U.S. patent application number 12/789582 was filed with the patent office on 2010-09-23 for pharmaceutical compositions for the administration of aptamers.
This patent application is currently assigned to IDEXX Laboratories, Inc.. Invention is credited to Yerramilli V.S.N. Murthy.
Application Number | 20100240741 12/789582 |
Document ID | / |
Family ID | 38041719 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240741 |
Kind Code |
A1 |
Murthy; Yerramilli V.S.N. |
September 23, 2010 |
PHARMACEUTICAL COMPOSITIONS FOR THE ADMINISTRATION OF APTAMERS
Abstract
Pharmaceutical compositions comprising an aptamer and an amino
acid ester or amide or an aptamer; a divalent metal cation; and a
carboxylic acid, a phospholipid, a phosphatidyl choline, or a
sphingomyelin. Methods of treating or preventing a condition in an
animal comprising administering to the animal the pharmaceutical
compositions.
Inventors: |
Murthy; Yerramilli V.S.N.;
(Falmouth, ME) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
IDEXX Laboratories, Inc.
Westbrook
ME
|
Family ID: |
38041719 |
Appl. No.: |
12/789582 |
Filed: |
May 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11400372 |
Apr 10, 2006 |
7754679 |
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12789582 |
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60736862 |
Nov 16, 2005 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61K 31/16 20130101;
A61K 31/16 20130101; A61K 31/685 20130101; A61K 31/4172 20130101;
A61K 31/685 20130101; A61K 31/22 20130101; A61K 31/22 20130101;
A61K 45/06 20130101; A61K 31/405 20130101; A61K 2300/00 20130101;
A61K 31/4172 20130101; A61K 2300/00 20130101; A61K 31/405 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
514/44.R |
International
Class: |
A61K 31/7088 20060101
A61K031/7088 |
Claims
1-84. (canceled)
85. A pharmaceutical composition comprising: (i) an amino acid
ester of formula: ##STR00033## wherein R is the amino acid side
chain; and R.sub.1 is a C.sub.1 to C.sub.22 hydrocarbon group; or
an amino acid amide of formula: ##STR00034## wherein R is the amino
acid side chain; R.sub.3 is a C.sub.1 to C.sub.22 hydrocarbon
group; and R.sub.4 is hydrogen or a C.sub.1 to C.sub.22 hydrocarbon
group; (ii) a protonated aptamer, optionally conjugated to a
polymer; and (iii) a pharmaceutically acceptable organic solvent,
wherein the protonated aptamer is dissolved in the pharmaceutically
acceptable organic solvent to provide a concentration of the
aptamer in the pharmaceutically acceptable organic solvent of at
least 2 percent by weight of the pharmaceutical composition or
about 2 percent by weight of the pharmaceutical composition, and
wherein the composition is suitable for administration to an animal
by injection.
86. The pharmaceutical composition of claim 85, wherein the
pharmaceutical composition forms a precipitate when injected into
water.
87. The pharmaceutical composition of claim 85, wherein the
pharmaceutical composition forms a depot when administered to an
animal.
88. The pharmaceutical composition of claim 85, wherein the
pharmaceutical composition does not form a precipitate when
injected into water.
89. The pharmaceutical composition of claim 85, further comprising
water.
90. The pharmaceutical composition of claim 85, wherein there is
more than 1 equivalent of basic groups on the amino acid ester or
amino acid amide per equivalent of acidic groups on the protonated
aptamer.
91. The pharmaceutical composition of claim 90, further comprising
at least one of a carboxylic acid, a phospholipid, a sphingomyelin,
or a phosphatidyl choline.
92. The pharmaceutical composition of claim 91, wherein the at
least one of a carboxylic acid, a phospholipid, a sphingomyelin, or
a phosphatidyl choline is a carboxylic acid.
93. The pharmaceutical composition of claim 92, wherein the
pharmaceutical composition forms a precipitate when injected into
water.
94. The pharmaceutical composition of claim 92, wherein the
pharmaceutical composition forms a depot when administered to an
animal.
95. The pharmaceutical composition of claim 92, wherein the
pharmaceutical composition does not form a precipitate when
injected into water.
96. The pharmaceutical composition of claim 92, wherein the
carboxylic acid is a fatty acid.
97. The pharmaceutical composition of claim 92, wherein the
carboxylic acid is a polycarboxylic acid.
98. The pharmaceutical composition of claim 85, wherein the amino
acid ester or amide is an ester or amide of lysine.
99. The pharmaceutical composition of claim 98, wherein the
pharmaceutical composition forms a precipitate when injected into
water.
100. The pharmaceutical composition of claim 98, wherein the
pharmaceutical composition forms a depot when administered to an
animal.
101. The pharmaceutical composition of claim 98, wherein the
pharmaceutical composition does not form a precipitate when
injected into water.
102. The pharmaceutical composition of claim 98, further comprising
water.
103. The pharmaceutical composition of claim 98, wherein there is
more than 1 equivalent of basic groups on the amino acid ester or
amino acid amide per equivalent of acidic groups on the protonated
aptamer.
104. The pharmaceutical composition of claim 103, further
comprising at least one of a carboxylic acid, a phospholipid, a
sphingomyelin, or a phosphatidyl choline.
105. The pharmaceutical composition of claim 104, wherein the at
least one of a carboxylic acid, a phospholipid, a sphingomyelin, or
a phosphatidyl choline is a carboxylic acid.
106. The pharmaceutical composition of claim 105, wherein the
pharmaceutical composition forms a precipitate when injected into
water.
107. The pharmaceutical composition of claim 105, wherein the
pharmaceutical composition forms a depot when administered to an
animal.
108. The pharmaceutical composition of claim 105, wherein the
pharmaceutical composition does not form a precipitate when
injected into water.
109. The pharmaceutical composition of claim 105, wherein the
carboxylic acid is a fatty acid.
110. The pharmaceutical composition of claim 105, wherein the
carboxylic acid is a polycarboxylic acid.
111. The pharmaceutical composition of claim 85, wherein the amino
acid ester or amide is an ester or amide of aspartic acid or
glutamic acid.
112. The pharmaceutical composition of claim 111, wherein the
pharmaceutical composition forms a precipitate when injected into
water.
113. The pharmaceutical composition of claim 111, wherein the
pharmaceutical composition forms a depot when administered to an
animal.
114. The pharmaceutical composition of claim 111, wherein the
pharmaceutical composition does not form a precipitate when
infected into water.
115. The pharmaceutical composition of claim 111, further
comprising water.
116. The pharmaceutical composition of claim 111, wherein there is
more than 1 equivalent of basic groups on the amino acid ester or
amino acid amide per equivalent of acidic groups on the protonated
aptamer.
117. The pharmaceutical composition of claim 116, further
comprising at least one of a carboxylic acid, a phospholipid, a
sphingomyelin, or a phosphatidyl choline.
118. The pharmaceutical composition of claim 117, wherein the at
least one of a carboxylic acid, a phospholipid, a sphingomyelin, or
a phosphatidyl choline is a carboxylic acid.
119. The pharmaceutical composition of claim 118, wherein the
pharmaceutical composition forms a precipitate when injected into
water.
120. The pharmaceutical composition of claim 118, wherein the
pharmaceutical composition forms a depot when administered to an
animal.
121. The pharmaceutical composition of claim 118, wherein the
pharmaceutical composition does not form a precipitate when
injected into water.
122. The pharmaceutical composition of claim 118, wherein the
carboxylic acid is a fatty acid.
123. The pharmaceutical composition of claim 118, wherein the
carboxylic acid is a polycarboxylic acid.
124. The pharmaceutical composition of claim 85, wherein the
pharmaceutically acceptable organic solvent is selected from the
group consisting of N-methyl-2-pyrrolidone, polyethylene glycol,
propylene glycol, glycerol formal, isosorbide dimethyl ether,
ethanol, dimethyl sulfoxide, tetraglycol, tetrahydrofurfuryl
alcohol, triacetin, propylene carbonate, dimethyl acetamide,
dimethyl formamide, dimethyl sulfoxide, and combinations
thereof.
125. The pharmaceutical composition of claim 98, wherein the
pharmaceutically acceptable organic solvent is selected from the
group consisting of N-methyl-2-pyrrolidone, polyethylene glycol,
propylene glycol, glycerol formal, isosorbide dimethyl ether,
ethanol, dimethyl sulfoxide, tetraglycol, tetrahydrofurfuryl
alcohol, triacetin, propylene carbonate, dimethyl acetamide,
dimethyl formamide, dimethyl sulfoxide, and combinations
thereof.
126. The pharmaceutical composition of claim 111, wherein the
pharmaceutically acceptable organic solvent is selected from the
group consisting of N-methyl-2-pyrrolidone, polyethylene glycol,
propylene glycol, glycerol formal, isosorbide dimethyl ether,
ethanol, dimethyl sulfoxide, tetraglycol, tetrahydrofurfuryl
alcohol, triacetin, propylene carbonate, dimethyl acetamide,
dimethyl formamide, dimethyl sulfoxide, and combinations
thereof.
127. The composition of claim 86, wherein the precipitate releases
the aptamer over time.
128. The composition of claim 85, wherein the aptamer is not
conjugated to a polymer.
129. The composition of claim 85, wherein the aptamer is conjugated
to a polymer.
130. A pharmaceutical composition comprising a complex between: (i)
an amino acid ester of formula: ##STR00035## wherein R is the amino
acid side chain; and R.sub.1 is a C.sub.1 to C.sub.22 hydrocarbon
group; or an amino acid amide of formula: ##STR00036## wherein R is
the amino acid side chain; R.sub.3 is a C.sub.1 to C.sub.n
hydrocarbon group; and R.sub.4 is hydrogen or a C.sub.i to C.sub.n
hydrocarbon group; and (ii) a protonated aptamer, optionally
conjugated to a polymer; and (iii) a pharmaceutically acceptable
organic solvent, wherein the composition forms micelle or a
liposome structures when injected into water.
131. The pharmaceutical composition of claim 130, further
comprising water.
132. The pharmaceutical composition of claim 130, wherein the
aptamer is present in an amount of at least 2 percent by weight of
the pharmaceutical composition or about 2 percent by weight of the
pharmaceutical composition.
133. The pharmaceutical composition of claim 130, wherein the
composition is suitable for administration to an animal by
injection.
134. The composition of claim 130, wherein the aptamer is not
conjugated to a polymer.
135. The composition of claim 130, wherein the aptamer is
conjugated to a polymer.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. provisional
application No. 60/736,862, filed Nov. 16, 2005, the contents of
which are incorporated herein by reference thereto.
2. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
[0002] Not Applicable.
3. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT
DISC
[0003] Not Applicable.
4. FIELD OF THE INVENTION
[0004] The invention relates to pharmaceutical compositions for
administering an aptamer to an animal in need thereof. In one
embodiment, the pharmaceutical compositions comprise (i) an aptamer
and (ii) an amino acid ester or amide. In another embodiment, the
pharmaceutical compositions comprise (i) an aptamer; (ii) a
divalent metal cation; and (iii) optionally a carboxylic acid, a
phospholipid, a phosphatidyl choline, or a sphingomyelin.
5. BACKGROUND OF THE INVENTION
[0005] Aptamers, are oligonucleotides, which can be synthetic or
natural, that bind to a particular target molecule, such as a
protein or metabolite. Typically, the binding is through
interactions other than classic Watson-Crick base pairing.
[0006] Aptamers represent a promising class of therapeutic agents
currently in pre-clinical and clinical development. Like biologics,
e.g., peptides or monoclonal antibodies, aptamers are capable of
binding specifically to molecular targets and, through binding,
inhibiting target function. A typical aptamer is 10-15 kDa in size
(i.e., 30-45 nucleotides), binds its target with sub-nanomolar
affinity, and discriminates among closely related targets (e.g.,
will typically not bind other proteins from the same gene family)
(Griffin, et al. (1993), Gene 137(1): 25-31; Jenison, et al.
(1998), Antisense Nucleic Acid Drug Dev. 8(4): 265-79; Bell, et al.
(1999), In Vitro Cell. Dev. Biol. Anim. 35(9): 533-42; Watson, et
al. (2000), Antisense Nucleic Acid Drug Dev. 10(2): 63-75; Daniels,
et al. (2002), Anal. Biochem. 305(2): 214-26; Chen, et al. (2003),
Proc. Natl. Acad. Sci. U.S.A. 100(16): 9226-31; Khati, et al.
(2003), J. Virol. 77(23): 12692-8; Vaish, et al. (2003),
Biochemistry 42(29): 8842-51).
[0007] Aptamers can be created by an entirely in vitro selection
process (Systematic Evaluation of Ligands by Experimental
Enrichment, i.e., SELEX.TM.) from libraries of random sequence
oligonucleotides as described in U.S. Pat. Nos. 5,475,096 and
5,270,163. Aptamers have been generated against numerous proteins
of therapeutic interest, including growth factors, enzymes,
immunoglobulins, and receptors (Ellington and Szostak (1990),
Nature 346(6287): 818-22; Tuerk and Gold (1990), Science 249(4968):
505-510).
[0008] Aptamers have a number of attractive characteristics for use
as therapeutics. In addition to high target affinity and
specificity, aptamers have shown little or no toxicity or
immunogenicity in standard assays (Wlotzka, et al. (2002), Proc.
Natl. Acad. Sci. U.S.A. 99(13): 8898-902). Indeed, several
therapeutic aptamers have been optimized and advanced through
varying stages of pre-clinical development, including
pharmacokinetic analysis, characterization of biological efficacy
in cellular and animal disease models, and preliminary safety
pharmacology assessment (Reyderman and Stavchansky (1998),
Pharmaceutical Research 15(6): 904-10; Tucker et al., (1999), J.
Chromatography B. 732: 203-212; Watson, et al. (2000), Antisense
Nucleic Acid Drug Dev. 10(2): 63-75).
[0009] It is important that the pharmacokinetic properties for all
oligonucleotide-based therapeutics, including aptamers, be tailored
to match the desired pharmaceutical application. While aptamers
directed against extracellular targets do not suffer from
difficulties associated with intracellular delivery (as is the case
with antisense and RNAi-based therapeutics), the aptamer must be
distributed to target organs and tissues, and remain in the body
(unmodified) for a period of time consistent with the desired
dosing regimen. Early work on nucleic acid-based therapeutics has
shown that, while unmodified oligonucleotides are degraded rapidly
by nuclease digestion, protective modifications at the 2'-position
of the sugar, and use of inverted terminal cap structures, e.g.,
[3'-3' dT], dramatically improve nucleic acid stability in vitro
and in vivo (Green, et al. (1995), Chem. Biol. 2(10): 683-95;
Jellinek, et al. (1995), Biochemistry 34(36): 11363-72; Rudman, et
al. (1998), J. Biol. Chem. 273(32): 20556-67; Uhlmann, et al.
(2000), Methods Enzymol. 313: 268-84). In some SELEX selections
(i.e., SELEX experiments or SELEX ions), starting pools of nucleic
acids from which aptamers are selected are typically pre-stabilized
by chemical modification, for example by incorporation of
2'-fluoropyrimidine (2'-F) substituted nucleotides, to enhance
resistance of aptamers against nuclease attack. Aptamers
incorporating 2'-O-methylpurine (2'-OMe purine) substituted
nucleotides have also been developed through post-SELEX
modification steps or, more recently, by enabling synthesis of
2'-OMe-containing random sequence libraries as an integral
component of the SELEX process itself.
[0010] In addition to clearance by nucleases, oligonucleotide
therapeutics are subject to elimination via renal filtration. As
such, a nuclease-resistant oligonucleotide administered
intravenously exhibits an in vivo half-life of <10 min, unless
filtration can be blocked. This can be accomplished by either
facilitating rapid distribution out of the blood stream into
tissues or by increasing the apparent molecular weight of the
oligonucleotide above the effective size cut-off for the
glomerulus. Conjugation to a PEG polymer ("PEGylation") can
dramatically lengthen residence times of aptamers in circulation,
thereby decreasing dosing frequency and enhancing effectiveness
against targets. Previous work in animals has examined the plasma
pharmacokinetic properties of PEG-conjugated aptamers (Reyderman
and Stavchansky (1998), Pharmaceutical Research 15(6): 904-10;
Watson, et al. (2000), Antisense Nucleic Acid Drug Dev. 10(2):
63-75)). Determining the extravasation of an aptamer therapeutic,
including aptamer therapeutics conjugated to a modifying moiety or
containing modified nucleotides and, in particular, determining the
potential of aptamers or their modified forms to access diseased
tissues (for example, sites of inflammation, or the interior of
tumors) define the spectrum of therapeutic opportunities for
aptamer intervention.
[0011] Typically, therapeutic aptamers are administered by
injection, for example, by subcutaneous injection. Accordingly, the
aptamer must be dissolved in a liquid vehicle for administration.
The relatively high molecular weight of aptamers, and in particular
aptamers that have been derivatized, for example by PEGylation,
however, often makes it difficult to obtain a pharmaceutical
composition wherein the aptamer is dissolved in a pharmaceutically
acceptable solvent at a sufficient concentration to provide a
pharmaceutical composition that is clinically useful for
administration to an animal.
[0012] U.S. published application no. 2005/0175708 discloses a
composition of matter that permits the sustained delivery of
aptamers to a mammal. The aptamers are administered as microspheres
that permit sustained release of the aptamers to the site of
interest so that the aptamers can exert their biological activity
over a prolonged period of time. The aptamers, can be anti-VEGF
aptamers.
[0013] P. Burmeister et al., (2004), Chemistry and Biology: 15,
25-33 disclose a method for generating a 2'-O-methyl aptamer
(ARC245) that binds to vascular endothelial growth factor, which
exhibits good stability.
[0014] Accordingly, there is a need in the art for improved
pharmaceutical compositions, wherein the therapeutic agent is an
aptamer. In particular, there is a need for pharmaceutical
composition wherein the aptamer can be dissolved in a
pharmaceutically acceptable solvent at a sufficient concentration
to provide a pharmaceutical composition that is clinically useful
for administration to an animal. The present invention addresses
this as well as other needs.
[0015] Citation of any reference in this application is not to be
construed as an admission that such reference is prior art to the
present application.
6. SUMMARY OF THE INVENTION
[0016] The invention is directed to a pharmaceutical composition
comprising:
[0017] (i) a salt formed between a protonated aptamer and a
pharmaceutically acceptable organic base; and
[0018] (ii) a pharmaceutically acceptable organic solvent.
[0019] In one embodiment, the solvent is a pharmaceutically
acceptable organic solvent. In one embodiment, the pharmaceutical
composition is a solution of the salt in the pharmaceutically
acceptable organic solvent.
[0020] The invention also relates to a pharmaceutical composition
comprising: [0021] (i) an amino acid ester of formula:
##STR00001##
[0021] wherein [0022] R is the amino acid side chain; and [0023]
R.sub.1 is a C.sub.1 to C.sub.22 hydrocarbon group; or an amino
acid amide of general formula:
##STR00002##
[0023] wherein [0024] R is the amino acid side chain; [0025]
R.sub.3 is a C.sub.1 to C.sub.22 hydrocarbon group; and [0026]
R.sub.4 is hydrogen or a C.sub.1 to C.sub.22 hydrocarbon group; and
[0027] (ii) a protonated aptamer.
[0028] In one embodiment, the amino acid ester or amide is an ester
or an amide of lysine and the pharmaceutical composition further
comprises one or more of a carboxylic acid, a phospholipid,
phosphatidyl choline, or a sphingomyelin.
[0029] In one embodiment, the amino acid ester or amide is a
diester or diamide of aspartic or glutamic acid.
[0030] In one embodiment, the pharmaceutical compositions further
comprises a solvent. In one embodiment, the solvent is a
pharmaceutically acceptable organic solvent.
[0031] The invention also relates to a pharmaceutical compositions
comprising [0032] (i) an aptamer; and [0033] (ii) a divalent metal
cation; and [0034] (iii) a pharmaceutically acceptable organic
solvent.
[0035] In one embodiment, the pharmaceutical composition further
comprises a carboxylate, a phospholipid, a phosphatidyl choline, or
a sphingomyelin.
[0036] The invention also relates to methods of administering an
aptamer to an animal comprising administering to the animal a
pharmaceutical composition of the invention.
[0037] The invention also relates to methods of treating or
preventing a condition in an animal comprising administering to the
animal a pharmaceutical composition of the invention.
7. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1. is a graphical representation of the viscosity of
pharmaceutical compositions of the invention containing an aptamer
at a concentration of 10% (w/v) and 1 equivalent of isoleucine
ethanoate, isoleucine butanoate, isoleucine hexanoate, isoleucine
octanoate, isoleucine decanoate, isoleucine dodecanoate, or
isoleucine hexadecanoate per equivalent of acidic groups on the
aptamer dissolved in N-methyl-2-pyrrolidone.
[0039] FIG. 2 is a graphical representation of the viscosity of
pharmaceutical compositions of the invention containing an aptamer
at a concentration of 10% (w/v) and 1, 2, or 6 equivalents of the
ester formed between isoleucine decanoate and a per equivalent of
acidic functional groups on the aptamer dissolved in
N-methyl-2-pyrrolidone.
