U.S. patent application number 13/994675 was filed with the patent office on 2013-12-05 for neuropeptide analogs, compositions, and methods for treating pain.
This patent application is currently assigned to Neuroadjuvants, Inc. The applicant listed for this patent is Grzegorz Bulaj, Brian Donald Klein, Daniel Ryan McDougle, Cameron Spencer Metcalf, Erika Adkins Scholl, Misty Danielle Smith, H. Steve White, Liuyin Zhang. Invention is credited to Grzegorz Bulaj, Brian Donald Klein, Daniel Ryan McDougle, Cameron Spencer Metcalf, Erika Adkins Scholl, Misty Danielle Smith, H. Steve White, Liuyin Zhang.
Application Number | 20130324467 13/994675 |
Document ID | / |
Family ID | 46245354 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130324467 |
Kind Code |
A1 |
White; H. Steve ; et
al. |
December 5, 2013 |
NEUROPEPTIDE ANALOGS, COMPOSITIONS, AND METHODS FOR TREATING
PAIN
Abstract
Neuropeptide analogs and compositions including neuropeptide
analogs are described herein. Also provided are methods of
producing and using the neuropeptide analogs and compositions
including one or more neuropeptide analogs.
Inventors: |
White; H. Steve; (Salt Lake
City, UT) ; Klein; Brian Donald; (Salt Lake City,
UT) ; Metcalf; Cameron Spencer; (Sandy, UT) ;
McDougle; Daniel Ryan; (Urbana, IL) ; Scholl; Erika
Adkins; (Sandy, UT) ; Smith; Misty Danielle;
(West Valley City, UT) ; Bulaj; Grzegorz; (Salt
Lake City, UT) ; Zhang; Liuyin; (Salt Lake City,
UT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
White; H. Steve
Klein; Brian Donald
Metcalf; Cameron Spencer
McDougle; Daniel Ryan
Scholl; Erika Adkins
Smith; Misty Danielle
Bulaj; Grzegorz
Zhang; Liuyin |
Salt Lake City
Salt Lake City
Sandy
Urbana
Sandy
West Valley City
Salt Lake City
Salt Lake City |
UT
UT
UT
IL
UT
UT
UT
UT |
US
US
US
US
US
US
US
US |
|
|
Assignee: |
Neuroadjuvants, Inc
Salt Lake City
UT
|
Family ID: |
46245354 |
Appl. No.: |
13/994675 |
Filed: |
December 14, 2011 |
PCT Filed: |
December 14, 2011 |
PCT NO: |
PCT/US11/64977 |
371 Date: |
August 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61423530 |
Dec 15, 2010 |
|
|
|
Current U.S.
Class: |
514/11.1 ;
514/9.7; 530/300; 530/311; 530/345 |
Current CPC
Class: |
A61K 38/2271 20130101;
A61P 25/04 20180101; C07K 14/57545 20130101; C07K 7/083 20130101;
A61K 47/60 20170801; A61P 25/00 20180101; A61K 38/10 20130101; A61K
38/31 20130101; C07K 14/655 20130101 |
Class at
Publication: |
514/11.1 ;
530/311; 530/300; 530/345; 514/9.7 |
International
Class: |
C07K 14/575 20060101
C07K014/575; C07K 7/08 20060101 C07K007/08; C07K 14/655 20060101
C07K014/655 |
Claims
1. A composition with increased metabolic stability, the
composition comprising: a neuropeptide analog wherein at least one
amino acid is covalently attached to a monodisperse oligoethylene
glycol unit; wherein the monodisperse oligoethylene glycol unit
attached to the amino acid of the neuropeptide analog increases the
metabolic stability of the composition compared to the metabolic
stability of the neuropeptide without the covalently attached
monodisperse oligoethylene glycol unit.
2. The composition of claim 1, wherein the neuropeptide analog
comprises at least one of a galanin analog, a neuropeptide Y
analog, a neurotensin analog, and a somatostatin analog.
3. The composition of claim 1, wherein the monodisperse
oligoethylene glycol unit attached to the amino acid of the
neuropeptide analog increases the metabolic stability of the
composition by increasing the half-life of the composition compared
to the half-life of a composition comprising the neuropeptide
without the covalently attached monodisperse oligoethylene glycol
unit.
4. The composition of claim 1, wherein the monodisperse
oligoethylene glycol unit comprises at least 2 ethylene glycol
repeats.
5. The composition of claim 1, wherein the monodisperse
oligoethylene glycol unit comprises at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, and 48 ethylene glycol repeats.
6. The composition of claim 1, wherein the neuropeptide analog is a
truncated neuropeptide.
7. The composition of claim 1, wherein the neuropeptide analog
comprises at least one terminal lysine.
8. The composition of claim 1, wherein the monodisperse
oligoethylene glycol unit is covalently linked to a lysine amino
acid.
9. The composition of claim 1, wherein the neuropeptide analog is a
truncated neuropeptide analog comprising at least 1, 2, 3, 4, or 5
terminal lysine residues.
10. The composition of claim 9, wherein the neuropeptide analog
comprises 4 terminal lysine residues and wherein the monodisperse
oligoethylene glycol unit is covalently attached to at least one of
the 4 terminal lysine residues.
11. The composition of claim 9, wherein the neuropeptide analog
comprises 5 terminal lysine residues and wherein the monodisperse
oligoethylene glycol unit is covalently attached to at least one of
the 5 terminal lysine residues.
12. A composition according to claim 7, wherein the monodisperse
oligoethylene glycol unit is covalently attached to the final
terminal lysine residue.
13. A composition according to claim 7, wherein the monodisperse
oligoethylene glycol unit is covalently attached to the penultimate
terminal lysine residue.
14. The composition of claim 1, wherein the neuropeptide analog is
selected from at least one of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, and 19.
15. The composition of claim 1, wherein the neuropeptide analog has
at least 95% homology with at least one of SEQ ID NO: 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19.
16. The composition of claim 1, wherein the composition has an
increased metabolic stability of at least 1%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a composition
comprising the wild-type neuropeptide.
17. The composition of claim 1, wherein the composition is
administered to a subject for the treatment of pain.
18. The composition of claim 1, wherein the composition does not
cross the blood brain barrier of a subject.
19. The composition of claim 1, wherein the monodisperse
oligoethylene glycol unit is a monodisperse polyethylene glycol
unit.
20. The composition of claim 1, wherein the neuropeptide analog is
N-methylated.
21. A method of making a neuropeptide analog with increased
metabolic stability, the method comprising: providing a
neuropeptide; covalently attaching at least one monodisperse
oligoethylene glycol unit to at least one amino acid of the
neuropeptide, thereby making a neuropeptide analog; wherein the
covalently attached monodisperse oligoethylene glycol unit
increases the metabolic stability of the neuropeptide analog in
comparison to the neuropeptide without the covalently attached
monodisperse oligoethylene glycol unit; and thereby making a
neuropeptide analog with increased metabolic stability.
22. The method of claim 21, wherein the neuropeptide analog
comprises at least one of a galanin analog, a neuropeptide Y
analog, a neurotensin analog, and a somatostatin analog.
23. The method of claim 21, wherein the neuropeptide analog
comprises at least one of a truncated galanin analog, a truncated
neuropeptide Y analog, a truncated neurotensin analog, and a
truncated somatostatin analog.
24. The method of claim 21, wherein the monodisperse oligoethylene
glycol unit covalently attached to the amino acid of the
neuropeptide analog increases the metabolic stability of the
composition by increasing the half-life of the composition compared
to the half-life of a composition comprising the neuropeptide
without the covalently attached monodisperse oligoethylene glycol
unit
25. The method of claim 21, wherein the neuropeptide analog is a
truncated neuropeptide analog comprising at least one terminal
lysine.
26. The method of claim 21, wherein the neuropeptide analog is
selected from at least one of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, and 19.
27. The method of claim 21, wherein the monodisperse oligoethylene
glycol unit comprises at least 2 ethylene glycol repeats.
28. The method of claim 21, wherein the monodisperse oligoethylene
glycol unit is covalently linked to a lysine amino acid.
29. The method of claim 21, wherein the neuropeptide analog has an
increased metabolic stability of at least 1%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the neuropeptide
without the covalently attached monodisperse oligoethylene glycol
unit.
30. The method of claim 21, wherein the neuropeptide analog is
administered to a subject for the treatment of pain.
31. The method of claim 21, wherein the neuropeptide analog does
not cross the blood brain barrier of a subject.
32. The method of claim 21, wherein the monodisperse oligoethylene
glycol unit is a monodisperse polyethylene glycol unit.
33. A method of making a neuropeptide analog that does not cross
the blood brain barrier, the method comprising: providing a
neuropeptide; covalently attaching at least one monodisperse
oligoethylene glycol unit to at least one amino acid of the
neuropeptide, thereby making a galanin analog; wherein the
covalently attached monodisperse oligoethylene glycol unit prevents
the galanin analog from crossing the blood brain barrier.
34. The method of claim 33, wherein covalently attaching at least
one monodisperse oligoethylene glycol unit to the at least one
amino acid of the neuropeptide analog comprises replacing a
lipoamino acid with the monodisperse oligoethylene glycol unit.
35. The method of claim 33, wherein the neuropeptide is at least
one of galanin, neuropeptide Y, somatostatin, and neurotensin.
36. A method of treating pain in a subject, the method comprising:
administering to the subject a pharmaceutically effective amount of
a compound comprising a neuropeptide analog, wherein the
neuropeptide analog comprises at least one amino acid covalently
attached to a monodisperse oligoethylene glycol unit.
37. The method of claim 36, wherein the neuropeptide analog does
not cross the blood brain barrier.
38. The method of claim 36, wherein the neuropeptide analog is
selected from at least one of SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, and 19.
39. The method of claim 36, wherein the neuropeptide analog has at
least 95% homology to at least one of SEQ ID NO: 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, and 19.
40. The method of claim 36, wherein the neuropeptide analog has an
increased metabolic stability of at least 1%, 5%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the neuropeptide
without the covalently attached monodisperse oligoethylene glycol
unit.
