U.S. patent application number 15/710375 was filed with the patent office on 2018-04-19 for analogs of sodium channel peptide toxin.
The applicant listed for this patent is Purdue Pharma L.P.. Invention is credited to Donald J. Kyle, Jae Hyun Park.
Application Number | 20180105561 15/710375 |
Document ID | / |
Family ID | 44504000 |
Filed Date | 2018-04-19 |
United States Patent
Application |
20180105561 |
Kind Code |
A1 |
Park; Jae Hyun ; et
al. |
April 19, 2018 |
ANALOGS OF SODIUM CHANNEL PEPTIDE TOXIN
Abstract
The present invention also relates to pharmaceutical
compositions useful for treating or preventing a disorder
responsive to the blockade of sodium ion channels, especially
Na.sub.v1.7 sodium ion channels. The present invention further
provides methods of treating a disorder responsive to the blockade
of sodium channels, and particularly Na.sub.v1.7 sodium channels,
in a mammal suffering from excess activity of said channels,
compositions and methods for providing analgesia by administering a
peptide of the invention.
Inventors: |
Park; Jae Hyun; (Chandler,
AZ) ; Kyle; Donald J.; (Yardley, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Purdue Pharma L.P. |
Stamford |
CT |
US |
|
|
Family ID: |
44504000 |
Appl. No.: |
15/710375 |
Filed: |
September 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15007566 |
Jan 27, 2016 |
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15710375 |
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13808504 |
May 29, 2013 |
9279003 |
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PCT/IB2011/001631 |
Jul 7, 2011 |
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15007566 |
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61362258 |
Jul 7, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/43518 20130101 |
International
Class: |
C07K 14/435 20060101
C07K014/435 |
Claims
1. An isolated peptide comprising the amino acid sequence
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Xaa.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Xaa.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Xaa.sub-
.28-Xaa.sub.29-Xaa.sub.30-Xaa.sub.31 (SEQ ID NO: 1), or a
pharmaceutically acceptable salt thereof, wherein each of
Xaa.sub.12, Xaa.sub.19, Xaa.sub.28 and Xaa.sub.29, is any natural
or modified amino acid residue; Xaa.sub.30 is any natural or
modified amino acid residue or is absent; and Xaa.sub.31 is any
natural or modified amino acid residue or is absent, and wherein
none of Xaa.sub.12, Xaa.sub.19, Xaa.sub.28, Xaa.sub.29, and
Xaa.sub.30 is an alanine residue; and wherein said isolated peptide
is not ProTx II (SEQ ID NO: 16).
2. The isolated peptide of claim 1, wherein Xaa.sub.31 is
absent.
3. The isolated peptide of claim 1, wherein Xaa.sub.30 and
Xaa.sub.31 are absent.
4. The isolated peptide of claim 1, wherein Xaa.sub.12 is a
glutamate residue.
5. The isolated peptide of claim 1, wherein Xaa.sub.19 is a leucine
residue or a methionine residue.
6. The isolated peptide of claim 1, wherein Xaa.sub.28 is a lysine
residue or an isoleucine residue.
7. The isolated peptide of claim 1, wherein Xaa.sub.29 is a leucine
residue, an isoleucine residue, an alpha-methylated leucine
residue, an N-methylated leucine residue, or a tryptophan
residue.
8. The isolated peptide of claim 1, wherein Xaa.sub.30 is present
and is tryptophan modified with an amino group, a tryptophan
residue, a leucine residue, or an isoleucine residue.
9-17. (canceled)
18. The isolated peptide of claim 1, wherein Xaa.sub.12 is a
glutamate residue, Xaa.sub.19 is a methionine residue, Xaa.sub.28is
a lysine residue, Xaa.sub.29 is a leucine residue, Xaa.sub.30 is
tryptophan modified with an amino group, and Xaa.sub.31 is absent
(SEQ ID NO: 2).
19. The isolated peptide of claim 1, wherein Xaa.sub.12 is a
glutamate residue, Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a
lysine residue, Xaa.sub.29 is a leucine residue, Xaa.sub.30 is a
tryptophan residue, and Xaa.sub.31 is absent (SEQ ID NO: 4).
20. The isolated peptide of claim 1, wherein Xaa.sub.12 is a
glutamate residue, Xaa.sub.19 is a methionine residue, Xaa.sub.28
is an isoleucine residue, Xaa.sub.29 is an isoleucine residue,
Xaa.sub.30 is absent, and Xaa.sub.31 is absent (SEQ ID NO: 5).
21-30. (canceled)
31. The isolated peptide of claim 1, wherein said isolated peptide
is a recombinant peptide.
32. The isolated peptide of claim 1, wherein said isolated peptide
is a chemically synthesized peptide.
33-34. (canceled)
35. The isolated peptide of claim 1, wherein said isolated peptide
contains three cystine bridges with the connectivity C.sub.2 to
C.sub.16, C.sub.9 to C.sub.21 and C.sub.15 to C.sub.25.
36. The isolated peptide of claim 1, wherein said isolated peptide
inhibits Nav1.7 ion channel activity.
37. The isolated peptide of claim 1, wherein said isolated peptide
selectively inhibits Nav1.7 ion channel activity relative to Nav1.2
ion channel activity.
38-40. (canceled)
41. A pharmaceutical composition comprising the isolated peptide of
claim 1 and a pharmaceutically acceptable carrier.
42-43. (canceled)
44. A method of treating pain, said method comprising administering
an effective amount of the isolated peptide of claim 1 to a subject
in need thereof.
45. The method of claim 44, wherein said pain is neuropathic pain,
chronic pain, acute pain, inflammatory pain, or surgical pain.
46-52. (canceled)
53. A polynucleotide molecule comprising a nucleotide sequence
encoding an amino acid sequence of
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Xaa.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Xaa.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Xaa.sub-
.28-Xaa.sub.29-Xaa.sub.30-Xaa.sub.31 (SEQ ID NO: 1), wherein each
of Xaa.sub.12, Xaa.sub.19, Xaa.sub.28 and Xaa.sub.29, is any
natural or modified amino acid residue; Xaa.sub.30 is any natural
or modified amino acid residue or is absent; and Xaa.sub.31 is any
natural or modified amino acid residue or is absent, and wherein
none of Xaa.sub.12, Xaa.sub.19, Xaa.sub.28, Xaa.sub.29, and
Xaa.sub.30 is an alanine residue; and wherein said amino acid
sequence is not ProTx II (SEQ ID NO: 16).
54. (canceled)
55. A host cell comprising a vector, wherein said vector comprises
a polynucleotide molecule, and wherein the polynucleotide molecule
comprises a nucleotide sequence encoding an amino acid sequence of
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Xaa.sub.12-Arg.sub.13-Lys.sub.14-Cys.sub-
.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Xaa.sub.19-Val.sub.20-Cys.sub.21-Arg.-
sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Xaa.sub.28-X-
aa.sub.29-Xaa.sub.30-Xaa.sub.31 (SEQ ID NO: 1), wherein each of
Xaa.sub.12, Xaa.sub.19, Xaa.sub.28 and Xaa.sub.29 is any natural or
modified amino acid residue. Xaa.sub.30 is any natural or modified
amino acid residue or is absent; and Xaa.sub.31 is any natural or
modified amino acid residue or is absent, and wherein none of
Xaa.sub.12, Xaa.sub.19, Xaa.sub.28, Xaa.sub.29, and Xaa.sub.30 is
an alanine residue; and wherein said amino acid sequence is not
ProTx II (SEQ ID NO: 16).
56. (canceled)
Description
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This invention is in the field of peptide chemistry. The
invention relates to novel peptides and the use of these peptides
as blockers of sodium (Na.sup.+) channels.
Background Art
[0002] Voltage-gated sodium channels (VGSCs) are found in all
excitable cells. In neuronal cells of the central nervous system
(CNS) and peripheral nervous system (PNS), sodium channels are
primarily responsible for generating the rapid upstroke of the
action potential. In this manner, sodium channels are essential to
the initiation and propagation of electrical signals in the nervous
system. Proper function of sodium channels is therefore necessary
for normal function of the neuron. Consequently, aberrant sodium
channel function is thought to underlie a variety of medical
disorders (See Hubner et al., Hum. Mol. Genet. 11:2435-2445 (2002)
for a general review of inherited ion channel disorders) including
epilepsy (Yogeeswari et al, Curr. Drug Target 5:589-602 (2004)),
arrhythmia (Noble, Proc. Natl. Acad. Sci. USA 99:5755-5756 (2002)),
myotonia (Cannon, Kidney Int. 57:772-779 (2000)), and pain (Wood et
al., J. Neurobiol., 61:55-71 (2004)).
[0003] VGSCs are composed of one .alpha.-subunit, which forms the
core of the channel and is responsible for voltage-dependent gating
and ion permeation, and several auxiliary p-subunits (see, e.g.,
Chahine et al., CNS & Neurological Disorders-Drug Targets
7:144-158 (2008) and Kyle and Ilyin., J. Med. Chem. 50:2583-2588
(2007)). .alpha.-Subunits are large proteins composed of four
homologous domains. Each domain contains six .alpha.-helical
transmembrane spanning segments. There are currently 9 known
members of the family of voltage-gated sodium channel
.alpha.-subunits. Names for this family include SCNx, SCNAx, and
Na.sub.vx.x (see Table 1, below). The VGSC family has been
phylogenetically divided into two subfamilies Na.sub.v1.x (all but
SCN6A) and Na.sub.v2.x (SCN6A). The Na.sub.v1.x subfamily can be
functionally subdivided into two groups, those which are sensitive
to blocking by tetrodotoxin (TTX-sensitive or TTX-s) and those
which are resistant to blocking by tetrodotoxin (TTX-resistant or
TTX-r).
[0004] There are three members of the subgroup of TTX-resistant
sodium channels. The SCN5A gene product (Na.sub.v1.5, HI) is almost
exclusively expressed in cardiac tissue and has been shown to
underlie a variety of cardiac arrhythmias and other conduction
disorders (Liu et al., Am. J. Pharmacogenomics 3:173-179 (2003)).
Consequently, blockers of Na.sub.v1.5 have found clinical utility
in treatment of such disorders (Srivatsa et al., Curr. Cardiol.
Rep. 4:401-410 (2002)). The remaining TTX-resistant sodium
channels, Na.sub.v1.8 (SCN10A, PN3, SNS) and Na.sub.v1.9 (SCN11A,
NaN, SNS2) are expressed in the peripheral nervous system and show
preferential expression in primary nociceptive neurons. Human
genetic variants of these channels have not been associated with
any inherited clinical disorder. However, aberrant expression of
Na.sub.v1.8 has been found in the CNS of human multiple sclerosis
(MS) patients and also in a rodent model of MS (Black et al., Proc.
Natl. Acad. Sci. USA 97:11598-115602 (2000)). Evidence for
involvement in nociception is both associative (preferential
expression in nociceptive neurons) and direct (genetic knockout).
Na.sub.v1.8-null mice exhibited typical nociceptive behavior in
response to acute noxious stimulation but had significant deficits
in referred pain and hyperalgesia (Laird et al., J. Neurosci.
22:8352-8356 (2002)).
TABLE-US-00001 TABLE 1 Voltage-gated sodium channel gene family
Gene Tissue TTX IC.sub.50 Disease Type Symbol Distribution (nM)
Association Indications Na.sub.v1.1 SCN1A CNS/PNS 10 Epilepsy Pain,
seizures, neurodegeneration Na.sub.v1.2 SCN2A CNS 10 Epilepsy
Epilepsy, neurodegeneration Na.sub.v1.3 SCN3A CNS 15 -- Pain
Na.sub.v1.4 SCN4A Skeletal muscle 25 Myotonia Myotonia Na.sub.v1.5
SCN5A Heart muscle 2,000 Arrhythmia Arrhythmia Na.sub.v1.6 SCN8A
CNS/PNS 6 -- Pain, movement disorders Na.sub.v1.7 SCN9A PNS 25
Erythermalgia Pain Na.sub.v1.8 SCN10A PNS 50,000 -- Pain
Na.sub.v1.9 SCN11A PNS 1,000 -- Pain
[0005] The Na.sub.v1.7 (PN1, SCN9A) VGSC is sensitive to blocking
by tetrodotoxin and is preferentially expressed in peripheral
sympathetic and sensory neurons. The SCN9A gene has been cloned
from a number of species, including human, rat, and rabbit and
shows .about.90% amino acid identity between the human and rat
genes (Toledo-Aral et al., Proc. Natl. Acad. Sci. USA 94:1527-1532
(1997)).
[0006] An increasing body of evidence suggests that Na.sub.v1.7 may
play a key role in various pain states, including acute,
inflammatory and/or neuropathic pain. Deletion of the SCN9A gene in
nociceptive neurons of mice led to an increase in mechanical and
thermal pain thresholds and reduction or abolition of inflammatory
pain responses (Nassar et al., Proc. Natl. Acad. Sci. USA
101:12706-12711 (2004)).
[0007] Sodium channel-blocking agents have been reported to be
effective in the treatment of various disease states, and have
found particular use as local anesthetics, e.g., lidocaine and
bupivacaine, and in the treatment of cardiac arrhythmias, e.g.,
propafenone and amiodarone, and epilepsy, e.g., lamotrigine,
phenytoin and carbamazepine (see Clare et al., Drug Discovery Today
5:506-510 (2000); Lai et al., Annu. Rev. Pharmacol. Toxicol.
44:371-397 (2004); Anger et al., J. Med. Chem. 44:115-137 (2001),
and Catterall, Trends Pharmacol. Sci. 8:57-65 (1987)). Each of
these agents is believed to act by interfering with the rapid
influx of sodium ions.
[0008] Other sodium channel blockers such as BW619C89 and
lifarizine have been shown to be neuroprotective in animal models
of global and focal ischemia (Graham et al., J. Pharmacol. Exp.
Ther. 269:854-859 (1994); Brown et al., British J. Pharmacol.
115:1425-1432 (1995)).
[0009] It has also been reported that sodium channel-blocking
agents may be useful in the treatment of pain, including acute,
chronic, inflammatory, neuropathic, and other types of pain such as
rectal, ocular, and submandibular pain typically associated with
paroxysmal extreme pain disorder; see, for example, Kyle and
Ilyin., J. Med. Chem. 50:2583-2588 (2007); Wood et al., J.