[0040] FIG. 3 is an HPLC chromatogram, obtained using the HPLC
parameters described in Example 11, of the supernatant (lower
trace) and the pellet (upper trace) formed when 50 .mu.L of the
pharmaceutical composition of Example 7B containing 10 equivalents
of lysine hexadecanoate is injected into 4 mL of water to provide a
precipitate and the resulting precipitate and supernatant are
separated by centrifugation as described in Example 8.
[0041] FIG. 4 is an HPLC chromatogram, obtained using the HPLC
parameters described in Example 11, of the pharmaceutical
composition of Example 7B containing 10 equivalents of lysine
hexadecanoate using the basic mobile phase (Trace A) and the acidic
mobile phase (Trace B). Trace C is the HPLC chromatogram of the
aptamer dissolved in methanol.
8. DETAILED DESCRIPTION OF THE INVENTION
[0042] The invention is directed to a pharmaceutical composition
comprising:
[0043] (i) a salt formed between a protonated aptamer and a
pharmaceutically acceptable organic base; and
[0044] (iii) a pharmaceutically acceptable organic solvent.
[0045] In one embodiment, the solvent is a pharmaceutically
acceptable organic solvent. In one embodiment, the pharmaceutical
composition is a solution of the salt in the pharmaceutically
acceptable organic solvent.
[0046] In one embodiment, the pharmaceutical compositions
comprises:
[0047] (i) an amino acid ester or an amino acid amide and
[0048] (ii) a protonated aptamer.
[0049] In one embodiment, the pharmaceutical composition further
comprises a solvent. In one embodiment, the solvent is a
pharmaceutically acceptable organic solvent.
[0050] In one embodiment, the pharmaceutical composition comprises:
[0051] (i) an ester or an amide of lysine; [0052] (ii) a protonated
aptamer; and [0053] (iii) a carboxylic acid.
[0054] In one embodiment, the pharmaceutical composition further
comprises a solvent. In one embodiment, the solvent is a
pharmaceutically acceptable organic solvent.
[0055] In one embodiment, the pharmaceutical composition comprises:
[0056] (i) an ester or an amide of lysine; [0057] (ii) a protonated
aptamer; and [0058] (iii) a phospholipid, phosphatidyl choline, or
a sphingomyelin.
[0059] In one embodiment, the pharmaceutical composition further
comprises a solvent. In one embodiment, the solvent is a
pharmaceutically acceptable organic solvent.
[0060] In one embodiment, the pharmaceutical composition comprises:
[0061] (i) a diester or diamide of aspartic or glutamic acid; and
[0062] (ii) a protonated aptamer.
[0063] In one embodiment, the pharmaceutical composition further
comprises a solvent. In one embodiment, the solvent is a
pharmaceutically acceptable organic solvent.
[0064] In another embodiment, the pharmaceutical compositions
comprises [0065] (i) an aptamer; and [0066] (ii) a divalent metal
cation; and [0067] (iii) a pharmaceutically acceptable organic
solvent.
[0068] In one embodiment, the pharmaceutical composition further
comprises a carboxylate, a phospholipid, a phosphatidyl choline, or
a sphingomyelin.
[0069] The invention also relates to methods of treating or
preventing a condition in an animal comprising administering to the
animal a pharmaceutical composition of the invention.
8.1 DEFINITIONS
[0070] As used herein, the following terms have the following
meaning:
[0071] The term "aptamer," as used herein, means an
oligonucleotide, which can be synthetic or natural, which can bind
to a particular target molecule, such as a protein or metabolite,
other than by Watson-Crick base pairing and have a pharmacological
effect in an animal. Aptamers can be synthesized using conventional
phosphodiester linked nucleotides and synthesized using standard
solid or solution phase synthesis techniques which are known to
those skilled in the art (See, for example, U.S. Pat. Nos.
5,475,096 and 5,270,163). The binding of aptamers to a target
polypeptide can be readily tested by assays known to those skilled
in the art. The term "protonated aptamer," as used herein, means an
aptamer wherein at least one of the phosphate groups of the aptamer
is protonated. In one embodiment, all of the phosphate groups of
the aptamer are protonated.
[0072] Typically, the pharmacological effect is treating or
preventing a condition in an animal.
[0073] The term "condition," as used herein means an interruption,
cessation, or disorder of a bodily function, system, or organ.
Representative conditions include, but are not limited to, diseases
such as cancer, inflammation, diabetes, and organ failure.
[0074] The phrase "treating," "treatment of," and the like includes
the amelioration or cessation of a specified condition.
[0075] The phrase "preventing," "prevention of," and the like
include the avoidance of the onset of a condition.
[0076] "C.sub.1-C.sub.22 hydrocarbon group" means a straight or
branched, saturated or unsaturated, cyclic or non-cyclic, aromatic
or non-aromatic, carbocyclic or heterocyclic group having from 1 to
22 carbon atoms. Similarly, phrases such as "C.sub.1-C.sub.22
hydrocarbon group," "C.sub.1-C.sub.16 hydrocarbon group,"
"C.sub.1-C.sub.10 hydrocarbon group," "C.sub.15 hydrocarbon group,"
"C.sub.1-C.sub.3 hydrocarbon group," "C.sub.16-C.sub.22 hydrocarbon
group," "C.sub.8-C.sub.18 hydrocarbon group," "C.sub.10-C.sub.18
hydrocarbon group," and "C.sub.16-C.sub.18 hydrocarbon group" means
a straight or branched, saturated or unsaturated, cyclic or
non-cyclic, aromatic or non-aromatic, carbocyclic or heterocyclic
group having from 1 to 21 carbon atoms, from 1 to 16 carbon atoms,
from 1 to 10 carbon atoms, from 1 to 5 carbon atoms, 1 to 3 carbon
atoms, 16 to 22 carbon atoms, 8 to 18 carbon atoms, 10 to 18 carbon
atoms, and 16 to 18 carbon atoms, respectively. Accordingly, the
phrase "an acyl group of formula --C(O)--R.sub.1, wherein R.sub.1
is a C.sub.1 to C.sub.21 group means an acyl group of formula
--C(O)--R.sub.1, wherein R.sub.1 is a straight or branched,
saturated or unsaturated, cyclic or non-cyclic, aromatic or
non-aromatic, carbocyclic or heterocyclic hydrocarbon group having
from 1 to 21 carbon atoms. Representative acyl groups of formula
--C(O)--R.sub.1, wherein R.sub.1 is an unsubstituted C.sub.1 to
C.sub.21 group include, but are not limited to, acetyl, propionyl,
butanoyl, hexanoyl, caproyl, laurolyl, myristoyl, palmitoyl,
stearoyl, palmioleoyl, oleoyl, linoleoyl, linolenoyl, and
benzoyl.
[0077] The term "lower alkyl," as used herein means a
C.sub.1-C.sub.6 hydrocarbon group.
[0078] The term "salt," as used herein, means two compounds that
are not covalently bound but are chemically bound by ionic
interactions.
[0079] The term "pharmaceutically acceptable," as used herein, when
referring to a component of a pharmaceutical composition means that
the component, when administered to an animal, does not have undue
adverse effects such as excessive toxicity, irritation, or allergic
response commensurate with a reasonable benefit/risk ratio.
Accordingly, the term "pharmaceutically acceptable organic
solvent," as used herein, means an organic solvent that when
administered to an animal does not have undue adverse effects such
as excessive toxicity, irritation, or allergic response
commensurate with a reasonable benefit/risk ratio. Preferably, the
pharmaceutically acceptable organic solvent is a solvent that is
generally recognized as safe ("GRAS") by the United States Food and
Drug Administration ("FDA"). Similarly, the term "pharmaceutically
acceptable organic base," as used herein, means an organic base
that when administered to an animal does not have undue adverse
effects such as excessive toxicity, irritation, or allergic
response commensurate with a reasonable benefit/risk ratio.
[0080] The term "water miscible organic solvent," as used herein,
means an organic solvent that is capable of mixing with water in
any ratio without separating into two phases.
[0081] The term "water soluble organic solvent," as used herein,
means an organic solvent that has a significant level of solubility
in water. Typically, a water soluble organic solvent is soluble in
water in an amount of at least about 5 percent by weight,
preferably at least about 10 percent by weight, more preferably at
least about 20 percent by weight, and most preferably at least
about 50 percent by weight. For example, triacetin is considered a
water soluble solvent since it is soluble in water at a ratio of
about 1:14.
[0082] The phrase "forms a precipitate," as used herein, means that
the pharmaceutical composition forms a precipitate, or solid, when
injected into water or into a physiological (in vivo) environment.
A precipitate is an insoluble solid formed in a solvent at room
temperature in vitro or in a physiological (in vivo) environment.
The precipitate can take many forms such as, for example, a solid,
a crystal, a gummy mass, or a gel. Preferably, the precipitate is a
gummy mass or a gel. A composition of the invention forms a
precipitate in water when at least 10% of the composition is
retained on a 0.22 .mu.m filter when the composition is mixed with
water and filtered at 98.degree. F. Typically, to form the
precipitate, about 50 .mu.L to 0.5 mL of the pharmaceutical
composition is injected into about 4-5 mL of water. In one
embodiment, about 50 .mu.L of the pharmaceutical composition is
injected into about 4 mL of water.
[0083] The term "fatty acid," as used herein means a carboxylic
acid of formula R--C(O)OH, wherein R a is C.sub.6-C.sub.22 linear
or branched, saturated or unsaturated, hydrocarbon group.
Representative fatty acids include, but are not limited to, caproic
acid, lauric acid, myristic acid, palmitic acid, stearic acid,
palmic acid, oleic acid, linoleic acid, and linolenic acid.
[0084] The term "polycarboxylic acid," as that term is used herein
means a polymeric compound having more than one --C(O)OH group. One
of ordinary skill in the art would readily recognize polymeric
compounds that have more than one --C(O)OH group. Representative
polycarboxylic acids include, but are not limited to, hyaluronic
acid, polyglutamic acid, polyaspartic acid, and polyacrylic
acid.
[0085] The phrase "injectable" or "injectable composition," as used
herein, means a composition that can be drawn into a syringe and
injected subcutaneously, intraperitoneally, or intramuscularly into
an animal without causing adverse effects due to the presence of
solid material in the composition. Solid materials include, but are
not limited to, crystals, gummy masses, and gels. Typically, a
formulation or composition is considered to be injectable when no
more than about 15%, preferably no more than about 10%, more
preferably no more than about 5%, even more preferably no more than
about 2%, and most preferably no more than about 1% of the
formulation is retained on a 0.22 .mu.m filter when the formulation
is filtered through the filter at 98.degree. F. There are, however,
some compositions of the invention, which are gels, that can be
easily dispensed from a syringe but will be retained on a 0.22
.mu.m filter. In one embodiment, the term "injectable," as used
herein, includes these gel compositions. In one embodiment, the
term "injectable," as used herein, further includes compositions
that when warmed to a temperature of up to about 40.degree. C. and
then filtered through a 0.22 .mu.m filter, no more than about 15%,
preferably no more than about 10%, more preferably no more than
about 5%, even more preferably no more than about 2%, and most
preferably no more than about 1% of the formulation is retained on
the filter. In one embodiment, an example of an injectable
pharmaceutical composition is a solution of a pharmaceutically
active compound (for example, an aptamer) in a pharmaceutically
acceptable solvent.
[0086] The term "solution," as used herein, means a uniformly
dispersed mixture at the molecular or ionic level of one or more
substances (solute), in one or more other substances (solvent),
typically a liquid.
[0087] The term "suspension," as used herein, means solid particles
that are evenly dispersed in a solvent, which can be aqueous or
non-aqueous.
[0088] The term "animal," as used herein, includes, but is not
limited to, humans, canines, felines, equines, bovines, ovines,
porcines, amphibians, reptiles, and avians. Representative animals
include, but are not limited to a cow, a horse, a sheep, a pig, an
ungulate, a chimpanzee, a monkey, a baboon, a chicken, a turkey, a
mouse, a rabbit, a rat, a guinea pig, a dog, a cat, and a human. In
one embodiment, the animal is a mammal. In one embodiment, the
animal is a human. In one embodiment, the animal is a non-human. In
one embodiment, the animal is a canine, a feline, an equine, a
bovine, an ovine, or a porcine.
[0089] The phrase "drug depot," as used herein means a precipitate,
which includes the aptamer, formed within the body of a treated
animal that releases the aptamer over time to provide a
pharmaceutically effective amount of the aptamer.
[0090] The phrase "substantially free of," as used herein, means
less than about 2 percent by weight. For example, the phrase "a
pharmaceutical composition substantially free of water" means that
the amount of water in the pharmaceutical composition is less than
about 2 percent by weight of the pharmaceutical composition.
[0091] The term "effective amount," as used herein, means an amount
sufficient to treat or prevent a condition in an animal.
[0092] The term "phospholipid," as used herein, means a compound
having the general formula:
##STR00003##
[0093] wherein [0094] R.sub.1 is O.sup.- or --OH; [0095] R.sub.2
is: [0096] (i) --H, or [0097] (ii) a C.sub.2-C.sub.36 saturated or
unsaturated, linear or branched acyl group; [0098] R.sub.3 is:
[0099] (i) --H, [0100] (ii) a C.sub.2-C.sub.36 saturated or
unsaturated, linear or branched acyl group; or [0101] (iii)
--C.dbd.C--R.sub.9 wherein R.sub.9 is a C.sub.1-C.sub.22 saturated
or unsaturated, linear or branched hydrocarbon group, optionally
substituted with one or more nitrogen containing groups; [0102] and
at least one of R.sub.2 or R.sub.3 is not --H; [0103] R.sub.4 is:
[0104] (i) --H; [0105] (i) --(CH.sub.2).sub.n--R.sub.5, [0106]
wherein R.sub.5 is --N(R.sub.6)(R.sub.7) or
--N.sup.+(R.sub.6)(R.sub.7)(R.sub.8), [0107] R.sub.6, R.sub.7, and
R.sub.8 are each independently --H, C.sub.1-C.sub.3 alkyl group,
or
[0108] R.sub.6 and R.sub.7 are connected to form a 5- or 6-membered
heterocyclic ring with the nitrogen,
[0109] and [0110] n is an integer ranging from 1 to 4, preferably
2; [0111] (iii) [0112] (iv)
[0112] ##STR00004## [0113] wherein each R.sub.10 is independently
--H or --P(O)(OH).sub.2; or [0114] (v)
--CH.sub.2CH(OH)CH.sub.2(OH).
[0115] The term "saturated or unsaturated, linear or branched
C.sub.2-C.sub.36 acyl group," as used herein, means a group of
formula --O--C(O)--R, wherein R is a C.sub.1-C.sub.35 hydrocarbon
group that can be saturated or unsaturated, linear or branched.
[0116] The term "sphingomyelin," as used herein, means a compound
having the general formula:
##STR00005##
[0117] wherein [0118] R.sub.1 is O.sup.- or --OH; [0119] R.sub.4
is: [0120] (i) --H; or [0121] (i) --(CH.sub.2).sub.n--R.sub.5,
[0122] wherein R.sub.5 is --N(R.sub.6)(R.sub.7) or
--N.sup.+(R.sub.6)(R.sub.7)(R.sub.8), [0123] R.sub.6, R.sub.7, and
R.sub.8 are each independently --H, C.sub.1-C.sub.3 alkyl, or
R.sub.6 and R.sub.7 are connected to form a 5- or 6-membered
heterocyclic ring with the nitrogen, and [0124] n is an integer
ranging from 1 to 4, preferably 2; and [0125] R.sub.11 is a
C.sub.1-C.sub.22 saturated or unsaturated, linear or branched
hydrocarbon group optionally substituted with one or more nitrogen
containing groups.
[0126] The term "about," as used herein to describe a range of
values, applies to both the upper limit and the lower limit of the
range. For example, the phrase "ranges from about 90:10 to 10:90"
has the same meaning as "ranges from about 90:10 to about
10:90."
8.2 The Aptamer
[0127] The aptamer can be any aptamer known to those skilled in the
art.
[0128] In one embodiment, the aptamer is a DNA strand. In one
embodiment, the DNA is double stranded DNA. In one embodiment, the
DNA is single stranded DNA.
[0129] In one embodiment, the aptamer is an RNA strand.
[0130] In one embodiment, the aptamer has a molecular weight of up
to 80 kD. In one embodiment, the molecular weight of the aptamer
ranges from about 15 kD to 80 Kd. In one embodiment, the molecular
weight of the aptamer ranges from about 10 kD to 80 Kd. In one
embodiment, the molecular weight of the aptamer ranges from about 5
kD to 80 Kd.
[0131] In one embodiment, the aptamer has a molecular weight of up
to 60 kD. In one embodiment, the molecular weight of the aptamer
ranges from about 15 kD to 60 Kd. In one embodiment, the molecular
weight of the aptamer ranges from about 10 kD to 60 Kd. In one
embodiment, the molecular weight of the aptamer ranges from about 5
kD to 60 Kd.
[0132] In one embodiment, the aptamer has a molecular weight of up
to 40 kD. In one embodiment, the molecular weight of the aptamer
ranges from about 15 kD to 40 Kd. In one embodiment, the molecular
weight of the aptamer ranges from about 10 kD to 40 Kd. In one
embodiment, the molecular weight of the aptamer ranges from about 5
kD to 40 Kd.
[0133] In one embodiment, the aptamer has a molecular weight of up
to 30 kD. In one embodiment, the molecular weight of the aptamer
ranges from about 15 kD to 30 Kd. In one embodiment, the molecular
weight of the aptamer ranges from about 10 kD to 30 Kd. In one
embodiment, the molecular weight of the aptamer ranges from about 5
kD to 30 Kd.
[0134] In one embodiment, the aptamer has a molecular weight of
more than 20 kD. In one embodiment, the molecular weight of the
aptamer ranges from about 10 kD to 20 Kd. In one embodiment, the
molecular weight of the aptamer ranges from about 5 kD to 20
Kd.
[0135] In one embodiment, the molecular weight of the aptamer
ranges from about 5 kD to 10 Kd.
[0136] The nucleotides that make up the aptamer can be modified to,
for example, improve their stability, i.e., improve their in vivo
half-life, and/or to reduce their rate of excretion when
administered to an animal. The term "modified" encompasses
nucleotides with a covalently modified base and/or sugar. For
example, modified nucleotides include nucleotides having sugars
which are covalently attached to low molecular weight organic
groups other than a hydroxyl group at the 3' position and other
than a phosphate group at the 5' position. Modified nucleotides may
also include 2' substituted sugars such as 2'-O-methyl-;
2'-O-alkyl; 2'-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halo
or 2'-azido-ribose; carbocyclic sugar analogues; .alpha.-anomeric
sugars; and epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, and sedoheptulose.
[0137] Modified nucleotides are known in the art and include, but
are not limited to, alkylated purines and/or pyrimidines; acylated
purines and/or pyrimidines; or other heterocycles. These classes of
pyrimidines and purines are known in the art and include,
pseudoisocytosine; N4, N4-ethanocytosine;
8-hydroxy-N-6-methyladenine; 4-acetylcytosine,
5-(carboxyhydroxylmethyl)uracil; 5-fluorouracil; 5-bromouracil;
5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyl
uracil; dihydrouracil; inosine; N6-isopentyl-adenine;
1-methyladenine; 1-methylpseudouracil; 1-methylguanine;
2,2-dimethylguanine; 2-methyladenine; 2-methylguanine;
3-methylcytosine; 5-methylcytosine; N6-methyladenine;
7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino
methyl-2-thiouracil; .beta.-D-mannosylqueosine;
5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2
methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl
ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil,
2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic
acid methylester; uracil 5-oxyacetic acid; queosine;
2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil;
5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine;
and 2,6,-diaminopurine; methylpseudouracil; 1-methylguanine; and
1-methylcytosine.
[0138] The aptamer can also be modified by replacing one or more
phosphodiester linkages with alternative linking groups.
Alternative linking groups include, but are not limited to
embodiments wherein P(O)O is replaced by P(O)S, P(S)S,
P(O)NR.sub.2, P(O)R, P(O)OR', CO, or CH.sub.2, wherein each R or R'
is independently H or a substituted or unsubstituted
C.sub.1-C.sub.20 alkyl. A preferred set of R substitutions for the
P(O)NR.sub.2 group are hydrogen and methoxyethyl. Linking groups
are typically attached to each adjacent nucleotide through an --O--
bond, but may be modified to include --N-- or --S-- bonds. Not all
linkages in an oligomer need to be identical.
[0139] The aptamer can also be modified by conjugating the aptamer
to a polymer, for example, to reduce the rate of excretion when
administered to an animal. For example, the aptamer can be
"PEGylated," i.e., conjugated to polyethylene glycol ("PEG"). In
one embodiment, the PEG has an average molecular weight ranging
from about 20 kD to 80 kD. Methods to conjugate an aptamer with a
polymer, such PEG, are well known to those skilled in the art (See,
e.g., Greg T. Hermanson, Bioconjugate Techniques, Academic Press,
1966)
[0140] As an example of a modified aptamer useful in the
compositions and methods of the invention see P. Burmeister et al.,
Direct In Vitro Selection of a 2'-O-methyl Aptamer to VEGF,
Chemistry and Biology, vol. 12, 25-33, January 2005.
[0141] In one embodiment, the aptamer is conjugated to a
polymer.
