydrochloride content,
preferred ranges in the mixed acid salt conjugate are from about 30
to 65 mole percent chloride, or from about 32 to 60 mole percent
chloride, or from about 35 to 57 mole percent chloride.
Example 7
Stress Stability Studies of 4-Arm-PEG-Gly-Irino-20K Materials
Having Differing Salt Ratios
[0354] Short term (4 week) stability studies were carried out on
4-arm-PEG20K-gly-irinotecan having various salt concentrations as
summarized in Table 4 below. "Pure" hydrochloride salt is shown in
the far left-hand column while the non-protonated, free base form
is shown in the far right column, with varying degradations
in-between. The studies were conducted essentially as described in
Example 3 over a range of temperatures (-20.degree. C. with no
humidity control, 5.degree. C. with no humidity control, 25.degree.
C. at 60% relative humidity, and 40.degree. C. at 75% relative
humidity.
TABLE-US-00005 TABLE 4 Sample Information REPRESEN- FREE SAMPLE HCl
INTERMED. TATIVE INTERMED. BASE LOT NO. Lot A Lot B Lot C Lot D Lot
E Cl 0.59% 0.43% 0.26% 0.11% NQ TFA NQ 0.25% 0.56% 0.07% NQ Cl (mol
%) 103.8% 75.6% 44.6% 18.9% NQ TFA mol % NQ 13.6% 30.6% 3.9% NQ
Total Salt 103.8% 89.2% 75.2% 22.8% 0% Mol %
[0355] For the HCl salt (Lot A), over the course of 4 weeks when
evaluated over the range of temperatures, total product related
species changed from 98.7% to 97.0% at 40.degree. C., while free
irinotecan changed from 0.4% to 1.25%. For the free base, (Lot D),
over the course of 4 weeks when evaluated over the range of
temperatures, the total product related species changed from 99.8%
to 62.5% at 40.degree. C., while free irinotecan changed from 0.3%
to 31.4%.
[0356] When evaluated under low temperature conditions, at
-20.degree. C. and 5.degree. C. over the course of 4 weeks, minimal
degradation was observed for each of the materials. When evaluated
at 25.degree. C., hydrolysis was observed in each of the species
tested with the free base material showing the most significant
hydrolytic release of drug. The same was observed at 40.degree. C.,
where the compositions having the greatest amount of free base
demonstrated a correspondingly faster rate of irinotecan
hydrolysis. Under the high temperature conditions, i.e., at
40.degree. C., cleavage of the PEG backbone was detected.
Example 8
Preparation of Pentaerythitol-Based 4-Arm-PEG-20K at 1.9 kg
Scale
[0357] Materials and Methods. A very high grade of ethylene oxide
having the lowest water content achievable should be used as water
content leads to polymeric diol impurities. CAUTION: Ethylene oxide
is a very reactive compound that can react explosively with
moisture, thus leaks in the reaction and transfer apparatus should
carefully avoided. Also, care should be taken in operations to
include having personnel work behind protective shields or in
bunkers.
[0358] Anhydrous toluene (4 L) was refluxed for two hours in a two
gallon jacketed stainless steel pressure reactor. Next, a part of
the solvent (3 L) was distilled off under atmospheric pressure. The
residual toluene was then discharged out and the reactor was dried
overnight by passing steam through the reactor jacket and applying
reduced pressure 3-5 mm Hg. Next the reactor was cooled to room
temperature, filled with anhydrous toluene (4 L) and pentaerythitol
based 4ARM-PEG-2K (SUNBRIGHT PTE.RTM.-2000 pentaerythritol,
molecular weight of about 2,000 Daltons, NOF Corporation; 200 g,
0.100 moles) was added. The solvent was distilled off under reduced
pressure, and then the reactor was cooled to 30.degree. C. under
dry nitrogen atmosphere. One liter of molecular sieves-dried
toluene (water content .about.5 ppm) and liquid sodium-potassium
alloy (Na 22%. K 78%; 1.2 g) were added to the reactor. The reactor
was warmed to 110.degree. C. and ethylene oxide (1,800 g) was
continuously added over three hours keeping the reaction
temperature at 110-120.degree. C. Next, the contents of the reactor
were heated for two hours at .about.100.degree. C., and then the
temperature was lowered to .about.70.degree. C. Excess ethylene
oxide and toluene were distilled off under reduced pressure. After
distillation, the contents of the reactor remained under reduced
pressure and a nitrogen sparge was performed to remove traces of
ethylene oxide. Phosphoric acid (IN) was added to neutralize the
basic residue and the product was dried under reduced pressure.
Finally the product was drained from the reactor and filtered
giving after cooling 1,900 g of white solid. Gel Filtration
Chromatography (GFC) was applied to characterize the alkoxylated
polymeric product, pentaerythitol based 4-ARM-PEG-20K. This
analytical method provided a chromatogram of the composition with
separation of the components according to molecular weight. An
Agilent 1100 HPLC system equipped with Shodex KW-803 GFC column
(300.times.8 mm) and differential refractometer detector was used.
The flow of the mobile phase (0. IM NaNO.sub.3) was 0.5 ml/min. The
GFC chromatogram is shown in FIG. 7.
[0359] GFC analysis showed that the 4ARM-PEG-20K product contained
the following: High MW product 0.42%, 4ARM-PEG-20K 99.14%,
HO-PEG(10K)-OH 0.44%.