Neurobiol. 61:55-71 (2004); Baker et al., TRENDS in Pharmacological
Sciences 22:27-31 (2001); and Lai et al., Current Opinion in
Neurobiology 13:291-297 (2003); the treatment of neurological
disorders such as epilepsy, seizures, epilepsy with febrile
seizures, epilepsy with benign familial neonatal infantile
seizures, inherited pain disorders, e.g., primary erythermalgia and
paroxysmal extreme pain disorder, familial hemiplegic migraine, and
movement disorder; and the treatment of other psychiatric disorders
such as autism, cerebeller atrophy, ataxia, and mental retardation;
see, for example, Chahine et al., CNS & Neurological
Disorders-Drug Targets 7:144-158 (2008) and Meisler and Kearney, J.
Clin. Invest. 115:2010-2017 (2005). In addition to the
above-mentioned clinical uses, carbamazepine, lidocaine and
phenytoin are occasionally used to treat neuropathic pain, such as
from trigeminal neuralgia, diabetic neuropathy and other forms of
nerve damage (Taylor and Meldrum, Trends Pharmacol. Sci. 16:309-316
(1995)). Furthermore, based on a number of similarities between
chronic pain and tinnitus (Moller, Am. J. Otol. 18:577-585 (1997);
Tonndorf, Hear. Res. 28:271-275 (1987)), it has been proposed that
tinnitus may be viewed as a form of chronic pain sensation
(Simpson, et al., Tip. 20:12-18 (1999)). Indeed, lidocaine and
carbamazepine have been shown to be efficacious in treating
tinnitus (Majumdar, B. et al., Clin. Otolaryngol. 8:175-180 (1983);
Donaldson, Laryngol. Otol. 95:947-951 (1981)).
[0010] The polypeptide toxins from the tarantula Thrixopelma
pruriens (protoxins) are members of the inhibitory cysteine-knot
family of protein toxins, which contain 30 to 35 amino acid
residues and three disulfide bridges. Protoxin I (ProTx I) and
Protoxin II (ProTx II) are T. pruriens peptide toxins that have
three cystine bridges in the connectivity pattern C.sub.2 to
C.sub.16, C.sub.9 to C.sub.21, and C.sub.15 to C.sub.25. For
example, Protoxin II (SEQ ID NO: 16) has cystine bridges as
follows:
##STR00001##
[0011] ProTx I and ProTx II inhibit activation of sodium channels
(Middleton, R. E. et al., Biochemistry 41: 14734-14747 (2002)).
ProTx I and ProTx II act as gating modifiers that prevent channel
activation via a voltage sensor-trapping mechanism (Edgerton, G. B.
et al., Toxicon 52: 489-500 (2008); Priest, B. T. et al., Toxicon
49: 194-201 (2007)). ProTx II inhibits Na.sub.v1.7 sodium channels
(Schmalhofer, W. A. et al., Molecular Pharm. 74: 1476-1481
(2008)).
[0012] Phrixotoxin I (PaTx I) is a Grammostola spatulata spider
toxin that acts as a gating modifier of Kv4 potassium channels
(Chagot, B. et al., Protein Science 13: 1197-1208 (2004)). GrTx1 is
a toxin isolated from Grammostola spatulata that blocks sodium
channels (Clement, H. et al., Toxicon 50: 65-74 (2007)).
[0013] Many patients with either acute or chronic pain disorders
respond poorly to current pain therapies, and developing resistance
or insensitivity to opiates is common. In addition, many of the
currently available treatments have undesirable side effects. Thus,
there remains a need for more effective and safer analgesics that
work by blocking sodium channels.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention provides the peptides disclosed
herein, and the pharmaceutically acceptable salts, prodrugs and
solvates thereof, which are useful as blockers of sodium (Na.sup.+)
channels, and particularly Na.sub.v1.7 channels. These peptides,
which comprise the amino acid sequence of SEQ ID NO: 1, show
selectivity as Na.sub.v1.7 channel blockers relative to
Na.sub.v1.2.
[0015] The present invention further provides pharmaceutical
compositions comprising an effective amount of a peptide comprising
an amino acid sequence of SEQ ID NO: 1, or a pharmaceutically
acceptable salt, prodrug or solvate thereof, in a mixture with one
or more pharmaceutically acceptable carriers. Pharmaceutical
compositions of the present invention are useful for treating or
preventing a disorder responsive to the blockade of sodium ion
channels, especially Na.sub.v1.7 sodium ion channels.
[0016] The present invention further provides methods of treating a
disorder responsive to the blockade of sodium channels, and
particularly Na.sub.v1.7 sodium channels, in a mammal suffering
from excess activity of said channels, said methods comprising
administering to said mammal an effective amount of a peptide
comprising the amino acid sequence of SEQ ID NO: 1, or a
pharmaceutically acceptable salt, prodrug or solvate thereof, as
described herein. In a preferred embodiment, the disorder being
treated is pain (e.g., acute pain, chronic pain, or inflammatory
pain, which includes but is not limited to, neuropathic pain and
surgical pain).
[0017] The present invention further provides a method of
preventing a disorder responsive to the blockade of sodium
channels, and particularly Na.sub.v1.7 sodium channels, in a mammal
at risk of suffering from excess activity of said channels, said
method comprising administering to said mammal an effective amount
of a peptide comprising the amino acid sequence of SEQ ID NO: 1, or
a pharmaceutically acceptable salt, prodrug or solvate thereof, as
described herein. In a preferred embodiment, the disorder being
prevented is pain (e.g., acute pain, chronic pain, or inflammatory
pain, which includes but is not limited to, neuropathic pain and
surgical pain).
[0018] The present invention further provides the use of a peptide
comprising the amino acid sequence of SEQ ID NO: 1, or a
pharmaceutically acceptable salt, prodrug or solvate thereof, in
the manufacture of a medicament useful to treat or prevent a
disorder responsive to the blockade of sodium channels, and
particularly Na.sub.v1.7 sodium channels. In a preferred
embodiment, the disorder being treated or prevented is pain (e.g.,
acute pain, chronic pain, or inflammatory pain, which includes but
is not limited to, neuropathic pain and surgical pain).
[0019] The present invention further provides a method of
modulating the activity of sodium ion channels, especially
Na.sub.v1.7 sodium ion channels, in a cell, or in a membrane
preparation, which method comprises administering to the cell or
membrane preparation an effective amount of a peptide comprising
the amino acid sequence of SEQ ID NO: 1, or a pharmaceutically
acceptable salt, prodrug or solvate thereof. In certain
embodiments, the method is carried out in an in vitro cellular or
membrane assay system. In other embodiments, the method is carried
out in an in vivo system, e.g., in a mammal such as a human.
[0020] The present invention further provides radiolabeled peptides
comprising the amino acid sequence of SEQ ID NO: 1, and the use of
such radio labeled peptides as radioligands for use in any
appropriately selected competitive binding assays and screening
methodologies Thus, the present invention further provides a method
for screening a candidate peptide for its ability to bind to a
sodium channel or sodium channel subunit using a radiolabeled
peptide of the present invention. In certain embodiments, the
peptide is radiolabeled with .sup.3H, .sup.11C or .sup.14C. This
competitive binding assay can be conducted using any appropriately
selected screening methodology. In one embodiment, the screening
methodology comprises: i) introducing a fixed concentration of the
radiolabeled peptide to an in vitro preparation comprising a
soluble or membrane-associated sodium channel, subunit or fragment
thereof under conditions that permit the radiolabeled peptide to
bind to the channel, subunit or fragment, respectively, to form a
conjugate; ii) titrating the mixture with a candidate peptide; and
iii) determining the ability of the candidate peptide to displace
the radiolabeled peptide from said channel, subunit or
fragment.
[0021] It is to be understood that both the foregoing summary and
the following detailed description are exemplary and explanatory
only and are not necessarily restrictive of the invention as
claimed.
DETAILED DESCRIPTION OF THE INVENTION
[0022] As used herein, the term "amino acid residue" refers to a
specific amino acid, usually dehydrated as a result of its
involvement in two peptide bonds or in a polypeptide backbone, but
also when the amino acid is involved in one peptide bond, as occurs
at each end of a linear polypeptide chain. The amino acid residues
may be referred to by the commonly accepted three-letter codes or
single-letter codes as known in the art. The amino acid residues
and amino acids as described herein may be in their D- or L-form,
and in one embodiment are in their L-form.
[0023] As used herein, a "prodrug" of a peptide of the present
invention is converted to the peptide of the present invention via
an enzymatic reaction, typically under physiological conditions in
the living body, that is, conversion from the prodrug to the
peptide occurs by enzymatically catalyzed oxidation, reduction, or
hydrolysis, etc. Methods for making peptide prodrugs are known in
the art. For example, see Oliyai, R., Advanced Drug Delivery
Reviews 19: 275-286 (1996); Oliyai, R. et al., Ann. Rev. Pharmcol.
Toxicol. 32: 521-44 (1993); Paulette, G. M. et al., Advanced Drug
Delivery Reviews 27: 235-256 (1997); Han, H.-K., AAPS Pharmsci 2:
1-11 (2000); Prokai, L. Expert Opinion On Therapeutic Patents 7:
233-245 (1997).
[0024] As used herein, the term "an isolated peptide comprising"
encompasses peptides containing the indicated amino acid sequence
(e.g. SEQ ID NO: 1) plus additional amino acids at the C-terminus
and/or N-terminus of said amino acid sequence, as well as peptides
consisting of the indicated amino acid sequence. In a specific
aspect, the peptide of present invention contains, for example, 10,
9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids in addition to the
indicated amino acid sequence. In a further specific aspect, the
peptide of present invention contains not more than 10, 9, 8, 7, 6,
5, 4, 3, 2, or 1 amino acids in addition to the indicated amino
acid sequence. In a further specific aspect, the peptide of present
invention consists of the indicated amino acid sequence, i.e. it
does not contain any additional amino acids.
[0025] As used in the context of present invention, the singular
forms of "a" and "an" also include the respective plurals unless
the context clearly dictates otherwise.
[0026] The term "about" in context with a numerical value or
parameter range denotes an interval of accuracy that the person
skilled in the art will understand to still ensure the technical
effect of the feature in question. The term typically indicates
deviation from the indicated numerical value of +/-10%, preferably
+/-5%.
[0027] The present invention is based on the use of peptides
comprising the amino acid sequence of SEQ ID NO: 1 and the
pharmaceutically acceptable salts, prodrugs and solvates thereof
(collectively referred to hereinafter as "peptides of the
invention"), as blockers of Na.sup.+ channels. In view of this
property, peptides of the invention are useful for treating or
preventing disorders that can be treated or prevented by the
blockade of sodium ion channels. In one aspect, peptides of the
invention selectively block Na.sub.v1.7 sodium ion channels
compared to other sodium channels, and are therefore useful for
treating or preventing disorders responsive to the selective
blockade of Na.sub.v1.7 sodium ion channels.
[0028] In a specific aspect, a peptide of the present invention
selectively blocks a Na.sub.v1.7 sodium ion channel, compared to a
Na.sub.v1.2 sodium channel. Selectivity of a peptide for a
Na.sub.v1.7 channel, compared to a Na.sub.v1.2 channel, means that
the peptide shows selectivity in one of the assays described
herein, e.g. a lower IC.sub.50 value. Thus, the ratio of binding to
Na.sub.v1.7 versus binding to Na.sub.v1.2 is greater than 1,
typically greater than about 2 and can reach values of about 40 and
more. In particular, the ratio may range from about 2 to about 500,
from about 2 to about 100, from about 3 to about 50, from about 8
to about 40, or have any numerical value within these ranges. For
example, the IC.sub.50 for Na.sub.v1.2 in comparison to the
IC.sub.50 for Na.sub.v1.7 may have these ratio ranges, as
exemplified in the Examples.
[0029] Likewise, when comparing the IC.sub.50, the phrase
"selectivity for a Na.sub.v1.7 sodium channel over a Na.sub.v1.2
sodium channel" is used herein to mean that the ratio of an
IC.sub.50 for Na.sub.v1.7 sodium channel blocking activity for a
peptide of the invention over an IC.sub.50 for Na.sub.v1.2 sodium
channel blocking activity for the same peptide is less than 1,
i.e., Na.sub.v1.7 IC.sub.50/Na.sub.v1.2 IC.sub.50<1. Preferably,
a peptide of the invention exhibits an Na.sub.v1.7
IC.sub.50/Na.sub.v1.2 IC.sub.50 ratio of about 1/2, 1/3, 1/4, 1/5,
1//6, 1/7, 1/8, 1/9, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40,
1/45, 1/50, 1/55, 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90, 1/95,
1/100, 1/125, 1/150, 1/175, 1/200, 1/225, 1/250, 1/275, 1/300,
1/325, 1/350, 1/375, 1/400, 1/425, 1/450, 1/475 or 1/500 or
less.
[0030] The peptides of the invention may be in their linear form or
they may contain one, two or three cystine bridges (disulfide
bridges). The connectivity of these one, two or three cystine
bridges is preferably selected from the group consisting of C.sub.2
to C.sub.16, C.sub.9 to C.sub.21 and C.sub.15 to C.sub.25 (i.e. the
Cys at position 2 is connected to the Cys at position 16 etc.). In
one embodiment, the peptides of the invention contain three cystine
bridges with the connectivity C.sub.2 to C.sub.16, C.sub.9 to
C.sub.21 and C.sub.15 to C.sub.25.
[0031] The present invention provides an isolated peptide
comprising the following amino acid sequence:
TABLE-US-00002 (SEQ ID NO: 1)
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr.-
sub.8-Cys.sub.9-Asp.sub.10-
Ser.sub.11-Xaa.sub.12-Arg.sub.13-Lys.sub.14-Cys.sub.15-Cys.sub.16-Glu.sub.-
17-Gly.sub.18-Xaa.sub.19-
Val.sub.20-Cys.sub.21-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.-
26-Lys.sub.27-Xaa.sub.28- Xaa.sub.29-Xaa.sub.30-Xaa.sub.31,
[0032] and the pharmaceutically acceptable salts, prodrugs and
solvates thereof,
[0033] wherein each of Xaa.sub.12, Xaa.sub.19, Xaa.sub.28 and
Xaa.sub.29 is any natural or modified amino acid residue;
[0034] Xaa.sub.30 is any natural or modified amino acid residue or
is absent; and
[0035] Xaa.sub.31 is any natural or modified amino acid residue or
is absent;
[0036] wherein the isolated peptide does not comprise the amino
acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ
ID NO: 28, or SEQ ID NO: 29, and wherein the isolated peptide does
not comprise the amino acid sequence of ProTx II (SEQ ID NO: 16) or
PaTx I (SEQ ID NO: 17).