[0142] In one embodiment, the aptamer is an RNA strand that has
been conjugated to a polymer.
[0143] In one embodiment, the aptamer is an DNA strand that has
been conjugated to a polymer.
[0144] In one embodiment, the aptamer is conjugated to PEG.
[0145] In one embodiment, the aptamer is an RNA strand that has
been conjugated to PEG.
[0146] In one embodiment, the aptamer is an DNA strand that has
been conjugated to PEG.
[0147] In one embodiment, the aptamer is a RNA strand wherein at
least one of the 2' hydroxyls on the sugars that make up the
aptamer are O-methylated.
[0148] In one embodiment, the aptamer is a RNA strand wherein at
least one of the 2' hydroxyls on the sugars that make up the
aptamer are O-methylated and wherein the RNA strand has been
conjugated to a polymer.
[0149] In one embodiment, the aptamer is a RNA strand wherein at
least one of the 2' hydroxyls on the nucleotides that make up the
aptamer are O-methylated and wherein the RNA strand has been
conjugated to PEG.
[0150] In one embodiment, the aptamer is an aptamer that binds to
VEGF (vascular endothelial growth factor).
[0151] In one embodiment, the aptamer is ARC224 identified in P.
Burmeister et al., Direct In Vitro Selection of a 2'-O-methyl
Aptamer to VEGF, Chemistry and Biology, vol. 12, 25-33, January
2005.
[0152] In one embodiment, the aptamer is ARC245 identified in P.
Burmeister et al., Direct In Vitro Selection of a 2'-O-methyl
Aptamer to VEGF, Chemistry and Biology, vol. 12, 25-33, January
2005.
[0153] In one embodiment, the aptamer is ARC225 identified in P.
Burmeister et al., Direct In Vitro Selection of a 2'-O-methyl
Aptamer to VEGF, Chemistry and Biology, vol. 12, 25-33, January
2005.
[0154] In one embodiment, the aptamer is ARC259 identified in P.
Burmeister et al., Direct In Vitro Selection of a 2'-O-methyl
Aptamer to VEGF, Chemistry and Biology, vol. 12, 25-33, January
2005.
[0155] In one embodiment, the aptamer is ARC259 identified in P.
Burmeister et al., Direct In Vitro Selection of a 2'-O-methyl
Aptamer to VEGF, Chemistry and Biology, vol. 12, 25-33, January
2005 wherein the 5' phosphate group of the aptamer has been
pegylated with:
##STR00006##
(referred to hereinafter as "pegylated ARC259").
8.3 The Organic Base
[0156] Any organic base known to those of ordinary skill in the art
can be used in the pharmaceutical compositions of the invention.
Preferably, the organic base is a pharmaceutically acceptable
organic base. Representative organic bases include, but are not
limited to, organic amines including, but are not limited to,
ammonia; unsubstituted or hydroxy-substituted mono-, di-, or
tri-alkylamines such as cyclohexylamine, cyclopentylamine,
cyclohexylamine, dicyclohexylamine; tributyl amine, N-methylamine,
N-ethylamine, diethylamine; dimethylamine, triethylamine, mono-,
bis-, or tris-(2-hydroxy-lower alkyl amines) (such as mono-, bis-,
or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, and
tris-(hydroxymethyl)methylamine), N,N,-di-lower alkyl-N-(hydroxy
lower alkyl)-amines (such as N,N,-dimethyl-N-(2-hydroxyethyl)amine
or N,N-dialkyl-N-tris-(2-hydroxyethyl)amines)); pyridine;
benzylamine; phenethylamine; N-methyl-D-glucamine;
N,N'-dibenzylethylenediamine; chloroprocaine; choline; procaine,
and amino acids such as arginine, lysine (See, also, Berge et al.,
J. Pharm. Sci., 1977, 66, 1).
[0157] The invention also contemplates pharmaceutical compositions
comprising a salt formed between the aptamer and a metal ion, such
as sodium, lithium, or potassium ion, and a pharmaceutically
acceptable organic solvent. Typically, these compositions are
useful when a low concentration, generally less than about 25
mg/mL, of the aptamer in the pharmaceutically acceptable organic
solvent is sufficient.
[0158] In one embodiment, the amine is an amino acid ester.
[0159] In one embodiment, the amine is an amino acid amide.
[0160] In one embodiment, the amine is a diamine (for example,
N,N'-dibenzylethylenediamine or an ester or amide of lysine).
[0161] In one embodiment, the amine is a diamine and the
pharmaceutical composition further comprises a carboxylic acid, a
phospholipid, a sphingomyelin, or phosphatidyl choline.
8.3.1 The Amino Acid Ester
[0162] The amino acid esters can be any ester of any amino acid,
i.e., an amino acid wherein the carboxylic acid group of the amino
acid is esterified with a C.sub.1-C.sub.22 alcohol. Accordingly,
the amino acid esters have the general formula (I):
##STR00007##
wherein [0163] R is the amino acid side chain; and [0164] R.sub.1
is a C.sub.1 to C.sub.22 hydrocarbon group.
[0165] As one of ordinary skill in the art would readily know, a
wide variety of groups are possible for the amino acid side, R. For
example, the amino acid side can be a hydrocarbon group that can be
optionally substituted. Suitable substituents include, but are not
limited to, halo, nitro, cyano, thiol, amino, hydroxy, carboxylic
acid, sulfonic acid, aromatic group, and aromatic or non-aromatic
heterocyclic group. Preferably the amino acid side chain is a
C.sub.1-C.sub.10 straight or branched chain hydrocarbon, optionally
substituted with a thiol, amino, hydroxy, carboxylic acid, aromatic
group, or aromatic or non-aromatic heterocyclic group.
[0166] The amino acid ester can be an ester of a naturally
occurring amino acid or a synthetically prepared amino acid. The
amino acid can be a D-amino acid or an L-amino acid. Preferably,
the amino acid ester is the ester of a naturally occurring amino
acid. More, preferably, the amino acid ester is an ester of an
amino acid selected from glycine, alanine, valine, leucine,
isoleucine, phenylalanine, asparagine, glutamine, tryptophane,
proline, serine, threonine, tyrosine, hydroxyproline, cysteine,
methionine, aspartic acid, glutamic acid, lysine, arginine, and
histidine.
[0167] The hydrocarbon group, R.sub.1, can be any C.sub.1 to
C.sub.22 hydrocarbon group. Representative C.sub.1 to C.sub.22
hydrocarbon groups include, but are not limited to, methyl, ethyl,
propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, allyl, cyclopentyl, cyclohexyl, cis-9-hexadecenyl,
cis-9-octadecenyl, cis, cis-9,12-octadecenyl, and cis, cis, cis-9,
12, 15-octadecatrienyl.
[0168] In one embodiment, R.sub.1 is a straight chain hydrocarbon
group.
[0169] In one embodiment, R.sub.1 is a branched chain hydrocarbon
group.
[0170] In one embodiment, R.sub.1 is a saturated hydrocarbon
group.
[0171] In one embodiment, R.sub.1 is an unsaturated hydrocarbon
group.
[0172] In one embodiment, R.sub.1 is a straight chain, saturated
hydrocarbon group.
[0173] In one embodiment, R.sub.1 is a straight chain, unsaturated
hydrocarbon group.
[0174] In one embodiment, R.sub.1 is a C.sub.1-C.sub.16 hydrocarbon
group.
[0175] In one embodiment, R.sub.1 is a C.sub.1-C.sub.10 hydrocarbon
group.
[0176] In one embodiment, R.sub.1 is a C.sub.1-C.sub.5 hydrocarbon
group.
[0177] In one embodiment, R.sub.1 is a C.sub.1-C.sub.3 hydrocarbon
group.
[0178] In one embodiment, R.sub.1 is a C.sub.6-C.sub.22 hydrocarbon
group.
[0179] In one embodiment, R.sub.1 is a C.sub.6-C.sub.18 hydrocarbon
group.
[0180] In one embodiment, R.sub.1 is a C.sub.8-C.sub.18 hydrocarbon
group.
[0181] In one embodiment, R.sub.1 is a C.sub.10-C.sub.18
hydrocarbon group.
[0182] In one embodiment, R.sub.1 is a C.sub.16-C.sub.18
hydrocarbon group.
[0183] In one embodiment, R.sub.1 is a C.sub.16-C.sub.22
hydrocarbon group.
[0184] In one embodiment, R.sub.1 is a C.sub.1-C.sub.16 straight
chain hydrocarbon group.
[0185] In one embodiment, R.sub.1 is a C.sub.1-C.sub.10 straight
chain hydrocarbon group.
[0186] In one embodiment, R.sub.1 is a C.sub.1-C.sub.5 straight
chain hydrocarbon group.
[0187] In one embodiment, R.sub.1 is a C.sub.1-C.sub.3 straight
chain hydrocarbon group.
[0188] In one embodiment, R.sub.1 is a C.sub.6-C.sub.22 straight
chain hydrocarbon group.
[0189] In one embodiment, R.sub.1 is a C.sub.6-C.sub.18 straight
chain hydrocarbon group.
[0190] In one embodiment, R.sub.1 is a C.sub.8-C.sub.18 straight
chain hydrocarbon group.
[0191] In one embodiment, R.sub.1 is a C.sub.10-C.sub.18 straight
chain hydrocarbon group.
[0192] In one embodiment, R.sub.1 is a C.sub.16-C.sub.18 straight
chain hydrocarbon group.
[0193] In one embodiment, R.sub.1 is a C.sub.16-C.sub.22 straight
chain hydrocarbon group.
[0194] In one embodiment, R.sub.1 is a C.sub.1-C.sub.16 branched
chain hydrocarbon group.
[0195] In one embodiment, R.sub.1 is a C.sub.1-C.sub.10 branched
chain hydrocarbon group.
[0196] In one embodiment, R.sub.1 is a C.sub.1-C.sub.5 branched
chain hydrocarbon group.
[0197] In one embodiment, R.sub.1 is a C.sub.1-C.sub.3 branched
chain hydrocarbon group.
[0198] In one embodiment, R.sub.1 is a C.sub.6-C.sub.22 branched
chain hydrocarbon group.
[0199] In one embodiment, R.sub.1 is a C.sub.6-C.sub.18 branched
chain hydrocarbon group.
[0200] In one embodiment, R.sub.1 is a C.sub.8-C.sub.18 branched
chain hydrocarbon group.
[0201] In one embodiment, R.sub.1 is a C.sub.10-C.sub.18 branched
chain hydrocarbon group.
[0202] In one embodiment, R.sub.1 is a C.sub.16-C.sub.18 branched
chain hydrocarbon group.
[0203] In one embodiment, R.sub.1 is a C.sub.16-C.sub.22 branched
chain hydrocarbon group.
[0204] In one embodiment, R.sub.1 is a C.sub.1-C.sub.16 straight
chain unsaturated hydrocarbon group.
[0205] In one embodiment, R.sub.1 is a C.sub.1-C.sub.10 straight
chain unsaturated hydrocarbon group.
[0206] In one embodiment, R.sub.1 is a C.sub.1-C.sub.5 straight
chain unsaturated hydrocarbon group.
[0207] In one embodiment, R.sub.1 is a C.sub.1-C.sub.3 straight
chain unsaturated hydrocarbon group.
[0208] In one embodiment, R.sub.1 is a C.sub.6-C.sub.22 straight
chain unsaturated hydrocarbon group.
[0209] In one embodiment, R.sub.1 is a C.sub.6-C.sub.18 straight
chain unsaturated hydrocarbon group.
[0210] In one embodiment, R.sub.1 is a C.sub.8-C.sub.18 straight
chain unsaturated hydrocarbon group.
[0211] In one embodiment, R.sub.1 is a C.sub.10-C.sub.18 straight
chain unsaturated hydrocarbon group.
[0212] In one embodiment, R.sub.1 is a C.sub.16-C.sub.18 straight
chain unsaturated hydrocarbon group.
[0213] In one embodiment, R.sub.1 is a C.sub.16-C.sub.22 straight
chain unsaturated hydrocarbon group.
[0214] As discussed later, by varying the structure of R.sub.1 it
is possible to vary the properties of the pharmaceutical
compositions.
[0215] The amino acid esters can be obtained by esterifying an
amino acid with an alcohol of formula R.sub.1--OH using methods
well known to those skilled in the art such as those described in
J. March, Advanced Organic Chemistry, Reaction Mechanisms and
Structure, 4.sup.th ed. John Wiley & Sons, NY, 1992, pp.
393-400. The amino acids and alcohols of formula R.sub.1--OH are
commercially available or can be prepared by methods well known to
those skilled in the art. When esterifying the amino acid with the
alcohol of formula R.sub.1--OH, it may be necessary to protect some
other functional group of the amino acid or the alcohol with a
protecting group that is subsequently removed after the
esterification reaction. One of ordinary skill in the art would
readily know what functional groups would need to be protected
before esterifying the amino acid with the alcohol of formula
R.sub.1--OH. Suitable protecting groups are known to those skilled
in the art such as those described in T. W. Greene, et al.
Protective Groups in Organic Synthesis, 3.sup.rd ed. (1999).
8.3.1 The Amino Acid Amide
[0216] The amino acid amides can be any amide of any amino acid,
i.e., an amino acid wherein the carboxylic acid group of the amino
acid is reacted with an amine of formula HN(R.sub.3)(R.sub.4),
wherein R.sub.3 and R.sub.4 are defined above, to provide an amide.
Accordingly, the amino acid amides have the general formula
(II):
##STR00008##
wherein [0217] R is the amino acid side chain; [0218] R.sub.3 is a
C.sub.1 to C.sub.22 hydrocarbon group; and [0219] R.sub.4 is
hydrogen or a C.sub.1 to C.sub.22 hydrocarbon group.
[0220] As one of ordinary skill in the art would readily know, a
wide variety of groups are possible for the amino acid side, R. For
example, the amino acid side can be a hydrocarbon group that can be
optionally substituted. Suitable substituents include, but are not
limited to, halo, nitro, cyano, thiol, amino, hydroxy, carboxylic
acid, sulfonic acid, aromatic group, and aromatic or non-aromatic
heterocyclic group. Preferably the amino acid side chain is a
C.sub.1-C.sub.10 straight or branched chain hydrocarbon, optionally
substituted with a thiol, amino, hydroxy, carboxylic acid, aromatic
group, or aromatic or non-aromatic heterocyclic group; an aromatic
group, or an aromatic or non-aromatic heterocyclic group.
[0221] The amino acid amide can be an amide of a naturally
occurring amino acid or a synthetically prepared amino acid. The
amino acid can be a D-amino acid or an L-amino acid. Preferably,
the amino acid ester is the ester of a naturally occurring amino
acid. More, preferably, the amino acid ester is an ester of an
amino acid selected from glycine, alanine, valine, leucine,
isoleucine, phenylalanine, asparagine, glutamine, tryptophane,
proline, serine, threonine, tyrosine, hydroxyproline, cysteine,
methionine, aspartic acid, glutamic acid, lysine, arginine, and
histidine.
[0222] The R.sub.3 group can be any C.sub.1 to C.sub.22 hydrocarbon
group. The R.sub.4 group can be hydrogen or any C.sub.1 to C.sub.22
hydrocarbon group. Representative C.sub.1 to C.sub.22 hydrocarbon
groups include, but are not limited to, methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,
dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl,
octadecyl, allyl, cyclopentyl, cyclohexyl, cis-9-hexadecenyl,
cis-9-octadecenyl, cis, cis-9,12-octadecenyl, and cis, cis,
cis-9,12,15-octadecatrienyl.
[0223] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
straight chain hydrocarbon group.
[0224] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
branched chain hydrocarbon group.
[0225] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
saturated hydrocarbon group.
[0226] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is an
unsaturated hydrocarbon group.
[0227] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
straight chain, saturated hydrocarbon group.
[0228] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
straight chain, unsaturated hydrocarbon group.
[0229] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.16 hydrocarbon group.
[0230] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.10 hydrocarbon group.
[0231] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.5 hydrocarbon group.
[0232] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.3 hydrocarbon group.
[0233] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.22 hydrocarbon group.
[0234] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.18 hydrocarbon group.
[0235] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.8-C.sub.18 hydrocarbon group.
[0236] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.10-C.sub.18 hydrocarbon group.
[0237] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.18 hydrocarbon group.
[0238] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.22 hydrocarbon group.
[0239] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.16 straight chain hydrocarbon group.
[0240] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.10 straight chain hydrocarbon group.
[0241] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.5 straight chain hydrocarbon group.
[0242] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.3 straight chain hydrocarbon group.
[0243] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.22 straight chain hydrocarbon group.
[0244] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.18 straight chain hydrocarbon group.
[0245] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.8-C.sub.18 straight chain hydrocarbon group.
[0246] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.10-C.sub.18 straight chain hydrocarbon group.
[0247] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.18 straight chain hydrocarbon group.
[0248] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.22 straight chain hydrocarbon group.
[0249] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.16 branched chain hydrocarbon group.
[0250] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.10 branched chain hydrocarbon group.
[0251] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.5 branched chain hydrocarbon group.
[0252] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.3 branched chain hydrocarbon group.
[0253] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.22 branched chain hydrocarbon group.
[0254] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.18 branched chain hydrocarbon group.
[0255] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.8-C.sub.18 branched chain hydrocarbon group.
[0256] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.10-C.sub.18 branched chain hydrocarbon group.
[0257] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.18 branched chain hydrocarbon group.
[0258] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.22 branched chain hydrocarbon group.
[0259] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.16 straight chain saturated hydrocarbon group.
[0260] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.10 straight chain saturated hydrocarbon group.
[0261] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.5 straight chain saturated hydrocarbon group.
[0262] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.1-C.sub.3 straight chain saturated hydrocarbon group.
[0263] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.22 straight chain saturated hydrocarbon group.
[0264] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.6-C.sub.18 straight chain saturated hydrocarbon group.
[0265] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.8-C.sub.18 straight chain saturated hydrocarbon group.
[0266] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.10-C.sub.18 straight chain saturated hydrocarbon group.
[0267] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.18 straight chain saturated hydrocarbon group.
[0268] In one embodiment, R.sub.4 is hydrogen and R.sub.3 is a
C.sub.16-C.sub.22 straight chain saturated hydrocarbon group.
[0269] In one embodiment, each of R.sub.3 and R.sub.4 are a
straight or branched chain, saturated or unsaturated hydrocarbon
group, wherein R.sub.3 and R.sub.4 may be the same or
different.
[0270] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.16 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0271] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.10 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0272] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.5 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0273] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.3 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0274] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.22 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0275] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.18 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0276] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.8-C.sub.18 hydrocarbon group, wherein R.sub.3 and R.sub.4 may
be the same or different.
[0277] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.10-C.sub.18 hydrocarbon group, wherein R.sub.3 and R.sub.4
may be the same or different.
[0278] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.18 hydrocarbon group, wherein R.sub.3 and R.sub.4
may be the same or different.
[0279] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.22 hydrocarbon group, wherein R.sub.3 and R.sub.4
may be the same or different.
[0280] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.16 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0281] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.10 straight chain hydrocarbon group,
[0282] wherein R.sub.3 and R.sub.4 may be the same or
different.
[0283] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.5 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0284] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.3 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0285] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.22 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0286] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.18 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0287] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.8-C.sub.18 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0288] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.10-C.sub.18 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0289] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.18 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0290] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.22 straight chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0291] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.16 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0292] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.10 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0293] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.5 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0294] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.3 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0295] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.22 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0296] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.18 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0297] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.8-C.sub.18 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0298] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.10-C.sub.18 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0299] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.18 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0300] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.22 branched chain hydrocarbon group, wherein R.sub.3
and R.sub.4 may be the same or different.
[0301] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.16 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0302] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.10 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0303] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.5 straight chain saturated hydrocarbon group, wherein
R.sub.3 and R.sub.4 may be the same or different.
[0304] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.1-C.sub.3 straight chain saturated hydrocarbon group, wherein
R.sub.3 and R.sub.4 may be the same or different.
[0305] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.22 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0306] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.6-C.sub.18 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0307] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.8-C.sub.18 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0308] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.10-C.sub.18 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0309] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.18 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0310] In one embodiment, each of R.sub.3 and R.sub.4 are a
C.sub.16-C.sub.22 straight chain saturated hydrocarbon group,
wherein R.sub.3 and R.sub.4 may be the same or different.
[0311] In one embodiment, the combined number of carbon atoms in
R.sub.3 and R.sub.4 is at least 6. In one embodiment, the combined
number of carbon atoms in R.sub.3 and R.sub.4 is at least 8. In one
embodiment, the combined number of carbon atoms in R.sub.3 and
R.sub.4 is at least 10. In one embodiment, the combined number of
carbon atoms in R.sub.3 and R.sub.4 is at least 12. In one
embodiment, the combined number of carbon atoms in R.sub.3 and
R.sub.4 is at least 18.
[0312] In one embodiment, the combined number of carbon atoms in
R.sub.3 and R.sub.4 is less than 6. In one embodiment, the combined
number of carbon atoms in R.sub.3 and R.sub.4 is less than 8. In
one embodiment, the combined number of carbon atoms in R.sub.3 and
R.sub.4 is less than 10. In one embodiment, the combined number of
carbon atoms in R.sub.3 and R.sub.4 is less than 12. In one
embodiment, the combined number of carbon atoms in R.sub.3 and
R.sub.4 is less than 18.