41. The method of claim 36, wherein the neuropeptide analog
comprises at least one of a galanin analog, a neuropeptide Y
analog, a neurotensin analog, and a somatostatin analog.
42. The method according to claim 36, wherein the monodisperse
oligoethylene glycol unit attached to the amino acid of the
neuropeptide analog increases the metabolic stability of the
composition by increasing the half-life of the composition by at
least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%
compared to the half-life of a composition comprising the
neuropeptide without the covalently attached monodisperse
oligoethylene glycol unit.
43. The method of claim 36, wherein the neuropeptide analog is a
truncated neuropeptide analog comprising at least one terminal
lysine residue.
44. The method of claim 43, wherein the neuropeptide analog is a
truncated neuropeptide analog comprising at least 1, 2, 3, 4, or 5
terminal lysine residues.
45. The method of claim 44, wherein the neuropeptide analog
comprises 4 terminal lysine residues and wherein the monodisperse
oligoethylene glycol unit is covalently attached to at least one of
the 4 terminal lysine residues.
46. The method of claim 44, wherein the neuropeptide analog
comprises 5 terminal lysine residues and wherein the monodisperse
oligoethylene glycol unit is covalently attached to at least one of
the 5 terminal lysine residues.
47. The method of claim 43, wherein the monodisperse oligoethylene
glycol unit is covalently attached to the final terminal lysine
residue.
48. The method of claim 43, wherein the monodisperse oligoethylene
glycol unit is covalently attached to the penultimate terminal
lysine residue.
49. The method of claim 36, wherein the monodisperse oligoethylene
glycol unit comprises at least 2 ethylene glycol repeats.
50. The method of claim 49, wherein the monodisperse oligoethylene
glycol unit comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,
47, and 48 ethylene glycol repeats.
51. The method of claim 36, wherein the monodisperse oligoethylene
glycol unit is a monodisperse polyethylene glycol unit.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0001] FIG. 1 shows the structures of analogs of MEGylated
neuropeptide analogs according to the present description.
[0002] FIG. 2 shows one embodiment of the synthesis of neuropeptide
analogs according to the present description.
[0003] FIG. 3 shows another embodiment of the synthesis of
neuropeptide analogs according to the present description.
[0004] FIG. 4 shows the structure of the MEGylated galanin analog
Gal-93, prepared according to the present description.
[0005] FIG. 5 is graph showing the analgesic activity of the
galanin analog Gal-93 demonstrated by the mouse abdominal
constriction assay.
[0006] FIG. 6 is graph showing the analgesic activity of the
neuropeptide Y analog NPY-B42 demonstrated by the mouse abdominal
constriction assay.
[0007] FIG. 7 is a graph showing the analgesic activity of the
galanin analog Gal-93 demonstrated by the rat partial sciatic nerve
ligation assay.
[0008] FIG. 8 is a graph displaying the analgesic activity of the
neuropeptide Y analog NPY-B42 demonstrated by the rat partial
sciatic nerve ligation assay.
[0009] FIG. 9 displays the results of the mouse carrageenan assay
using the neuropeptide analog Gal-93.
[0010] FIG. 10 displays the results of the mouse carrageenan assay
using the neuropeptide analog NPY-B42.
DETAILED DESCRIPTION
[0011] Neuropeptide analogs are described herein. In certain
examples, neuropeptide analogs such as analogs of galanin,
neuropeptide Y, somatostatin, and neurotensin are described herein.
In other particular embodiments, the analogs described herein
exhibit favorable pharmacological characteristics. For example, in
certain such embodiments, the neuropeptide analogs described herein
are metabolically stable. In other such embodiments, neuropeptide
analogs exhibit activity in the peripheral nervous system when
administered systemically, but do not show significant activity in
the central nervous system. In still other embodiments,
neuropeptide analogs described herein do not exhibit cardiovascular
toxicity. In yet further such embodiments, the neuropeptide analogs
described herein provide an analgesic effect. In specific
embodiments, neuropeptide analogs disclosed herein exhibit one or
all of the following characteristics: metabolic stability; activity
in the peripheral nervous system when administered systemically
combined with a lack of measureable activity in the central nervous
system; a lack of cardiovascular toxicity; and an analgesic effect.
In specific embodiments, neuropeptide analogs described herein
comprise at least one amino acid attached to a monodisperse
oligoethylene glycol unit (i.e., a MEGylated amino acid, or
MEG-AA).
[0012] In addition to neuropeptide analogs, compositions and
methods including such analogs are described herein. For example,
in particular embodiments, analgesic compositions including one or
more neuropeptide analog according to the present description are
provided, and methods of using such analgesic compositions are
described herein. In particular embodiments, methods of treating
pain are provided, with such methods including administering a
therapeutically effective amount of an analgesic composition
comprising a neuropeptide analog as described herein to a subject
in need thereof.
[0013] It is understood that when combinations, subsets,
interactions, groups, etc. of these compositions and methods are
disclosed, that while specific reference of each various individual
and collective combinations and permutation of these compositions
may not be explicitly disclosed, each is specifically contemplated
and described herein. For example, if a polypeptide is disclosed
and discussed and a number of modifications that can be made to a
number of molecules including the polypeptide are discussed, each
and every combination and permutation of polypeptide and the
modifications that are possible are specifically contemplated
unless specifically indicated to the contrary. Thus, if a class of
molecules A, B, and C are disclosed as well as a class of molecules
D, E, and F and an example of a combination molecule, A-D is
disclosed, then even if each is not individually recited, each is
individually and collectively contemplated. Thus, in this example,
each of the combinations. A-E, A-F, B-D, B-E, B-F, C-D, C-E, and
C-F are specifically contemplated and should be considered
disclosed from disclosure of A, B, and C; D, E, and F; and the
example combination A-D. Likewise, any subset or combination of
these is also specifically contemplated and disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E are specifically
contemplated and should be considered disclosed from disclosure of
A, B, and C; D, E, and F; and the example combination A-D. This
concept applies to all aspects of this application including, but
not limited to, steps in methods of making and using the disclosed
compositions. Thus, if there are a variety of additional steps that
can be performed it is understood that each of these additional
steps can be performed with any specific embodiment or combination
of embodiments of the disclosed methods, and that each such
combination is specifically contemplated and should be considered
disclosed.
[0014] It is understood that the disclosed methods and compositions
are not limited to the particular methodology, protocols, and
reagents described, as these may vary. It is also to be understood
that the terminology used herein is for the purpose of describing
particular embodiments only, and is not intended to limit the scope
of the present invention which will be limited only by the appended
claims. Unless defined otherwise, all technical and scientific
terms used herein have the meanings that would be commonly
understood by one of skill in the art in the context of the present
specification.
[0015] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a peptide" includes a plurality of such
peptides; reference to "the peptide" is a reference to one or more
peptides and equivalents thereof known to those skilled in the art,
and so forth.
[0016] "Optional" or "optionally" means that the subsequently
described event, circumstance, or material may or may not occur or
be present, and that the description includes instances where the
event, circumstance, or material occurs or is present and instances
where it does not occur or is not present.
[0017] Ranges can be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint. It is
also understood that there are a number of values disclosed herein,
and that each value is also herein disclosed as "about" that
particular value in addition to the value itself. For example, if
the value "10" is disclosed, then "about 10" is also disclosed. It
is also understood that when a value is disclosed that "less than
or equal to" the value, "greater than or equal to the value" and
possible ranges between values are also disclosed, as appropriately
understood by the skilled artisan. For example, if the value "10"
is disclosed the "less than or equal to 10" as well as "greater
than or equal to 10" is also disclosed. It is also understood that
the throughout the application, data is provided in a number of
different formats, and that this data, represents endpoints and
starting points, and ranges for any combination of the data points.
For example, if a particular data point "10" and a particular data
point 15 are disclosed, it is understood that greater than, greater
than or equal to, less than, less than or equal to, and equal to 10
and 15 are considered disclosed as well as between 10 and 15. It is
also understood that each unit between two particular units are
also disclosed. For example, if 10 and 15 are disclosed, then 11,
12, 13, and 14 are also disclosed.
[0018] As the terms are used herein, "protein" and "peptide" are
used simply refer to polypeptide molecules generally and are not
used to refer to polypeptide molecules of any specific size, length
or molecular weight. Protein variants and derivatives are well
understood to those of skill in the art and can involve amino acid
sequence modifications. Amino acid substitutions may include one or
more residues and can occur at a number of different locations at
once. Substitutions, deletions, insertions or any combination
thereof may be combined to arrive at a final construct.
Substitutional variants are those in which at least one residue has
been removed and a different residue inserted in its place.
[0019] It is understood that, as discussed herein, the use of the
terms "homology" and "identity" mean the same thing as
"similarity." Thus, for example, if the use of the word homology is
used between two sequences it is understood that this is not
necessarily indicating an evolutionary relationship between these
two sequences, but rather refers to the percent similarity or
relatedness between their nucleic acid sequences. For example, a
peptide may have at least approximately 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, and 99% homology with a reference amino acid
sequence. Many of the methods for determining homology between two
evolutionarily related molecules are routinely applied to any two
or more nucleic acid sequences or amino acid sequences for the
purpose of measuring sequence identity or similarity, regardless of
whether such molecules are evolutionarily related.
[0020] It is understood that one way to define the, analogs,
variants, and derivatives of the MEGylated neuropeptide analogs
disclosed herein is through defining the analogs, variants, and
derivatives in terms of identity to specific known, native, and
unmodified peptide sequences or their analogs not containing
MEG-AA. Disclosed herein are neuropeptide analogs having at least
40, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
or 99 percent identity to a reference amino acid sequence or the
native amino acid sequence, such as, for purposes of example only,
an unmodified galanin polypeptide sequence (e.g., SEQ ID NO: 1),
and wherein the neuropeptide analog comprises at least one, at
least two, at least three, at least four, at least five, or at
least six or more of any of the substitutions, deletions,
additions, or extensions disclosed herein.