[0037] In one embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
[0038] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue; each of Xaa.sub.19,
Xaa.sub.28 and Xaa.sub.29 is any natural or modified amino acid
residue; Xaa.sub.30 is any natural or modified amino acid residue
or is absent; and Xaa.sub.31 is absent.
[0039] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.19 is a leucine residue; each of Xaa.sub.12,
Xaa.sub.28 and Xaa.sub.29 is any natural or modified amino acid
residue; Xaa.sub.30 is any natural or modified amino acid residue
or is absent; and Xaa.sub.31 is absent.
[0040] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue; Xaa.sub.19 is a
leucine residue; each of Xaa.sub.28 and Xaa.sub.29 is any natural
or modified amino acid residue; Xaa.sub.30 is any natural or
modified amino acid residue or is absent; and Xaa.sub.31 is
absent.
[0041] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is a glutamate residue, Xaa.sub.19 is a
methionine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is a
leucine residue, Xaa.sub.30 is tryptophan modified with an amino
group, and Xaa.sub.31 is absent (SEQ ID NO: 2).
[0042] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is a glutamate residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is a
leucine residue, Xaa.sub.30 is a tryptophan residue, and Xaa.sub.31
is absent (SEQ ID NO: 4).
[0043] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is a glutamate residue, Xaa.sub.19 is a
methionine residue, Xaa.sub.28 is an isoleucine residue, Xaa.sub.29
is an isoleucine residue, Xaa.sub.30 is absent, and Xaa.sub.31 is
absent (SEQ ID NO: 5).
[0044] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is a
leucine residue, Xaa.sub.30 is a tryptophan residue, and Xaa.sub.31
is absent (SEQ ID NO: 6).
[0045] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is an
alpha-methylated leucine residue, Xaa.sub.30 is a tryptophan
residue, and Xaa.sub.31 is absent (SEQ ID NO: 7).
[0046] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is an
N-methylated leucine residue, Xaa.sub.30 is a tryptophan residue,
and Xaa.sub.31 is absent (SEQ ID NO: 8).
[0047] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is an
isoleucine residue, Xaa.sub.30 is a leucine residue, and Xaa.sub.31
is a tryptophan residue (SEQ ID NO: 9).
[0048] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is a
leucine residue, Xaa.sub.30 is an isoleucine residue, and
Xaa.sub.31 is absent (SEQ ID NO: 10).
[0049] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is an
alpha-methylated leucine residue, Xaa.sub.30 is an isoleucine
residue, and Xaa.sub.31 is absent (SEQ ID NO: 11).
[0050] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is an
N-methylated leucine residue, Xaa.sub.30 is an isoleucine residue,
and Xaa.sub.31 is absent (SEQ ID NO: 12).
[0051] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is an isoleucine residue, Xaa.sub.29 is
a leucine residue, Xaa.sub.30 is a tryptophan residue, and
Xaa.sub.31 is absent (SEQ ID NO: 13).
[0052] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is an isoleucine residue, Xaa.sub.29 is
an isoleucine residue, Xaa.sub.30 is a tryptophan residue, and
Xaa.sub.31 is absent (SEQ ID NO: 14).
[0053] In another embodiment, the present invention provides an
isolated peptide comprising the amino acid sequence of SEQ ID NO:
1, wherein Xaa.sub.12 is an alanine residue, Xaa.sub.19 is a
leucine residue, Xaa.sub.28 is a lysine residue, Xaa.sub.29 is a
tryptophan residue, Xaa.sub.30 is absent, and Xaa.sub.31 is absent
(SEQ ID NO: 15).
[0054] In one embodiment, Xaa.sub.31 is absent. In another
embodiment, Xaa.sub.30 and Xaa.sub.31 are both absent.
[0055] In another embodiment, the peptide of the invention is 31
amino acid residues in length. In another embodiment, the peptide
of the invention is 30 amino acid residues in length. In another
embodiment, the peptide of the invention is 29 amino acid residues
in length. In another embodiment, the peptide of the invention is
more than 31 amino acid residues in length, e.g. 32, 33, 34, 35,
36, 37, 38, 39, 40, or 41 residues in length. In this embodiment,
the peptide contains additional amino acids at the C-terminus
and/or N-terminus of the sequences described herein.
[0056] In another embodiment, the peptide of the invention consists
of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid
sequence is 29, 30 or 31 amino acid residues in length.
[0057] In one embodiment, at least one of Xaa.sub.12, Xaa.sub.19,
Xaa.sub.28, Xaa.sub.29 and Xaa.sub.30 is a canonical amino acid
residue. A "canonical" amino acid is an amino acid listed in the
following table.
TABLE-US-00003 Amino Acid 3 Letter Code 1 Letter Code Alanine Ala A
Glutamine Gln Q Leucine Leu L Serine Ser S Arginine Arg R Glutamic
Glu E Acid/Glutamate Lysine Lys K Threonine Thr T Asparagine Asn N
Glycine Gly G Methionine Met M Tryptophan Trp W Aspartic Asp D
Acid/Aspartate Histidine His H Phenylalanine Phe F Tyrosine Tyr Y
Cysteine Cys C Isoleucine Ile I Proline Pro P Valine Val V
[0058] As used herein, the terms "canonical amino acid" and
"natural amino acid" are synonymous. As also used herein, the terms
"canonical amino acid residue" and "natural amino acid residue" are
synonymous.
[0059] In another embodiment, at least one of Xaa.sub.12,
Xaa.sub.19, Xaa.sub.28, Xaa.sub.29 and Xaa.sub.30 is a modified
canonical amino acid residue, which is a canonical amino acid
residue as defined above to which a chemical moiety, e.g., an amino
group or a methyl group, is covalently attached to the canonical
amino acid residue. Such chemical moieties may be one or more
moieties known in the art for amino acid modification, e.g.
C.sub.1-C.sub.4 alkyl groups, halogens like F or Cl, amino groups,
C.sub.1-C.sub.4 amide groups, C.sub.1-C.sub.4 ether or ester
groups, or any combination thereof. In a particular embodiment,
there is only one chemical moiety present, typically an amino group
or a C.sub.1-C.sub.4 alkyl group. Examples for modified canonical
residues are N-Me-Leu (nml; this is also a non-canonical amino acid
residue, see below), .alpha.-Me-Leu (aml) and Trp-NH.sub.2. In
specific embodiments, such modified canonical amino acid residue is
a canonical amino acid residue to which a methyl group is
covalently attached, e.g. nml or aml. The latter modified amino
acids can, e.g., be present at position Xaa.sub.29.
[0060] In another embodiment, at least one of Xaa.sub.12,
Xaa.sub.19, Xaa.sub.28, Xaa.sub.29 and Xaa.sub.30 is a
non-canonical amino acid residue. As used herein, a "non-canonical
amino acid residue" is an amino acid residue in D- or L-form that
is not among the 20 canonical amino acids in the Table above.
Non-limiting examples of non-canonical amino acid residues include
.beta.-amino acids, homoamino acids, cyclic amino acids and amino
acids with derivatized side chains. Examples include (in the L-form
or D-form): citrulline (Cit), homocitrulline (hCit),
N-methylcitrulline (NMeCit), N-methylhomocitrulline (NMeHoCit),
ornithine (Orn or O), N-Methylomithine (NMeOrn), sarcosine (Sar),
homolysine (hK or Hlys), homoarginine (hR or hArg), homoglutamine
(hQ), N-methylarginine (NMeR), N-methylleucine (NmeL; nml),
N-methylhomolysine (NMeHoK), N-methylglutamine (NMeQ), norleucine
(Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic),
nitrophenylalanine (nitrophe), aminophenylalanine (aminophe),
benzylphenyalanine (benzylphe), .gamma.-carboxyglutamic acid
(.gamma.-carboxyglu), hydroxyproline (hydroxypro),
p-carboxyl-phenylalanine (Cpa), .alpha.-aminoadipic acid (Aad),
acetylarginine (acetylarg), .alpha.,.beta.-diaminopropionoic acid
(Dpr), .alpha.,.gamma.-diaminobutyric acid (Dab), diaminopropionic
acid (Dap), .beta.-(1-Naphthyl)-alanine (1Nal),
.beta.-(2-Naphthyl)-alanine (2Nal), cyclohexylalanine (Cha),
4-methyl-phenylalanine (MePhe), .beta.,.beta.-diphenyl-alanine
(BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (4Bip),
.alpha.-amino-isobutyric acid (Aib), and derivatized forms of any
of these as described herein. Nomenclature and symbolism for amino
acids and peptides by the UPAC-IUB Joint Commission on Biochemical
Nomenclature (JCBN) have been published in the following documents:
Biochem. 1 219: 345-373 (1984); Eur. J. Biochem. 138: 9-37 (1984);
Internat. J. Pept. Prot. Res. 24:, 84 (1984); J. Biol. Chem. 260:
14-42 (1985); and Amino Acids and Peptides 16: 387-410 (1985).
[0061] In one embodiment, an amino acid residue is an "acidic
residue," which is an amino acid residue in D- or L-form having a
sidechain comprising an acidic group. Exemplary acidic residues
include aspartate (D) residues and glutamate (E) residues.
[0062] In another embodiment, an amino acid residue is an "amide
residue," which is an amino acid residue in D- or L-form having a
sidechain comprising an amide derivative of an acidic group.
Exemplary residues include asparagine (N) residues and a glutamine
(Q) residues.
[0063] In another embodiment, an amino acid residue is an "aromatic
residue," which is an amino acid residue in D- or L-form having a
sidechain comprising an aromatic group. Exemplary aromatic residues
include phenylalanine (F) residues, tyrosine (Y) residues, and
tryptophan (W) residues.
[0064] In another embodiment, an amino acid residue is a "basic
residue," which is an amino acid residue in D- or L-form having a
sidechain comprising a basic group. Exemplary basic residues
include histidine (H) residues, lysine (K) residues, arginine (R)
residues, N-methyl-arginine residues, .omega.-aminoarginine
residues, .omega.-methyl-arginine residues, 1-methyl-histidine
residues, 3-methyl-histidine residues, and homoarginine (hR)
residues.
[0065] In another embodiment, an amino acid residue is a
"hydrophilic residue," which is an amino acid residue in D- or
L-form having a sidechain comprising a polar group. Exemplary
hydrophilic residues include histidine (H) residues, cysteine (C)
residues, serine (S) residues, threonine (T) residues, asparagine
(N) residues, glutamine (Q) residues, aspartate (D) residues,
glutamate (E) residues, lysine (K) residues, proline (P) residues,
and citrulline (Cit) residues.
[0066] In another embodiment, the amino acid residue is an amino
acid residue in D- or L-form having a sidechain that lacks an
acidic, basic, or aromatic group. Such amino acid residues include
methionine (M) residues, glycine (G) residues, alanine (A)
residues, valine (V) residues, isoleucine (I) residues, leucine (L)
residues and norleucine (Nle) residues.
[0067] In another embodiment, an amino acid residue is a "neutral
residue," which is an amino acid residue in D- or L-form having a
sidechain that lacks a basic, acidic, or polar group. Exemplary
neutral polar amino acid residues include alanine (A) residues,
valine (V) residues, leucine (L) residues, isoleucine (I) residues,
proline (P) residues, tryptophan (W) residues, and phenylalanine
(F) residues.
[0068] In another embodiment, an amino acid residue is a
"hydrophobic residue," which is a hydrophobic amino acid residue in
D- or L-form having a sidechain that lacks a basic or an acidic
groups. Exemplary hydrophobic amino acid residues include alanine
(A) residues, valine (V) residues, leucine (L) residues, isoleucine
(I) residues, tryptophan (W) residues, methionine (M) residues,
phenylalanine (F) residues, threonine (T) residues, glycine (G)
residues and tyrosine (Y) residues.
[0069] As used herein, the term "amino" or "amino group" refers to
--NH.sub.2.
[0070] In another embodiment, at least one of Xaa.sub.12,
Xaa.sub.19, Xaa.sub.28, Xaa.sub.29 and Xaa.sub.30 is a modified
non-canonical amino acid residue, which is a non-canonical amino
acid residue to which a chemical moiety, e.g., an amino group or a
methyl group, is covalently attached to the non-canonical amino
acid residue. Such chemical moieties may be one or more moieties
known in the art for amino acid modification, e.g. C.sub.1-C.sub.4
alkyl groups, halogens like F or Cl, amino groups, C.sub.1-C.sub.4
amide groups, C.sub.1-C.sub.4 ether or ester groups, or any
combination thereof. In a particular embodiment, there is only one
chemical moiety present, typically an amino group or a
C.sub.1-C.sub.4 alkyl group.
[0071] In one embodiment, at least one of Xaa.sub.12, Xaa.sub.19,
Xaa.sub.28, Xaa.sub.29 and Xaa.sub.30 is a hydrophilic amino acid
residue. In another embodiment, at least one of Xaa.sub.12,
Xaa.sub.19, Xaa.sub.28, Xaa.sub.29 and Xaa.sub.30 is a hydrophobic
amino acid residue. In another embodiment, at least one of
Xaa.sub.12, Xaa.sub.19, Xm.sub.28, Xaa.sub.29 and Xaa.sub.30 is a
neutral amino acid residue.
[0072] In another embodiment, Xaa.sub.12 is a neutral residue or an
acidic residue.
[0073] In another embodiment, Xaa.sub.19 is a residue which lacks
acidic, basic or aromatic groups, or a neutral residue. In another
embodiment, Xaa.sub.28 is a hydrophilic or neutral residue. In
another embodiment, Xaa.sub.29 is a neutral residue. In another
embodiment, Xaa.sub.30 is a neutral or aromatic residue. In another
embodiment, Xaa.sub.31 is an aromatic residue. In another
embodiment, a combination of these definitions for Xaa.sub.12,
Xaa.sub.19, Xaa.sub.28, Xaa.sub.29, Xaa.sub.30 and Xaa.sub.31 is
realized. For example, in one embodiment, Xaa.sub.12 is a neutral
or acidic residue, e.g. alanine or glutamate, and Xaa.sub.19 is a
residue which lacks acidic, basic or aromatic groups, or a neutral
residue, e.g. leucine or methionine.
[0074] In another embodiment, Xaa.sub.12 is an alanine residue. In
another embodiment, Xaa.sub.19 is a leucine residue.
[0075] In another embodiment, Xaa.sub.12 is an alanine residue and
Xaa.sub.19 is a leucine residue.