[0313] In one embodiment, the combined number of carbon atoms in
R.sub.3 and R.sub.4 ranges from about 1 to 16. In one embodiment,
the combined number of carbon atoms in R.sub.3 and R.sub.4 ranges
from about 1 to 10. In one embodiment, the combined number of
carbon atoms in R.sub.3 and R.sub.4 ranges from about 1 to 5. In
one embodiment, the combined number of carbon atoms in R.sub.3 and
R.sub.4 ranges from about 1 to 3. In one embodiment, the combined
number of carbon atoms in R.sub.3 and R.sub.4 ranges from about 16
to 22. In one embodiment, the combined number of carbon atoms in
R.sub.3 and R.sub.4 ranges from about 16 to 18. In one embodiment,
the combined number of carbon atoms in R.sub.3 and R.sub.4 ranges
from about 8 to 18. In one embodiment, the combined number of
carbon atoms in R.sub.3 and R.sub.4 ranges from about 10 to 18. In
one embodiment, the combined number of carbon atoms in R.sub.3 and
R.sub.4 ranges from about 12 to 18. In one embodiment, the combined
number of carbon atoms in R.sub.3 and R.sub.4 ranges from about 6
to 30. In one embodiment, the combined number of carbon atoms in
R.sub.3 and R.sub.4 ranges from about 22 to 30.
[0314] As discussed later, by varying the structure of R.sub.3 and
R.sub.4 is possible to vary the properties of the pharmaceutical
compositions.
[0315] The amino acid amides can be obtained by converting the
carboxylic acid group of the amino acid to an amide group using
methods well known to those skilled in the art such as those
described in J. March, Advanced Organic Chemistry, Reaction
Mechanisms and Structure, 4.sup.th ed. John Wiley & Sons, NY,
1992, pp. 417-427. Typically, the amino acid is converted to an
amino acid derivative such as an amino acid ester or an acid
chloride of the amino acid and the amino acid derivative is then
reacted with an amine of formula NHR.sub.3R.sub.4 to provide the
amino acid amide. The amino acids and amines of formula
NHR.sub.3R.sub.4 are commercially available or can be prepared by
methods well known to those skilled in the art. When forming the
derivative of the amino acid or reacting the amino acid derivative
with an amine of formula NHR.sub.3R.sub.4, it may be necessary to
protect some other functional group of the amino acid derivative or
the amine with a protecting group that is subsequently removed
after the amidation reaction. One of ordinary skill in the art
would readily know what functional groups would need to be
protected before reacting the derivative of the amino acid with the
amine of formula NHR.sub.3R.sub.4. Suitable protecting groups are
known to those skilled in the art such as those described in T. W.
Greene, et al. Protective Groups in Organic Synthesis, 3.sup.rd ed.
(1999).
8.4 Examples of Pharmaceutical Compositions of the Invention
8.4.1 Pharmaceutical Compositions Comprising (i) a Pharmaceutically
Acceptable Organic Base and (ii) a Protonated Aptamer
[0316] In one embodiment, the pharmaceutical composition comprises
(i) a protonated aptamer and an (ii) a pharmaceutically acceptable
organic base. Without wishing to be bound by theory, it is believed
that the acidic phosphate groups of the a protonated aptamer
protonate the amine group of the pharmaceutically acceptable
organic base to form a salt between one or more pharmaceutically
acceptable organic base molecules and the aptamer as illustrated
schematically below for a pharmaceutically acceptable organic base
of formula Base-NH.sub.2 and a protonated aptamer.
##STR00009##
wherein B is a nucleotide, S is a sugar, and Base-NH.sub.3.sup.+ is
a protonated pharmaceutically acceptable organic base. It is not
necessary, however, that every phosphate group be ionically bound
to a pharmaceutically acceptable organic base molecule.
[0317] Any pharmaceutically acceptable organic base described above
can be used in the pharmaceutical compositions.
[0318] Any aptamer described above can be used in the
pharmaceutical compositions.
[0319] In one embodiment, the pharmaceutical composition further
comprises a solvent.
[0320] In one embodiment, the solvent comprises water.
[0321] In one embodiment, the solvent comprises a pharmaceutically
acceptable organic solvent. Any of the pharmaceutically acceptable
organic solvents described herein can be used in the compositions
of the invention.
[0322] In one embodiment, the pharmaceutical composition is a
solution of the salt in the pharmaceutically acceptable organic
solvent.
[0323] In one embodiment, the pharmaceutical composition comprises
a pharmaceutically acceptable organic solvent and further comprises
a phospholipid, a sphingomyelin, or phosphatidyl choline. Without
wishing to be bound by theory, it is believed that the
phospholipid, sphingomyelin, or phosphatidyl choline facilitates
formation of a precipitate when the pharmaceutical composition is
injected into water and can also facilitate controlled release of
the aptamer from the resulting precipitate. Typically, the
phospholipid, sphingomyelin, or phosphatidyl choline is present in
an amount ranging from greater than 0 to 10 percent by weight of
the pharmaceutical composition. In one embodiment, the
phospholipid, sphingomyelin, or phosphatidyl choline is present in
an amount ranging from about 0.1 to 10 percent by weight of the
pharmaceutical composition. In one embodiment, the phospholipid,
sphingomyelin, or phosphatidyl choline is present in an amount
ranging from about 1 to 7.5 percent by weight of the pharmaceutical
composition. In one embodiment, the phospholipid, sphingomyelin, or
phosphatidyl choline is present in an amount ranging from about 1.5
to 5 percent by weight of the pharmaceutical composition. In one
embodiment, the phospholipid, sphingomyelin, or phosphatidyl
choline is present in an amount ranging from about 2 to 4 percent
by weight of the pharmaceutical composition.
[0324] The molar ratio of acidic groups on the aptamer to basic
groups on the a pharmaceutically acceptable organic base typically
ranges from about 2:1 to 1:2. In one embodiment, the molar ratio of
acidic groups on the aptamer to basic groups on the
pharmaceutically acceptable organic base ranges about 1.5:1 to
1:1.5. In one embodiment, the molar ratio of acidic groups on the
aptamer to basic groups on the pharmaceutically acceptable organic
base ranges about 1.25:1 to 1:1.25. In one embodiment, the molar
ratio of acidic groups on the aptamer to basic groups on the
pharmaceutically acceptable organic base ranges about 1.1:1. to
1:1.1. In one embodiment, the molar ratio of acidic groups on the
aptamer to basic groups on the pharmaceutically acceptable organic
base is about 1:1. A wider range for the molar ratio of acidic
groups on the aptamer to basic groups on the pharmaceutically
acceptable organic base, however, is also possible. For example,
the molar ratio of acidic groups on the aptamer to basic groups on
the pharmaceutically acceptable organic base can range from about
15:1 to 1:15.
8.4.1 (i) Pharmaceutical Compositions Comprising (i) an Amino Acid
Ester or Amino Acid Amide and (ii) a Protonated Aptamer
[0325] Without wishing to be bound by theory, it is believed that
the acidic phosphate groups of the protonated aptamer protonate the
amine group of the amino acid ester or amide to form a salt between
one or more amino acid ester or amide molecules and the aptamer as
illustrated schematically below for an amino acid ester and an
aptamer:
##STR00010##
wherein B, S, R, and R.sub.1 have the meaning described above. It
is not necessary, however, that every phosphate group be ionically
bound to an amino acid ester or amino acid amide.
[0326] Any amino acid or amino acid ester described above can be
used in the pharmaceutical compositions.
[0327] Any aptamer described above can be used in the
pharmaceutical compositions.
[0328] In one embodiment, the pharmaceutical composition further
comprises a solvent.
[0329] In one embodiment, the solvent comprises water.
[0330] In one embodiment, the solvent comprises a pharmaceutically
acceptable organic solvent. Any of the pharmaceutically acceptable
organic solvents described herein can be used in the compositions
of the invention.
[0331] In one embodiment, the pharmaceutical composition comprises
a pharmaceutically acceptable organic solvent and further comprises
a phospholipid, a sphingomyelin, or phosphatidyl choline. Without
wishing to be bound by theory, it is believed that the
phospholipid, sphingomyelin, or phosphatidyl choline facilitates
formation of a precipitate when the pharmaceutical composition is
injected into water and can also facilitate controlled release of
the aptamer from the resulting precipitate. Typically, the
phospholipid, sphingomyelin, or phosphatidyl choline is present in
an amount ranging from greater than 0 to 10 percent by weight of
the pharmaceutical composition. In one embodiment, the
phospholipid, sphingomyelin, or phosphatidyl choline is present in
an amount ranging from about 0.1 to 10 percent by weight of the
pharmaceutical composition. In one embodiment, the phospholipid,
sphingomyelin, or phosphatidyl choline is present in an amount
ranging from about 1 to 7.5 percent by weight of the pharmaceutical
composition. In one embodiment, the phospholipid, sphingomyelin, or
phosphatidyl choline is present in an amount ranging from about 1.5
to 5 percent by weight of the pharmaceutical composition. In one
embodiment, the phospholipid, sphingomyelin, or phosphatidyl
choline is present in an amount ranging from about 2 to 4 percent
by weight of the pharmaceutical composition.
[0332] The molar ratio of acidic groups on the aptamer to basic
groups on the amino acid ester or amino acid amide typically ranges
from about 2:1 to 1:2. In one embodiment, the molar ratio of acidic
groups on the aptamer to basic groups on the amino acid ester or
amino acid amide ranges from about 1.5:1 to 1:1.5. In one
embodiment, the molar ratio of acidic groups on the aptamer to
basic groups on the amino acid ester or amino acid amide ranges
from about 1.25:1 to 1:1.25. In one embodiment, the molar ratio of
acidic groups on the aptamer to basic groups on the amino acid
ester or amino acid amide ranges from about 1.1:1. to 1:1.1. In one
embodiment, the molar ratio of acidic groups on the aptamer to
basic groups on the amino acid ester or amino acid amide is about
1:1. A wider range for the molar ratio of acidic groups on the
aptamer to basic groups on the amino acid ester or amino acid,
however, is also possible. For example, the molar ratio of acidic
groups on the aptamer to basic groups on the amino acid ester or
amino acid can range from about 15:1 to 1:15.
8.4.1 (i)(a) Pharmaceutical Compositions Wherein the Amino Acid
Ester or Amide is an Amino Acid Ester or Amide of Lysine
[0333] In one embodiment, the pharmaceutical composition comprises
an ester or amide of lysine.
[0334] In one embodiment, there is less than a molar equivalent of
lysine molecules relative to acidic phosphate groups on the
aptamer, i.e., there is an excess of acidic phosphate groups on the
aptamer relative to amino acid ester or amide molecules.
[0335] Without wishing to be bound by theory it is believed that
the amino acid ester or amide of lysine cross-links two protonated
aptamer molecules as depicted below:
##STR00011##
wherein B, S, and R.sub.1 have the meaning described above.
Pharmaceutical Compositions Comprising an Ester or Amide of Lysine,
a Protonated Aptamer, and a Carboxylic Acid
[0336] In one embodiment, the amino acid ester or amide is an ester
or amide of lysine and the pharmaceutical composition further
comprises a carboxylic acid. Without wishing to be bound by theory,
it is believed that the carboxylic acid protonates the
.epsilon.-amine group of lysine to provide a structure as depicted
below:
##STR00012##
wherein B, S, and R.sub.1 are defined above and R.sub.9 is a
C.sub.1-C.sub.21 hydrocarbon.
[0337] The combined molar ratio of acidic groups on the aptamer and
acid groups on the carboxylic acid to basic groups on the amino
acid ester or amino acid amide typically ranges from about 2:1 to
1:2. In one embodiment, the combined molar ratio of acidic groups
on the aptamer and acid groups on the carboxylic acid to basic
groups on the amino acid ester or amino acid amide ranges from
about 1.5:1 to 1:1.5. In one embodiment, the combined molar ratio
of acidic groups on the aptamer and acid groups on the carboxylic
acid to basic groups on the amino acid ester or amino acid amide
ranges from about 1.25:1 to 1:1.25. In one embodiment, the combined
molar ratio of acidic groups on the aptamer and acid groups on the
carboxylic acid to basic groups on the amino acid ester or amino
acid amide ranges from about 1.1:1. to 1:1.1. In one embodiment,
the combined molar ratio of acidic groups on the aptamer and acid
groups on the carboxylic acid to basic groups on the amino acid
ester or amino acid amide is about 1:1. A wider range for the molar
ratio of acidic groups on the aptamer and acid groups on the
carboxylic acid to basic groups on the amino acid ester or amino
acid amide, however, is also possible. For example, the molar ratio
of acidic groups on the aptamer and acid groups on the carboxylic
acid to basic groups on the amino acid ester or amino acid amide
can range from about 15:1 to 1:15.
[0338] Generally, the molar ratio of acidic groups on the aptamer
to acid groups on the carboxylic acid ranges from about 20:1 to
1:20. In one embodiment, the molar ratio of acidic groups on the
aptamer to acid groups on the carboxylic acid ranges from about
15:1 to 1:15. In one embodiment, the molar ratio of acidic groups
on the aptamer to acid groups on the carboxylic acid ranges from
about 10:1 to 1:10. In one embodiment, the molar ratio of acidic
groups on the aptamer to acid groups on the carboxylic acid ranges
from about 5:1 to 1:5. In one embodiment, the molar ratio of acidic
groups on the aptamer to acid groups on the carboxylic acid ranges
from about 2:1 to 1:2.
The Carboxylic Acid
[0339] The carboxylic acid can be any pharmaceutically acceptable
carboxylic acid. Typically, the carboxylic acid is a
C.sub.1-C.sub.22 carboxylic acid. Suitable carboxylic acids
include, but are not limited to, acetic acid, propanoic acid,
butanoic acid, pentanoic acid, decanoic acid, hexanoic acid,
benzoic acid, caproic acid, lauric acid, myristic acid, palmitic
acid, stearic acid, palmic acid, oleic acid, linoleic acid, and
linolenic acid.
[0340] In one embodiment, the carboxylic acid is a C.sub.1-C.sub.16
carboxylic acid.
[0341] In one embodiment, the carboxylic acid is a C.sub.1-C.sub.10
carboxylic acid.
[0342] In one embodiment, the carboxylic acid is a C.sub.1-C.sub.5
carboxylic acid.
[0343] In one embodiment, the carboxylic acid is a C.sub.1-C.sub.3
carboxylic acid.
[0344] In one embodiment, the carboxylic acid is a C.sub.6-C.sub.22
carboxylic acid.
[0345] In one embodiment, the carboxylic acid is a C.sub.6-C.sub.18
carboxylic acid.
[0346] In one embodiment, the carboxylic acid is a C.sub.8-C.sub.18
carboxylic acid.
[0347] In one embodiment, the carboxylic acid is a
C.sub.10-C.sub.18 carboxylic acid.
[0348] In one embodiment, the carboxylic acid is a C.sub.6-C.sub.18
carboxylic acid.
[0349] In one embodiment, the carboxylic acid is a
C.sub.16-C.sub.22 carboxylic acid.
[0350] In one embodiment, the carboxylic acid is a saturated or
unsaturated fatty acid.
[0351] In one embodiment, the carboxylic acid is a saturated fatty
acid.
[0352] In one embodiment, the carboxylic acid is an unsaturated
fatty acid.
[0353] In one embodiment, the carboxylic acid is a dicarboxylic
acid. Suitable dicarboxylic acids include, but are not limited to,
oxalic acid, malonic aid, succinic acid, glutamic acid, adipic
acid, and pimelic acid.
[0354] In one embodiment, the carboxylic acid is a polycarboxylic
acid.
[0355] The carboxylic acids are commercially available or can be
prepared by methods well known to those skilled in the art.
[0356] In one embodiment, the carboxylic acid is an N-acyl amino
acid. The N-acyl amino acids have the following general formula
(III):
##STR00013##
wherein:
[0357] R is the amino acid side chain and is defined above; and
[0358] R.sub.2 is an acyl group of formula --C(O)--R.sub.5, wherein
R.sub.5 is a substituted C.sub.1 to C.sub.21 hydrocarbon group,
i.e., the acyl group, R.sub.2, is a C.sub.1- to C.sub.22 acyl
group. Representative acyl groups of formula --C(O)--R.sub.5
include, but are not limited to, acetyl, propionyl, butanoyl,
hexanoyl, caproyl, heptoyl, octoyl, nonoyl, decoyl, undecoyl,
dodecoyl, tridecoyl, tetradecoyl, pentadecoyl, hexadecoyl,
heptadecoyl, octadecoyl, laurolyl, myristoyl, palmitoyl, stearoyl,
palmioleoyl, oleoyl, linoleoyl, linolenoyl, and benzoyl.
[0359] In one embodiment, R.sub.5 is a C.sub.1-C.sub.15 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.2-C.sub.16 acyl group.
[0360] In one embodiment, R.sub.5 is a C.sub.1-C.sub.9 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.2-C.sub.10 acyl group.
[0361] In one embodiment, R.sub.5 is a C.sub.1-C.sub.5 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.2-C.sub.6 acyl group.
[0362] In one embodiment, R.sub.5 is a C.sub.1-C.sub.3 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.2-C.sub.4 acyl group.
[0363] In one embodiment, R.sub.5 is a C.sub.5-C.sub.21 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.6-C.sub.22 acyl group.
[0364] In one embodiment, R.sub.5 is a C.sub.5-C.sub.17 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.6-C.sub.18 acyl group.
[0365] In one embodiment, R.sub.5 is a C.sub.7-C.sub.17 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.8-C.sub.18 acyl group.
[0366] In one embodiment, R.sub.5 is a C.sub.9-C.sub.17 hydrocarbon
group, i.e., the acyl group of formula --C(O)--R.sub.5 is a
C.sub.10-C.sub.18 acyl group.
[0367] In one embodiment, R.sub.5 is a C.sub.15-C.sub.21
hydrocarbon group, i.e., the acyl group of formula --C(O)--R.sub.5
is a C.sub.16-C.sub.22 acyl group.
[0368] In one embodiment, the acyl group of formula --C(O)--R.sub.5
is obtained from a saturated or unsaturated fatty acid.
[0369] In one embodiment, the acyl group of formula --C(O)--R.sub.5
is a caproyl, laurolyl, myristoyl, palmitoyl, stearoyl,
palmioleoyl, oleoyl, linoleoyl, or linolenoyl group.
[0370] The N-acylated amino acids can be obtained by methods well
known to those skilled in the art. For example, the N-acylated
amino acids can be obtained by reacting an amino acid with an acid
halide of formula T-C(O)--R.sub.5, wherein T is a halide,
preferably chloride, and R.sub.1 is as defined above, using methods
well known to those skilled in the art. When N-acylating the amino
acid with the acid halide of formula T-C(O)--R.sub.5, it may be
necessary to protect some other functional group of the amino acid
or the acid halide with a protecting group that is subsequently
removed after the acylation reaction. One of ordinary skill in the
art would readily know what functional groups would need to be
protected before acylating the amino acid with the acid halide of
formula T-C(O)--R.sub.5. Suitable protecting groups are known to
those skilled in the art such as those described in T. W. Greene,
et al. Protective Groups in Organic Synthesis, 3.sup.rd ed.
(1999).
[0371] Acid halides can be obtained using methods well known to
those skilled in the art such as those described in J. March,
Advanced Organic Chemistry, Reaction Mechanisms and Structure,
4.sup.th ed. John Wiley & Sons, NY, 1992, pp. 437-8. For
example, acid halides can be prepared by reacting a carboxylic acid
with thionyl chloride, bromide, or iodide. Acid chlorides and
bromides can also be prepared by reacting a carboxylic acid with
phosphorous trichloride or phosphorous tribromide, respectively.
Acid chlorides can also be prepared by reacting a carboxylic acid
with Ph.sub.3P in carbon tetrachloride. Acid fluorides can be
prepared by reacting a carboxylic acid with cyanuric fluoride.
[0372] As discussed later, by varying the structure of carboxylic
acid it is possible to vary the properties of the pharmaceutical
compositions.
Pharmaceutical Compositions Comprising an Ester or Amide of Lysine,
a Protonated Aptamer, and a Phospholipid, Phosphatidyl Choline, or
a Sphingomyelin
[0373] In another embodiment, the amino acid ester or amide is an
ester or amide of lysine and the pharmaceutical composition further
comprises a phospholipid, phosphatidyl choline, or a sphingomyelin.
Without wishing to be bound by theory, it is believed that
protonated phosphate groups on the phospholipid, phosphatidyl
choline, or sphingomyelin protonates the E-amine group of lysine to
provide a structure as depicted below for a phospholipid:
##STR00014##
wherein B, S, R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are defined
above.