[0021] Methods of calculating percentage identity of one or more
nucleotide or polypeptide sequences are known by those of skill in
the art. For example, the percent identity can be calculated after
aligning the two sequences so that the identity is at its highest
level. Another way of calculating sequence similarity or identity
can be performed by published algorithms. Optimal alignment of
sequences for comparison may be conducted by the local homology
algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by
the homology alignment algorithm of Needleman and Wunsch, J. Mol.
Biol. 48: 443 (1970), by the search for similarity method of
Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988),
by computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection.
[0022] The same types of identity and similarity can be obtained
for nucleic acids by for example the algorithms disclosed in Zuker,
M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci.
USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306,
1989, which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used, and that in certain
instances, the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity, and be disclosed herein.
[0023] Substantial changes in peptide function or immunological
identity may be made by selecting amino acid substitutions that
differ in their effect on maintaining, for example, (a) the
structure of the polypeptide backbone in the area of the
substitution, for example as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site or
(c) the bulk of the side chain. The substitutions which may produce
changes in the protein properties can include those in which (a) a
hydrophilic residue, e.g. seryl or threonyl, is substituted for (or
by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,
valyl or alanyl; (b) a cysteine or proline is substituted for (or
by) any other residue; (c) a residue having an electropositive side
chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or
by) an electronegative residue, e.g., glutamyl or aspartyl; or (d)
a residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) one not having a side chain, e.g., glycine,
in this case, (e) by increasing the number of sites for sulfation
and/or glycosylation.
[0024] As this specification discusses various proteins and protein
sequences, it is understood that the nucleic acids that can encode
those protein sequences are also disclosed. This would include all
degenerate sequences related to a specific protein sequence, i.e.,
all nucleic acids having a sequence that encodes one particular
protein sequence as well as all nucleic acids, including degenerate
nucleic acids, encoding the disclosed variants and derivatives of
the protein sequences. Thus, while each particular nucleic acid
sequence may not be written out herein, it is understood that each
and every sequence is in fact disclosed and described herein
through the disclosed protein sequence.
[0025] It is understood that there are numerous amino acid and
peptide analogs which can be incorporated into the disclosed
compositions. For example, there are numerous D amino acids or
amino acids which have a different functional substituent. The
opposite stereo isomers of naturally occurring peptides are
disclosed, as well as the stereo isomers of peptide analogs. These
amino acids can readily be incorporated into polypeptide chains by
charging tRNA molecules with the amino acid of choice and
engineering genetic constructs that utilize, for example, amber
codons, to insert the analog amino acid into a peptide chain in a
site specific way (Thorson et al., Methods in Molec. Biol. 77:43-73
(1991), Zoller, Current Opinion in Biotechnology, 3:348-354 (1992);
Ibba, Biotechnology & Genetic Engineering Reviews 13:197-216
(1995), Cahill et al., TIBS, 14(10):400-403 (1989); Benner, TIB
Tech, 12:158-163 (1994); Ibba and Hennecke, Bio/technology,
12:678-682 (1994) all of which are herein incorporated by reference
at least for material related to amino acid analogs).
[0026] D-amino acids can be used to generate more stable peptides,
because D-amino acids are not recognized by peptidases and such.
Systematic substitution of one or more amino acids of a consensus
sequence with a D-amino acid of the same type (e.g., D-lysine in
place of L-lysine) can be used to generate more stable peptides.
Cysteine residues can be used to cyclize or attach two or more
peptides together. This can be beneficial to constrain peptides
into particular conformations. (Rizo and Gierasch Ann. Rev.
Biochem. 61:387 (1992), incorporated herein by reference).
[0027] The term "neuropeptide" as used herein is used to refer to
several types of polypeptide molecules found in neural tissues
including those found in the brain, the spinal cord, and the gut.
Neuropeptides are involved in many nerve functions including
analgesia, nociception, waking and sleep regulation, cognition,
feeding, regulation of mood, and regulation of blood, etc. Examples
of specific neuropeptides are galanin, neuropeptide Y, neurotensin,
and somatostatin. Galanin is a 30-amino acid neuropeptide encoded
by the GAL gene and is expressed in the CNS and other tissues of
humans and other mammals (see, e.g., SEQ ID NO: 1). Neuropeptide Y
is a 36-amino acid neuropeptide encoded by the NPY gene and found
in many tissues of the body including the nervous system (see e.g.,
SEQ ID NO: 2). Neurotensin is a 13-amino acid neuropeptide found in
the nervous system and the gut (see, e.g., SEQ ID NO: 3).
Somatostatin is a neuropeptide with a 14-amino acid form and is
expressed in the nervous system and the gut (see, e.g., SEQ ID NO:
4).
[0028] The neuropeptide analogs described herein have at least one
MEGylated amino acid. A MEGylated amino acid, as used herein,
denotes the attachment of at least one monodisperse oligoethylene
glycol unit to an amino acid side chain of a peptide. In certain
embodiments, one or more amino acids included in the reference
amino acid sequence of a neuropeptide are substituted with a
MEGylated amino acid. In some embodiments, at least one amino acid
included in the reference amino acid sequence of the neuropeptide
is modified such that it is covalently linked with one or more
monodisperse oligoethylene glycol units. Examples of neuropeptides
and analogs thereof that may be MEGylated as disclosed herein may
be found in U.S. Patent Application Publication No. US
2009/0281031, the entirety of which is incorporated herein by
reference. More specifically, examples of neuropeptides that may be
MEGylated as disclosed herein comprise galanin, neuropeptide Y,
neurotensin, and somatostatin.
[0029] The process of MEGylation as described herein is the process
of covalently attaching a monodispersed oligoethylene glycol to an
amino acid of a peptide. MEGylation as used herein is also meant to
include PEGylation. In one embodiment, MEGylation as disclosed
herein may include the attachment of one or more monodispersed
polyethylene glycol (MPEG) units to one or more amino acids in a
peptide. In certain embodiments, the MEGylation process disclosed
herein is similar to the process of PEGylation, a process that is
well known by those of skill in the art.
[0030] The at least one monodisperse oligoethylene glycol unit used
to form a MEGylated amino acid, as included in the neuropeptide
analogs disclosed herein, includes 2 or more ethylene glycol
repeats. In one embodiment, a neuropeptide analog according to the
present disclosure may include one or more amino acids having a
monodisperse oligoethylene glycol unit comprising at least 2 to 48
ethylene glycol repeats. In one such embodiment, a neuropeptide
analog may include one or more MEGylated amino acids, wherein each
MEGylated amino acid comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45 46, 47, and 48 or more ethylene glycol repeats. In another
embodiment, the neuropeptide analogs disclosed herein may comprise
a MEGylated amino acid having a monodisperse oligoethylene glycol
unit comprising from 2 to 48 monodispersed polyethylene glycol
(MPEG.sub.n=2-48) repeats. In one such embodiment, a neuropeptide
analog as described herein may comprise at least one MEGylated
amino acid having a monodisperse polyethylene glycol unit that
includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, and 48
MPEG repeats.
[0031] A neuropeptide analog as provided herein may be MEGylated at
one or more of any of its amino acid positions. In one embodiment,
a galanin analog as provided herein may be MEGylated at one or more
of the amino acid positions of the galanin neuropeptide. In a
particular embodiment, the galanin analog may be MEGylated at any
one of amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
and 30, as numbered from N- (amino) terminus to C- (carboxy)
terminus of the galanin analog. In certain embodiments, a
neurotensin analog as provided herein may be MEGylated at any one
of the amino acid positions of the neurotensin analog. More
specifically, the neurotensin analog may be MEGylated at any one of
amino acid positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13.
In another embodiment, a neuropeptide Y analog as provided herein
may be MEGylated at any one of the amino acid positions of the
neuropeptide Y analog. In one such embodiment, the neuropeptide Y
analog may be MEGylated at any one of amino acid positions 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36. In
yet another embodiment, a somatostatin analog, such as the native
peptide or its non-natural analogs, for example octreotide, may be
MEGylated at one or more of the amino acid positions of the
somatostatin neuropeptide. In a particular embodiment, the
somatostatin analog may be MEGylated at any one of amino acid
positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.
[0032] In certain embodiments, the neuropeptide analogs as
disclosed herein may be MEGylated at one or more amino acids
located in the C-terminus, the N-terminus, or optionally in the
C-terminus and the N-terminus of the neuropeptide. In particular
embodiments, the MEGylated neuropeptide analog may comprise a
full-length peptide or, alternatively, a truncated peptide, wherein
any one of the amino acids in the peptide may be MEGylated.
[0033] In one embodiment, a truncated galanin analog, such as, by
way of example only, the galanin analog of SEQ ID NO: 5 can be used
with the compositions and methods disclosed herein. In another
embodiment, a truncated galanin analog may comprise a Gly.sup.1
residue that has been replaced by N-methyl-Gly (sarcosine, SAR).
The N-methylation of Gly.sup.1 may protect the peptide from
accelerated proteolytic degradation from the N-terminus, thereby
improving the metabolic stability of the galanin analog. In another
embodiment, a truncated galanin analog, such as a galanin analog as
described herein, such as, by way of example only, the galanin
analog of SEQ ID NO: 6, may comprise a C-terminal extension or
addition.
[0034] In one embodiment, the MEGylated neuropeptide analogs as
disclosed herein may include one or more terminal lysine (Lys),
homo-Lys, and/or ornithine amino acids. In certain embodiments, the
MEGylated neuropeptide analogs described herein include one or more
terminal Lys amino acids. In one such embodiment, the neuropeptide
analog may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Lys
amino acids at the C-terminus of the galanin analog. In another
such embodiment of a galanin analog as described herein, the one or
more terminal Lys amino acids may comprise a monodispersed
oligoethylene glycol unit covalently attached to the one or more
terminal Lys amino acids.
[0035] In certain embodiments, the neuropeptide analog disclosed
herein is a MEGylated galanin analog including at least 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10 or more Lys amino acids at the C-terminus of
the galanin analog. In one such embodiment, the one or more
terminal Lys amino acids may comprise a monodispersed oligoethylene
glycol unit covalently attached to the one or more terminal Lys
amino acids.