[0076] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue; Xaa.sub.28 is any natural or
modified amino acid residue, Xaa.sub.29 is any natural or modified
amino acid residue, and Xaa.sub.30 is any natural or modified amino
acid residue or is absent.
[0077] In another embodiment, Xaa.sub.12 is a glutamate residue,
Xaa.sub.19 is a methionine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is a leucine residue, Xaa.sub.30 is a tryptophan residue
modified with an amino group, and Xaa.sub.31 is absent (SEQ ID NO:
2).
[0078] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a methionine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is a leucine residue, Xaa.sub.30 is a tryptophan
residue, and Xaa.sub.31 is absent (SEQ ID NO: 3).
[0079] In another embodiment, Xaa.sub.12 is a glutamate residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is a leucine residue, Xaa.sub.30 is a tryptophan
residue, and Xaa.sub.31 is absent (SEQ ID NO: 4).
[0080] In another embodiment, Xaa.sub.12 is a glutamate residue,
Xaa.sub.19 is a methionine residue, Xaa.sub.28 is an isoleucine
residue, Xaa.sub.29 is an isoleucine residue, Xaa.sub.30 is absent,
and Xaa.sub.31 is absent (SEQ ID NO: 5).
[0081] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is a leucine residue, Xaa.sub.30 is a tryptophan
residue, and Xaa.sub.31 is absent (SEQ ID NO: 6).
[0082] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is an alpha-methylated leucine residue, Xaa.sub.30 is a
tryptophan residue, and Xaa.sub.31 is absent (SEQ ID NO: 7).
[0083] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is an N-methylated leucine residue, Xaa.sub.30 is a
tryptophan residue, and Xaa.sub.31 is absent (SEQ ID NO: 8).
[0084] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is an isoleucine residue, Xaa.sub.30 is a leucine
residue, and Xaa.sub.31 is a tryptophan residue (SEQ ID NO: 9).
[0085] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is a leucine residue, Xaa.sub.30 is an isoleucine
residue, and Xaa.sub.31 is absent (SEQ ID NO: 10).
[0086] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is an alpha-methylated leucine residue, Xaa.sub.30 is an
isoleucine residue, and Xaa.sub.31 is absent (SEQ ID NO: 11).
[0087] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a lysine residue,
Xaa.sub.29 is an N-methylated leucine residue, Xaa.sub.30 is an
isoleucine residue, and Xaa.sub.31 is absent (SEQ ID NO: 12).
[0088] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is an isoleucine
residue, Xaa.sub.29 is a leucine residue, Xaa.sub.30 is a
tryptophan residue, and Xaa.sub.31 is absent (SEQ ID NO: 13).
[0089] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is an isoleucine
residue, Xaa.sub.29 is an isoleucine residue, Xaa.sub.30 is a
tryptophan residue, and Xaa.sub.31 is absent (SEQ ID NO: 14).
[0090] In another embodiment, Xaa.sub.12 is an alanine residue,
Xaa.sub.19 is a leucine residue, Xaa.sub.28 is a leucine residue,
Xaa.sub.29 is a tryptophan residue, Xaa.sub.30 is absent, and
Xaa.sub.31 is absent (SEQ ID NO: 15).
[0091] In one embodiment, the peptide of the present invention does
not comprise the amino acid sequence of ProTx II (SEQ ID NO: 16),
PaTx I (SEQ ID NO: 17), JTx XII (SEQ ID NO: 18), GsAF I (SEQ ID NO:
19), JzTx V (SEQ ID NO: 20), VsTx II (SEQ ID NO: 21), GsAF II (SEQ
ID NO: 22), GrTx I (SEQ ID NO: 23), or GsMTx II/PaTx II (SEQ ID NO:
24), SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 or
SEQ ID NO: 29.
[0092] In another embodiment, the peptide of the present invention
does not comprise the amino acid sequence of a peptide disclosed in
Smith, J. J. et al., J. Biol. Chem. 287: 12687-12697 (2007), i.e.,
the peptide of the invention does not comprise any of SEQ ID NOS:
25-29, and does not comprise any of the Y1A, Q3A, K4Q, W5A, M6A,
W7A, T8A, D10A, S11A, E12A, R13Q, K14A E17A, M19A, V20A, R22A,
L23A, W24L, K26Q, K27Q, K28A, L29A or W30A mutations of ProTx II
(SEQ ID NO: 16).
[0093] In another embodiment, a peptide of the present invention
does not comprise the amino acid sequence of SEQ ID NO: 12 and SEQ
ID NO: 15.
[0094] In one embodiment, if Xaa.sub.12 in SEQ ID NO: 1 is an
alanine residue and Xaa.sub.31 is absent, then the amino acid
sequence is not
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30(SEQ ID NO: 25).
[0095] In another embodiment, if Xaa.sub.19 in SEQ ID NO: 1 is an
alanine residue and Xaa.sub.31 is absent, then the amino acid
sequence is not
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Ala.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.20-Trp.sub.30(SEQ ID NO: 26).
[0096] In another embodiment, if Xaa.sub.28 in SEQ ID NO: 1 is an
alanine residue and Xaa.sub.31 is absent, then the amino acid
sequence is not
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Ala.sub-
.28-Leu.sub.29-Trp.sub.30-Xaa.sub.31(SEQ ID NO: 27).
[0097] In another embodiment, if Xaa.sub.29 in SEQ ID NO: 1 is an
alanine residue and Xaa.sub.31 is absent, then the amino acid
sequence is not
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Ala.sub.20-Trp.sub.30 (SEQ ID NO: 28);
[0098] In another embodiment, if Xaa.sub.30 in SEQ ID NO: 1 is an
alanine residue and Xaa.sub.31 is absent, then the amino acid
sequence is not
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Ala.sub.30 (SEQ ID NO: 29).
[0099] In another embodiment, the isolated peptide is not one in
which (a) one of Xaa.sub.12, Xaa.sub.19, Xaa.sub.28, Xaa.sub.29, or
Xaa.sub.30 is an alanine residue, and (b) the amino acid residues
at the other variable Xaa positions are the same amino acid
residues as in ProTx II (SEQ ID NO: 16).
[0100] In one embodiment, the isolated peptide is a recombinantly
expressed peptide. In another embodiment, the isolated peptide is a
chemically synthesized peptide.
[0101] The present invention also provides a composition comprising
an isolated peptide of SEQ ID NO: 1. In one embodiment, the
composition is a sterile composition.
[0102] The present invention also provides a container comprising
an isolated peptide of SEQ ID NO: 1. In one embodiment, the
container is a vial. In another embodiment, the container is an
intravenous fluid delivery container, e.g., a bag, a length of
tubing, or a cartridge, each of which can be adapted for use with a
mechanized analgesic delivery system, such as a pump.
[0103] The present invention also provides a pharmaceutical
composition comprising an isolated peptide comprising the amino
acid sequence of SEQ ID NO: 1 or a salt, prodrug or solvate
thereof, and a pharmaceutically acceptable carrier or diluent. The
present invention may further comprise a sterile container
comprising the pharmaceutical composition of the present
invention.
[0104] The present invention also provides an article of
manufacture comprising a plurality of containers, each of which
contains a pharmaceutical composition of the present invention.
[0105] The present invention also provides a polynucleotide
molecule comprising a nucleotide sequence encoding the amino acid
sequence of SEQ ID NO: 1. In cases where the amino acid sequence
contains non-canonical amino acids or modified amino acids, the
nucleotide sequence encodes its natural occurring counterpart. In
one embodiment, the polynucleotide molecule is a deoxyribonucleic
acid (DNA), such as cDNA. In another embodiment, the polynucleotide
molecule is a ribonucleic acid (RNA), such as messenger RNA
(mRNA).
[0106] The present invention also provides a recombinant vector
comprising a polynucleotide molecule of the invention. In one
embodiment, the vector is an expression vector. In a preferred
embodiment, the recombinant vector comprises the polynucleotide
molecule of the invention in operative association with one or more
control elements necessary to enable the expression of the
polynucleotide molecule under appropriate conditions.
[0107] The present invention also provides a host cell comprising a
vector of the present invention. As used herein, the term "host
cell" refers to either a single host cell or a plurality of host
cells. In one embodiment, the host cell is a eukaryotic host cell,
e.g., a mammalian cell, a plant cell, a yeast cell or an insect
cell. In one embodiment, the mammalian cell is a human cell or a
cell of human origin. In another embodiment, the host cell is a
prokaryotic cell. In one embodiment, the prokaryotic cell is a
bacterial cell.
[0108] The present invention also provides a recombinant method of
making a peptide comprising the amino acid of SEQ ID NO: 1, said
method comprising culturing a host cell comprising a polynucleotide
molecule of the present invention in culture medium and under
conditions suitable to induce expression of the peptide; and then
isolating the peptide from the host cell or culture medium.
[0109] The invention disclosed herein also encompasses any of the
disclosed peptides being isotopically-labelled (i.e., radiolabeled)
by having one or more atoms thereof replaced by an atom having a
different atomic mass or mass number. Examples of isotopes that can
be incorporated into the disclosed peptides of the present
invention include isotopes of hydrogen, carbon, nitrogen, oxygen,
phosphorous, fluorine and chlorine, such as .sup.2H, .sup.3H,
.sup.11C, .sup.13C, .sup.14C, .sup.15N, .sup.18O, .sup.17O,
.sup.31P, .sup.32P, .sup.35S, .sup.18F, and .sup.36Cl,
respectively, and preferably .sup.3H, .sup.11C, and .sup.14C.
Isotopically-labeled peptides of SEQ ID NO: 1 can be prepared by
methods known in the art in view of this disclosure.
[0110] The invention disclosed herein is also intended to encompass
peptides of the present invention that have been fluorescently
labeled or labeled with Europium or a Europium-based label.
[0111] The present invention also provides the use of any of the
radiolabeled or fluorescently labeled peptides of the invention as
detectably labeled ligands to bind to the sodium channel. One use
of such labeled peptides is the characterization of specific
receptor binding. Another use of such labeled peptides is as an
alternative to animal testing for the evaluation of chemical
structure-activity relationships. For example, the receptor assay
can be performed at a fixed concentration of a labeled peptide of
SEQ ID NO: 1 and at increasing concentrations of a candidate
peptide in a competition assay. For example, a radiolabeled peptide
such as a tritiated peptide of SEQ ID NO: 1 can be prepared by
introducing tritium into the particular peptide, for example, by
catalytic dehalogenation with tritium. This method may include
reacting a suitably halogen-substituted precursor of the peptide
with tritium gas in the presence of a suitable catalyst, for
example Pd/C, in the presence or absence of a base. Other suitable
methods for preparing tritiated peptides can be found in Filer,
Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled
Compounds (Part A), Chapter 6 (1987). .sup.14C-labeled peptides can
be prepared by employing starting materials having a .sup.14C
carbon.
[0112] The invention disclosed herein also encompasses the
preparation and use of salts of the disclosed peptides, including
all pharmaceutically acceptable salts of the disclosed peptides.
Examples of pharmaceutically acceptable addition salts include
inorganic and organic acid addition salts and basic salts. The
pharmaceutically acceptable salts include, but are not limited to,
metal salts such as sodium salt, potassium salt, cesium salt and
the like; alkaline earth metals such as calcium salt, magnesium
salt and the like; organic amine salts such as triethylamine salt,
pyridine salt, picoline salt, ethanolamine salt, triethanolamine
salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt and
the like; inorganic acid salts such as hydrochloride, hydrobromide,
phosphate, sulphate and the like; organic acid salts such as
citrate, lactate, tartrate, maleate, fumarate, mandelate, acetate,
dichloroacetate, trifluoroacetate, oxalate, formate and the like;
sulfonates such as methanesulfonate, benzenesulfonate,
p-toluenesulfonate and the like; and amino acid salts such as
arginate, asparginate, glutamate or the like.
[0113] Acid addition salts can be formed by mixing a solution of
the particular peptide of the invention with a solution of a
pharmaceutically acceptable non-toxic acid such as hydrochloric
acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric
acid, tartaric acid, carbonic acid, phosphoric acid, oxalic acid,
dichloroacetic acid, or the like. Basic salts can be formed by
mixing a solution of the peptide of SEQ ID NO: 1 with a solution of
a pharmaceutically acceptable non-toxic base such as sodium
hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate
or the like.
[0114] The invention disclosed herein is also meant to encompass
solvates of any of the disclosed peptides. Solvates typically do
not significantly alter the physiological activity or toxicity of
the peptides, and as such may function as pharmacological
equivalents. The term "solvate" as used herein is a combination,
physical association and/or solvation of a peptide of the invention
with a solvent molecule such as, e.g. a disolvate, monosolvate or
hemisolvate, where the ratio of solvent molecule to peptide is
typically 2:1, 1:1 or 1:2, respectively. This physical association
involves varying degrees of ionic and covalent bonding, including
hydrogen bonding. In certain instances, the solvate can be
isolated, such as when one or more solvent molecules are
incorporated into the crystal lattice of a crystalline solid. Thus,
"solvate" encompasses both solution-phase and isolatable solvates.
Peptides of the invention may be unsolvated, or may be solvated
with a pharmaceutically acceptable solvent such as water, methanol,
ethanol, and the like. One type of solvate is a hydrate. A
"hydrate" relates to a particular subgroup of solvates where the
solvent molecule is water. Methods for preparing solvates are
generally known in the art. See, for example, M. Caira et al., J.
Pharmaceut. Sci., 93(3):601-611 (2004), which describes the
preparation of solvates of fluconazole with ethyl acetate, and with
water. Similar preparation of solvates, hemisolvates, hydrates, and
the like are described by E. C. van Tonder et al., AAPS Pharm. Sci.
Tech., 5(1):Article 12 (2004), and A. L. Bingham et al., Chem.
Commun.: 603-604 (2001). A typical, non-limiting, process of
preparing a solvate would involve dissolving a peptide of the
invention in a desired solvent (organic, water, or a mixture
thereof) at temperatures above about 20.degree. C. to about
25.degree. C., then cooling the solution at a rate sufficient to
form crystals, and isolating the crystals by known methods, e.g.,
filtration. Analytical techniques such as infrared spectroscopy can
be used to confirm the presence of the solvent in a crystal of the
solvate.