[0374] The combined molar ratio of acidic groups on the aptamer and
acidic groups on the phospholipid, phosphatidyl choline, or
sphingomyelin to basic groups on the amino acid ester or amino acid
amide typically ranges from about 2:1 to 1:2. In one embodiment,
the combined molar ratio of acidic groups on the aptamer and acidic
groups on the phospholipid, phosphatidyl choline, or sphingomyelin
to basic groups on the amino acid ester or amino acid amide ranges
from about 1.5:1 to 1:1.5. In one embodiment, the combined molar
ratio of acidic groups on the aptamer and acidic groups on the
phospholipid, phosphatidyl choline, or sphingomyelin to basic
groups on the amino acid ester or amino acid amide ranges from
about 1.25:1 to 1:1.25. In one embodiment, the combined molar ratio
of acidic groups on the aptamer and acidic groups on the
phospholipid, phosphatidyl choline, or sphingomyelin to basic
groups on the amino acid ester or amino acid amide ranges from
about 1.1:1. to 1:1.1. In one embodiment, the combined molar ratio
of acidic groups on the aptamer and acidic groups on the
phospholipid, phosphatidyl choline, or sphingomyelin to basic
groups on the amino acid ester or amino acid amide is about 1:1. A
wider range for the molar ratio of acidic groups on the aptamer and
acidic groups on the phospholipid, phosphatidyl choline, or
sphingomyelin to basic groups on the amino acid ester or amino acid
amide, however, is also possible. For example, the molar ratio of
acidic groups on the aptamer and acidic groups on the phospholipid,
phosphatidyl choline, or sphingomyelin to basic groups on the amino
acid ester or amino acid amide can range from about 15:1 to
1:15.
[0375] Generally, the molar ratio of acidic groups on the aptamer
to acidic groups on the phospholipid, phosphatidyl choline, or
sphingomyelin ranges from about 20:1 to 1:20. In one embodiment,
the molar ratio of acidic groups on the aptamer to acidic groups on
the phospholipid, phosphatidyl choline, or sphingomyelin ranges
from about 15:1 to 1:15. In one embodiment, the molar ratio of
acidic groups on the aptamer to acidic groups on the phospholipid,
phosphatidyl choline, or sphingomyelin ranges from about 10:1 to
1:10. In one embodiment, the molar ratio of acidic groups on the
aptamer to acidic groups on the phospholipid, phosphatidyl choline,
or sphingomyelin ranges from about 5:1 to 1:5. In one embodiment,
the molar ratio of acidic groups on the aptamer to acidic groups on
the phospholipid, phosphatidyl choline, or sphingomyelin ranges
from about 2:1 to 1:2.
[0376] As discussed later, by varying the structure of
phospholipid, phosphatidyl choline, or sphingomyelin it is possible
to vary the properties of the pharmaceutical compositions.
The Phospholipid
[0377] Any pharmaceutically acceptable phospholipid can be used in
the pharmaceutical compositions of the invention.
[0378] Representative, pharmaceutically acceptable phospholipids
include, but are not limited to:
[0379] phosphatidic acids of general formula:
##STR00015##
wherein R.sub.1, R.sub.2, and R.sub.3 are defined above. Suitable
phosphatidic acids suitable for use in the compositions and methods
of the invention include, but are not limited to, the
1-acyl-2-acyl-sn-glycero-3-phosphates and the
1,2-diacyl-sn-glycero-3-phosphates commercially available from
Avanti Polar Lipids Inc. of Alabaster, Ala.
[0380] phosphatidylethanolamines of general formula
##STR00016##
wherein R.sub.1, R.sub.2, and R.sub.3 are defined above. Suitable
phosphatidylethanolamines suitable for use in the compositions and
methods of the invention include, but are not limited to, the
1-acyl-2-acyl-sn-glycero-3-phosphoethanolamines and the
1,2-diacyl-sn-glycero-3-phosphoethanolamines commercially available
from Avanti Polar Lipids Inc. of Alabaster, Ala.
[0381] phosphatidylcholines of general formula
##STR00017##
wherein R.sub.1, R.sub.2, and R.sub.3 are defined above. Suitable
phosphatidylcholines suitable for use in the compositions and
methods of the invention include, but are not limited to, the
1-acyl-2-acyl-sn-glycero-3-phosphocholines, the
1,2-diacyl-sn-glycero-3-phosphoethanolamines (saturated series),
and the 1,2-diacyl-sn-glycero-3-phosphoethanolamines (unsaturated
series), commercially available from Avanti Polar Lipids Inc. of
Alabaster, Ala. and Phospholipon.RTM.-50PG,
Phospholipon.RTM.-53MCT, Phospholipon.RTM.-75SA,
Phospholipon.RTM.-80, Phospholipon.RTM.-90NG,
Phospholipon.RTM.-90H, and Phospholipon.RTM.-100H, commercially
available from Phospholipid GmbH of Cologne, Germany. In one
embodiment, the phospholipid is Phospholipon.RTM.-90H.
[0382] phosphatidylserines of general formula
##STR00018##
wherein R.sub.1, R.sub.2, and R.sub.3 are defined above. Suitable
phosphatidylserines suitable for use in the compositions and
methods of the invention include, but are not limited to, the
1-acyl-2-acyl-sn-glycero-3-[phospho-L-serine]s and the
1,2-diacyl-sn-glycero-3-[phospho-L-serine]s commercially available
from Avanti Polar Lipids Inc. of Alabaster, Ala.
[0383] plasmalogens of general formula
##STR00019##
wherein R.sub.1 and R.sub.2 are defined above and R.sub.3 is
--C.dbd.C--R.sub.9, wherein R.sub.9 is defined above. Suitable
plasmalogens suitable for use in the compositions and methods of
the invention include, but are not limited to, C16(Plasm)-12:0 NBD
PC, C16(Plasm)-18:1 PC, C16(Plasm)-20:4 PC, C16(Plasm)-22:6 PC,
C16(Plasm)-18:1 PC, C16(Plasm)-20:4 PE, and C16(Plasm)-22:6 PE,
commercially available from Avanti Polar Lipids Inc. of Alabaster,
Ala.
[0384] phosphatidylglycerols of general formula
##STR00020##
wherein R.sub.1, R.sub.2, and R.sub.3 are defined above. Suitable
phosphatidylglycerols suitable for use in the compositions and
methods of the invention include, but are not limited to, the
1-acyl-2-acyl-sn-glycero-3-[phospho-rac-(1-glycerol)]s and the
1,2-diacyl-sn-glycero-3-[phospho-rac-(1-glycerol)]s, commercially
available from Avanti Polar Lipids Inc. of Alabaster, Ala.
[0385] phosphatidylinositols of general formula
##STR00021##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.10 are defined above.
Suitable phosphatidylinositols suitable for use in the compositions
and methods of the invention include, but are not limited to,
phosphatidylinositol, phosphatidylinositol-4-phosphate, and
phosphatidylinositol-4,5-bisphosphate, commercially available from
Avanti Polar Lipids Inc. of Alabaster, Ala.
[0386] The phospholipids are commercially available or can be
obtained by methods well known to those skilled in the art.
Representative methods for obtaining phospholipids are described in
Sandra Pesch et al., Properties of Unusual Phospholipids Bearing
Acetylenic Fatty Acids, Tettrahedron, vol. 15, no. 43, 14,627-14634
(1997); Sepp D. Kohlwein, Phospholipid Synthesis, Sorting,
Subcellular Traffic--The Yeast Approach, Trends in Cell Biology,
vol. 6, 260-266 (1996), Serguei V. Vinogradov, Synthesis of
Phospholipids--Oligodeoxyribonucleotide Conjugates, Tett. Lett.,
vol. 36, no. 14, 2493-2496 (1995), and references cited
therein.
[0387] In one embodiment, the phospholipid is
Phospholipon.RTM.-E:80 (commercially available from Phospholipid
GmbH of Cologne, Germany or American Lecithin Company of Oxford,
Conn.).
[0388] In one embodiment, the phospholipid is Phospholipon.RTM.-80G
(commercially available from Phospholipid GmbH of Cologne, Germany
or American Lecithin Company of Oxford, Conn.).
[0389] In one embodiment, the phospholipid is Phospholipon.RTM.-85G
(commercially available from Phospholipid GmbH of Cologne, Germany
or American Lecithin Company of Oxford, Conn.).
[0390] In one embodiment, the phospholipid is
Phospholipon.RTM.-100H (commercially available from Phospholipid
GmbH of Cologne, Germany or American Lecithin Company of Oxford,
Conn.).
The Sphingomyelin
[0391] Any pharmaceutically acceptable sphingomyelin can be used in
the pharmaceutical compositions of the invention.
[0392] In one embodiment, the sphingomyelin is
##STR00022##
wherein R.sub.11 is a C.sub.1-C.sub.24 linear, saturated or
unsaturated hydrocarbon and R.sub.4 is
--CH.sub.2CH.sub.2N(CH.sub.3).sub.3.sup.+. In another embodiment,
R.sub.11 is a C.sub.8-C.sub.24 linear, saturated or unsaturated
hydrocarbon and R.sub.4 is
--CH.sub.2CH.sub.2N(CH.sub.3).sub.3.sup.+. In another embodiment,
R.sub.11 is a C.sub.16-C.sub.24 linear, saturated or unsaturated
hydrocarbon and R.sub.4 is
--CH.sub.2CH.sub.2N(CH.sub.3).sub.3.sup.+.
[0393] Suitable sphingomyelins include, but are not limited to,
C2-Sphingomyelin, C6-Sphingomyelin, C18-Sphingomyelin,
C6-NBD-Sphingomyelin, and C12-NBD Sphingomyelin, commercially
available from Avanti Polar Lipids Inc. of Alabaster, Ala.
[0394] Similarly, in another embodiment, the amino acid ester or
amide is an ester or amide of lysine and the pharmaceutical
composition further comprises a phosphatidyl choline. Without
wishing to be bound by theory, it is believed that protonated
phosphate groups on the phosphatidyl choline protonates the
.epsilon.-amine group of lysine to provide a structure as depicted
below:
##STR00023##
wherein S, B, and R.sub.1 are defined above.
[0395] Without wishing to be bound by theory it is also believed
that pharmaceutical compositions that comprise an amino acid ester
or amide of lysine and further comprise a phospholipid,
phosphatidyl choline, or a sphingomyelin that the ester or amide of
lysine also forms structures wherein each amino group of the lysine
ester or amide is protonated by a phospholipid, phosphatidyl
choline, or sphingomyelin molecule. Such a structure is depicted
below for a phospholipid:
##STR00024##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are defined
above.
[0396] The invention also includes pharmaceutical compositions such
as those described above that include an ester or amide of lysine,
wherein the ester or amide of lysine is replaced with another
diamine such as, for example N,N'-dibenzylethylenediamine.
8.4.1 (i)(b) Pharmaceutical Compositions Comprising a Diester or
Diamide of Aspartic Acid or Glutamic Acid and a Protonated
Aptamer
[0397] In another embodiment, the amino acid ester or amide is an
ester or amide of aspartic acid or glutamic acid and the side chain
carboxylic acid group of the aspartic acid or glutamic acid is also
esterified or amidated, i.e., a diester or diamide of aspartic acid
or glutamic acid. Without wishing to be bound by theory it is
believed that the acidic phosphate groups of the aptamer protonate
the amine group of the diester or diamide of aspartic acid or
glutamic acid to form a salt between diester or diamide of aspartic
acid or glutamic acid and the aptamer as illustrated below for a
diester of aspartic acid that is protonated by an aptamer to
provide a structure as depicted below:
##STR00025##
wherein S, B, and R.sub.1 are defined above and R.sub.6 is defined
below.
[0398] The diesters of aspartic acid and glutamic acid have the
structures:
##STR00026##
respectively, wherein R.sub.1 is defined above and R.sub.6 is the
same as R.sub.1. R.sub.1 and R.sub.6 can be the same or different.
Typically, however, R.sub.1 and R.sub.6 are the same.
[0399] The diamides of aspartic acid and glutamic acid have the
structures:
##STR00027##
respectively, wherein R.sub.3 and R.sub.4 are defined above,
R.sub.7 is the same as R.sub.3, and R.sub.8 is the same as R.sub.4.
The amide groups --N(R.sub.3)(R.sub.4) and --N(R.sub.7)(R.sub.8)
can be the same or different. Typically, however, the amide groups
--N(R.sub.3)(R.sub.4) and --N(R.sub.7)(R.sub.8) are the same.
[0400] The molar ratio of acidic groups on the aptamer to the
diester or diamide of aspartic acid or glutamic acid typically
ranges from about 2:1 to 1:2. In one embodiment, the molar ratio of
acidic groups on the aptamer to the diester or diamide of aspartic
acid or glutamic acid ranges from about 1.5:1 to 1:1.5. In one
embodiment, the molar ratio of acidic groups on the aptamer to the
diester or diamide of aspartic acid or glutamic acid ranges from
about 1.25:1 to 1:1.25. In one embodiment, the molar ratio of
acidic groups on the aptamer to the diester or diamide of aspartic
acid or glutamic acid ranges from about 1.1:1. to 1:1.1. In one
embodiment, the molar ratio of acidic groups on the aptamer to the
diester or diamide of aspartic acid or glutamic acid is about 1:1.
A wider range for molar ratio of acidic groups on the aptamer to
the diester or diamide of aspartic acid or glutamic acid, however,
is also possible. For example, the molar ratio of acidic groups on
the aptamer to the diester or diamide of aspartic acid or glutamic
acid can range from about 15:1 to 1:15.
[0401] As discussed later, by varying the structure of diester or
diamide of aspartic acid or glutamic acid, i.e., R.sub.1 and
R.sub.6 of the diester and R.sub.3, R.sub.4, R.sub.7, and R.sub.8
of the diamide, it is possible to vary the properties of the
pharmaceutical compositions.
8.4.1 (ii) Pharmaceutical Compositions Comprising (i) a Protonated
Aptamer, and (ii) a Polylysine
[0402] In another embodiment, the pharmaceutical compositions
comprise a protonated aptamer and polylysine.
[0403] Any of the aptamers described above can be used in the
pharmaceutical compositions.
[0404] Any polylysine (for example, any of the polylysines
commercially available from Sigma-Aldrich of Milwaukee, Wis. as the
hydrobromide salt, which can be converted to polylysine as
described later) can be used in the pharmaceutical compositions. In
one embodiment, the polylysine has a molecular weight range of from
about 1,000 to 4,000. In one embodiment, the polylysine has a
molecular weight range of from about 4,000 to 15,000. In one
embodiment, the polylysine has a molecular weight range of from
about 15,000 to 30,000. In one embodiment, the polylysine has a
molecular weight range of from about 30,000 to 70,000. In one
embodiment, the polylysine has a molecular weight range of from
about 70,000 to 150,000. In one embodiment, the polylysine has a
molecular weight range of from about 150,000 to 300,000.
[0405] Without wishing to be bound by theory, it is believed that
the amine groups on the polylysine are protonated by acidic
phosphate groups on the aptamer.
[0406] Typically, the amount of polylysine relative to the amount
of the aptamer is an amount sufficient to provide a solution of the
pharmaceutical composition (for example, a methanol or aqueous
solution) having a pH value ranging from about 3 to 10. In one
embodiment, a solution of the pharmaceutical composition has a pH
value ranging from about 5 to 9. In one embodiment, a solution of
the pharmaceutical composition has a pH value ranging from about 6
to 8. In one embodiment, a solution of the pharmaceutical
composition has a pH value of about 7. Other pH ranges, however,
are also within the scope of the invention. For example, in one
embodiment, a solution of the pharmaceutical composition has a pH
value ranging from about 3 to 7 and in another embodiment a
solution of the pharmaceutical composition has a pH value ranging
from about 7 to 10.
[0407] The pH can be readily measured by dissolving the
pharmaceutical composition in a solvent (for example methanol or
water) and removing a few microliters of the resulting solution and
applying it to a wet pH test strip (such as commercially available
from Sigma-Aldrich of Milwaukee, Wis.) that indicates the pH of the
solution by the color of the test strip after the solution is
applied.
[0408] In one embodiment, the pharmaceutical composition comprising
a protonated aptamer and polylysine further comprises a solvent. In
one embodiment, the solvent comprises water. In one embodiment, the
solvent is water. In one embodiment, the solvent comprises a
pharmaceutically acceptable organic solvent. In one embodiment, the
solvent is a pharmaceutically acceptable organic solvent. In one
embodiment, the solvent comprises N-methylpyrrolidone. In one
embodiment, the solvent is N-methylpyrrolidone.
[0409] Advantageously, the pharmaceutical compositions comprising a
protonated aptamer and polylysine have increased solubility in
water and organic solvents. For example, the pharmaceutical
composition formed between pegylated ARC259 and polylysine having
an average molecular weight of about 13,000 is soluble in water and
N-methylpyrrolidone at a concentration of up to about 12% (w/v). In
contrast, polylysine having an average molecular weight of about
13,000 (obtained as described later from the commercially available
hydrobromide salt) and the protonated aptamer are both essentially
insoluble in water and N-methylpyrrolidone.
8.4.2 Pharmaceutical Compositions Comprising (i) an Aptamer, (ii) a
Divalent Metal Cation, and (iii) optionally a carboxylate, a
phospholipid, a phosphatidyl choline, or a Sphingomyelin
[0410] In another embodiment, the pharmaceutical compositions
comprise (i) an aptamer, (ii) a divalent metal cation and (iii)
optionally a carboxylate, a phospholipid, a phosphatidyl choline,
or a sphingomyelin. Without wishing to be bound by theory, it is
believed that the divalent metal cation interacts with the
phosphate groups on the aptamer to form a structure as depicted
below:
##STR00028##
wherein M.sup.+2 is a divalent metal cation and B and S are defined
above.
[0411] Without wishing to be bound by theory, it is believed that
when the pharmaceutical composition includes the optional
carboxylate, phospholipid, phosphatidyl choline, or sphingomyelin
the divalent metal cation interacts with the phosphate groups on
the aptamer and the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin to form a structure as depicted below for
a carboxylate:
##STR00029##
wherein M.sup.+2, B, S, and R.sub.9 are defined above. Without
wishing to be bound by theory, it is believed that the structures
are similar to the structures formed between an aptamer; the amino
acid lysine; and a carboxylic acid, a phospholipid, phosphatidyl
choline, or a sphingomyelin, described above, except that the
divalent metal cation replaces the lysine.
[0412] Without wishing to be bound by theory it is also believed
that when the pharmaceutical composition includes the optional
carboxylate, phospholipid, phosphatidyl choline, or sphingomyelin
the divalent metal cation interacts with more than one carboxylate,
phospholipid, phosphatidyl choline, or sphingomyelin to form a
structure as depicted below for a carboxylate:
##STR00030##
wherein M.sup.+2 and R.sub.9 are defined above.
[0413] In one embodiment, the pharmaceutical composition comprises
a carboxylate.
[0414] In one embodiment, the pharmaceutical composition comprises
a phospholipid.
[0415] In one embodiment, the pharmaceutical composition comprises
phosphatidyl choline.
[0416] In one embodiment, the pharmaceutical composition comprises
a sphingomyelin.
[0417] Any of the aptamers described above can be used in the
pharmaceutical compositions.
[0418] The carboxylate can be obtained from any pharmaceutically
acceptable carboxylic acid. Any of the carboxylic acids described
herein can be used to provide the carboxylate.
[0419] In one embodiment, the carboxylic acid is an N-acyl amino
acid of general formula (III). Any N-acyl amino acid of general
formula (III) described above can be used in the pharmaceutical
compositions.
[0420] Any of the phospholipids described above can be used in the
pharmaceutical compositions.
[0421] Any of the sphingomyelins described above can be used in the
pharmaceutical compositions.
[0422] Suitable divalent metal cations include, but are not limited
to, the alkaline earth metal cations, Mg.sup.+2, Zn.sup.+2,
Cu.sup.+2, and Fe.sup.+2. Preferred divalent metal cations are
Ca.sup.+2, Mg.sup.+2, Zn.sup.+2, Cu.sup.+2, and Fe.sup.+2.
[0423] The combined molar ratio of anionic groups on the aptamer
and anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin to the divalent metal cation typically
ranges from about 4:1 to 1:4. In one embodiment, the combined molar
ratio of anionic groups on the aptamer and anionic groups on the
carboxylate, phospholipid, phosphatidyl choline, or sphingomyelin
to the divalent metal cation ranges from about 3:1 to 1:3. In one
embodiment, the combined molar ratio of anionic groups on the
aptamer and anionic groups on the carboxylate, phospholipid,
phosphatidyl choline, or sphingomyelin to the divalent metal cation
ranges from about 2.5:1 to 1:2.5. In one embodiment, the combined
molar ratio of anionic groups on the aptamer and anionic groups on
the carboxylate, phospholipid, phosphatidyl choline, or
sphingomyelin to the divalent metal cation ranges from about 2:1.
to 1:2. In one embodiment, the combined molar ratio of anionic
groups on the aptamer and anionic groups on the carboxylate,
phospholipid, phosphatidyl choline, or sphingomyelin to the
divalent metal cation is about 2:1. A wider range for the molar
ratio of anionic groups on the aptamer and anionic groups on the
carboxylate, phospholipid, phosphatidyl choline, or sphingomyelin
to the divalent metal cation, however, is also possible. For
example, the molar ratio of anionic groups on the aptamer and
anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin to the divalent metal cation can range
from about 15:1 to 1:15.