[0036] In other certain embodiments, the neuropeptide analog
disclosed herein is a MEGylated neuropeptide Y analog including at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Lys amino acids at
the N-terminus of the neuropeptide Y analog. In one such
embodiment, the one or more terminal Lys amino acids may comprise a
monodispersed oligoethylene glycol unit covalently attached to the
one or more terminal Lys amino acids (see, e.g., SEQ ID NO: 17 in
FIG. 1).
[0037] In still other certain embodiments, the neuropeptide analog
disclosed herein is a MEGylated neurotensin analog including at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Lys amino acids at
the C-terminus of the galanin analog. In one such embodiment, the
one or more terminal Lys amino acids may comprise a monodispersed
oligoethylene glycol unit covalently attached to the one or more
terminal Lys amino acids (see, e.g., SEQ ID NO: 18 in FIG. 1).
[0038] Also disclosed herein are neuropeptide analogs comprising
amino acid substitutions and additions, wherein the substitution or
addition is of a naturally or non-naturally occurring substance.
Examples include, but are not limited to, sarcosine (Sar),
diaminobutyric acid (DAB), diaminopropionic acid (DAP),
Lys-palmityoyl (Lys-Palm), Lys-.alpha.-Linolenic acid
(Lys-.alpha.-Lnn), Chloro-phe, aminohexanoic acid (AHX),
perfluorohexanoic acid (PerFHX), 8-amino-3,6,-dioxaoctanic acid,
oligo-Lys, and tert-leucine.
[0039] In particular embodiments, the neuropeptide analogs
disclosed herein are metabolically stable. As used herein, the
terms "metabolic stability" and "metabolically stable" refer to a
neuropeptide analog that is more resistant to degradation and has a
longer circulating half-life when compared with a reference
sequence, the wild type peptide, non-altered, unmodified, or native
peptide, or with a control composition. For example, the rate of
increased metabolic stability, as measured by half-life in serum or
in vitro, can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,
76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275,
300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700,
750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500,
2750, 3000, 3500, or 4000 percent when compared with the control,
unmodified, native, or wild-type neuropeptide or composition.
[0040] In certain embodiments, MEGylated neuropeptide analogs as
disclosed herein exhibit activity in the peripheral nervous system
but exhibit no measureable central nervous system activity or
penetration of the blood brain barrier. For example, in certain
embodiments, the neuropeptide analogs described herein exhibit
analgesic activity when administered systemically, while exhibiting
no measureable central nervous system activity or penetration of
the blood brain barrier. Without being bound by a particular
theory, it is presently thought that MEGylation of the amino acid
side chains of the neuropeptide analogs described herein prevents
the neuropeptide analog from crossing the blood brain barrier and
acting on the central nervous system. Therefore, such neuropeptide
analogs reduce or eliminate potential toxicity or side effects
associated with penetration into the CNS. In particular
embodiments, the ability of a peptide to penetrate the blood brain
barrier may be assessed using an in-vivo model of epilepsy.
[0041] Methods for producing the neuropeptide analogs described
herein are provided. For certain embodiments of the MEGylated
neuropeptide analogs described herein, the modification of amino
acids as disclosed herein can be introduced during solid-phase
peptide synthesis using an automated peptide synthesizer. In one
such embodiment, a method of producing the disclosed neuropeptide
analogs includes linking two or more peptides or polypeptides
together by protein chemistry techniques. For example, peptides or
polypeptides can be chemically synthesized using currently
available laboratory equipment using either Fmoc
(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)
chemistry (Applied Biosystems, Inc., Foster City, Calif.). One
skilled in the art can readily appreciate that a peptide or
polypeptide corresponding to the disclosed neuropeptide analogs,
for example, can be synthesized by standard chemical reactions. For
example, a peptide or polypeptide can be synthesized and not
cleaved from its synthesis resin whereas the other fragment of a
peptide or protein can be synthesized and subsequently cleaved from
the resin, thereby exposing a terminal group which is functionally
blocked on the other fragment. By peptide condensation reactions,
these two fragments can be covalently joined via a peptide bond at
their carboxyl and amino termini, respectively, to form a protein,
or fragment thereof. (Grant G A (1992) Synthetic Peptides: A User
Guide. W. H. Freeman and Co., N.Y. (1992); Bodansky M and Trost B.,
Ed. (1993) Principles of Peptide Synthesis. Springer-Verlag Inc.,
NY (which is herein incorporated by reference at least for material
related to peptide synthesis). Optionally, the peptide or
polypeptide may be independently synthesized in vivo. Once
isolated, these independent peptides or polypeptides may be linked
to form a peptide or fragment thereof via similar peptide
condensation reactions.
[0042] In another embodiment, the MEGylated neuropeptide analogs
may be synthesized according to enzymatic ligation of cloned or
synthetic peptide segments, thereby allowing relatively short
peptide fragments to be joined to produce larger peptide fragments,
polypeptides or whole protein domains (Abrahmsen L et al.,
Biochemistry, 30:4151 (1991)). Optionally, native chemical ligation
of synthetic peptides can be utilized to synthetically construct
large peptides or polypeptides from shorter peptide fragments. This
method consists of a two step chemical reaction (Dawson et al.
Synthesis of Proteins by Native Chemical Ligation. Science,
266:776-779 (1994)). The first step is the chemoselective reaction
of an unprotected synthetic peptide--thioester with another
unprotected peptide segment containing an amino-terminal Cys
residue to give a thioester-linked intermediate as the initial
covalent product. Without a change in the `reaction conditions,
this intermediate undergoes spontaneous, rapid intramolecular
reaction to form a native peptide bond at the ligation site
(Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et
al., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,
Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry
33:6623-30 (1994)).
[0043] Optionally, MEGylated neuropeptide analogs may be produced
according to the process wherein unprotected peptide segments are
chemically linked where the bond formed between the peptide
segments as a result of the chemical ligation is an unnatural
(non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)).
This technique has been used to synthesize analogs of protein
domains as well as large amounts of relatively pure proteins with
full biological activity (deLisle Milton R C et al., Techniques in
Protein Chemistry IV. Academic Press, New York, pp. 257-267
(1992)).
[0044] Analgesic compositions can be prepared to include one or
more neuropeptide analogs according to the present description. In
particular embodiments, the analgesic compositions described herein
are provided as pharmaceutical compositions, and can include, for
example, one or more MEGylated neuropeptide analogs as described
herein in combination with a pharmaceutically acceptable carrier.
In one such embodiment, an analgesic composition as disclosed
herein may include a MEGylated galanin analog. In another such
embodiment, an analgesic composition as disclosed herein may
include a MEGylated neuropeptide Y analog. In yet another such
embodiment, an analgesic composition as disclosed herein may
include a MEGylated neurotensin analog. In still another
embodiment, an analgesic composition as disclosed herein may
include a MEGylated somatostatin analog.
[0045] As used herein, the term "pharmaceutically acceptable"
refers to a material that is not biologically or otherwise
undesirable, i.e., the material may be administered to a subject,
without causing any undesirable biological effects or interacting
in a deleterious manner with any of the other components of the
pharmaceutical composition in which it is contained. Examples of
carriers suitable for administration to human and animal subjects
include solutions such as sterile water, saline, and buffered
solutions at physiological pH. The carrier would naturally be
selected to minimize any degradation of the one or more
neuropeptide analogs and to minimize any adverse side effects in
the subject. Pharmaceutically acceptable carriers, excipients and
diluents suitable for therapeutic use are well known in the
pharmaceutical art, and are described, for example, in Remington's
Pharmaceutical Sciences, Maack Publishing Co. (A. R. Gennaro (Ed.)
1985).
[0046] Administration of analgesic compositions as described herein
may be accomplished by any effective route, e.g., orally or
parenterally. Methods of parenteral delivery include, for example,
intra-arterial, subcutaneous, intramedullary, intravenous,
intramuscular, intrasternal, intracavernous, intrathecal,
intrameatal, intraurethral injection or infusion techniques, as
well as intranasal, sublingual, buccal, rectal, and vaginal
administration.
[0047] Analgesic compositions as described herein for oral
administration can be formulated using pharmaceutically acceptable
carriers well known in the art, in dosages suitable for oral
administration. Such carriers enable the analgesic compositions to
be formulated as tablets, pills, dragees, capsules, liquids, gels,
syrups, slurries, suspensions, etc., suitable for ingestion by a
subject.
[0048] Analgesic compositions for oral administration can be
obtained, for example, through combination of one or more
neuropeptide analog compounds with a solid excipient through, for
instance, known granulation processes for providing compositions
suitable for tableting or for inclusion in a capsule. In other
embodiments, analgesic compositions for oral administration as
described herein can be obtained, through combination of one or
more neuropeptide analog compounds with a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding suitable additional compounds, if
desired, to obtain tablets or dragee cores. Examples of excipients
suitable for formulating analgesic compositions for oral
administration include carbohydrate or protein fillers. Such
excipients include, but are not limited to: sugars, including
lactose, sucrose, mannitol, or sorbitol, starch from corn, wheat,
rice, potato, or other plants; cellulose such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
and gums including arabic and tragacanth; as well as proteins, such
as gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium
alginate. Where pharmaceutical formulations of the analgesic
compositions described herein are formulated using dragee cores,
such cores may be provided with suitable coatings, such as
concentrated sugar solutions, which may also contain, for example,
gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene
glycol, and/or titanium dioxide, lacquer solutions, and suitable
organic solvents or solvent mixtures.
[0049] In further embodiments, analgesic compositions suited for
oral administration can be formulated, for example, as push-fit
capsules made of gelatin, as well as soft, sealed capsules made of
gelatin and a coating such as glycerol or sorbitol. Push-fit
capsules can contain one or more analgesic compounds mixed with,
for example, filler or binders such as lactose or starches,
lubricants such as talc or magnesium stearate, and, optionally,
stabilizers. In soft capsules, the one or more neuropeptide analogs
may be dissolved or suspended in suitable liquids, such as fatty
oils, liquid paraffin, or liquid polyethylene glycol with or
without stabilizers.