[0115] The present invention also provides N-terminal and
C-terminal derivatives of the peptides of the invention. For
example, the N-terminal amino acid residue and/or the C-terminal
amino acid residue of the peptide can be derivatized with an amino
acid residue or another chemical moiety. In one embodiment, the
N-terminal amino acid residue and/or the C-terminal amino acid
residue of the peptide is derivatized with another chemical moiety,
i.e. a moiety which is not an amoino acid. Non-limiting examples of
moieties with which the N-terminal (first) amino acid can be
derivatized include an alkyl group (such as C.sub.1-C.sub.4 alkyl),
a methyl group, a carboxy group, an acetyl group, and a substituted
acetyl group. Non-limiting examples of chemical moieties with which
the C-terminal (last) amino acid can be derivatized include an
alkyl group (such as C.sub.1-C.sub.4 alkyl, e.g. a methyl or ethyl
group); an aryl group or aryl alkyl group, such as phenyl or
benzyl; a halogen, such as a fluoro or chloro; an alkoxy group; and
an amino group.
[0116] The present invention also provides a method for treating or
preventing a disorder responsive to the blockade of sodium
channels, and particularly the selective blockade of Na.sub.v1.7
sodium channels, in a subject suffering from, or at risk of
suffering from, the disorder, the method comprising administering
to the subject an effective amount of a peptide of the
invention.
[0117] In one embodiment, the present invention provides a method
of treating pain (palliative treatment). In another embodiment, the
present invention provides a method of preventing pain (pre-emptive
treatment). In one embodiment, the type of pain treated is chronic
pain. In another embodiment, the type of pain treated is acute
pain. In another embodiment, the type of pain treated is
neuropathic pain. In another embodiment, the type of pain treated
is inflammatory pain. In another embodiment, the type of pain
treated is surgical pain. In each instance, such method of
treatment or prevention requires administering to a subject in need
of such treatment or prevention an amount of a peptide of the
invention that is therapeutically effective in achieving said
result. In one embodiment, the amount of such peptide is the amount
that is effective to substantially block sodium channels in
vivo.
[0118] Chronic pain includes, but is not limited to, inflammatory
pain, neuropathic pain, postoperative pain, cancer pain,
osteoarthritis pain associated with metastatic cancer, trigeminal
neuralgia, acute herpetic and postherpetic neuralgia, diabetic
neuropathy, causalgia, brachial plexus avulsion, occipital
neuralgia, reflex sympathetic dystrophy, fibromyalgia, gout,
phantom limb pain, burn pain, and other forms of neuralgia,
neuropathic, and idiopathic pain syndromes.
[0119] The methods of the present invention may be used to treat or
prevent chronic somatic pain, which generally results from
inflammatory responses to tissue injury such as nerve entrapment,
surgical procedures, cancer or arthritis (Brower, Nature
Biotechnology 2000; 18: 387-391).
[0120] Inflammatory pain includes, but is not limited to, pain
associated with osteoarthritis and rheumatoid arthritis.
[0121] The methods of the present invention may be used to treat or
prevent chronic neuropathic pain, which is a heterogenous disease
state with an unclear etiology. In chronic neuropathic pain, the
pain can be mediated by multiple mechanisms. This type of pain
generally arises from injury to the peripheral or central nervous
tissue. The syndromes include pain associated with spinal cord
injury, multiple sclerosis, post-herpetic neuralgia, trigeminal
neuralgia, phantom pain, causalgia, and reflex sympathetic
dystrophy and lower back pain. Chronic pain is different from acute
pain in that patients suffering from chronic pain suffer the
abnormal pain sensations that can be described as spontaneous pain,
continuous superficial burning and/or deep aching pain. The pain
can be evoked by heat-, cold-, and mechano-hyperalgesia or by
heat-, cold-, or mechano-allodynia.
[0122] The methods of the present invention may be used to treat or
prevent neuropathic pain, which can be caused by injury or
infection of peripheral sensory nerves. It includes, but is not
limited to, pain from peripheral nerve trauma, herpes virus
infection, diabetes mellitus, causalgia, plexus avulsion, neuroma,
limb amputation, and vasculitis. Neuropathic pain is also caused by
nerve damage from chronic alcoholism, human immunodeficiency virus
infection, hypothyroidism, uremia, or vitamin deficiencies. Stroke
(spinal or brain) and spinal cord injury can also induce
neuropathic pain. Cancer-related neuropathic pain results from
tumor growth compression of adjacent nerves, brain, or spinal cord.
In addition, cancer treatments, including chemotherapy and
radiation therapy, can also cause nerve injury. Neuropathic pain
includes but is not limited to pain caused by nerve injury such as,
for example, the pain from which diabetics suffer.
[0123] The methods of the present invention may also be used to
treat or prevent epilepsy, seizures, epilepsy with febrile
seizures, epilepsy with benign familial neonatal infantile
seizures, inherited pain disorders, e.g., primary erythermalgia and
paroxysmal extreme pain disorder, familial hemiplegic migraine,
movement disorder, psychiatric disorders (such as autism,
cerebeller atrophy, ataxia, and mental
retardation/neurodegeneration), global or focal ischemia, myotonia,
a movement disorder, erythermalgia, cardiac arrhythmias or other
conduction disorders, including supraventricular tachycardia,
ventricular tachycardia, symptomatic ventricular premature beats,
and prevention of ventricular fibrillationventricular fibrillation,
and to provide local anesthesia.
[0124] In one embodiment, the subject being treated by a method of
the present invention is a mammal. In another embodiment, the
mammal is a human, or other primate (e.g., a chimpanzee, orangutan,
gorilla, or lemur), or a canine (e.g., a dog, fox, wolf, or
coyote), feline (e.g., a cat, lion, tiger, bobcat, leopard,
cheetah, panther), equine (e.g., a horse, llama, alpaca, zebra,
deer, moose, elk, mule or donkey), bovine (e.g., a cow, a bull, a
buffalo or a bison), or a pig, marine mammal (e.g., a seal, walrus,
otter, sea lion, manatee, dolphin, porpoise or whale), rodent
(e.g., a rat, a mouse, ferret or guinea pig), or any other
mammal.
[0125] In one embodiment, a peptide of the invention is
administered to the subject by any suitable route of
administration, including by one or more of the oral, buccal,
mucosal, sublingual, parenteral, subcutaneous, intramuscular,
intraperitoneal, intrathecal, intranasal, inhalation, transdermal,
rectal or vaginal routes of administration.
[0126] The present invention is also directed to the use of a
peptide of the invention in the manufacture of a medicament for
modulating sodium channels, especially Na.sub.v1.7 sodium channels,
in an in vitro or in vivo system.
[0127] The present invention is also directed to the use of a
peptide of the invention in the manufacture of a medicament for
treating a disorder or providing preemptive or palliative treatment
of a disorder that is responsive to the blockade of sodium channels
(e.g., any of the disorders listed above) in a subject suffering
from said disorder. In one embodiment, the disorder is responsive
to the selective blockade of Na.sub.v1.7 sodium channels.
[0128] Synthesis of Peptides
[0129] The peptides of the invention can be synthesized using
peptide synthesis methodologies, in which amino acids are linked by
peptide bonds, and other chemical synthetic procedures, as known in
the art and in view of this disclosure. For example, see
Pennington, M. W., Ed., "Peptide Synthesis Protocols," in Methods
in Molecular Biology 35, Humana Press; and Atherton, E. and
Sheppard, R. C., "Solid Phase peptide synthesis: a practical
approach," IRL Press. (1989). Non-limiting examples of synthetic
methods are liquid-phase synthesis and solid-phase synthesis.
Non-limiting examples of solid-phase synthesis are Fmoc solid-phase
synthesis (e.g., the syntheses used in the Examples), and t-boc
solid phase synthesis.
[0130] For forming cystine bridges, any conventional method can be
used, e.g. an oxidation method using GSSG like the method described
in the examples. Typically, there are 3 cystine bridges present in
the peptide of the invention. They typically have the connectivity
C.sub.2 to C.sub.16, C.sub.9 to C.sub.21 and C.sub.15 to
C.sub.25.
[0131] Testing of Peptides
[0132] Representative peptides of the invention can be assessed by
electrophysiological assays testing for sodium channel activity.
One aspect of the present invention is based on the use of the
peptides herein described as sodium channel blockers. In one aspect
of the present invention, it has been found that certain peptides
show selectivity as Na.sub.v1.7 sodium channel blockers. Based upon
this property, these peptides are considered useful in treating
pain.
[0133] More specifically, the present invention is directed to
peptides that are blockers of sodium channels. In one embodiment,
peptides having preferred sodium channel blocking properties
exhibit an IC.sub.50 of about 100 .mu.M or less in one or more of
the sodium electrophysiological assays described herein, or an
IC.sub.50 of 10 .mu.M or less, or an IC.sub.50 of about 6 .mu.M or
less, or an IC.sub.50 of about 1.0 .mu.M or less, or an IC.sub.50
of about 500 nM or less, or an IC.sub.50 of about 100 nM or
less.
[0134] Peptides of the invention can be tested for their sodium
channel blocking activity using electrophysiological assays known
in the art, such as the assay disclosed herein. For example, see
Clare, J. J. et al., Drug Discovery Today 5: 506-520 (2000).
[0135] In one embodiment, peptides useful in the present invention
are those represented by SEQ ID NO: 1 that exhibit selectivity for
Na.sub.v1.7 sodium channels over Na.sub.v1.2 sodium channels in
electrophysiological assays described herein. The phrase
"selectivity for Na.sub.v1.7 sodium channels over Na.sub.v1.2
sodium channels" is used herein to mean that the ratio of an
IC.sub.50 for Na.sub.v1.7 sodium channel blocking activity for a
peptide of the invention over an IC.sub.50 for Na.sub.v1.2 sodium
channel blocking activity for the same peptide is less than 1,
i.e., Na.sub.v1.7 IC.sub.50/Na.sub.v1.2 IC.sub.50<1. Preferably,
a peptide of SEQ ID NO: 1 exhibits an Na.sub.v1.7
IC.sub.50/Na.sub.v1.2 IC.sub.50 ratio of about 1/2, 1/3, 1/4, 1/5,
1//6, 1/7, 1/8, 1/9, 1/10, 1/15, 1/20, 1/25, 1/30, 1/35, 1/40,
1/45, 1/50, 1/55, 1/60, 1/65, 1/70, 1/75, 1/80, 1/85, 1/90, 1/95,
1/100, 1/125, 1/150, 1/175, 1/200, 1/225, 1/250, 1/275, 1/300,
1/325, 1/350, 1/375, 1/400, 1/425, 1/450, 1/475 or 1/500 or
less.
[0136] In Vitro Assay Protocols:
FLIPR.RTM. Assays:
[0137] Recombinant Na.sub.v1.7 Cell Line: In vitro assays were
performed in a recombinant cell line expressing cDNA encoding the
alpha subunit (Na.sub.v1.7, SCN9a, PN1, NE) of human Na.sub.v1.7
(Accession No. NM_002977). The cell line was provided by
investigators at Yale University (Cummins et al, J. Neurosci.
18(23): 9607-9619 (1998)). For dominant selection of the
Na.sub.v1.7-expressing clones, the expression plasmid co-expressed
the neomycin resistance gene. The cell line was constructed in the
human embryonic kidney cell line, HEK293, under the influence of
the CMV major late promoter, and stable clones were selected using
limiting dilution cloning and antibiotic selection using the
neomycin analogue, G418. Recombinant beta and gamma subunits were
not introduced into this cell line. Additional cell lines
expressing recombinant Na.sub.v1.7 cloned from other species can
also be used, alone or in combination with various beta subunits,
gamma subunits or chaperones.
[0138] Non-recombinant Cell Lines Expressing Native Na.sub.v1.7:
Alternatively, in vitro assays can be performed in a cell line
expressing native, non-recombinant Na.sub.v1.7, such as the ND7
mouse neuroblastoma X rat dorsal root ganglion (DRG) hybrid cell
line ND7/23, available from the European Cell Culture Collection
(Cat. No. 92090903, Salisbury, Wiltshire, United Kingdom). The
assays can also be performed in other cell lines expressing native,
non-recombinant Na.sub.v1.7 from various species, or in cultures of
fresh or preserved sensory neurons, such as dorsal root ganglion
(DRG) cells, isolated from various species. Primary screens or
counter-screens of other voltage-gated sodium channels can also be
performed, and the cell lines can be constructed using methods
known in the art, purchased from collaborators or commercial
establishments, and they can express either recombinant or native
channels. The primary counter-screen is for one of the central
neuronal sodium channels, Na.sub.v1.2 (rBIIa), expressed in HEK293
host cells (Ilyin et al., Br. J. Pharmacol. 144:801-812 (2005)).
Pharmacological profiling for these counter-screens is carried out
under conditions similar to the primary or alternative Na.sub.v1.7
assays described below.
[0139] Cell maintenance: Unless otherwise noted, cell culture
reagents were purchased from Mediatech of Herndon, Va. The
recombinant Na.sub.v1.7/HEK293 cells were routinely cultured in
growth medium consisting of Dulbecco's minimum essential medium
containing 10% fetal bovine serum (FBS, Hyclone, Thermo Fisher
Scientific, Logan, Utah), 100 U/mL penicillin, 100 .mu.g/mL
streptomycin, 2-4 mM L-glutamine, and 500 mg/mL G418. For natural,
non-recombinant cell lines, the selective antibiotic was omitted,
and additional media formulations can be applied as needed.
[0140] Assay Buffer: The assay buffer was formulated by removing
120 mL from a 1 L bottle of fresh, sterile dH.sub.2O (Mediatech,
Herndon, Va.) and adding 100 mL of 10.times. HBSS that does not
contain Ca.sup.++ or Mg.sup.++ (Gibco, Invitrogen, Grand Island,
N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific,
BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3,
1.261 mM CaCl.sub.2, 0.493 mM MgCl.sub.2, 0.407 mM Mg(SO).sub.4,
5.33 mM KCl, 0.441 mM KH.sub.2PO.sub.4, 137 mM NaCl, 0.336 mM
Na.sub.2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp.
Biol. Med. 71:196 (1949)), and the simple formulation was typically
the basic buffer throughout the assay (i.e., all wash and addition
steps).
[0141] CoroNa.TM. Green AM Na.sup.+ Dye for Primary Fluorescence
Assay: The fluorescence indicator used in the primary fluorescence
assay was the cell permeant version of CoroNa.TM. Green
(Invitrogen, Molecular Probes, Eugene, Oreg.), a dye that emits
light in the fluorescence range (Harootunian et al., J. Biol. Chem.