Example 9
Analysis of Commercially Available 4ARM-PEG-20K
[0360] NOF Corporation is a current leader in providing commercial
PEGs. Thus a fresh commercially available pentaerythritol-based
4ARM-PEG-20K (SUNBRIGHT PTE.RTM.-20,000, molecular weight of about
20,000 Daltons, NOF Corporation) was obtained and analyzed using
Gel Filtration Chromatography (GFC). An Agilent 1100 HPLC system
equipped with Shodex KW-803 GFC column (300.times.8 mm) and
differential refractometer detector was used. The flow of the
mobile phase (0.1M NaNO.sub.3) was 0.5 ml/min. The GFC chromatogram
is shown in FIG. 8.
[0361] GFC analysis showed that this commercial 4ARM-PEG-20K
product contained: High MW products 3.93%, 4ARM-PEG-20K 88.56%,
HO-PEG(10K)-OH 3.93%, HO-PEG(5K)-OH 3.58%.
Example 10
Preparation of Alkoxylatable Oligomer: Pentaerythritol-Based
4-Arm-PEG-2K at 15 kg Scale
[0362] A twenty gallon jacketed stainless steel pressure reactor
was washed two times with 95 kg of deionized water at 95.degree. C.
The wash water was removed and the reactor was dried overnight by
passing steam through the reactor jacket and applying reduced
pressure (3-5 mm Hg). The reactor was filled with 25 kg of
anhydrous toluene and a part of the solvent was distilled off under
reduced pressure. The residual toluene was then discharged out and
the reactor was kept under reduced pressure. Next the reactor was
cooled to room temperature, filled with anhydrous toluene (15 L)
and pentaerythritol (1,020 g) was added. Part of the solvent
(.about.8 L) was distilled off under reduced pressure, and then the
reactor was cooled to 30.degree. C. under dry nitrogen atmosphere.
Liquid sodium-potassium alloy (Na 22%, K 78%; 2.2 g) was added to
the reactor. Anhydrous ethylene oxide (14,080 g) was continuously
added over three hours keeping the reaction temperature at
150-155.degree. C. Next, the contents of the reactor were heated
for 30 min at .about.150.degree. C., and then the temperature was
lowered to 70.degree. C. Excess ethylene oxide and toluene were
distilled off under reduced pressure. After distillation, the
contents of the reactor remained under reduced pressure and a
nitrogen sparge was performed to remove traces of ethylene oxide.
Finally the product was drained from the reactor giving 14,200 g of
viscous liquid. Gel Filtration Chromatography (GFC) was applied to
characterize the product, pentaerythritol based 4-ARM-PEG-2K. This
analytical method provided a chromatogram of the composition with
separation of the components according to molecular weight. An
Agilent 1100 HPLC system equipped with Shodex KW-803 GFC column
(300.times.8 mm) and differential refractometer detector was used.
The flow of the mobile phase (0.1 M NaNO.sub.3) was 0.5 ml/min.
[0363] GFC analysis showed that the 4ARM-PEG-2K product was
.about.100% pure with low or high molecular weight impurities below
detectable limits.
Example 11
Preparation of Pentaerythritol-Based 4-Arm-PEG-20K at 20 kg
Scale
[0364] A twenty gallon jacketed stainless steel pressure reactor
was washed two times with 95 kg of deionized water at 95.degree. C.
Water was discharged out and the reactor was dried overnight by
passing steam through the reactor jacket and applying reduced
pressure 3-5 mm Hg. The reactor was filled with 25 kg of toluene
and a part of the solvent was distilled off under reduced pressure.
The residual toluene was then discharged out and the reactor was
kept under reduced pressure. Next the reactor was cooled to room
temperature, filled with anhydrous toluene (21 L) and previously
isolated alkoxylatable oligomer: pentaerythritol based 4ARM-PEG-2K
from the Example 10 (2,064 g) was added. Part of the solvent (16 L)
was distilled off under reduced pressure, and then the reactor was
cooled to 30.degree. C. under dry nitrogen atmosphere. Four liter
of molecular sieves-dried toluene (water content .about.5 ppm) and
liquid sodium-potassium alloy (Na 22%, K 78%; 1.7 g) were added,
and the reactor was warmed to 110.degree. C. Next ethylene oxide
(19,300 g) was continuously added over five hours keeping the
reaction temperature at 145-150.degree. C. Next, the contents of
the reactor were heated for 30 min at .about.140.degree. C., and
then the temperature was lowered to .about.100.degree. C. Glacial
acidic acid (100 g) was added to neutralize the catalyst. Excess
ethylene oxide and toluene were distilled off under reduced
pressure. After distillation, the contents of the reactor remained
under reduced pressure and a nitrogen sparge was performed to
remove traces of ethylene oxide. Finally the product was drained
from the reactor giving 20.100 g of white solid. Gel Filtration
Chromatography (GFC) was applied to characterize the alkoxylated
polymer product, pentaerythritol based 4-ARM-PEG-20K. This
analytical method provided a chromatogram of the composition with
separation of the components according to molecular weight. An
Agilent 1100 HPLC system equipped with Shodex KW-803 GFC column
(300.times.8 mm) and differential refractometer detector was used.
The flow of the mobile phase (0. IM NaNO.sub.3) was 0.5 ml/min.
[0365] GFC analysis showed that the 4ARM-PEG-20K product contained
the following: High MW product 0.75%, 4ARM-PEG-20K 97.92%,
HO-PEG(10K)-OH 1.08%, HO-PEG(5K)-OH 0.48%.
[0366] The invention(s) set forth herein has been described with
respect to particular exemplified embodiments. However, the
foregoing description is not intended to limit the invention to the
exemplified embodiments, and the skilled artisan should recognize
that variations can be made within the spirit and scope of the
invention as described in the foregoing specification.
* * * * *