[0050] Where the analgesic compositions are provided as
pharmaceutical compositions or dosage forms for oral
administration, such compositions may optionally include one or
more pharmaceutically acceptable sweetening agents, preservatives,
dyestuffs, flavorings, or any combination thereof. When the
composition is in the form of a solid, unit dosage form, such as a
tablet, the compositions may include a core formulation covered in
one or more of a protective, functional or cosmetic coating, as is
well known in the art. Moreover, in particular embodiments,
dyestuffs or pigments may be added to a dosage form for oral
administration or a coating included in or provided over such
dosage form for purposes of product identification or to
characterize the quantity of active compound (i.e., dosage).
[0051] In specific embodiments, analgesic compositions for
parenteral administration include one or more MEGylated
neuropeptide analog compounds. For injection, the analgesic
compositions described herein may be formulated in aqueous
solutions, preferably in physiologically compatible buffers such as
Hank's solution, Ringer's solution, or physiologically buffered
saline. Aqueous injection suspensions may contain substances, which
increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Additionally,
suspensions may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Optionally, where an
analgesic composition is formulated as a suspension, the
composition may also contain suitable stabilizers or agents, which
increase the solubility of one or more neuropeptide analog
compounds to allow for the preparation of highly concentrated
formulations.
[0052] Analgesic compositions according to the present description
may be manufactured according to techniques known in the art (e.g.,
by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes). In particular embodiments, the
analgesic compositions described herein may also be modified to
provide appropriate release characteristics, e.g., sustained
release or targeted release, by conventional means (e.g., through
the use of a functional coating and/or known matrices or materials
providing sustained or targeted release of active agents). After
the analgesic compositions described herein have been prepared,
they can be placed in an appropriate container and labeled for
use.
[0053] Methods of treating pain and other neurological disorders
are provided herein. In particular embodiments, the methods
described herein comprising administering a therapeutically
effective amount of an analgesic composition according to the
present description to a subject in need thereof. In certain
embodiments, the methods may further include the step of
identifying a subject in pain, identifying a subject at risk of
suffering pain or discomfort, or identifying a subject suffering
from a disease or disorder that causes pain or discomfort, such as,
for example, those described herein, followed by administering a
therapeutically effective amount of an analgesic composition
according to the present description.
[0054] The amount of an analgesic composition actually administered
in a given method will be dependent upon the individual to which
treatment is to be applied, the nature of the condition to be
treated, and the amount of neuropeptide analog compound material
included in the composition. The amount of analgesic composition
administered may be an optimized amount, such that a desired
therapeutic effect is achieved without an unacceptable level of
side-effects. With the benefit of the teachings provided herein,
determination of a therapeutically effective dose is well within
the capability of those skilled in the art. Of course, the skilled
person will realize that divided and partial doses are also within
the scope of the methods described herein.
[0055] Therapeutic efficacy and possible toxicity of analgesic
compositions described herein can be determined by standard
pharmaceutical procedures, in cell cultures or experimental animals
(e.g., ED.sub.50, the dose therapeutically effective in 50% of the
population; and LD.sub.50, the dose lethal to 50% of the
population). The dose ratio between therapeutic and toxic effects
is the therapeutic index and can be expressed as the ratio
ED.sub.50/LD.sub.50. Analgesic compositions which exhibit large
therapeutic indices may be selected for administration to subjects.
Data obtained from cell culture assays and animal studies may be
used in formulating a range of dosages for use in an intended
subject or class of subjects (e.g., humans). In particular
embodiments, the amount of an analgesic composition administered to
a subject provides a dose of the one or more neuropeptide analog
compounds that result in a circulating concentration that lies
within a range of circulating concentrations that include the
ED.sub.50, while exhibiting little or no toxicity. The dosage of a
given neuropeptide analog compound may vary within this range,
depending, for example, upon the dosage form employed, sensitivity
of the subject, and the route of administration selected.
[0056] Methods for treating pain as described herein include
methods of treating specific diseases and disorders that result in
or are associated with discomfort or pain. For example, the methods
described herein can be used to treat one or more diseases and
disorders selected from chronic back pain, spinal cord injuries,
peripheral nerve injuries, traumatic brain injuries,
neurodegenerative disorders, fibromyalgia, postherpetic neuralgia,
diabetic neuropathy, traumatic mononeuropathy, complex regional
pain syndrome, adjuvant analgesic, rhizotomy/nerve ablation,
preamptive analgesia/amputations, chemical exposure,
chemotherapy-induced neuropathy, cancer, opioid withdrawal, and
chronic neuropathic pain.
[0057] The methods and analgesic compositions disclosed herein can
be used in combination with other compositions or treatment
methods. As used herein, the phrase "in combination with" refers to
a method by which at least one or more compositions in addition to
the analgesic compositions as disclosed herein is administered to
the subject. In certain embodiments, therefore, a method involving
administration of a combination of compositions comprises,
administering an analgesic composition as described herein in
combination with at least one of the following: opioids and opioid
peptides, morphine, hydroxymorphine, fentanyl, oxycodone, codeine,
capsaicin, antiepileptic drugs (e.g., carbamazepine, primidone,
gabapentin, pregabalin, diazepam, felbamate, fluorofelbamate,
lamotrigine, lacosamide, levetiracetam, phenobarbital, phenyloin,
fos-phenyloin, topiramate, valproate, vigabatrin, zonisamide, and
oxcarbazepine), nonsteroidal anti-inflammatory drugs (NSAIDs),
local anesthetics (e.g., lidocaine), glutamate receptor
antagonists, NMDA antagonists, alpha-adrenoceptor agonists and
antagonists, adenosine, cannabinoids, NK-1 antagonist (e.g.,
CI-021), antidepressants (e.g., amitriptyline, desipramine,
imipramine), analogs and derivatives of galanin, somatostatin,
neurotensin, neuropeptide Y, delta-sleep inducing peptide,
enkephalins, oxytocin, cholecystikinin, calcitonin, cortistatin,
nociceptin and other neuropeptide-based therapeutics. In another
embodiment, the analgesic compositions as disclosed herein may be
administered to the subject in combination with two or more
additional compositions.
[0058] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
Examples
Example 1
[0059] Several MEGylated galanin analogs were chemically
synthesized using solid-phase peptide synthesis protocols. In this
example, two strategies were used to introduce MEGylated amino
acids into a truncated galanin analog. One strategy was to replace
a lipoamino acid with a MEGylated amino acid, as seen in Gal-BX
shown in FIG. 1. Another strategy included replacing an amino acid
with a MEGylated amino acid, as seen in Gal-B92 shown in FIG. 1.
Table 1 summarizes structures of example galanin-based analogs that
contain MEGylated amino acids that vary by the number of
monodispersed polyethylene glycol (MPEG.sub.n) repeats ranging from
4 to 24. As seen in Table 1, Sar is sarcosine, and N-Methyl-Trp is
N-methyl-tryptophan.
TABLE-US-00001 TABLE 1 Analog Sequence Gal-58
(Sar)WTLNSAGYLLGPKK(Lys-MPEG.sub.4)K-NH.sub.2 (SEQ ID NO: 5)
Gal-103 WTLNSAGYLLGPKK(Lys-MPEG.sub.4)K-NH.sub.2 (SEQ ID NO: 6)
Gal-104 (N-Methyl-Trp)TLNSAGYLLGPKK(Lys-MPEG.sub.4)K- NH.sub.2 (SEQ
ID NO: 7) Gal-50 (Sar)WTLNSAGYLLGPKK(Lys-MPEG.sub.12)K-NH.sub.2
(SEQ ID NO: 8) Gal-75
(Sar)WTLNSAGYLLGPKK(Lys-Pal)K(Lys-MPEG.sub.12)- NH.sub.2 (SEQ ID
NO: 9) Gal-93 WTLNSAGYLLGPKK(Lys-MPEG.sub.24)K-NH.sub.2 (SEQ ID NO:
10) Gal-B92 (Sar)WTLNSAGYLLGPKKKK(Lys-MPEG.sub.24)-NH.sub.2 (SEQ ID
NO: 11) Gal-B81 (Sar)WTLNSAGYLLGPKK(Lys-Pal)K(Lys-MPEG.sub.24)-
NH.sub.2 (SEQ ID NO: 12) Gal-91
WTLNSAGYLLGPKK(Lys-Pal)K(Lys-MPEG.sub.24)-NH.sub.2 (SEQ ID NO: 13)
Gal-100 (Sar)WTLNSAGYLLGPKK(Lys-.alpha.-Lnn)K(Lys-
MPEG.sub.24)-NH.sub.2 (SEQ ID NO: 14) Gal-105
WTLNSAGYLLGPKK(Lys-.alpha.-Lnn)K(Lys-MPEG.sub.24)- NH.sub.2 (SEQ ID
NO: 15) Gal-82 (Sar)WTLNSAGYLLGPKK(Lys-Pal)K(Lys-
(MPEG.sub.24-NH-PEG.sub.24))-NH.sub.2 (SEQ ID NO: 19)
[0060] Gal-58, Gal-93, Gal-103, and Gal-104 were synthesized with
preloaded Fmoc-Lys (Boc)-Clear Rink Amide resin and Fmoc-Lys
(Mmt)-OH was coupled to the peptide's second position. After
coupling with all the remaining amino acids, an Mmt-group was
removed by HAc/TFE/DCM (1:2:7), and then the resin was neutralized
by 10% DIEA/DCM, different MPEG-acids were coupled to give the
desired products.
[0061] The synthesis of Gal-75, Gal-78, Gal-B81, Gal-82, and Gal-91
was accomplished from the same intermediate, as shown in FIG. 2.
Weak acid liable TG Sieber resin was selected to conjugate PEG acid
on C-terminal or in peptide chain.
[0062] The synthesis of Gal-100 and Gal-105 involved stepwise
deprotection/conjugation methods as shown in FIG. 3.