264(32):19458-19467 (1989)). The intensity of this emission, but
not the wavelength range, is increased when the dye is exposed to
Na.sup.+ ions, which it can bind with partial selectivity. Cells
expressing Na.sub.v1.7 or other sodium channels were loaded with
the CoroNa.TM. Green dye immediately in advance of the fluorescence
assay, and then, after agonist stimulation, the mobilization of
Na.sup.+ ions was detected as the Na.sup.+ ions flowed from the
extracellular fluid into the cytoplasm through the activated sodium
channel pores. The dye was stored in the dark as a lyophilized
powder, and then an aliquot was dissolved immediately before the
cell loading procedure, according to the instructions of the
manufacturer to a stock concentration of 10 mM in DMSO. It was then
diluted in the assay buffer to a 4.times. concentrated working
solution, so that the final concentration of dye in the cell
loading buffer was 5 .mu.M.
[0142] Membrane Potential Dye for Alternative Fluorescence Assays:
A fluorescence indicator that can be used in alternative
fluorescence assays is the blue version membrane potential dye
(MDS, Molecular Devices, Sunnyvale, Calif.), a dye that detects
changes in molecules following a change in membrane potential. An
increase in fluorescence is expected if agonist stimulation
provokes a change in membrane potential. Cells expressing
Na.sub.v1.7 or other sodium channels are incubated with the
membrane potential dye 30-60 minutes before the fluorescence assay.
In the case of the KCl pre-stimulation version of the assay, the
dye and all other components are washed out immediately before the
assay, and the dye is then replaced. In the version lacking KCl
pre-stimulation, the dye remains on the cells and is not washed out
or replaced. The dye is stored in the dark as a lyophilized powder,
and then an aliquot is dissolved in assay buffer to form a
20.times.-concentrated stock solution that can be used for several
weeks.
[0143] Agonists: In the fluorescence assays, two agonists were used
in combination, namely 1) veratridine, and 2) the venom from the
yellow scorpion, Leiurus quinquestriatus hebraeus. Veratridine is
an alkaloid small molecule that facilitates the capture of channel
openings by inhibiting inactivation, and the scorpion venom is a
natural preparation that includes peptide toxins selective for
different subsets of voltage-gated sodium channels. These scorpion
toxins inhibit the fast inactivation of their cognate target
channels. Stock solutions of the agonists were prepared to 40 mM in
DMSO (veratridine) and 1 mg/mL in dH.sub.2O (scorpion venom), and
then diluted to make a 4.times. or 2.times. stock (depending on the
particular assay) in assay buffer, the final concentration being
100 .mu.M (veratridine) and 10 .mu.g/mL (scorpion venom). Both of
the agonists were purchased from Sigma Aldrich, St. Louis, Mo.
[0144] Test Compounds: Test compounds were dissolved in DMSO to
yield 10 mM stock solutions. The stock solutions were further
diluted using DMSO in 1:3 serial dilution steps with 10 points
(10,000 .mu.M, 3,333 .mu.M, 1,111 .mu.M, 370 .mu.M, 123 .mu.M, 41
.mu.M, 14 .mu.M, 4.6 .mu.M, 1.5 .mu.M and 0.5 .mu.M). The stock
solutions were further diluted in assay buffer (1:125) as 4.times.
stock serial dilutions with a DMSO concentration of 0.8% (final
[DMSO], in the assay, from the compounds component=0.2%), so that
the compounds' final concentrations in the assay were 20 .mu.M, 6.7
.mu.M, 2.2 .mu.M, 0.74 .mu.M, 0.25 .mu.M, 0.08 .mu.M, 0.03 .mu.M,
0.01 .mu.M, 0.003 .mu.M and 0.001 .mu.M. If a particular test
article appeared to be especially potent, then the concentration
curve was adjusted, e.g., to 10-fold lower concentrations, in order
to perform the dose-response in a more relevant concentration
range. Compound dilutions were added during the dye-loading and
pre-stimulation step, and then again during the fluorescence assay,
early in the kinetic read. Compound dilutions were added in
duplicate rows across the middle 80 wells of the 96-well plate,
whereas the fully stimulated and the fully inhibited controls
(positive and negative) were located in the top 4 side wells and
the bottom 4 side wells, respectively, on the left and right sides
of the assay plate.
[0145] Data Analysis: The data were analyzed according to methods
known in the art or using the GraphPad.RTM. Prism 4.0 Program
(available from GraphPad Software, San Diego, Calif.) to determine
the IC.sub.50 value for the test article. At least one standard
reference compound was evaluated during each experiment.
[0146] FLIPR.RTM. or FLIPR.sup.TETRA.RTM. sodium dye assay with KCl
and test article pre-incubation: Cells were prepared by plating the
recombinant HEK293 cells or other host cells expressing either
recombinant or non-recombinant, native Na.sub.v1.7 alpha subunit,
alone or in combination with various beta and gamma subunits at a
density of 40,000 cells/well into a 96-well black, clear-bottom,
PDL-coated plate. The assay can be adapted to 384-well or
1,536-well format, if desired, using proportionately less cells and
media. The plate was then incubated in growth media, with or
without selective antibiotic, overnight at 37.degree. C. at 5%
CO.sub.2, 95% humidity, in preparation for the assay. For
counter-screens of other voltage-gated sodium channels, the
procedure was very similar, though optimal densities of cells,
media and subsequent assay components can be fine-tuned for the
particular cell line or isoform.
[0147] The next day, at the start of the assay, the media was
flicked from the cells and the wells were washed once with 50
.mu.L/well assay buffer (1.times. Hank's balanced salt solution
without sodium bicarbonate or phenol red, 20 mM Hepes, pH 7.3) and
then pre-incubated with the test articles, CoroNa.TM. Green AM
sodium dye (for cell loading) and KCl for re-polarization and
synchronization of the channels in the entire population of cells.
For this dye-loading and pre-stimulation step, the components were
added as follows, immediately after the wash step: 1) the compound
dilutions and controls were added as 4.times. concentrates in assay
buffer at 50 .mu.L/well; 2) CoroNa.TM. Green AM dye was diluted
from the stock solution to 20 .mu.M in assay buffer (4.times.
concentrate) and added to the plate at 50 .mu.L/well; and 3) a
solution of 180 mM KCl (2.times.) was prepared by diluting a 2M
stock solution into assay buffer and the solution was added to the
cells at 100 .mu.L/well. The cells were incubated at 25.degree. C.
in the dark for 30 min. before their fluorescence was measured.
[0148] The plates containing dye-loaded cells were then flicked to
remove the pre-incubation components and washed once with 100
.mu.L/well assay buffer. A 100 .mu.L/well aliquot of assay buffer
was added back to the plate, and the real-time assay was commenced.
The fluorescence of cells was measured using a fluorescence plate
reader (FLIPR.sup.TETRA.RTM. or FLIPR384.RTM., MDS, Molecular
Devices, Sunnyvale, Calif.). Samples were excited by either a laser
or a PMT light source (Excitation wavelength=470-495 nM) and the
emissions were filtered (Emission wavelength=515-575 nM). The
additions of compound and the channel activators in this
cell-based, medium-to-high throughput assay were performed on the
fluorescence plate reader and the results (expressed as relative
fluorescence units) were captured by means of camera shots every
1-3 sec., then displayed in real-time and stored. Generally, there
was a 15 sec. base line, with camera shots taken every 1.5 sec.,
then the test compounds were added, then another 120 sec. baseline
was conducted, with camera shots taken every 3 sec.; and finally,
the agonist solution (containing veratridine and scorpion venom)
was added. The amplitude of fluorescence increase, resulting from
the binding of Na.sup.+ ions to the CoroNa.TM. Green dye, was
captured for .about.180 sec. thereafter. Results were expressed in
relative fluorescence units (RFU) and can be determined by using
the maximum signal during the latter part of the stimulation; or
the maximum minus the minimum during the whole agonist stimulation
period; or by taking the area under the curve for the whole
stimulation period.
[0149] The assay can be performed as a screening assay as well as
with the test articles present in standard amounts (e.g., 10 .mu.M)
in only one or two wells of a multi-well plate during the primary
screen. Hits in this screen were typically profiled more
exhaustively (multiple times), subjected to dose-response or
competition assays and tested in counter screens against other
voltage-gate sodium channels or other biologically relevant target
molecules.
[0150] FLIPR.RTM. or FLIPR.sup.TETRA.RTM. membrane potential assay
with KCl and test article pre-incubation: Cells are prepared by
plating the recombinant HEK293 cells or other host cells expressing
either recombinant or non-recombinant, native Na.sub.v1.7 alpha
subunit, alone or in combination with various beta and gamma
subunits at a density of .about.40,000 cells/well into a 96-well
black, clear-bottom, PDL-coated plate. The assay can be adapted to
384-well or 1,536-well format, if desired, using proportionately
less cells and media. The plate is then incubated in growth media,
with or without selective antibiotic, overnight at 37.degree. C. at
5% CO.sub.2, 95% humidity, in preparation for the assay (see, e.g.,
Benjamin et. al., J. Biomol. Screen 10(4):365-373 (2005)). For
screens and counter-screens of other voltage-gated sodium channels,
the assay protocol is similar, though optimal densities of cells,
media and subsequent assay components can be fine-tuned for the
particular cell line or sodium channel isoform being tested.
[0151] The next day, at the start of the assay, the media is
flicked from the cells and the wells are washed once with 50
.mu.L/well assay buffer (1.times. Hank's balanced salt solution
without sodium bicarbonate or phenol red, 20 mM Hepes, pH 7.3) and
then pre-incubated with the test articles, the membrane potential
dye (for cell loading), and the KCl for re-polarization and
synchronization of the channels in the entire population of cells.
For this dye-loading and pre-stimulation step, the components are
added as follows, immediately after the wash step: 1) first, the
compound dilutions and controls are added as 4.times. concentrates
in assay buffer at 50 .mu.L/well; 2) membrane potential dye is
diluted from the stock solution in assay buffer (4.times.
concentrate) and added to the plate at 50 .mu.L/well; and 3) a
solution of 180 mM KCl (2.times.) is prepared by diluting a 2M
stock solution into assay buffer and the solution added to the
cells at 100 .mu.L/well. The cells are incubated at 37.degree. C.
in the dark for 30-60 min. before their fluorescence is
measured.
[0152] The plates containing dye-loaded cells are then flicked to
remove the pre-incubation components and washed once with 50
.mu.L/well assay buffer. A 50 .mu.L/well aliquot of membrane
potential dye is added back to the plate, and the real-time assay
is commenced. The fluorescence of cells is measured using a
fluorescence plate reader (FLIPR.sup.TETRA.RTM. or FLIPR384.RTM.,
MDS, Molecular Devices, Sunnyvale, Calif.). Samples are excited by
either a laser or a PMT light source (Excitation wavelength=510-545
nM) and the emissions are filtered (Emission wavelength=565-625
nM). The additions of the compounds (first) and then the channel
activators (later) in this are performed on the fluorescence plate
reader and the results, expressed as relative fluorescence units
(RFU), are captured by means of camera shots every 1-3 sec., then
displayed in real-time and stored. Generally, there is a 15 sec.
base line, with camera shots taken every 1.5 sec., then the test
compounds are added, then another 120 sec. baseline is conducted,
with camera shots taken every 3 sec. Finally, the agonist solution
(containing veratridine and scorpion venom) is added. The amplitude
of fluorescence increase, resulting from the detection of membrane
potential change, is captured for .about.120 sec. thereafter.
Results are expressed in relative fluorescence units (RFU) and can
be determined by using the maximum signal during the latter part of
the stimulation; or the maximum minus the minimum during the whole
stimulation period; or by taking the area under the curve for the
whole stimulation period.
[0153] The assay can be performed as a screening assay as well with
the test articles present in standard amounts (e.g., 10 .mu.M) in
only one or two wells of a multi-well plate during the primary
screen. Hits in this screen are typically profiled more
exhaustively (multiple times), subjected to dose-response or
competition assays and tested in counter screens against other
voltage-gate sodium channels or other biologically relevant target
molecules.
[0154] FLIPR.RTM. or FLIPR.sup.TETRA.RTM. sodium dye assay without
KCl and test article pre-incubation: Cells are prepared by plating
the recombinant HEK293 cells or other host cells expressing either
recombinant or non-recombinant, native, Na.sub.v1.7 alpha subunit,
alone or in combination with various beta and gamma subunits at a
density of .about.40,000 cells/well into a 96-well black,
clear-bottom, PDL-coated plate. The assay can be adapted to
384-well or 1,536-well format, if desired, using proportionately
less cells and media. The plate is then incubated in growth media,
with or without selective antibiotic, overnight at 37.degree. C. at
5% CO.sub.2, 95% humidity, in preparation for the assay. For
counter-screens of other voltage-gated sodium channels, the
procedure is very similar, though optimal densities of cells, media
and subsequent assay components can be fine-tuned for the
particular cell line or isoform.
[0155] The next day, at the start of the assay, the media is
flicked from the cells and the wells washed once with 50 .mu.L/well
assay buffer (1.times. Hank's balanced salt solution without sodium
bicarbonate or phenol red, 20 mM Hepes, pH 7.3). Membrane potential
dye is then added to each well of the 96-well plate (50
.mu.L/well), from a freshly diluted sample of the stock (now at
4.times. concentration) in the assay buffer. The cells are
incubated at 37.degree. C. in the dark for 30-60 min. before their
fluorescence is measured.
[0156] In this standard membrane potential assay, the 96-well plate
containing dye-loaded cells is then loaded directly onto the plate
reader without aspirating the dye solution and without any further
washing of the cells. The fluorescence of cells is measured using a
fluorescence plate reader (FLIPR.sup.TETRA.RTM. or FLIPR384.RTM.,
MDS, Molecular Devices, Sunnyvale, Calif.). Samples are excited by
either a laser or a PMT light source (Excitation wavelength=510-545
nM) and the emissions are filtered (Emission wavelength=565-625
nM). The additions of the compounds (first, 50 .mu.L/well from a
4.times. stock plate) and then the channel activators (later, 100
.mu.L/well from a 2.times. stock solution) in this kinetic assay
are performed on the fluorescence plate reader and the results,
expressed as relative fluorescence units (RFU), are captured by
means of camera shots every 1-3 sec., then displayed in real-time
and stored. Generally, there is a 15 sec. base line, with camera
shots taken every 1.5 sec., then the test compounds are added, then
another 120 sec. baseline is conducted, with camera shots taken
every 3 sec. Finally, the agonist solution (containing veratridine
and scorpion venom) is added. The amplitude of fluorescence
increase, resulting from the detection of membrane potential
change, is captured for .about.120 sec. thereafter. Results are
expressed in relative fluorescence units (RFU) and can be
determined by using the maximum signal during the latter part of
the stimulation; or the maximum minus the minimum during the whole
stimulation period; or by taking the area under the curve for the
whole stimulation period.