[0424] Generally, the molar ratio of anionic groups on the aptamer
to anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin ranges from about 20:1 to 1:20. In one
embodiment, the molar ratio of anionic groups on the aptamer to
anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin ranges from about 15:1 to 1:15. In one
embodiment, the molar ratio of anionic groups on the aptamer to
anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin ranges from about 10:1 to 1:10. In one
embodiment, the molar ratio of anionic groups on the aptamer to
anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin ranges from about 5:1 to 1:5. In one
embodiment, the molar ratio of anionic groups on the aptamer to
anionic groups on the carboxylate, phospholipid, phosphatidyl
choline, or sphingomyelin ranges from about 2:1 to 1:2.
[0425] By varying the structure of the carboxylate, phospholipid,
phosphatidyl choline, or sphingomyelin it is possible to vary the
properties of the pharmaceutical compositions, as is discussed
later.
8.5 Optional Additives
[0426] The pharmaceutical compositions can optionally comprise one
or more additional excipients or additives to provide a dosage form
suitable for administration to an animal. When administered to an
animal, the aptamer containing pharmaceutical compositions are
typically administered as a component of a composition that
comprises a pharmaceutically acceptable carrier or excipient so as
to provide the form for proper administration to the animal.
Suitable pharmaceutical excipients are described in Remington's
Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro ed., 19th ed.
1995), incorporated herein by reference. The pharmaceutical
compositions can take the form of solutions, suspensions, emulsion,
tablets, pills, pellets, capsules, capsules containing liquids,
powders, suppositories, emulsions, aerosols, sprays, suspensions,
or any other form suitable for use.
[0427] In one embodiment, the pharmaceutical compositions are
formulated for intravenous or parenteral administration. Typically,
compositions for intravenous or parenteral administration comprise
a suitable sterile solvent, which may be an isotonic aqueous buffer
or pharmaceutically acceptable organic solvent. Where necessary,
the compositions can also include a solubilizing agent.
Compositions for intravenous administration can optionally include
a local anesthetic such as lidocaine to lessen pain at the site of
the injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where aptamer containing pharmaceutical
compositions are to be administered by infusion, they can be
dispensed, for example, with an infusion bottle containing, for
example, sterile pharmaceutical grade water or saline. Where the
pharmaceutical compositions are administered by injection, an
ampoule of sterile water for injection, saline, or other solvent
such as a pharmaceutically acceptable organic solvent can be
provided so that the ingredients can be mixed prior to
administration.
[0428] In another embodiment, the pharmaceutical compositions are
formulated in accordance with routine procedures as a composition
adapted for oral administration. Compositions for oral delivery can
be in the form of tablets, lozenges, aqueous or oily suspensions,
granules, powders, emulsions, capsules, syrups, or elixirs, for
example. Oral compositions can include standard excipients such as
mannitol, lactose, starch, magnesium stearate, sodium saccharin,
cellulose, and magnesium carbonate. Typically, the excipients are
of pharmaceutical grade. Orally administered compositions can also
contain one or more agents, for example, sweetening agents such as
fructose, aspartame or saccharin; flavoring agents such as
peppermint, oil of wintergreen, or cherry; coloring agents; and
preserving agents, to provide a pharmaceutically palatable
preparation. Moreover, when in tablet or pill form, the
compositions can be coated to delay disintegration and absorption
in the gastrointestinal tract thereby providing a sustained action
over an extended period of time. Selectively permeable membranes
surrounding an osmotically active driving compound are also
suitable for orally administered compositions. A time-delay
material such as glycerol monostearate or glycerol stearate can
also be used.
[0429] The pharmaceutical compositions further comprising a solvent
can optionally comprise a suitable amount of a pharmaceutically
acceptable preservative, if desired, so as to provide additional
protection against microbial growth. Examples of preservatives
useful in the pharmaceutical compositions of the invention include,
but are not limited to, potassium sorbate, methylparaben,
propylparaben, benzoic acid and its salts, other esters of
parahydroxybenzoic acid such as butylparaben, alcohols such as
ethyl or benzyl alcohol, phenolic compounds such as phenol, or
quaternary compounds such as benzalkonium chlorides (e.g.,
benzethonium chloride).
[0430] In one embodiment, the pharmaceutical compositions of the
invention optionally contain a suitable amount of a
pharmaceutically acceptable polymer. The polymer can increase the
viscosity of the pharmaceutical composition. Suitable polymers for
use in the compositions and methods of the invention include, but
are not limited to, hydroxypropylcellulose,
hydroxypropylmethylcellulose (HPMC), chitosan, polyacrylic acid,
and polymethacrylic acid.
[0431] Typically, the polymer is present in an amount ranging from
greater than 0 to 10 percent by weight of the pharmaceutical
composition.
[0432] In one embodiment, the polymer is present in an amount
ranging from about 0.1 to 10 percent by weight of the
pharmaceutical composition.
[0433] In one embodiment, the polymer is present in an amount
ranging from about 1 to 7.5 percent by weight of the pharmaceutical
composition.
[0434] In one embodiment, the polymer is present in an amount
ranging from about 1.5 to 5 percent by weight of the pharmaceutical
composition.
[0435] In one embodiment, the polymer is present in an amount
ranging from about 2 to 4 percent by weight of the pharmaceutical
composition.
[0436] In one embodiment, the pharmaceutical compositions of the
invention are substantially free of polymers.
[0437] In one embodiment, any additional components added to the
pharmaceutical compositions of the invention are designated as GRAS
by the FDA for use or consumption by animals.
[0438] In one embodiment, any additional components added to the
pharmaceutical compositions of the invention are designated as GRAS
by the FDA for use or consumption by humans.
[0439] The components of the pharmaceutical composition (the
solvents and any other optional components) are preferably
biocompatible and non-toxic and, over time, are simply absorbed
and/or metabolized by the body.
8.5.1 Pharmaceutical Compositions Further Comprising a Solvent
[0440] As described above, the pharmaceutical compositions of the
invention can further comprise a solvent.
[0441] In one embodiment, the solvent comprises water.
[0442] In one embodiment, the solvent comprises a pharmaceutically
acceptable organic solvent.
[0443] Typically, aptamers are available as the salt of a metal
cation, for example, as the potassium or sodium salt. These salts,
however, have low solubility in aqueous solvents and/or organic
solvents, typically, less than about 25 mg/mL. The pharmaceutical
compositions of the invention comprising (i) an amino acid ester or
amino acid amide and (ii) a protonated aptamer, however, are
significantly more soluble in aqueous solvents and/or organic
solvents. Without wishing to be bound by theory, it is believed
that the amino acid ester or amino acid amide and the protonated
aptamer form a salt, such as illustrated above, and the salt is
soluble in aqueous and/or organic solvents.
[0444] Similarly, without wishing to be bound by theory, it is
believed that the pharmaceutical compositions comprising (i) an
aptamer; (ii) a divalent metal cation; and (iii) optionally a
carboxylate, a phospholipid, a phosphatidyl choline, or a
sphingomyelin form a salt, such as illustrated above, and the salt
is soluble in aqueous and/or organic solvents.
[0445] In one embodiment, the concentration of the aptamer in the
solvent is greater than about 2 percent by weight of the
pharmaceutical composition. In one embodiment, the concentration of
the aptamer in the solvent is greater than about 5 percent by
weight of the pharmaceutical composition. In one embodiment, the
concentration of the aptamer in the solvent is greater than about
7.5 percent by weight of the pharmaceutical composition. In one
embodiment, the concentration of the aptamer in the solvent is
greater than about 10 percent by weight of the pharmaceutical
composition. In one embodiment, the concentration of the aptamer in
the solvent is greater than about 12 percent by weight of the
pharmaceutical composition. In one embodiment, the concentration of
the aptamer in the solvent is greater than about 15 percent by
weight of the pharmaceutical composition. In one embodiment, the
concentration of the aptamer in the solvent is ranges from about 2
percent to 5 percent by weight of the pharmaceutical composition.
In one embodiment, the concentration of the aptamer in the solvent
is ranges from about 2 percent to 7.5 percent by weight of the
pharmaceutical composition. In one embodiment, the concentration of
the aptamer in the solvent ranges from about 2 percent to 10
percent by weight of the pharmaceutical composition. In one
embodiment, the concentration of the aptamer in the solvent is
ranges from about 2 percent to 12 percent by weight of the
pharmaceutical composition. In one embodiment, the concentration of
the aptamer in the solvent is ranges from about 2 percent to 15
percent by weight of the pharmaceutical composition. In one
embodiment, the concentration of the aptamer in the solvent is
ranges from about 2 percent to 20 percent by weight of the
pharmaceutical composition.
[0446] Any pharmaceutically acceptable organic solvent can be used
in the pharmaceutical compositions of the invention.
Representative, pharmaceutically acceptable organic solvents
include, but are not limited to, pyrrolidone,
N-methyl-2-pyrrolidone, polyethylene glycol, propylene glycol
(i.e., 1,3-propylene glycol), glycerol formal, isosorbide dimethyl
ether, ethanol, dimethyl sulfoxide, tetraglycol, tetrahydrofurfuryl
alcohol, triacetin, propylene carbonate, dimethyl acetamide,
dimethyl formamide, dimethyl sulfoxide, and combinations
thereof.
[0447] In one embodiment, the pharmaceutically acceptable organic
solvent is a water soluble solvent. A representative
pharmaceutically acceptable water soluble organic solvents is
triacetin.
[0448] In one embodiment, the pharmaceutically acceptable organic
solvent is a water miscible solvent. Representative
pharmaceutically acceptable water miscible organic solvents
include, but are not limited to, glycerol formal, polyethylene
glycol, and propylene glycol.
[0449] In one embodiment, the pharmaceutically acceptable organic
solvent comprises pyrrolidone. In one embodiment, the
pharmaceutically acceptable organic solvent is pyrrolidone
substantially free of another organic solvent.
[0450] In one embodiment, the pharmaceutically acceptable organic
solvent comprises N-methyl-2-pyrrolidone. In one embodiment, the
pharmaceutically acceptable organic solvent is
N-methyl-2-pyrrolidone substantially free of another organic
solvent.
[0451] In one embodiment, the pharmaceutically acceptable organic
solvent comprises polyethylene glycol. In one embodiment, the
pharmaceutically acceptable organic solvent is polyethylene glycol
substantially free of another organic solvent.
[0452] In one embodiment, the pharmaceutically acceptable organic
solvent comprises propylene glycol. In one embodiment, the
pharmaceutically acceptable organic solvent is propylene glycol
substantially free of another organic solvent.
[0453] In one embodiment, the pharmaceutically acceptable organic
solvent comprises glycerol formal. In one embodiment, the
pharmaceutically acceptable organic solvent is glycerol formal
substantially free of another organic solvent.
[0454] In one embodiment, the pharmaceutically acceptable organic
solvent comprises isosorbide dimethyl ether. In one embodiment, the
pharmaceutically acceptable organic solvent is isosorbide dimethyl
ether substantially free of another organic solvent.
[0455] In one embodiment, the pharmaceutically acceptable organic
solvent comprises ethanol. In one embodiment, the pharmaceutically
acceptable organic solvent is ethanol substantially free of another
organic solvent.
[0456] In one embodiment, the pharmaceutically acceptable organic
solvent comprises dimethyl sulfoxide. In one embodiment, the
pharmaceutically acceptable organic solvent is dimethyl sulfoxide
substantially free of another organic solvent.
[0457] In one embodiment, the pharmaceutically acceptable organic
solvent comprises tetraglycol. In one embodiment, the
pharmaceutically acceptable organic solvent is tetraglycol
substantially free of another organic solvent.
[0458] In one embodiment, the pharmaceutically acceptable organic
solvent comprises tetrahydrofurfuryl alcohol. In one embodiment,
the pharmaceutically acceptable organic solvent is
tetrahydrofurfuryl alcohol substantially free of another organic
solvent.
[0459] In one embodiment, the pharmaceutically acceptable organic
solvent comprises triacetin. In one embodiment, the
pharmaceutically acceptable organic solvent is triacetin
substantially free of another organic solvent.
[0460] In one embodiment, the pharmaceutically acceptable organic
solvent comprises propylene carbonate. In one embodiment, the
pharmaceutically acceptable organic solvent is propylene carbonate
substantially free of another organic solvent.
[0461] In one embodiment, the pharmaceutically acceptable organic
solvent comprises dimethyl acetamide. In one embodiment, the
pharmaceutically acceptable organic solvent is dimethyl acetamide
substantially free of another organic solvent.
[0462] In one embodiment, the pharmaceutically acceptable organic
solvent comprises dimethyl formamide. In one embodiment, the
pharmaceutically acceptable organic solvent is dimethyl formamide
substantially free of another organic solvent.
[0463] In one embodiment, the pharmaceutically acceptable organic
solvent comprises at least two pharmaceutically acceptable organic
solvents.
[0464] In one embodiment, the pharmaceutically acceptable organic
solvent comprises N-methyl-2-pyrrolidone and glycerol formal. In
one embodiment, the pharmaceutically acceptable organic solvent is
N-methyl-2-pyrrolidone and glycerol formal. In one embodiment, the
ratio of N-methyl-2-pyrrolidone to glycerol formal ranges from
about 90:10 to 10:90.
[0465] In one embodiment, the pharmaceutically acceptable organic
solvent comprises propylene glycol and glycerol formal. In one
embodiment, the pharmaceutically acceptable organic solvent is
propylene glycol and glycerol formal. In one embodiment, the ratio
of propylene glycol to glycerol formal ranges from about 90:10 to
10:90.
[0466] In one embodiment, the pharmaceutically acceptable organic
solvent is a solvent that is recognized as GRAS by the FDA for
administration or consumption by animals.
[0467] In one embodiment, the pharmaceutically acceptable organic
solvent is a solvent that is recognized as GRAS by the FDA for
administration or consumption by humans.
[0468] In one embodiment, the pharmaceutically acceptable organic
solvent is substantially free of water. In one embodiment, the
pharmaceutically acceptable organic solvent contains less than
about 1 percent by weight of water. In one embodiment, the
pharmaceutically acceptable organic solvent contains less about 0.5
percent by weight of water. In one embodiment, the pharmaceutically
acceptable organic solvent contains less about 0.2 percent by
weight of water. Pharmaceutically acceptable organic solvents that
are substantially free of water are advantageous since they are not
conducive to bacterial growth. Accordingly, it is typically not
necessary to include a preservative in pharmaceutical compositions
that are substantially free of water. Another advantage of
pharmaceutical compositions that use a pharmaceutically acceptable
organic solvent, preferably substantially free of water, as the
solvent is that hydrolysis of the aptamer is minimized. Typically,
the more water present in the solvent the more readily the aptamer
can be hydrolyzed. Accordingly, aptamer containing pharmaceutical
compositions that use a pharmaceutically acceptable organic solvent
as the solvent can be more stable than aptamer containing
pharmaceutical compositions that use water as the solvent.
[0469] In one embodiment, comprising a pharmaceutically acceptable
organic solvent, the pharmaceutical composition is injectable.
[0470] In one embodiment, the injectable pharmaceutical
compositions are of sufficiently low viscosity that they can be
easily drawn into a 20 gauge and needle and then easily expelled
from the 20 gauge needle. Typically, the viscosity of the
injectable pharmaceutical compositions are less than about 1,200
cps. In one embodiment, the viscosity of the injectable
pharmaceutical compositions are less than about 1,000 cps. In one
embodiment, the viscosity of the injectable pharmaceutical
compositions are less than about 800 cps. In one embodiment, the
viscosity of the injectable pharmaceutical compositions are less
than about 500 cps. Injectable pharmaceutical compositions having a
viscosity greater than about 1,200 cps and even greater than about
2,000 cps (for example gels) are also within the scope of the
invention provided that the compositions can be expelled through an
18 to 24 gauge needle.
[0471] In one embodiment, comprising a pharmaceutically acceptable
organic solvent, the pharmaceutical composition is injectable and
does not form a precipitate when injected into water.
[0472] In one embodiment, comprising a pharmaceutically acceptable
organic solvent, the pharmaceutical composition is injectable and
forms a precipitate when injected into water. Without wishing to be
bound by theory, it is believed, for pharmaceutical compositions
that comprise a protonated aptamer and an amino acid ester or
amide, that the .alpha.-amino group of the amino acid ester or
amino acid amide is protonated by the aptamer to form a salt, such
as illustrated above, which is soluble in the pharmaceutically
acceptable organic solvent but insoluble in water. Similarly, when
the pharmaceutical composition comprises (i) an aptamer; (ii) a
divalent metal cation; and (iii) optionally a carboxylate, a
phospholipid, a phosphatidyl choline, or a sphingomyelin, it is
believed that the components of the composition form a salt, such
as illustrated above, which is soluble in the pharmaceutically
acceptable organic solvent but insoluble in water. Accordingly,
when the pharmaceutical compositions are injected into an animal,
at least a portion of the pharmaceutical composition precipitates
at the injection site to provide a drug depot. Without wishing to
be bound by theory, it is believed that when the pharmaceutically
compositions are injected into an animal, the pharmaceutically
acceptable organic solvent diffuses away from the injection site
and aqueous bodily fluids diffuse towards the injection site,
resulting in an increase in concentration of water at the injection
site, that causes at least a portion of the composition to
precipitate and form a drug depot. The precipitate can take the
form of a solid, a crystal, a gummy mass, or a gel. The
precipitate, however, provides a depot of the aptamer at the
injection site that releases the aptamer over time. The components
of the pharmaceutical composition, i.e., the amino acid ester or
amino acid amide, the pharmaceutically acceptable organic solvent,
and any other components are biocompatible and non-toxic and, over
time, are simply absorbed and/or metabolized by the body.
[0473] In one embodiment, comprising a pharmaceutically acceptable
organic solvent, the pharmaceutical composition is injectable and
forms liposomal or micellar structures when injected into water
(typically about 500 .mu.L are injected into about 4 mL of water).
The formation of liposomal or micellar structures are most often
formed when the pharmaceutical composition includes a phospholipid.
Without wishing to be bound by theory, it is believed that the
aptamer in the form of a salt, which can be a salt formed with an
amino acid ester or amide or can be a salt with a divalent metal
cation and optionally a carboxylate, a phospholipid, a phosphatidyl
choline, or a sphingomyelin, that is trapped within the liposomal
or micellar structure. Without wishing to be bound by theory, it is
believed that when these pharmaceutically compositions are injected
into an animal, the liposomal or micellar structures release the
aptamer over time.
[0474] In one embodiment, the pharmaceutical composition further
comprising a pharmaceutically acceptable organic solvent is a
suspension of solid particles in the pharmaceutically acceptable
organic solvent. Without wishing to be bound by theory, it is
believed that the solid particles comprise a salt formed between
the amino acid ester or amino acid amide and the protonated aptamer
wherein the acidic phosphate groups of the aptamer protonates the
amino group of the amino acid ester or amino acid amide, such as
illustrated above, or comprises a salt formed between the aptamer;
divalent metal cation; and optional carboxylate, phospholipid,
phosphatidyl choline, or sphingomyelin, as illustrated above.
Pharmaceutical compositions that are suspensions can also form drug
depots when injected into an animal.
[0475] By varying the lipophilicity and/or molecular weight of the
amino acid ester or amino acid amide it is possible to vary the
properties of pharmaceutical compositions that include these
components and further comprise an organic solvent. The
lipophilicity and/or molecular weight of the amino acid ester or
amino acid amide can be varied by varying the amino acid and/or the
alcohol (or amine) used to form the amino acid ester (or amino acid
amide). For example, the lipophilicity and/or molecular weight of
the amino acid ester can be varied by varying the R.sub.1
hydrocarbon group of the amino acid ester. Typically, increasing
the molecular weight of R.sub.1 increase the lipophilicity of the
amino acid ester. Similarly, the lipophilicity and/or molecular
weight of the amino acid amide can be varied by varying the R.sub.3
or R.sub.4 groups of the amino acid amide.
[0476] For example, by varying the lipophilicity and/or molecular
weight of the amino acid ester or amino acid amide it is possible
to vary the solubility of the aptamer in water, to vary the
solubility of the aptamer in the organic solvent, vary the
viscosity of the pharmaceutical composition comprising a solvent,
and vary the ease at which the pharmaceutical composition can be
drawn into a 20 gauge needle and then expelled from the 20 gauge
needle.
[0477] Furthermore, by varying the lipophilicity and/or molecular
weight of the amino acid ester or amino acid amide (i.e., by
varying R.sub.1 of the amino acid ester or R.sub.3 and R.sub.4 of
the amino acid amide) it is possible to control whether the
pharmaceutical composition that further comprises an organic
solvent will form a precipitate when injected into water. Although
different aptamers exhibit different solubility and behavior,
generally the higher the molecular weight of the amino acid ester
or amino acid amide, the more likely it is that the salt of the
protonated aptamer and the amino acid ester of the amide will form
a precipitate when injected into water. Typically, when R.sub.1 of
the amino acid ester is a hydrocarbon of about C.sub.16 or higher
the pharmaceutical composition will form a precipitate when
injected into water and when R.sub.1 of the amino acid ester is a
hydrocarbon of about C.sub.12 or less the pharmaceutical
composition will not form a precipitate when injected into water.