.alpha.-Linolenic acid is a light and air sensitive unsaturated
fatty acid, so the conjugation of .alpha.-Linolenic acid was
designed in the last step. Aloc/Mmt orthogonal protecting groups
were selected to fulfill the modification of peptides with
PEGylation and lipidization. Since alloc-deprotection uses acetic
acid as scavenger, which will remove Mmt-simultaneously, Mmt needed
to be removed firstly. So the side chain of C-terminal lysine was
protected with Mmt-, the other Lysine at position 3 was protected
with Aloc-. After the peptide was synthesized on resin, the
Mmt-group was deprotected with HAc/TFE/DCM (1:2:7) and then
PEGylated with MPEG.sub.24-acid. Alloc was removed with Pd
(P(Ph.sub.3)).sub.4/HAc/NMM/DCM and then .alpha.-Linolenic acid was
conjugated to give the target product. In the cleavage of the
unsaturated fatty acids modified peptide (such as
.alpha.-Linolenyl), common cleavage reagents such as reagent K
without dithioethane (TFA/Thioanisole/PhOH/H.sub.2O) or
TFA/TIPS/H.sub.2O, all gave a large quantity of isomer, especially
with long cleavage times, probably attributing to the double bound
(cis/trans) isomers which can be induced at strong acid (TFA)
conditions. This isomerization can be inhibited at low temperatures
(0.degree. C)., but the peptides are not fully deprotected at this
condition. Therefore, room temperature was selected. Reagent K
without dithioethane (TFA/Thioanisole/PhOH/H.sub.2O 85/5/5/5) was
used as the cleavage recipe, cleavage time was monitored with HPLC.
The cleavage reaction was completed after 40 minutes, accompanied
with 30% isomer.
[0063] The general synthetic procedures for the galanin analogs
described herein are as follows: TG Sieber resin (0.19 meq) was
purchased from Novabiochem. m-dPEG.TM.-24 acid and
N-Fmoc-Amido-dPEG.TM.-24 acid were purchased from Quanta biodesign
Limited. Fmoc-N-methyl-Trp (Boc)-OH was purchased from Bachem Inc.
All other reagents were purchased from Chemimpex International
Inc.
[0064] Reactions were performed under N.sub.2 atmosphere, unless
otherwise indicated. Automatic solid phase peptide synthesis (SPPS)
was operated in a Symphony peptide synthesizer. Preparative HPLC
was performed on a Waters 600 pump system equipped with a Waters
2487 dual wavelength detector (Al) 220 nm, A2) 280 nm) and a
preparative Vydac diphenyl column (219TP101522). Analytical HPLC
used an analytical Vydac diphenyl column (219TP54). The HPLC mobile
phases are buffer A, 100% water (0.1% TFA), and buffer B, 90%
acetonitrile (0.1% TFA). MALDI-TOF MS was conducted at the
University of Utah Core Facility.
[0065] For the synthesis of Gal-58, 2-fold of Fmoc-Lys (Mmt)-OH was
manually coupled to the preloaded Lys (Boc)-Rink Amide Clear resin
by PyBop method. After coupling with all the remaining amino acids,
10 mL HAc/TFE/DCM (1:2:7) was added to the resin with shaking for 6
to 10 min to remove Mmt group. The deprotecting process was
monitored to check for the solution to change color from yellow to
clear. After neutralization with 10% DIEA/DCM, MPEG4-acid
(MeO(CH.sub.2CH.sub.2O).sub.4CH.sub.2COOH) was coupled to the resin
using PyBop method. The peptide was cleaved with Reagent K
(TFA-phenol-water-thioanisole-1,2-dithioethane 82.5:5:5:5:2.5),
precipitated out from MTBE and purified with preparative HPLC
(Vydac diphenyl column).
[0066] For Gal-93 and Gal-103, 2-fold of Boc-Trp (Boc)-OH was
coupled instead of Fmoc-Trp (Boc)-OH in the peptides synthesis,
following the same procedure as described in the synthesis of
Gal-58.
[0067] For Gal-104, 2-fold of Fmoc-N-methyl-Trp (Boc)-OH was
coupled manually for 24 h, and then Fmoc was removed with 20%
piperidine/NMP, following the same procedure as Gal-58.
[0068] For Gal-91 and Gal-92, Fmoc-Lys (Mmt)-OH was coupled to Rink
Amid clear resin, following by the same procedure as Gal-58.
[0069] For the general intermediate synthesis of Gal-75, Gal-78,
Gal-B81, and Gal-82, TG Sieber resin was selected for the flexible
modification on N-Lysine of galanin analogues. Fmoc-based PyBop
coupling protocols were used as previously described. 5-fold
Fmoc-amino acids/PyBop/DIEA (1:1:2, molar ratio) were applied in
peptide synthesis. Firstly, Fmoc-Lys (Mmt)-OH was coupled to Sieber
resin, followed by the coupling of all the remaining Fmoc-protected
amino acids. The N-terminal amino acid was coupled with Boc-capped
Sarcosine to facilitate Lysine side chain modification for the
synthesis of Gal-82, which uses Fmoc-protected PEG24 acid to
synthesize the PEG48 moiety. Fmoc-Lys (Palmitoyl)-OH was coupled
manually. After the coupling was finished, the Mmt-group of
N-Lysine at the C-terminal end was removed by the reagents
HAc/CF.sub.3CH.sub.2OH/CH.sub.2Cl.sub.2 (1:2:7) for 6.times.10 min.
The resin was neutralized with 10% DIEA in CH.sub.2Cl.sub.2 for 5
min, and then washed with CH.sub.2Cl.sub.2 to give the general
intermediate resin for the synthesis of Gal-75, Gal-78, Gal-B81,
and Gal-82.
[0070] Additionally, for Gal-81, 1.5-fold of m-dPEG.TM.-24
acid/PyBop/HOBt/DIEA (1:0.98:1:2) was added to Mmt-deprotected
resin with shaking for 24 h until ninhydrin test was negative. The
peptide was cleaved from the resin with Reagent K
(TFA-phenol-water-thioanisole-1,2-dithioethane 82.5:5:5:5:2.5) for
2 h. After evaporation of TFA, the residues were precipitated with
MTBE and purified by RP-HPLC to give the PEGylated galanin analogue
Gal-81. Gal-75 was made using the same method as described in
Gal-B81; however MPEG12-acid was coupled instead of m-dPEG.TM.-24
acid. Alternative methods for the syntheses of Gal-75 and Gal-81
including removing Mmt and cleaving the protected intermediate from
Sieber resin simultaneously with 1% TFA in 95% DCM/5% TES
(triethylsilane) as indicated in FIG. 2; then conjugating the
intermediate with dPEG-acids by PyBop method to get the final
target peptides, after neutralized with 10% DIEA in DCM.
[0071] For Gal-82, 1.5-fold of N-Fmoc-Amido-dPEG.TM.24
acid/PyBop/HOBt/DIEA (1:0.98:1:2) was added to Mmt-deprotected
resin for 24 h, and then Fmoc was removed. m-dPEG.TM.-24
acid/PyBop/HOBt/DIEA (1:0.98/1:2) was coupled to the resin,
following the same cleavage and purification method as Gal-B81 to
give Gal-82.
[0072] For Gal-100, 2-fold of Fmoc-Lys (Mmt)-OH was coupled
manually by PyBop method to Rink Amide Clear resin, followed with
the coupling of Fmoc-Lys (Boc)-OH and Fmoc-Lys (Aloc)-OH. After all
the remaining amino acids were coupled, HAc/TFE/DCM (1:2:7) was
added to the resin to remove the Mmt group, and then the resin was
neutralized with 10% DIEA/CH.sub.2Cl.sub.2, and m-dPEG.TM.-24 acid
was coupled using same method as disclosed for Gal-B81. Aloc was
deprotected with tetrakis(triphenylphosphine)palladium(0)
(Pd(PPh.sub.3).sub.4, 0.23 g, 0.2 mmol) in 2.78 mL
DCM/AcOH/N-methylmorpholine (NMM) for 2 h. The resin was then
washed with CH.sub.2Cl.sub.2, 0.5% DIEA/CH.sub.2Cl.sub.2 to remove
AcOH, 0.02 M sodium diethyldithiocarbamate solution in NMP and to
remove Palladium residues, CH.sub.2Cl.sub.2. .alpha.-Linolenic acid
was coupled for 20 h. 5 ml TFA/PhOH/Thioanisole/H.sub.2O (85/5/5/5)
was added to the resin under N.sub.2 and protected from light.
After 40 min, TFA was evaporated in vacuum and MTBE was added to
precipitate the products. The crude peptide was purified by
preparative RP-HPLC to give the target peptide Gal-100. For
Gal-105, the same synthetic method for Gal-100 was followed, only
Boc-Trp (Boc)-OH was used at the N-terminal amino acid.
Example 2
[0073] The galanin analog Gal-93 (SEQ ID NO: 10), as shown in FIG.
4, was tested in animal models for its affect on pain and epilepsy.
As shown in Table 2, the biological testing of Gal-93 revealed that
this analog had no apparent antiepileptic activity in the 6 Hz
model of epilepsy; however it displayed analgesic activities in
several pain models as disclosed herein. Under identical screening
conditions, the lipoamino acid-containing compound, Gal-B2 (not
shown), exhibited the anticonvulsant activity with 4/4 mice
protected at time points 30 min, 60 min and 120 min (Bulaj et al,
2008, J Med Chem, vol 51, p. 8038-8047).
TABLE-US-00002 TABLE 2 # of protected mice from seizures (from
groups of 4 mice), at a dose 4 mg/kg of Gal-93, following
intraperitoneal administration time 15 min 30 min 60 min 120 min
240 min # mice 0/4 0/4 0/4 0/4 0/4
[0074] Gal-93 and other MEGylated analogs were tested using the
formalin test of acute and chronic hyperalgesia in mice. A summary
of the results is presented in Table 3. The formalin test was
performed according to the method described by Tjolsen et al.
(Tjolsen A, Berge O G, Hunskaar S, Rosland J H, Hole K. The
Formalin Test: An Evaluation of the Method. Pain 53(2), 237; 1992).