[0157] The assay can be performed as a screening assay as well,
with the test articles present in standard amounts (e.g. 10 .mu.M)
in only one or two wells of a multi-well plate during the primary
screen. Hits in this screen are typically profiled more
exhaustively (multiple times), subjected to dose-response or
competition assays and tested in counter screens against other
voltage-gate sodium channels or other biologically relevant target
molecules.
[0158] Electrophysiology Assay
[0159] Cells: The hNa.sub.v1.7 expressing HEK-293 cells were plated
on 35 mm culture dishes pre-coated with poly-D-lysine in standard
DMEM culture media (Mediatech, Inc., Herndon, Va.) and incubated in
a 5% CO.sub.2 incubator at 37.degree. C. Cultured cells were used
approximately 12-48 hours after plating.
[0160] Electrophysiology: On the day of experimentation, the 35 mm
dish was placed on the stage of an inverted microscope equipped
with a perfusion system that continuously perfuses the culture dish
with fresh recording media. A gravity driven superfusion system was
used to apply test solutions directly to the cell under evaluation.
This system consists of an array of glass pipettes connected to a
motorized horizontal translator. The outlet of the shooter was
positioned approximately 100 .mu.m from the cell of interest.
[0161] Whole cell currents were recorded using the whole-cell patch
clamp configuration using an Axopatch 200B amplifier (Axon
Instruments, Foster City Calif.), 1322A A/D converter (Axon
Instruments) and pClamp software (v. 8; Axon Instruments) and
stored on a personal computer. Gigaseals were formed and the
whole-cell configuration was established in voltage clamp mode, and
membrane currents generated by hNa.sub.v1.7 were recorded in
gap-free mode. Borosilicate glass pipettes have resistance values
between 1.5 and 2.0 M.OMEGA. when filled with pipette solution and
series resistance (<5 M.OMEGA.) was compensated 75-80%. Signals
were sampled at 50 kHz and low pass filtered at 3 kHz.
[0162] The voltage clamp protocol to examine hNa.sub.v1.7 currents
was as follows. First, the standard current-voltage relationship
was tested by pulsing the cell from the holding voltage (V.sub.h)
of -120 mV by a series of 5 msec long square-shaped test pulses
incrementing in +10 mV steps over the membrane voltage range of -90
mV to +60 mV at the pace of stimulation of 0.5 Hz. This procedure
determines the voltage that elicits the maximal current
(V.sub.max). Second, V.sub.h was re-set to -120 mV and a
steady-state inactivation (SSIN) curve was taken by the standard
double-pulse protocol: 100 ms depolarizing pre-pulse was
incremented in steps of +10 mV (voltage range from -90mV to 0 mV)
immediately followed by the 5ms long test pulse to -10 mV at the
pace of stimulation of 0.2 Hz. This procedure determines the
voltage of full inactivation (V.sub.full). Third, the cell was
repeatedly stimulated with the following protocol, first in the
absence of the test compound then in its presence. The protocol
consisted of depolarizing the cell from the holding potential of
-120 mV to the V.sub.full value for 4.5 seconds then repolarizing
the cell to the holding potential for 10 ms before applying the
test pulse to the V.sub.max for 5 ms. The amount of inhibition
produced by the test compound was determined by comparing the
current amplitude elicited by the test pulse in the absence and
presence of the compound.
[0163] Solutions and chemicals: For electrophysiological recordings
the external solution was either standard, DMEM supplemented with
10 mM HEPES (pH adjusted to 7.34 with NaOH and the osmolarity
adjusted to 320) or Tyrodes salt solution (Sigma, USA) supplemented
with 10 mM HEPES (pH adjusted to 7.4 with NaOH; osmolarity=320).
The internal pipette solution contained (in mM): NaCl (10), CsF
(140), CaCl.sub.2 (1), MgCl.sub.2 (5), EGTA (11), HEPES (10: pH
7.4, 305 mOsm). Compounds were prepared first as series of stock
solutions in DMSO and then dissolved in external solution; DMSO
content in final dilutions did not exceed 0.3%. At this
concentration, DMSO did not affect sodium currents. Vehicle
solution used to establish base line was also contacting 0.3%
DMSO.
[0164] Data analysis: Data was analyzed off-line using Clampfit
software (pClamp, v. 8; Axon Instruments) and graphed using
GraphPad Prizm (v. 4.0) software.
[0165] In Vivo Assay for Pain
[0166] The peptides of the invention can be tested for their
antinociceptive activity in the formalin model as described in
Hunskaar et al., J. Neurosci. Methods 14: 69-76 (1985). Male Swiss
Webster NIH mice (20-30 g; Harlan, San Diego, Calif.) can be used
in all experiments. Food is withdrawn on the day of experiment.
Mice are placed in Plexiglass jars for at least 1 hour to acclimate
to the environment. Following the acclimation period, mice are
weighed and given either the compound of interest administered i.p.
or p.o., or the appropriate volume of vehicle (for example, 10%
Tween-80 or 0.9% saline, and other pharmaceutically acceptable
vehicles) as control. Fifteen minutes after the i.p. dosing, and 30
minutes after the p.o. dosing, mice are injected with formalin (20
.mu.L of 5% formaldehyde solution in saline) into the dorsal
surface of the right hind paw. Mice are transferred to the
Plexiglass jars and monitored for the amount of time spent licking
or biting the injected paw. Periods of licking and biting are
recorded in 5-minute intervals for 1 hour after the formalin
injection. All experiments are done in a blinded manner during the
light cycle. The early phase of the formalin response is measured
as licking/biting between 0-5 minutes, and the late phase is
measured from 15-50 minutes. Differences between vehicle and drug
treated groups can be analyzed by one-way analysis of variance
(ANOVA). A P value<0.05 is considered significant. Peptides are
considered to be efficacious for treating acute and chronic pain if
they have activity in blocking both the early and second phase of
formalin-induced paw-licking activity.
[0167] In Vivo Assays for Inflammatory or Neuropathic Pain
[0168] Test Animals: Each experiment uses rats weighing between
200-260 g at the start of the experiment. The rats are group-housed
and have free access to food and water at all times, except prior
to oral administration of a test compound when food is removed for
16 hours before dosing. A control group acts as a comparison to
rats treated with test peptides. The control group is administered
the carrier as used for the test compound. The volume of carrier
administered to the control group is the same as the volume of
carrier and test compound administered to the test group.
[0169] Inflammatory Pain: To assess the actions of test peptides on
the treatment of inflammatory pain, the Freund's complete adjuvant
("FCA") model of inflammatory pain is used. FCA-induced
inflammation of the rat hind paw is associated with the development
of persistent inflammatory mechanical and thermal hyperalgesia and
provides reliable prediction of the anti-hyperalgesic action of
clinically useful analgesic drugs (Bartho et al.,
Naunyn-Schmiedeberg's Archives of Pharmacol. 342:666.sup.-670
(1990)). The left hind paw of each animal is administered a 50
.mu.L intraplantar injection of 50% FCA. 24-hour post-injection,
each animal is assessed for response to noxious mechanical stimuli
by determining the paw withdrawal threshold (PWT), or to noxious
thermal stimuli by determining the paw withdrawal latency (PWL), as
described below. Rats are then administered a single injection of
either a test peptide or 30 mg/Kg of a positive control compound
(indomethacin). Responses to noxious mechanical or thermal stimuli
are then determined 1, 3, 5 and 24 hours post administration
(admin). Percentage reversal of hyperalgesia for each animal is
defined as:
% reversal = [ ( post administration P W T or P W L ) - ( pre -
administration P W T or P W L ) ] [ ( baseline P W T or P W L ) - (
pre - administration P W T or P W L ) ] .times. 100
##EQU00001##
[0170] Neuropathic Pain: To assess the actions of the test
compounds for the treatment of neuropathic pain the Seltzer model
or the Chung model can be used.
[0171] In the Seltzer model, the partial sciatic nerve ligation
model of neuropathic pain is used to produce neuropathic
hyperalgesia in rats (Seltzer et al., Pain 43:205-218 (1990)).
Partial ligation of the left sciatic nerve is performed under
isoflurane/O.sub.2 inhalation anaesthesia. Following induction of
anaesthesia, the left thigh of the rat is shaved and the sciatic
nerve exposed at high thigh level through a small incision and is
carefully cleared of surrounding connective tissues at a site near
the trocanther just distal to the point at which the posterior
biceps semitendinosus nerve branches off of the common sciatic
nerve. A 7-0 silk suture is inserted into the nerve with a 3/8
curved, reversed-cutting mini-needle and tightly ligated so that
the dorsal 1/3 to 1/2 of the nerve thickness is held within the
ligature. The wound is closed with a single muscle suture (4-0
nylon (Vicryl)) and vetbond tissue glue. Following surgery, the
wound area is dusted with antibiotic powder. Sham-treated rats
undergo an identical surgical procedure except that the sciatic
nerve is not manipulated. Following surgery, animals are weighed
and placed on a warm pad until they recover from anaesthesia.
Animals are then returned to their home cages until behavioral
testing begins. The animals are assessed for response to noxious
mechanical stimuli by determining PWT, as described below, prior to
surgery (baseline), then immediately prior to and 1, 3, and 5 hours
after drug administration for rear paw of the animal. Percentage
reversal of neuropathic hyperalgesia is defined as:
% reversal = [ ( post administration P W T ) - ( pre -
administration P W T ) ] [ ( baseline P W T ) - ( pre -
administration P W T ) ] .times. 100 ##EQU00002##
[0172] In the Chung model, the spinal nerve ligation model of
neuropathic pain is used to produce mechanical hyperalgesia,
thermal hyperalgesia and tactile allodynia in rats. Surgery is
performed under isoflurane/O.sub.2 inhalation anaesthesia.
Following induction of anaesthesia, a 3 cm incision is made and the
left paraspinal muscles are separated from the spinous process at
the L.sub.4-S.sub.2 levels. The L.sub.6 transverse process is
carefully removed with a pair of small rongeurs to identify
visually the L.sub.4-L.sub.6 spinal nerves. The left L.sub.5 (or
L.sub.5 and L.sub.6) spinal nerve(s) is (are) isolated and tightly
ligated with silk thread. A complete hemostasis is confirmed and
the wound is sutured using non-absorbable sutures, such as nylon
sutures or stainless steel staples. Sham-treated rats undergo an
identical surgical procedure except that the spinal nerve(s) is
(are) not manipulated. Following surgery, animals are weighed,
administered a subcutaneous (s.c.) injection of saline or ringers
lactate, the wound area is dusted with antibiotic powder and they
are kept on a warm pad until they recover from the anaesthesia.
Animals are then returned to their home cages until behavioral
testing begins. The animals are assessed for response to noxious
mechanical stimuli by determining PWT, as described below, prior to
surgery (baseline), then immediately prior to and 1, 3, and 5 hours
after being administered a peptide to the left rear paw of the
animal. The animals can also be assessed for response to noxious
thermal stimuli or for tactile allodynia, as described below. The
Chung model for neuropathic pain is described in Kim et al., Pain
50(3):355-363 (1992).
[0173] Tactile Allodynia: Sensitivity to non-noxious mechanical
stimuli can be measured in animals to assess tactile allodynia.
Rats are transferred to an elevated testing cage with a wire mesh
floor and allowed to acclimate for five to ten minutes. A series of
von Frey monofilaments are applied to the plantar surface of the
hindpaw to determine the animal's withdrawal threshold. The first
filament used possesses a buckling weight of 9.1 gms (0.96 log
value) and is applied up to five times to see if it elicits a
withdrawal response. If the animal has a withdrawal response, then
the next lightest filament in the series would be applied up to
five times to determine if it also could elicit a response. This
procedure is repeated with subsequent lesser filaments until there
is no response and the identity of the lightest filament that
elicits a response is recorded. If the animal does not have a
withdrawal response from the initial 9.1 gms filament, then
subsequent filaments of increased weight are applied until a
filament elicits a response and the identity of this filament is
recorded. For each animal, three measurements are made at every
time point to produce an average withdrawal threshold
determination. Tests can be performed prior to, and at 1, 2, 4 and
24 hours post drug administration.
[0174] Mechanical Hyperalgesia: Sensitivity to noxious mechanical
stimuli can be measured in animals using the paw pressure test to
assess mechanical hyperalgesia. In rats, hind paw withdrawal
thresholds ("PWT"), measured in grams, in response to a noxious
mechanical stimulus are determined using an analgesymeter (Model
7200, commercially available from Ugo Basile of Italy), as
described in Stein (Biochemistry & Behavior 31: 451-455
(1988)). The rat's paw is placed on a small platform, and weight is
applied in a graded manner up to a maximum of 250 grams. The
endpoint is taken as the weight at which the paw is completely
withdrawn. PWT is determined once for each rat at each time point.
PWT can be measured only in the injured paw, or in both injured and
non-injured paws. In one non-limiting embodiment, mechanical
hyperalgesia associated with nerve injury induced pain (neuropathic
pain) can be assessed in rats. Rats are tested prior to surgery to
determine a baseline, or normal, PWT. Rats are tested again 2 to 3
weeks post-surgery, prior to, and at different times after (e.g. 1,
3, 5 and 24 hr) drug administration. An increase in PWT following
drug administration indicates that the test compound reduces
mechanical hyperalgesia.
[0175] Thermal Hyperalgesia: The plantar test can be used to assess
thermal hyperalgesia. For this test, hind paw withdrawal latencies
to a noxious thermal stimulus are determined using a plantar test
apparatus (commercially available from Ugo Basile of Italy)
following the technique described by K. Hargreaves et al., "A New
and Sensitive Method for Measuring Thermal Nociception in Cutaneous
Hyperalgesia," Pain 32(1): 77-88 (1988). The maximum exposure time
is set at 32 seconds to avoid tissue damage and any directed paw
withdrawal from the heat source is taken as the end point. Three
latencies are determined at each time point and averaged. Only the
affected (ipsilateral) paw is tested.