Indeed, with amino acid esters wherein R.sub.1 is a hydrocarbon of
about C.sub.12 or less, the salt of the protonated aptamer and the
amino acid ester is, in many cases, soluble in water. Similarly,
with amino acid amides, if the combined number of carbons in
R.sub.3 and R.sub.4 is 16 or more the pharmaceutical composition
will typically form a precipitate when injected into water and if
the combined number of carbons in R.sub.3 and R.sub.4 is 12 or less
the pharmaceutical composition will not form a precipitate when
injected into water. Whether or not a pharmaceutical composition
that further comprises a pharmaceutically acceptable organic
solvent will form a precipitate when injected into water can
readily be determined by injecting about 0.05 mL of the
pharmaceutical composition into about 4 mL of water at about
98.degree. F. and determining how much material is retained on a
0.22 .mu.m filter after the composition is mixed with water and
filtered. Typically, a formulation or composition is considered to
be injectable when no more than 10% of the formulation is retained
on the filter. In one embodiment, no more than 5% of the
formulation is retained on the filter. In one embodiment, no more
than 2% of the formulation is retained on the filter. In one
embodiment, no more than 1% of the formulation is retained on the
filter.
[0478] Similarly, in pharmaceutical compositions that comprise a
protonated aptamer and a diester or diamide of aspartic or glutamic
acid, it is possible to vary the properties of pharmaceutical
compositions by varying the amount and/or lipophilicity and/or
molecular weight of the diester or diamide of aspartic or glutamic
acid. Similarly, in pharmaceutical compositions that comprise an
aptamer; a divalent metal cation; and a carboxylate, a
phospholipid, a phosphatidyl choline, or a sphingomyelin, it is
possible to vary the properties of pharmaceutical compositions by
varying the amount and/or lipophilicity and/or molecular weight of
the carboxylate, phospholipid, phosphatidyl choline, or
sphingomyelin.
[0479] Further, when the pharmaceutical compositions that further
comprises an organic solvent form a depot when administered to an
animal, it is also possible to vary the rate at which the aptamer
is released from the drug depot by varying the lipophilicity and/or
molecular weight of the amino acid ester or amino acid amide.
Generally, the more lipophilic the amino acid ester or amino acid
amide, the more slowly the aptamer is released from the depot.
Similarly, when the pharmaceutical compositions that further
comprises an organic solvent and also further comprise a
carboxylate, phospholipid, phosphatidyl choline, sphingomyelin, or
a diester or diamide of aspartic or glutamic acid and form a depot
when administered to an animal, it is possible to vary the rate at
which the aptamer is released from the drug depot by varying the
amount and/or lipophilicity and/or molecular weight of the
carboxylate, phospholipid, phosphatidyl choline, sphingomyelin, or
the diester or diamide of aspartic or glutamic acid.
[0480] Release rates from a precipitate can be measured injecting
about 50 .mu.L of the pharmaceutical composition into about 4 mL of
deionized water in a centrifuge tube. The time that the
pharmaceutical composition is injected into the water is recorded
as T=0. After a specified amount of time, T, the sample is cooled
to about -9.degree. C. and spun on a centrifuge at about 13,000 rpm
for about 20 min. The resulting supernatant is then analyzed by
HPLC to determine the amount of aptamer present in the aqueous
solution. The amount of aptamer in the pellet resulting from the
centrifugation can also be determined by collecting the pellet,
dissolving the pellet in about 10 .mu.L of methanol, and analyzing
the methanol solution by HPLC to determine the amount of aptamer in
the precipitate. The amount of aptamer in the aqueous solution and
the amount of aptamer in the precipitate are determined by
comparing the peak area for the HPLC peak corresponding to the
aptamer against a standard curve of aptamer peak area against
concentration of aptamer. Suitable HPLC conditions can be readily
determined by one of ordinary skill in the art.
8.6 Methods of Preparing the Aptamer Containing Pharmaceutical
Compositions
[0481] The pharmaceutical compositions can be prepared by
dissolving an inorganic salt of the aptamer, typically a potassium
or sodium salt, in a solvent in which it is soluble, for example
methanol or water, and adjusting the pH of the resulting solution
to a value of between about 2 and 3 with an organic acid, such as
formic acid, as depicted below:
##STR00031##
wherein S and B are defined above and M.sup.+ is a metal ion, to
provide a solution of the protonated aptamer.
[0482] The resulting solution of protonated aptamer is then
dialyzed against water to remove excess formic acid and formate
salts and if, for example, the neutralization is conducted in a
methanol solvent, to replace the methanol with water. The water can
then be removed from the aqueous solution of the protonated aptamer
by lyophilization to provide the protonated aptamer or,
alternatively, the aqueous solution of the protonated aptamer can
be dialyzed against methanol to replace the water with methanol and
then simply removing the methanol under reduced pressure to provide
the protonated aptamer.
[0483] A solution of the protonated aptamer can also be prepared
using a cation exchange resin. Any cationion exchange resin known
to one skilled in the art can be used, for example, a Strata.RTM.
SCX cation exchange resin (commercially available from Phenomenex
of Torrance, Calif.) or a DOWEX.RTM. cation exchange resin, such as
DOWEX.RTM. 50 (commercially available from Dow Chemical Company of
Midland, Mich.) can be used. Typically, a column containing the
cation exchange resin is first washed with an acidic solution to
protonate the resin and then a solution of the inorganic salt of
the aptamer, typically a potassium or sodium salt, in a solvent,
for example methanol or water, is passed through the resin to
provide, as the eluant, a solution of the protonated aptamer.
[0484] To prepare the pharmaceutical compositions comprising a
protonated aptamer and an a pharmaceutically acceptable organic
base (using an amino acid ester or amide as a representative
pharmaceutically acceptable organic base), the protonated aptamer
is dissolved in a solvent, such as methanol, typically with
stirring, and to the resulting solution is then added the amino
acid ester or amide, as depicted below:
##STR00032##
wherein S, B, R, and R.sub.1 are defined above.
[0485] Any other components of the pharmaceutical composition, such
as a carboxylic acid, phospholipid, phosphatidyl choline,
sphingomyelin, or diester or diamide of aspartic or glutamic acid
are then added to the resulting solution.
[0486] Typically, sufficient amino acid ester or amide, and any
other components, are added to provide a solution having a pH value
ranging from about 5 to 9. In one embodiment, sufficient amino acid
ester or amide, and any other components, are added to provide a
solution having a pH value ranging from about 6 to 8. In one
embodiment, sufficient amino acid ester or amide, and any other
components, are added to provide a solution having a pH value of
about 7. The pH can be readily measured by removing a few
microliters of the solution and applying it to a wet pH test strip
(such as commercially available from Sigma-Aldrich of Milwaukee,
Wis.) that indicates the pH of the solution by the color of the
test strip after the solution is applied. The solvent is then
removed under reduced pressure to provide the pharmaceutical
composition comprising the amino acid ester or amino acid amide and
the aptamer. The resulting composition can then be dissolved in a
pharmaceutically acceptable organic solvent to provide the
pharmaceutical composition comprising the amino acid ester or amino
acid amide, the protonated aptamer, and a pharmaceutically
acceptable organic solvent. Alternatively, the pharmaceutical
compositions comprising a protonated aptamer, an amino acid ester
or amide, and any other components, and a pharmaceutically
acceptable organic solvent can be prepared by dissolving the
protonated aptamer in the pharmaceutically acceptable solvent and
adding the amino acid ester or amide and any other components to
the resulting solution, preferably with stirring, to provide the
pharmaceutical composition.
[0487] To prepare the pharmaceutical compositions comprising an
aptamer; a divalent metal cation; and a carboxylate, a
phospholipid, a phosphatidyl choline, or a sphingomyelin, the
protonated aptamer is dissolved in a solvent, such as methanol, and
to the resulting solution is added a metal salt, such as a metal
acetate, or a metal hydroxide, preferably with stirring. To the
resulting solution is then added the carboxylic acid, phospholipid,
phosphatidyl choline, or sphingomyelin, preferably with stirring.
The solvent is then removed under reduced pressure to provide the
pharmaceutical composition comprising the aptamer; a divalent metal
cation; and a carboxylate, a phospholipid, a phosphatidyl choline,
or a sphingomyelin. The resulting composition can then be dissolved
in a pharmaceutically acceptable organic solvent to provide the
pharmaceutical composition comprising the aptamer; a divalent metal
cation; and a carboxylate, a phospholipid, a phosphatidyl choline,
or a sphingomyelin; and a pharmaceutically acceptable organic
solvent. Alternatively, the pharmaceutical compositions comprising
an aptamer; a divalent metal cation; and a carboxylate, a
phospholipid, a phosphatidyl choline, or a sphingomyelin; and a
pharmaceutically acceptable organic solvent can be prepared by
dissolving the protonated aptamer in the pharmaceutically
acceptable solvent; adding a metal salt, such as a metal acetate,
or a metal hydroxide to the resulting solution, preferably with
stirring; and then adding the carboxylic acid, phospholipid,
phosphatidyl choline, or sphingomyelin, preferably with stirring,
to provide the pharmaceutical composition.
[0488] To prepare the pharmaceutical compositions comprising a
protonated aptamer and polylysine, a polylysine solution (such as a
methanol solution) is slowly added to a solution (such as a
methanol solution) of the protonated aptamer, preferably with
stirring, and the pH of the resulting solution monitored to provide
a solution having the desired pH value. The methanol is then
removed under reduced pressure to provide the pharmaceutical
composition comprising a protonated aptamer and polylysine.
[0489] The polylysine is obtained from commercially available
polylysine hydrobromide (commercially available from Sigma-Aldrich,
St. Louis, Mo.) by simply neutralizing a solution (such as a
methanol or water solution) of the polylysine hydrobromide with
ammonium hydroxide to provide a solution having a pH value ranging
from about 10 to 12. The resulting solution of polylysine is then
dialyzed against water to remove excess ammonium bromide and
ammonium hydroxide and if, for example, the neutralization is
conducted in a methanol solvent, to replace the methanol with
water. The water can then be removed from the aqueous solution of
the polylysine by lyophilization to provide the polylysine or,
alternatively, the aqueous solution of the polylysine can be
dialyzed against methanol to replace the water with methanol and
then the methanol simply removed under reduced pressure to provide
the polylysine.
8.7 Methods of Treating a Condition in an Animal
[0490] The pharmaceutical compositions of the invention are useful
in human medicine and veterinary medicine. Accordingly, the
invention further relates to a method of treating or preventing a
condition in an animal comprising administering to the animal an
effective amount of the pharmaceutical composition of the
invention.
[0491] In one embodiment, the invention relates to methods of
treating a condition in an animal comprising administering to an
animal in need thereof an effective amount of a pharmaceutical
composition of the invention.
[0492] In one embodiment, the invention relates to methods of
preventing a condition in an animal comprising administering to an
animal in need thereof an effective amount of a pharmaceutical
composition of the invention.
[0493] Methods of administration include, but are not limited to,
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, oral, sublingual,
intracerebral, intravaginal, transdermal, rectal, by inhalation, or
topical. The mode of administration is left to the discretion of
the practitioner. In most instances, administration will result in
the release of the aptamer into the bloodstream.
[0494] In one embodiment, the method of treating or preventing a
condition in an animal comprises administering to the animal in
need thereof an effective amount of an aptamer by parenterally
administering the pharmaceutical composition of the invention. In
one embodiment, the pharmaceutical compositions are administered by
infusion or bolus injection. In one embodiment, the pharmaceutical
composition is administered subcutaneously.
[0495] In one embodiment, the method of treating or preventing a
condition in an animal comprises administering to the animal in
need thereof an effective amount of an aptamer by orally
administering the pharmaceutical composition of the invention. In
one embodiment, the composition is in the form of a capsule or
tablet.
[0496] The pharmaceutical compositions can also be administered by
any other convenient route, for example, topically, by absorption
through epithelial or mucocutaneous linings (e.g., oral, rectal,
and intestinal mucosa, etc.).
[0497] The pharmaceutical compositions can be administered
systemically or locally.
[0498] The pharmaceutical compositions can be administered together
with another biologically active agent.
[0499] In one embodiment, the animal is a mammal.
[0500] In one embodiment the animal is a human.
[0501] In one embodiment, the animal is a non-human animal.
[0502] In one embodiment, the animal is a canine, a feline, an
equine, a bovine, an ovine, or a porcine.
[0503] The effective amount administered to the animal depends on a
variety of factors including, but not limited to the type of animal
being treated, the condition being treated, the severity of the
condition, and the specific aptamer being administered. One of
ordinary skill in the art will readily know what is an effective
amount of the pharmaceutical composition to treat a condition in an
animal.
[0504] In one embodiment, the aptamer is a anti-Vascular
Endothelial Growth Factor (VEGF) aptamer. In one embodiment, the
aptamer is a anti-Vascular Endothelial Growth Factor (VEGF) aptamer
and the disorder is an ocular disorder. Representative ocular
disorders include, but are not limited to, age-related macular
degeneration, optic disc neovascularization, iris
neovascularization, retinal neovascularization, choroidal
neovascularization, corneal neovascularization, vitreal
neovascularization, glaucoma, pannus, pterygium, macular edema,
vascular retinopathy, retinal degeneration, uveitis, inflammatory
diseases of the retina, or proliferative vitreoretinopathy.
Virtually any method of delivering a medication to the eye may be
used for the delivery of the pharmaceutical compositions of the
invention. In one embodiment, the pharmaceutical composition is
administered intravitreally, for example, via intravitreal
injection. In one embodiment, the pharmaceutical composition is
administered transclerally.
[0505] In one embodiment, the aptamer is an aptamer that inhibits
angiogenesis.
[0506] In one embodiment, the aptamer is an aptamer that inhibits
angiogenesis and the disease being treated is cancer. In one
embodiment, the aptamer is an aptamer that inhibits angiogenesis
and the disease being treated is a solid tumor.
[0507] The following examples are set forth to assist in
understanding the invention and should not be construed as
specifically limiting the invention described and claimed herein.
Such variations of the invention, including the substitution of all
equivalents now known or later developed, which would be within the
purview of those skilled in the art, and changes in formulation or
minor changes in experimental design, are to be considered to fall
within the scope of the invention incorporated herein.
9 EXAMPLES
Example 1
Preparation of Amino Acid Esters
[0508] Tryptophane butanoate: 1 g of tryptophane butanoate
hydrochloride salt (commercially available from Sigma-Aldrich, St.
Louis, Mo.) was suspended in 25 mL of dichloromethane and 600 .mu.l
of triethylamine was added to the suspension with stirring.
Stirring was continued for 15 min and the resulting solution was
transferred to a separatory funnel. The organic solution was washed
twice with 25 mL of water followed by 25 mL of saturated aqueous
sodium bicarbonate. The organic layer was then dried over anhydrous
sodium sulfate and concentrated under reduced pressure to provide
tryptophane butanoate. The structure was confirmed using mass
spectroscopy.
[0509] Tryptophane octanoate: 4 g of tryptophane butanoate
hydrochloride salt (commercially available from Sigma-Aldrich, St.
Louis, Mo. (www.sima-aldrich.com)) was suspended in 100 mL of
dichloromethane and 3 ml of triethylamine was added to the
suspension with stirring. Stirring was continued for 15 min and the
resulting solution was transferred to a separatory funnel. The
organic solution was washed twice with 25 mL of water followed by
25 mL of saturated aqueous sodium bicarbonate. The organic layer
was then dried over anhydrous sodium sulfate and concentrated under
reduced pressure to provide tryptophane octanoate. The structure
was confirmed using mass spectroscopy.
[0510] Tyrosine butanoate: 18.19 g of tyrosine was suspended in a
solution of 9.8 g of concentrated sulfuric acid, 40 mL water, 40 mL
of butanol, and 200 mL of toluene in a 500 mL round bottom flask
equipped with a condenser and a Dean-Stark apparatus. The resulting
solution was heated at reflux temperature until no more water could
be distilled. The resulting solution was cooled in an ice bath,
which caused the solution to separate into two phases. The upper
phase was discarded and the lower phase, an oily syrup, was
retained. The syrup was mixed with sufficient 5% aqueous sodium
bicarbonate solution to neutralize acidic impurities to provide a
solid that was collected by filtration and washed with cold water.
The resulting solid was re-crystallized in ethyl acetate.
[0511] Isoleucine butyrate: 26.23 g of isoleucine was dissolved in
a solution of 20 g of concentrated sulfuric acid, 20 mL water, 40
mL of butanol, and 200 mL of toluene in a 500 mL round bottom flask
equipped with a condenser and a Dean-Stark apparatus. The resulting
solution was heated at reflux temperature until no more water could
be distilled. The resulting solution was then cooled to room
temperature and washed with saturated aqueous sodium bicarbonate to
neutralize acidic impurities, washed with saturated brine, and
dried over anhydrous sodium sulfate. The solvent was removed under
reduced pressure and the resulting liquid distilled under vacuum to
provide isoleucine butyrate as a colorless liquid.
[0512] Phenylalanine butyrate: 16.52 g of isoleucine was dissolved
in a solution of 10 g of concentrated sulfuric acid, 20 mL water,
20 mL of butanol, and 200 mL of toluene in a 500 mL round bottom
flask equipped with a condenser and a Dean-Stark apparatus. The
resulting solution was heated at reflux temperature until no more
water could be distilled. The resulting solution was then cooled to
room temperature and washed with saturated aqueous sodium
bicarbonate to neutralize acidic impurities, washed with saturated
brine, and dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure and the resulting liquid distilled
under vacuum to provide phenylalanine butyrate.
[0513] Phenylalanine octanoate: 16.52 g of phenylalanine was
dissolved in a solution of 10 g of concentrated sulfuric acid, 20
mL water, 20 mL of octanol, and 120 mL of toluene in a 500 mL round
bottom flask equipped with a condenser and a Dean-Stark apparatus.
The resulting solution was heated at reflux temperature until no
more water could be distilled. The resulting solution was then
cooled to room temperature and washed with saturated aqueous sodium
bicarbonate to neutralize acidic impurities, washed with saturated
brine, and dried over anhydrous sodium sulfate. The solvent was
then removed under reduced pressure to provide phenylalanine
octanoate as a white solid that was purified using a silica gel
column eluted with a 1:9 methanol:dichloromethane mixture.
[0514] Phenylalanine dodecanoate: 16.52 g of phenylalanine was
dissolved in a solution of 10 g of concentrated sulfuric acid, 20
mL water, 20 mL of dodecanol, and 120 mL of toluene in a 500 mL
round bottom flask equipped with a condenser and a Dean-Stark
apparatus. The resulting solution was heated at reflux temperature
until no more water could be distilled. The resulting solution was
then cooled to room temperature and washed with saturated aqueous
sodium bicarbonate to neutralize acidic impurities, washed with
saturated brine, and dried over anhydrous sodium sulfate. The
solvent was then removed under reduced pressure to provide
phenylalanine dodecanoate as a solid that was purified using a
silica gel column eluted with a 1:9 methanol:dichloromethane
mixture.
[0515] Tyrosine octanoate: 9.06 g of tyrosine was dissolved in a
solution of 10 g of concentrated sulfuric acid, 20 mL water, 10 mL
of octanol, and 200 mL of toluene in a 500 mL round bottom flask
equipped with a condenser and a Dean-Stark apparatus. The resulting
solution was heated at reflux temperature until no more water could
be distilled. The resulting solution was then cooled to room
temperature and washed with saturated aqueous sodium bicarbonate to
neutralize acidic impurities to provide an emulsion. About 150 mL
of ethyl acetate was added to the emulsion to provide two phases.
The aqueous phase was discarded and the organic phase washed with
saturated Brine and dried over anhydrous sodium sulfate. The
solvent was the removed under reduced pressure to provide tyrosine
octanoate as a white solid that was purified using a silica gel
column eluted with a 1:9 methanol:dichloromethane mixture.
[0516] Isoleucine octanoate: 13.1 g of isoleucine was dissolved in
a solution of 10 g of concentrated sulfuric acid, 20 mL water, 20
mL of octanol, and 200 mL of toluene in a 500 mL round bottom flask
equipped with a condenser and a Dean-Stark apparatus placed in an
oil bath. The resulting solution was heated at reflux temperature
until no more water could be distilled. The resulting solution was
then cooled to room temperature, diluted with 120 mL of ethyl
acetate and the organic layer washed with saturated aqueous sodium
bicarbonate to neutralize acidic impurities, washed with saturated
Brine, and dried over anhydrous sodium sulfate. The solvent was
removed under reduced pressure and the resulting liquid distilled
to provide isoleucine octanoate as a colorless liquid.
[0517] Proline butanoate: 34.5 g of proline was suspended in a
solution of 35 g of concentrated sulfuric acid, 40 mL water, 120 mL
of butanol, and 200 mL of toluene in a 500 mL round bottom flask
equipped with a condenser and a Dean-Stark apparatus. The resulting
solution was heated at reflux temperature until no more water could
be distilled. The resulting solution was then cooled to room
temperature, washed with saturated aqueous sodium bicarbonate to
neutralize acidic impurities, washed with saturated Brine, and
dried over anhydrous sodium sulfate. The solvent was removed under
reduced pressure and the resulting liquid distilled to provide
proline butanoate as a colorless liquid.