More specifically, an injection of 0.5% formalin is made into the
plantar region of the right hind paw of a mouse. This elicits a
distinct behavioral profile in response to the formalin injection
characterized by the mouse licking the affected paw. The behavior
is characteristically biphasic in nature. For example, immediately
following the injection the mouse intensely licks the paw for
approximately 5-10 min. This initial behavior is considered phase 1
(acute) and is thought to be mediated primarily by chemical
activation of local C-fibers. The acute phase is followed by a
brief latent (usually <5 min) period where there is little or no
behavioral activity. A more prolonged (about 20 to 30 min) period
of licking then ensues which constitutes phase 2 of the response
(inflammatory). Prior to the administration of the test drug or
vehicle, each mouse undergoes a 15-min conditioning period in one
of several 6" tall plexiglass cages (4'' diameter) that are placed
in front of a mirror. It is in these tubes that mice are observed
for the licking activity for the duration of the experiment:
Following conditioning, the test substance is dosed i.p. after
which the mouse is returned to its home tube. At the TPE of the
test substance, formalin is injected sub-dermally into the plantar
surface of the right hind foot in a volume of 20 .mu.l with a 27
gauge stainless steel needle attached to a Hamilton syringe. The
bevel of the needle is placed facing down toward the skin
surface.
[0075] Following the injection of the formalin, each mouse was
observed for the first 2 min of 5-min epochs until 45 min had
elapsed since the administration of the test drug. The cumulative
length of licking for each 2-min time period was recorded. An
animal receiving the requisite volume of vehicle was alternated
with each mouse given the test peptide. Area under the curve (AUC)
and subsequent percent of control for drug-treated animal groups
(n=8) was determined using the GraphPad Prism Version 3.03. Total
AUC was calculated for both the test substance and control groups
for both the acute and inflammatory phases. The AUC for individual
animals for each phase was also calculated and converted to
percentage of total AUC of control. The average and S.E.M. for both
the drug treated and control percentages were then calculated and
tested for significant differences. The results showed that Gal-93
and Gal-81 significantly reduced the duration of licking,
suggesting analgesic activity, in both the acute and inflammatory
phases of the mouse formalin test. Gal-100, Gal104 and Gal105
significantly reduced the duration of licking, suggesting analgesic
activity, in the inflammatory phase of the mouse formalin test.
TABLE-US-00003 TABLE 3 AUC Analog AUC Inflam- Name Sequence Acute
matory Gal-103 WTLNSAGYLLGPKK(Lys-MPEG.sub.4)K-NH.sub.2 66.3 .+-.
75.6 .+-. (SEQ ID NO: 6) 14.6 8.9* Gal-81 (Sar)WTLNSAGYLLGPKK(Lys-
19.7 .+-. 2.9 .+-. palmitoyl)K(Lys-MPEG.sub.24) 7.2** 2.2** (SEQ ID
NO: 12) Gal-93 WTLNSAGYLLGPKKKK(Lys-MPEG.sub.24) 34.9 .+-. 49.6
.+-. (SEQ ID NO: 10) 6.6** 3.5** Gal-100
(Sar)WTLNSAGYLLGPKK(Lys-.alpha.-Lnn) K(Lys- 83.08 .+-. 33.78 .+-.
MPEG.sub.24) 13.34 3.04** (SEQ ID NO: 14) Gal-105
WTLNSAGYLLGPKK(Lys-.alpha.-Lnn)K(Lys- 108.3 .+-. 69.9 .+-.
MPEG.sub.24) 24.8 6** (SEQ ID NO: 15) *P < 0.05, **P < 0.01
compared with vehicle treated control mice
[0076] Table 4 summarizes a dose-response study of the analgesic
activity of Gal-93 in the rat formalin assay following intravenous
administration into the femoral vein of the rats. Prior to the
administration of formalin, each rat underwent a 30-min
conditioning period in one of several 30.5 cm tall plexiglass tubes
(15 cm diameter). Prior to placement in the plexiglass tubes, a
metal band was fitted on to the right hind leg and secured with a
drop of superglue as such, animals acclimate to both the tube and
the metal band. It is in these plexiglass cylinders that rats were
later observed for the flinching behavior that accompanies hind-paw
formalin injection. Following a 30 min conditioning period, 50
.mu.l of 2.5% formalin was injected sub-dermally into the plantar
surface of the right hind foot in a volume of 50 .mu.l using a 27
gauge stainless steel needle attached to a Hamilton syringe. The
bevel of the needle was placed facing down toward the skin surface.
Following the injection of the formalin each animal was placed in a
new plexiglass cylinder on top of a detection unit, and the
Automated Nociception Analyzer (Dept. of Anesthesiology, Univ.
California, San Diego) is initiated. The number of flinches was
collected for every minute for the duration of the 60 minute
experiment. In these studies Gal-93 was administered i.v. at 5
mg/kg and formalin was injected into the paw at 10 min, 30 min or
60 min following Gal-93 administration. The number of flinches
recorded over the 60 min following formalin injected was calculated
as area-under-the-curve (AUC) as described for the mouse formalin
assay. These studies showed a peak activity for Gal-93 at 60 min
following i.v. administration in the rat formalin test.
TABLE-US-00004 TABLE 4 Dose i.v. & Time of formalin
administration Acute Phase Inflammatory Phase Number of
post-treatment AUC AUC Rats 5 mg/kg, 10 min 64.5 .+-. 17.9 91.3
.+-. 6.5 2 5 mg/kg, 30 min 68.4 .+-. 8.6 70.8 .+-. 19.8 4 5 mg/kg,
60 min 34.3 .+-. 5.1 55.8 .+-. 11.0 2
Example 3
[0077] An acetic acid induced abdominal constriction (writhing)
assay was used to test the analgesic effect of neuropeptide
analogs. In this assay of chemical nociception, a 0.6% acetic acid
solution is injected into the peritoneal cavity of adult male CF-1
mice where it directly activates nociceptors and produces
inflammation of both the visceral (subdiaphragmatic) and
subcutaneous (muscle wall) tissues. This results in a
characteristic "writhing response" in the mouse involving
lengthwise stretching of the torso and concave arching of the back
and extensions of the hind-limbs (Jensen T S, Yaksh T L. Effects of
an intrathecal dopamine agonist, apomorphine, on thermal and
chemical evoked noxious responses in rats. Brain Res. 1984 Apr. 2;
296(2):285-93). The writhing behaviors were observed while the mice
were kept in 6'' tall plexiglass cages (4'' diameter).
[0078] Following a 15 minute conditioning period, the test
neuropeptide analog compound was administered intraperitoneally
(i.p.) and the mouse returned to its home tube. One hour after
injection of the test neuropeptide analog compound, the acetic acid
solution (0.6% v/v) is injected i.p. at a volume of 0.1 ml/10 g
body weight using a 1 ml syringe with a 26G 3/8 bevel needle.
Following the injection of the acetic acid, the total number of
abdominal constrictions was recorded over a 30 minute observation
period. An animal receiving an equivalent volume of vehicle was
observed side by side with the animal receiving the test compound.
One writhe is considered to have occurred with the adoption of the
typical posture and to have terminated upon resumption of a normal
posture. The average number of abdominal constrictions was compared
between groups using the Student's t-test comparison.
[0079] As shown in FIGS. 5 and 6, Gal-93 (SEQ ID NO: 10) and
NPY-B42 (SEQ ID NO: 17) reduced the number of abdominal
constrictions, as compared to vehicle alone, with a peak effect
post i.p. administration (4 mg/kg, n=3-4 per group) of 60 minutes
and 30 minutes, respectively.
Example 4
[0080] The rat partial sciatic nerve ligation was used as a model
of neuropathic pain. Briefly, a small incision is made unilaterally
in the upper thigh of anesthetized rats and approximately 1/3 to
1/2 of the sciatic nerve is tied off by passing a "taper by 130-4''
needle attached to size 8 nylon sutures through the nerve. This
ligation is performed dorsal to where the sciatic nerve bifurcates
and only a portion of the sciatic nerve is tied off to maintain
some motor response. After a 7 day recovery period, the rats are
tested for the development of consistent, mechanical allodynia
(pain response to a non-noxious stimulus). The animals are each put
in a bottomless plexiglass box placed on a 1/4'' wire mesh
(stainless steel or galvanized) platform. After at least a 30-min
conditioning period, a baseline mechanical sensitivity is
determined. This procedure is done by applying a series of
calibrated Von Frey fibers perpendicularly to the plantar surface
of each hind paw in between the pads or further back toward the
heel. The 50% threshold for foot withdrawal is determined by using
the step procedure. That is, after a positive response (withdrawal
of the foot) is noted a weaker fiber is applied. If there is no
recoil the next highest/stiffer/thicker fiber is again applied and
so forth. This is repeated for 5 steps.
[0081] Following the determination of the initial baseline
sensitivity the rats were given an i.p. injection of the
neuropeptide analog test compound and the mechanical threshold was
assessed at 1, 2, 4, 6, and 24 hrs post-injection to determine the
duration of action of the test compound and its time of peak
effect. The withdrawal threshold for each animal at each time point
was computed using the "xoxox" procedure (Chaplan S R, Bach F W,
Pogrel J W, Chung J M, Yaksh T L. Quantitative assessment of
tactile allodynia in the rat paw. J Neurosci Methods. 1994
53(1):55-63). The average and S.E.M., of the pre-drug withdrawal
threshold were calculated and compared to the average withdrawal
threshold of the group at each time point following drug treatment.
The average and S.E.M. for both the drug treated and control
percentages were calculated and tested for significant
difference.
[0082] As shown in FIG. 7, the mice treated with the galanin analog
Gal-93 had a higher 50% paw withdrawal threshold (PVVT) when
compared to the pre-drug untreated controls, with peak activity at
1 hr post i.p. administration of Gal-93 (2 mg/kg, n=4 per group).
As shown in FIG. 8, mice treated with NPY-B42 (SEQ ID NO: 17) had a
higher 50% PWT when compared to the pre-drug untreated controls,
with peak activity at 2 hr post i.p. administration of NPY-B42 (8
mg/kg, n=8 per group).
Example 5
[0083] The mouse carrageenan assay was used as a model of
chemically induced inflammatory pain. For this model, male CF-1
mice weighing 25-35 g were injected with 25 ul of carrageenan (2%
in 0.9% NaCI, lambda carrageenan) into the plantar surface of the
right hind paw. Latency to paw withdrawal was tested 3 h following
carrageenan administration. Briefly, mice are placed on a glass
surface heated to 30.degree. C. Radiant heat is applied to the
plantar surface of the paw, through the glass plate, until a
withdrawal of the paw from the glass surface occurs (Dirig et al.