[0518] Lysine hexadecanoate: BOC protected lysine (6.25 g, 0.018
mole) was dissolved in about 40 mL of tetrahydrofuran under a
nitrogen atmosphere. The solution was cooled to about 0.degree. C.
using an ice-water bath and carbonyl diimidazole (2.93 g, 0.018
mole) was added to the cooled solution. The reaction mixture was
then allowed to stir for about 5 min. at about 5.degree. C. and
then for about 30 min. at room temperature. To the resulting
solution was then added by dropwise addition a solution of
hexadecanol (4.38 g, 0.018 mole) in about 10 mL of tetrahydrofuran.
The resulting solution was then warmed to about 45.degree. C. and
allowed to stir for about 12 h. After stirring, the solvent was
evaporated under reduced pressure; the resulting residue dissolved
in ethyl acetate; the ethyl acetate washed with 0.1 N hydrochloric
acid (3 times), saturated aqueous sodium hydrogen carbonate (3
times), and brine (3 times); and the organic phase dried
(Na.sub.2SO.sub.4). The ethyl acetate was then removed under
reduced pressure to provide crude BOC protected lysine
hexadecanoate that was purified using silica gel column
chromatography eluted with 0 to 20 percent ethyl acetate in hexane.
The solvent was then evaporated under reduced pressure to provide
purified BOC protected lysine hexadecanoate. Trifluoroacetic acid
(20 mL) was added to the purified BOC protected lysine
hexadecanoate and the resulting reaction mixture stirred for about
5 h. Excess trifluoroacetic acid was removed under reduced
pressure. The resulting residue was then dissolved in methanol and
passed through a Dowex 550A(OH) resin (50 g) (commercially
available from Dow Chemical Company of Midland Mich.) and the
solvent removed under reduced pressure to provide lysine
hexadecanoate that was dried under vacuum to provide dried lysine
hexadecanoate (3.6 g).
Example 2
Pharmaceutical Composition of the Invention
[0519] A pharmaceutical compositions containing pegylated ARC259
was prepared by adding 108 mg of protonated pegylated ARC259 to 800
.mu.L of N-methyl-2-pyrrolidone and sonicating the resulting
mixture for about 25 min. to provide a clear thick solution. To the
clear thick solution was then added 120 .mu.L of a solution of
isoleucine butyrate in N-methyl-2-pyrrolidone (about 71.5 mg/mL)
and the resulting clear solution made up to a volume of 1 mL with
N-methyl-2-pyrrolidone to provide the pharmaceutical
composition.
[0520] 50 .mu.L of the pharmaceutical composition was then injected
in 4 mL of water. No precipitate was observed to form when the
pharmaceutical composition was injected into the water.
Example 3
Pharmaceutical Composition of the Invention
[0521] A pharmaceutical compositions containing pegylated ARC259
was prepared by adding 108 mg of protonated pegylated ARC259 to 800
.mu.L of N-methyl-2-pyrrolidone and allowing the resulting mixture
to be shaken for about 14 h. using an automatic shaker to provide a
clear thick solution. To the clear thick solution was then added
120 .mu.L of a solution of isoleucine butyrate in
N-methyl-2-pyrrolidone (about 71.5 mg/mL) and the resulting clear
solution made up to a volume of 1 mL with N-methyl-2-pyrrolidone to
provide the pharmaceutical composition.
[0522] 50 .mu.L of the pharmaceutical composition was then injected
in 4 mL of water. No precipitate was observed to form when the
pharmaceutical composition was injected into the water.
Example 4
Pharmaceutical Composition of the Invention
[0523] A pharmaceutical compositions containing pegylated ARC259
was prepared by adding 108 mg of protonated pegylated ARC259 to 800
.mu.L of glycerol formal and sonicating the resulting mixture for
about 25 min. to provide a clear thick solution. To the clear thick
solution was then added 120 .mu.L of a solution of isoleucine
butyrate in glycerol formal (about 71.5 mg/mL) and the resulting
clear solution made up to a volume of 1 mL with glycerol formal to
provide the pharmaceutical composition.
[0524] 50 .mu.L of the pharmaceutical composition was then injected
in 4 mL of water. No precipitate was observed to form when the
pharmaceutical composition was injected into the water.
Example 5
Viscosity of Pharmaceutical Compositions Containing an Aptamer and
Amino Acid Ester in an Organic Solvent as a Function of Ester Chain
Length
[0525] Pharmaceutical compositions containing pegylated ARC259 at a
concentration of about 10% (w/v) and 1 equivalent of isoleucine
ethanoate, isoleucine butanoate, isoleucine hexanoate, isoleucine
octanoate, isoleucine decanoate, isoleucine dodecanoate, or
isoleucine hexadecanoate per acidic groups on the aptamer dissolved
in N-methyl-2-pyrrolidone were prepared. The pharmaceutical
compositions were prepared by adding 75 mg of protonated aptamer to
0.7 mL of N-methyl-2-pyrrolidone and then adding an appropriate
amount of the isoleucine ester as indicated below:
TABLE-US-00001 isoleucine ethanoate 6.3 mg (6.8 .mu.L) isoleucine
butanoate 7.4 mg (8.46 .mu.L) isoleucine hexanoate 8.4 mg (9.6
.mu.L) isoleucine octanoate 9.6 mg (11.3 .mu.L) isoleucine
decanoate 10.6 mg (12.7 .mu.L) isoleucine dodecanoate 11.7 mg (14.5
.mu.L) isoleucine hexadecanoate 13.9 mg (17.8 .mu.L)
The volume of the solution was then made up to a volume of 0.75 mL
with N-methyl-2-pyrrolidone, if necessary, to provide a clear
solution.
[0526] The viscosity of the resulting pharmaceutical compositions
was then determined using a Brookfield DV-II-PRO viscometer
(commercially available from Brookfield of Marlboro, Mass.) with a
cone and plate sampler, a CPE-40 spindle, a sample size of 0.5 mL,
a speed of 3 rpm, and a temperature controlled to be 25.degree.
C.
[0527] FIG. 1 shows a graphical representation of the viscosity of
the pharmaceutical composition v. number of carbons in the alcohol
group of the isoleucine ester. The results show that, in general,
increasing the number of carbons in the alcohol group of the ester
decreases the viscosity of the pharmaceutical composition up to 8
carbons in the alcohol group of the ester. The C.sub.12 ester,
however, has a viscosity that is less than the C.sub.16 ester.
Example 6
Viscosity of Pharmaceutical Compositions Containing an Amino Acid
Ester and an Aptamer in an Organic Solvent as a Function of the
Equivalents of Ester Per Equivalents of Acidic Functional Groups on
the Aptamer
[0528] Pharmaceutical compositions containing pegylated ARC259 at a
concentration of about 10% (w/v) and 1 equivalent, 2 equivalents,
or 6 equivalents of isoleucine decanoate per equivalent of acidic
groups on the aptamer dissolved in N-methyl-2-pyrrolidone were
prepared. The compositions were prepared by adding 75 mg of
protonated aptamer to 0.7 mL of N-methyl-2-pyrrolidone and then
adding 1 equivalent (10.6 mg, 12.7 .mu.L), 2 equivalents (21.2 mg,
25.4 .mu.L), or 3 equivalents (31.8 mg, 38.1 .mu.L) of isoleucine
decanoate. The volume of the solution was then made to a volume of
0.75 mL with N-methyl-2-pyrrolidone, if necessary, to provide a
clear solution.
[0529] The viscosity of the resulting compositions was then
determined using the method described above.
[0530] FIG. 2 shows a graphical representation of the viscosity of
the pharmaceutical composition v. equivalents of isoleucine
decanoate per equivalent of acidic functional groups on the
aptamer. The results show that the viscosity of the pharmaceutical
composition decreases as the number of equivalents of isoleucine
decanoate is increased up to about 2 equivalents of isoleucine
decanoate per equivalent of acidic functional groups on the
aptamer. Thereafter the viscosity appears to remain unchanged up to
6 equivalents of isoleucine decanoate per equivalent of acidic
functional groups on the aptamer.
Example 7
In Vitro Depot Formation of Pharmaceutical Compositions Containing
an Aptamer and an Amino Acid Ester in an Organic Solvent
[0531] A. Pharmaceutical compositions containing pegylated ARC259
at a concentration of about 10% (w/v) and 4 equivalents or 6
equivalents of isoleucine decanoate, isoleucine dodecanoate, or
isoleucine hexadecanoate per equivalent of acidic groups on the
aptamer dissolved in N-methyl-2-pyrrolidone were prepared. The
compositions were prepared by adding 75 mg of protonated aptamer to
0.7 mL of N-methyl-2-pyrrolidone and then adding an appropriate
amount of the ester as indicated below:
TABLE-US-00002 4 equivalent of isoleucine decanoate 42.4 mg (50.8
.mu.L) 6 equivalents of isoleucine decanoate 63.6 mg (76.2 .mu.L) 4
equivalents of isoleucine dodecanoate 46.8 mg (58 .mu.L) 6
equivalents of isoleucine dodecanoate 70.2 mg (87 .mu.L) 4
equivalents of isoleucine hexadecanoate 55. 6 mg (71.2 .mu.L) 6
equivalents of isoleucine hexadecanoate 83.4 mg (106.8 .mu.L)
The volume of the solution was then made to a volume of 0.75 mL
with N-methyl-2-pyrrolidone, if necessary, to provide a clear
solution.
[0532] 50 .mu.L of each pharmaceutical composition was then
injected into 4 mL of water. In each case, a precipitate was
observed to form when the pharmaceutical composition was injected
into the water.
[0533] B. Pharmaceutical compositions containing pegylated ARC259
at a concentration of about 10% (w/v) and 1, 2, 4, 6, 8, or 10
equivalents of lysine hexadecanoate per equivalent of acidic groups
on the aptamer dissolved in N-methyl-2-pyrrolidone were also
prepared following the same procedure described above to provide a
clear solution. 50 .mu.L of each composition was then injected in 4
mL of water. In each case, a precipitate was observed to form when
the pharmaceutical composition was injected into the water.
[0534] When the pharmaceutical compositions having between 1 and 4
equivalents of lysine hexadecanoate per equivalent of acidic groups
on the aptamer were injected into the water, an oily precipitate
formed that could be made to dissolve in the water with
shaking.
[0535] When the pharmaceutical compositions having between 4 and 10
equivalents of lysine hexadecanoate per equivalent of acidic groups
on the aptamer were injected into the water, a gel like precipitate
formed in the water that would not dissolve with shaking.
Similarly, when the pharmaceutical compositions having between 4
and 10 equivalents of lysine hexadecanoate per equivalent of acidic
groups on the aptamer were injected into phosphate buffered saline
(PBS) or into water containing about 0.643 .mu.M bovine serum
albumen (BSA), a gel like precipitate formed in the aqueous media
that would not dissolve with shaking. The greater the number of
equivalents of lysine hexadecanoate per equivalent of acidic groups
on the aptamer, the longer the precipitate remained before
dissolving. For example, for pharmaceutical compositions having 4
equivalents of lysine hexadecanoate per equivalent of acidic groups
on the aptamer, the precipitate remained for about 2 days before
dissolving. For pharmaceutical compositions having 6 equivalents
and 10 equivalents of lysine hexadecanoate per equivalent of acidic
groups on the aptamer, the precipitate remained for about 4 days
and 6 days, respectively, before dissolving.
Example 8
Pharmaceutical Compositions Containing an Aptamer and a Lysine
Ester and a Fatty Acid in an Organic Solvent
[0536] A pharmaceutical composition was prepared by adding 100 mg
of pegylated ARC259 and 9.6 mg of the ester formed between lysine
hexadecanoate (about 1 eq. per equivalent of acidic groups on the
aptamer) to 650 .mu.L of N-methyl-2-pyrrolidone. An additional 30.4
mg of the lysine hexadecanoate was then added to the resulting
solution followed by 15 mg of lauric acid. The volume of the
resulting solution was then made up to 1 mL with
N-methyl-2-pyrrolidone to provide a clear solution. When 50 .mu.L
of the pharmaceutical composition was injected into 4 mL of water,
a precipitate was observed to form.
Example 9
Pharmaceutical Compositions Containing an Aptamer, an Isoleucine
Ester, and a Phospholipid
[0537] A solution was prepared by dissolving 307 mg of
Phospholipon.RTM. 80 (commercially available from Phospholipid GmbH
of Cologne, Germany or American Lecithin Company of Oxford, Conn.)
in 5 mL of N-methyl-2-pyrrolidone to provide "solution A." 108 mg
of pegylated ARC259 was then dissolved in 800 .mu.L of solution A
followed by 11.5 .mu.L of isoleucine butyrate. The resulting
mixture was then sonicated to provide a clear solution and the
volume of the solution was made up to 1 mL with solution A to
provide the pharmaceutical composition as a clear solution. When 50
.mu.L of the pharmaceutical composition was injected into 4 mL of
water, a gel like precipitate was observed to form. When the
solution of the precipitate in water was shaken, liposomal and
micellar structures were also observed which may not be retained on
a 0.22 .mu.m filter.
[0538] Similar pharmaceutical compositions can be made using other
esters or amides of amino acids, other organic solvents, and/or
other phospholipids.
Example 10
Pharmaceutical Compositions Containing an Aptamer, a Divalent Metal
Ion, and a Phospholipid
[0539] A pharmaceutical composition was prepared by dissolving 19
mg of pegylated ARC259 in 0.5 mL of N-methyl-2-pyrrolidone
containing 10% (w/v) of Phospholipon.RTM. 80 (commercially
available from Phospholipid GmbH of Cologne, Germany or American
Lecithin Company of Oxford, Conn.). To the resulting solution was
added 0.4 mL of neat N-methyl-2-pyrrolidone followed by 25 mg of
zinc acetate with mixing to provide a clear solution. When 50 .mu.L
of the pharmaceutical composition was injected into 4 mL of water,
a gel like precipitate was observed to form. When the solution of
the precipitate in water was shaken, liposomal and micellar
structures were also observed which may not be retained on a 0.22
.mu.m filter.
[0540] Similar pharmaceutical compositions can be made using other
esters or amides of amino acids, other divalent metal ions, other
organic solvents, and/or other phospholipids.
Example 11
HPLC Analysis of the Pharmaceutical Compositions of the Invention
and Method for Measuring Rate of Release of the Aptamer from the
Pharmaceutical Compositions of the Invention
[0541] The amount of aptamer released from a precipitate as a
function of time can be measured by injecting about 50 .mu.L of the
pharmaceutical composition into about 4 mL of deionized water in a
centrifuge tube to form the precipitate. The time that the
pharmaceutical composition is injected into the water is recorded
as T=0. After a specified amount of time, T, the sample, optionally
cooled to about -9.degree. C., is spun on a centrifuge at about
13,000 rpm for about 20 min. to provide a pellet and a supernatant
liquid that can be easily separated by decanting the supernatant.
The resulting supernatant is then analyzed by a suitable HPLC
method to determine the amount of aptamer present in the aqueous
solution. The amount of aptamer in the pellet can also be
determined by dissolving the pellet in about 3 mL of methanol and
analyzing the methanol solution by a suitable HPLC method to
determine the amount of aptamer in the precipitate. The amount of
aptamer in the aqueous solution and the amount of aptamer in the
precipitate can be determined by comparing the peak area for the
HPLC peak corresponding to the aptamer against a standard curve of
aptamer peak area against concentration of aptamer. Suitable HPLC
methods can be readily determined by one of ordinary skill in the
art. For example for the aptamer used in the above experiments
(i.e., pegylated ARC259) the following HPLC method can be used.
TABLE-US-00003 Column: Jupiter 5.mu. C4 300A, 30x4.6 mm (Part #
00A-4167-EO). Flow rate: 2.0 mL/min. Injection volume: 20 .mu.L
Detector setting: 258 nm Run Time: 10 min. Pump A: Option 1 (Acidic
mobile phase): 25 mM Ammonium Acetate-Trifluoroacetic Acid (TFA),
pH 4.76 or Pump A: Option 2 (Basic mobile phase): 50 mM
Triethanolamine-HCl, pH 7.8 Pump B: Methanol Initial Conditions: 0%
pump B 100% pump A
[0542] The HPLC column is eluted using the following gradient
elution profile:
TABLE-US-00004 Time (min) Module Function Value Duration (min) 0.00
pump % B 90.00 3.00 6.00 pump % B 0.00 0.50 6.00 pump Flow Rate
4.00 0.00 10.00 Detector stop acquiring data
[0543] Under these conditions the aptamer has a retention time of
about 3 min.
[0544] 50 .mu.L of the pharmaceutical composition of Example 7B
containing 10 equivalents of lysine hexadecanoate was injected into
4 mL of water to provide a precipitate and the precipitate and
supernatant were separated by centrifugation following the
procedure described above to provide a pellet and a supernatant
liquid. The pellet was dissolved in about 3 mL of methanol. The
supernatant and the methanol solution of the pellet were then
analyzed by HPLC using the conditions described above using the
basic mobile phase.
[0545] FIG. 3 shows an HPLC chromatogram of the supernatant (lower
trace) and an HPLC chromatogram of the methanol solution of the
pellet (upper trace). The HPLC chromatogram shows that about 5% of
the aptamer was in the supernatant and about 95% of the aptamer was
in the pellet.
[0546] FIG. 4 shows HPLC analysis of the pharmaceutical composition
of Example 4B containing 10 equivalents of lysine hexadecanoate. 50
.mu.L of the pharmaceutical composition was injected into about 3
mL of methanol and the resulting methanol solution analyzed by HPLC
using the HPLC parameters described above. Trace A is the HPLC
chromatogram the pharmaceutical composition obtained using the
basic mobile phase. Trace B is the HPLC chromatogram the
pharmaceutical composition obtained using the acidic mobile phase.
Trace C is the HPLC chromatogram of the aptamer dissolved in
methanol using the basic mobile phase.
[0547] FIG. 4 also shows that when the pharmaceutical composition
is analyzed using the acidic mobile phase a less sharp peak at a
later retention time is obtained (Trace B) compared to analysis
using the basic mobile phase. Without wishing to be bound by
theory, it is believed that when using the acidic mobile, the
aptamer and the lysine hexadecanoate remain associated resulting in
the peak corresponding to the aptamer eluting later and being a
less sharp peak. When using the basic mobile phase, however, the
aptamer and the lysine hexadecanoate are not associated resulting
in the peak corresponding to the aptamer eluting earlier as a
sharper peak. FIG. 4 shows that the basic mobile phase is better
for analyzing the aptamer by HPLC.
[0548] FIG. 4 also shows that the complex between the aptamer and
the amino acid (in this example lysine hexadecanoate) remain
associated under acidic conditions. Accordingly, this suggests that
when a pharmaceutical composition of the invention is orally
administered to an animal, it is likely that the complex between
the aptamer and the amino acid will remain associated in the acidic
environment of the stomach, which could result in extended release
of the aptamer and/or better absorption of the aptamer.
Example 12
Pharmaceutical Compositions Containing an Aptamer a Lysine Ester
and a Fatty Acid in an Organic Solvent, Wherein the Lysine Ester is
Present in an Excess Relative to the Aptamer
[0549] A pharmaceutical composition was prepared by adding 100 mg
of pegylated ARC259 to 1 mL of N-methyl-2-pyrrolidone. The
resulting mixture was mixed using a vortex mixer and occasionally
sonicated to provide a clear viscous solution. To the clear viscous
solution was added 40 mg of lysine hexadecanoate and the resulting
mixture mixed using a vortex mixer to provide a clear solution. The
pH of the resulting solution, determined as described above using a
wet pH test strip (such as commercially available from
Sigma-Aldrich of Milwaukee, Wis.), was basic. To the basic solution
was added 15 mg of lauric acid and the resulting mixture mixed
using a vortex mixer to provide a clear solution. The pH of the
clear solution, determined as described above using a wet pH test
strip was neutral, i.e., about pH 7. When 50 .mu.L of the
pharmaceutical composition was injected into 4 mL of water, a
precipitate was observed to form.
Example 13
Pharmaceutical Compositions Containing an Aptamer and a
Polycarboxylic Acid
[0550] A pharmaceutical composition was prepared by adding 100 mg
of pegylated ARC259 to 1 mL of N-methyl-2-pyrrolidone. The
resulting mixture was mixed using a vortex mixer and occasionally
sonicated to provide a clear viscous solution. To the clear viscous
solution was added 8 mg of polyacrylic acid (20,000 molecular
weight, commercially available from Sigma-Aldrich of Milwaukee,
Wis.) and the resulting mixture mixed using a vortex mixer to
provide a clear solution. The pH of the resulting solution,
determined as described above using a wet pH test strip (such as
commercially available from Sigma-Aldrich of Milwaukee, Wis.), was
slightly basic. To the slightly basic solution was added a small
amount of the polyacrylic acid, the mixture mixed well using a
vortex mixer, and the pH checked again. Additional small amounts of
the of polyacrylic acid were added with mixing and the pH of the
resulting solution checked until the pH of the resulting solution
was about pH 7. When 50 .mu.L of the pharmaceutical composition was
injected into 4 mL of water, a precipitate was observed to
form.
[0551] The present invention is not to be limited in scope by the
specific embodiments disclosed in the examples which are intended
as illustrations of a few aspects of the invention and any
embodiments that are functionally equivalent are within the scope
of this invention. Indeed, various modifications of the invention
in addition to those shown and described herein will become
apparent to those skilled in the art and are intended to fall
within the scope of the appended claims.
[0552] A number of references have been cited, the entire
disclosure of which are incorporated.
* * * * *