1997, Hargreaves et al. 1988). Latency to paw withdrawal is
measured from the onset of heat application until a full paw
withdrawal occurs. Two measurements are taken from each paw
(injected and non-injected), with at least 1 min between
measurements, which are then averaged. The mean withdrawal latency
from the non-injected paw is subtracted from the
carrageenan-injected paw to obtain a withdrawal latency difference
for each animal. Experimental conditions, including animal
habituation, glass plate temperature, and thermal stimulus
intensity have been optimized such that withdrawal latency
differences for carrageenan-injected/vehicle-treated animals are
approximately 4 s.
[0084] Experimental neuropeptide analog compounds were dissolved in
1% Tween20/0.9% NaCI and administered via intraperitoneal injection
at varying doses 1 h (Gal-93) or 2 h (NPY-B42) prior to withdrawal
latency testing. The neuropeptide analog compounds were considered
to have full analgesic efficacy when the withdrawal latency
difference was zero. All data are presented as means.+-.standard
error. Comparisons between two means were performed using a
Student's t-test. Comparisons between multiple means were performed
using a one- or two-way ANOVA followed by a Newman-Keuls or
Bonferroni test, respectively.
[0085] As shown in FIG. 9, the galanin analog Gal-93 increased the
withdrawal latency difference when compared to the carrageenan
controls. The 6 mg/kg and higher doses of Gal-93 tested showed
significant reversal of the carrageenan induced hyperalgesia
relative to the controls. As shown in FIG. 10, the NPY-B42 analog
increased the withdrawal latency when compared to the carrageenan
controls. The 4 mg/kg dose of NPY-B42 showed significant reversal
of the carrageenan induced hyperalgesia relative to the
controls.
Example 6
[0086] In this example, the cardiovascular effects of neuropeptide
analog compounds were assessed following i.v. administration in
rats. Male Sprague-Dawley rats weighing between 250 and 350 g were
anesthetized and implanted with femoral vein and artery catheters.
On the following day, the arterial catheter was connected to a
pulse pressure transducer for continuous monitoring of blood
pressure (BP) and heart rate (HR). The venous catheter is connected
to a remote syringe for intravenous (iv) infusions. The galanin
analog compound Gal-93 was dissolved in 1% Tween 20/0.9% NaCI and
administered over approximately 1 min (0.25 mg/kg iv, 1 ml infusion
volume). Mean BP/HR samples were taken at baseline, dosing, and at
1, 5, 10, 20, 30, 40, 50, and 60 minutes after dosing. Prior to
dosing and 60 min after dosing, baroreceptor reflex sensitivity was
assessed by infusion of phenylephrine (9 ug, 0.05 ml infusion
volume), which elicits a BP increase of 40-60 mmHg and a
corresponding bradycardia of 50-150 beats/min (in vehicle-treated
animals). In addition, blood samples were collected from the
arterial catheter at baseline, 30 min, and 60 min after dosing for
the determination of hematocrit, plasma protein, and blood glucose.
Body temperature was also monitored, with samples taken at
baseline, 15 min, 30 min, and 60 min after dosing. Mean BP and HR
were obtained from 30-60 s digitized pulse pressure recording
segments at the previously mentioned time points. For determination
of baroreceptor reflex sensitivity, BP and HR were obtained at the
highest and lowest points, respectively, following phenylephrine
infusion.
[0087] The results of the cardiovascular safety evaluation of
Gal-93 and NPY-B42 (n=3 rats) are shown in Table 5. All data are
presented as means.+-.standard error. For blood pressure (BP) and
heart rate (HR) the maximal change observed during the time-points
(1-60 minutes) is recorded. Comparisons between the two means were
performed using a Student's t-test. Comparisons between multiple
means were performed using a one- or two-way ANOVA followed by a
Newman-Keuls or Bonferroni test, respectively. In these studies,
neither Gal-93 or NPY-B42 showed statistically significant
difference from vehicle treated rats with any of the cardiovascular
parameters measured.
TABLE-US-00005 TABLE 5 Phenylephrine post-treatment response
glucose HCT plasma protein temperature NAX# BP min HR max BP max HR
min 30 min 60 min 30 min 60 min 30 min 60 min 30 min 60 min Vehicle
111 .+-. 5 427 .+-. 10 158 .+-. 3 334 .+-. 8 81 .+-. 16 83 .+-. 13
40 .+-. 2 43 .+-. 3 6.4 .+-. 0.2 7.1 .+-. 0.2 37.6 .+-. 0.2 37.5
.+-. 0.1 GalBBB93 113 .+-. 3 455 .+-. 39 164 .+-. 7 268 .+-. 20 70
.+-. 16 61 .+-. 11 43 .+-. 2 37 .+-. 3 7.5 .+-. 0.1 6.8 .+-. 0.4
37.6 .+-. 0.2 37.2 .+-. 0.3 NPY-B42 108 .+-. 5 451 .+-. 14 161 .+-.
4 291 .+-. 26 78 .+-. 5 73 .+-. 4 42 .+-. 2 42 .+-. 1 6.5 .+-. 0.4
6.8 .+-. 0.3 37.2 .+-. 0.2 36.9 .+-. 0.4
Example 7
[0088] Metabolic stability assay: Peptide stability was assessed in
a rat serum assay. One mL of 25% rat serum was incubated at
37.degree. C. for 10 min, prior to addition of the analogs.
Reactions were prepared by adding each analog, dissolved in
nanopure H.sub.2O, to a solution containing 25% rat blood serum and
0.1 M Tris-HCI, pH 7.5 to a final peptide concentration of 20
.mu.M. At appropriate time intervals (ranging up to 8 h), 200 .mu.L
aliquots were withdrawn and added to 100 .mu.L "quenching solution"
(15% trichloroacetic acid in 40% isopropanol). Isopropanol (40%,
aqueous solution) was added to quenching mixture (this step
improved recovery of the Gal-B2 and other analogs). Upon
precipitation with the quenching mixture, the samples were
incubated at -20.degree. C. for 15 min and centrifuged at 12,000
rpm. The supernatant was analyzed using HPLC separation with an YMC
ODS-A.TM. M 5 .mu.m 120 .ANG. column (Waters, Cat#: AA12S052503WT).
In cases where analog peaks overlapped with peaks observed in the
"serum-only" control samples, the gradient was optimized by
changing the composition of the mobile phases, column temperature
or HPLC column (for example C.sub.8 rather than diphenyl column).
Recovery of the analogs was assessed by spiking "serum-only"
control samples after the trichloroacetic acid precipitation with
known amounts of the analog. Metabolic stability was assessed by
monitoring the disappearance of the analogs over a period of 8 h.
This was accomplished by comparison the area under the curve for
the peak corresponding to the intact analog at each time point.
Half-time, t.sub.1/2, for each analog was calculated using the
average of three independent experiments for each time point.
Results were plotted on a log-scale plot using the Kaleidagraph
software. Linear curve-fit analysis was used to fit the time-course
of the degradation of the analogs according to the following
formula: t.sub.1/2(h)=(Ln(50)-b)/(m), where "m" represents the
slope of the line and "b" is the y-intercept.
[0089] It will be obvious to those having skill in the art that
many changes may be made to the details of the above-described
embodiments without departing from the underlying principles of the
invention. The scope of the present invention should, therefore, be
determined only by the following claims.
Sequence CWU 1
1
19130PRTHomo sapiens 1Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu
Gly Pro His Ala Val 1 5 10 15 Gly Asn His Arg Ser Phe Ser Asp Lys
Asn Gly Leu Thr Ser 20 25 30 236PRTHomo sapiens 2Tyr Pro Ser Lys
Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala
Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 20 25 30
Arg Gln Arg Tyr 35 313PRTHomo sapiens 3Glx Leu Tyr Glu Asn Lys Pro
Arg Arg Pro Tyr Ile Leu 1 5 10 414PRTHomo sapiens 4Ala Gly Cys Lys
Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 1 5 10 517PRTArtificial
SequenceNeuropeptide analog 5Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys 616PRTArtificial
SequenceNeuropeptide analog 6Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Gly Pro Lys Lys Xaa Lys 1 5 10 15 716PRTArtificial
SequenceNeuropeptide analog 7Xaa Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Gly Pro Lys Lys Xaa Lys 1 5 10 15 817PRTArtificial
SequenceNeuropeptide analog 8Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys 918PRTArtificial
SequenceNeuropeptide analog 9Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys Xaa 1016PRTArtificial
SequenceNeuropeptide analog 10Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Gly Pro Lys Lys Xaa Lys 1 5 10 15 1118PRTArtificial
SequenceNeuropeptide analog 11Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Lys 1 5 10 15 Lys Xaa 1218PRTArtificial
SequenceNeuropeptide analog 12Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys Xaa 1317PRTArtificial
SequenceNeuropeptide analog 13Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Gly Pro Lys Lys Xaa Lys 1 5 10 15 Xaa 1418PRTArtificial
SequenceNueropeptide analog 14Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys Xaa 1517PRTArtificial
SequenceNeuropeptide analog 15Trp Thr Leu Asn Ser Ala Gly Tyr Leu
Leu Gly Pro Lys Lys Xaa Lys 1 5 10 15 Xaa 1617PRTArtificial
SequenceNeuropeptide analog 16Xaa Trp Thr Leu Asn Ser Ala Gly Tyr
Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys 1718PRTArtificial
SequenceNueropeptide analog 17Tyr Lys Lys Xaa Xaa Ala Arg His Tyr
Ile Asn Leu Ile Thr Arg Gln 1 5 10 15 Arg Tyr 188PRTArtificial
SequenceNeuropeptide analog 18Lys Xaa Lys Lys Pro Tyr Ile Leu 1 5
1918PRTArtificial SequenceNuropeptide analog 19Xaa Trp Thr Leu Asn
Ser Ala Gly Tyr Leu Leu Gly Pro Lys Lys Xaa 1 5 10 15 Lys Xaa
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