[0176] In Vivo Assay for Anticonvulsant Activity
[0177] The compounds of the present invention can be tested for in
vivo anticonvulsant activity after i.v., p.o., or i.p. injection
using any of a number of anticonvulsant tests in mice, including
the maximum electroshock seizure test (MES). Maximum electroshock
seizures are induced in male NSA mice weighing between 15-20 g and
in male Sprague-Dawley rats weighing between 200-225 g by
application of current (for mice: 50 mA, 60 pulses/sec, 0.8 msec
pulse width, 1 sec duration, D.C.; for rats: 99 mA, 125 pulses/sec,
0.8 msec pulse width, 2 sec duration, D.C.) using a Ugo Basile ECT
device (Model 7801). Mice are restrained by gripping the loose skin
on their dorsal surface and saline-coated corneal electrodes are
held lightly against the two corneae. Rats are allowed free
movement on the bench top and ear-clip electrodes are used. Current
is applied and animals are observed for a period of up to 30
seconds for the occurrence of a tonic hindlimb extensor response. A
tonic seizure is defined as a hindlimb extension in excess of 90
degrees from the plane of the body. Results can be treated in a
quantal manner.
Pharmaceutical Compositions
[0178] Although a peptide of the invention may be administered to a
subject in the form of a raw chemical without any other components
present, the peptide is preferably administered as part of a
pharmaceutical composition containing the peptide combined with a
suitable pharmaceutically acceptable carrier. Such a carrier can be
selected from pharmaceutically acceptable excipients and
auxiliaries appropriate for peptide compositions.
[0179] Pharmaceutical "compositions within the scope of the present
invention include all compositions where a peptide of the invention
is combined with a pharmaceutically acceptable carrier. In a
preferred embodiment, the peptide is present in the composition in
an amount that is effective to achieve its intended therapeutic
purpose. While individual subject needs may vary, a determination
of optimal ranges of effective amounts of each peptide is within
the skill of the art. Typically, the peptides may be administered
to a subject, e.g., a human, at a dosage of about 0.01, 0.05, 0.1,
0.5, 1, 5, 10, 50, 100, or 500 .mu.g/kg body weight, depending on
the specific peptide selected, the desired therapeutic response,
the route of administration, the formulation, the medical condition
of the subject, and other factors known to those of skill in the
art.
[0180] A pharmaceutical composition of the present invention can be
administered to any subject that may experience the beneficial
effects of a peptide of the invention. Foremost among such subjects
are mammals, especially humans and companion animals, although the
invention is not intended to be so limited.
[0181] A pharmaceutical composition of the present invention is
preferably manufactured in a manner which itself will be known in
view of the instant disclosure, for example, by means of
conventional mixing, dissolving, formulating or lyophilizing
processes.
[0182] A pharmaceutical composition of the present invention can
contain from about 0.01 to 99 percent by weight, and preferably
from about 0.25 to 75 percent by weight, of active peptide(s).
[0183] A method of the present invention, such as a method for
treating a disorder or providing preemptive or palliative treatment
of a disorder responsive to the blockade of sodium channels in a
subject in need thereof, can further comprise administering a
second therapeutic agent to the subject in combination with a
peptide of the present invention. The other therapeutic agent is
preferably administered in an effective amount.
[0184] Effective amounts of the other therapeutic agents will
generally be known to or readily ascertainable by those skilled in
the art. It is well within the skilled artisan's purview to
determine the other therapeutic agent's optimal effective-amount
range.
[0185] A peptide of the invention (i.e., the first therapeutic
agent) and the second therapeutic agent can act additively or
synergistically. Alternatively, the second therapeutic agent can be
used to treat a disorder or condition that is different from the
disorder or condition for which the first therapeutic agent is
being administered. In one embodiment, a peptide of the invention
is administered concurrently with a second therapeutic agent; for
example, a single composition comprising both an effective amount
of a peptide of the invention, and an effective amount of the
second therapeutic agent can be administered. Accordingly, the
present invention further provides a pharmaceutical composition
comprising a combination of a peptide of the invention, the second
therapeutic agent, and a pharmaceutically acceptable carrier.
Alternatively, a first pharmaceutical composition comprising an
effective amount of a peptide of the invention and a second
pharmaceutical composition comprising an effective amount of the
second therapeutic agent can be concurrently administered in two
different compositions. In another embodiment, an effective amount
of a peptide of the invention is administered prior or subsequent
to administration of an effective amount of the second therapeutic
agent. In this embodiment, the peptide of the invention is
administered while the second therapeutic agent exerts its
therapeutic effect, or the second therapeutic agent is administered
while the peptide of the invention exerts its therapeutic effect
for treating a disorder or condition or providing preemptive or
palliative treatment of a disorder or condition.
[0186] The second therapeutic agent can be an opioid agonist, a
non-opioid analgesic, a non-steroidal anti-inflammatory agent, an
antimigraine agent, a Cox-II inhibitor, a .beta.-adrenergic
blocker, an anticonvulsant, an antidepressant, an anticancer agent,
an agent for treating addictive disorder, an agent for treating
Parkinson's disease and parkinsonism, an agent for treating
anxiety, an agent for treating epilepsy, an agent for treating a
seizure, an agent for treating a stroke, an agent for treating a
pruritic condition, an agent for treating psychosis, an agent for
treating ALS, an agent for treating a cognitive disorder, an agent
for treating a migraine, an agent for treating vomiting, an agent
for treating dyskinesia, or an agent for treating depression, or a
mixture thereof.
[0187] Examples of useful opioid agonists include, but are not
limited to, alfentanil, allylprodine, alphaprodine, anileridine,
benzylmorphine, bezitramide, buprenorphine, butorphanol,
clonitazene, codeine, desomorphine, dextromoramide, dezocine,
diampromide, diamorphone, dihydrocodeine, dihydromorphine,
dimenoxadol, dimepheptanol, dimethylthiambutene, dioxaphetyl
butyrate, dipipanone, eptazocine, ethoheptazine,
ethylmethylthiambutene, ethylmorphine, etonitazene, fentanyl,
heroin, hydrocodone, hydromorphone, hydroxypethidine, isomethadone,
ketobemidone, levorphanol, levophenacylmorphan, lofentanil,
meperidine, meptazinol, metazocine, methadone, metopon, morphine,
myrophine, nalbuphine, narceine, nicomorphine, norlevorphanol,
normethadone, nalorphine, normorphine, norpipanone, opium,
oxycodone, oxymorphone, papaveretum, pentazocine, phenadoxone,
phenomorphan, phenazocine, phenoperidine, piminodine, piritramide,
proheptazine, promedol, properidine, propiram, propoxyphene,
sufentanil, tilidine, tramadol, pharmaceutically acceptable salts
thereof; and mixtures thereof.
[0188] In certain embodiments, the opioid agonist is selected from
codeine, hydromorphone, hydrocodone, oxycodone, dihydrocodeine,
dihydromorphine, morphine, tramadol, oxymorphone, pharmaceutically
acceptable salts thereof, and mixtures thereof.
[0189] Examples of useful non-opioid analgesics include
non-steroidal anti-inflammatory agents, such as aspirin, ibuprofen,
diclofenac, naproxen, benoxaprofen, flurbiprofen, fenoprofen,
flubufen, ketoprofen, indoprofen, piroprofen, carprofen, oxaprozin,
pramoprofen, muroprofen, trioxaprofen, suprofen, aminoprofen,
tiaprofenic acid, fluprofen, bucloxic acid, indomethacin, sulindac,
tolmetin, zomepirac, tiopinac, zidometacin, acemetacin, fentiazac,
clidanac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic
acid, niflumic acid, tolfenamic acid, diflurisal, flufenisal,
piroxicam, sudoxicam, isoxicam, and pharmaceutically acceptable
salts thereof, and mixtures thereof. Examples of other suitable
non-opioid analgesics include the following, non limiting, chemical
classes of analgesic, antipyretic, nonsteroidal antiinflammatory
drugs: salicylic acid derivatives, including aspirin, sodium
salicylate, choline magnesium trisalicylate, salsalate, diflunisal,
salicylsalicylic acid, sulfasalazine, and olsalazin; para
aminophennol derivatives including acetaminophen and phenacetin;
indole and indene acetic acids, including indomethacin, sulindac,
and etodolac; heteroaryl acetic acids, including tolmetin,
diclofenac, and ketorolac; anthranilic acids (fenamates), including
mefenamic acid, and meclofenamic acid; enolic acids, including
oxicams (piroxicam, tenoxicam), and pyrazolidinediones
(phenylbutazone, oxyphenthartazone); and alkanones, including
nabumetone. For a more detailed description of the NSAIDs, see Paul
A. Insel, Analgesic Antipyretic and Antiinflammatory Agents and
Drugs Employed in the Treatment of Gout, in Goodman & Gilman's
The Pharmacological Basis of Therapeutics 617-57 (Perry B.
Molinhoff and Raymond W. Ruddon eds., 9th ed 1996) and Glen R.
Hanson, Analgesic, Antipyretic and Anti Inflammatory Drugs in
Remington: The Science and Practice of Pharmacy Vol. II 1196-1221
(A. R. Gennaro ed. 19th ed. 1995) which are hereby incorporated by
reference in their entireties. Suitable Cox-II inhibitors and
5-lipoxygenase inhibitors, as well as combinations thereof, are
described in U.S. Pat. No. 6,136,839, which is hereby incorporated
by reference in its entirety. Examples of useful Cox II inhibitors
include, but are not limited to, rofecoxib and celecoxib.
[0190] Examples of useful antimigraine agents include, but are not
limited to, alpiropride, bromocriptine, dihydroergotamine,
dolasetron, ergocornine, ergocominine, ergocryptine, ergonovine,
ergot, ergotamine, flumedroxone acetate, fonazine, ketanserin,
lisuride, lomerizine, methylergonovine, methysergide, metoprolol,
naratriptan, oxetorone, pizotyline, propranolol, risperidone,
rizatriptan, sumatriptan, timolol, trazodone, zolmitriptan, and
mixtures thereof.
[0191] Examples of useful .beta.-adrenergic blockers include, but
are not limited to, acebutolol, alprenolol, amosulabol, arotinolol,
atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol,
bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, butidrine
hydrochloride, butofilolol, carazolol, carteolol, carvedilol,
celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol,
indenolol, labetalol, levobunolol, mepindolol, metipranolol,
metoprolol, moprolol, nadolol, nadoxolol, nebivalol, nifenalol,
nipradilol, oxprenolol, penbutolol, pindolol, practolol,
pronethalol, propranolol, sotalol, sulfinalol, talinolol,
tertatolol, tilisolol, timolol, toliprolol, and xibenolol.
[0192] Examples of useful anticonvulsants include, but are not
limited to, acetylpheneturide, albutoin, aloxidone,
aminoglutethimide, 4-amino-3-hydroxybutyric acid, atrolactamide,
beclamide, buramate, calcium bromide, carbamazepine, cinromide,
clomethiazole, clonazepam, decimemide, diethadione, dimethadione,
doxenitroin, eterobarb, ethadione, ethosuximide, ethotoin,
felbamate, fluoresone, gabapentin, 5-hydroxytryptophan,
lamotrigine, magnesium bromide, magnesium sulfate, mephenytoin,
mephobarbital, metharbital, methetoin, methsuximide,
5-methyl-5-(3-phenanthryl)-hydantoin, 3-methyl-5-phenylhydantoin,
narcobarbital, nimetazepam, nitrazepam, oxcarbazepine,
paramethadione, phenacemide, phenetharbital, pheneturide,
phenobarbital, phensuximide, phenylmethylbarbituric acid,
phenytoin, phethenylate sodium, potassium bromide, pregabaline,
primidone, progabide, sodium bromide, solanum, strontium bromide,
suclofenide, sulthiame, tetrantoin, tiagabine, topiramate,
trimethadione, valproic acid, valpromide, vigabatrin, and
zonisamide.
[0193] Examples of useful antidepressants include, but are not
limited to, binedaline, caroxazone, citalopram, (S)-citalopram,
dimethazan, fencamine, indalpine, indeloxazine hydrocholoride,
nefopam, nomifensine, oxitriptan, oxypertine, paroxetine,
sertraline, thiazesim, trazodone, benmoxine, iproclozide,
iproniazid, isocarboxazid, nialamide, octamoxin, phenelzine,
cotinine, rolicyprine, rolipram, maprotiline, metralindole,
mianserin, mirtazepine, adinazolam, amitriptyline,
amitriptylinoxide, amoxapine, butriptyline, clomipramine,
demexiptiline, desipramine, dibenzepin, dimetacrine, dothiepin,
doxepin, fluacizine, imipramine, imipramine N-oxide, iprindole,
lofepramine, melitracen, metapramine, nortriptyline, noxiptilin,
opipramol, pizotyline, propizepine, protriptyline, quinupramine,
tianeptine, trimipramine, adrafinil, benactyzine, bupropion,
butacetin, dioxadrol, duloxetine, etoperidone, febarbamate,
femoxetine, fenpentadiol, fluoxetine, fluvoxamine, hematoporphyrin,
hypericin, levophacetoperane, medifoxamine, milnacipran, minaprine,
moclobemide, nefazodone, oxaflozane, piberaline, prolintane,
pyrisuccideanol, ritanserin, roxindole, rubidium chloride,
sulpiride, tandospirone, thozalinone, tofenacin, toloxatone,
tranylcypromine, L-tryptophan, venlafaxine, viloxazine, and
zimeldine.
[0194] Examples of useful anticancer agents include, but are not
limited to, acivicin, aclarubicin, acodazole hydrochloride,
acronine, adozelesin, aldesleukin, altretamine, ambomycin,
ametantrone acetate, aminoglutethimide, amsacrine, anastrozole,
anthramycin, asparaginase, asperlin, azacitidine, azetepa,
azotomycin, batimastat, benzodepa, bicalutamide, bisantrene
hydrochloride, bisnafide dimesylate, bizelesin, bleomycin sulfate,
brequinar sodium, bropirimine, busulfan, cactinomycin, calusterone,
caracemide, carbetimer, carboplatin, carmustine, carubicin
hydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin,
and cisplatin.
[0195] Therapeutic agents useful for treating an addictive disorder
include, but are not limited to, methadone, desipramine,
amantadine, fluoxetine, buprenorphine, an opiate agonist,
3-phenoxypyridine, or a serotonin antagonist.
[0196] Examples of useful therapeutic agents for treating
Parkinson's disease and parkinsonism include, but are not limited
to, carbidopa/levodopa, pergolide, bromocriptine, ropinirole,
pramipexole, entacapone, tolcapone, selegiline, amantadine, and
trihexyphenidyl hydrochloride.
[0197] Examples of useful therapeutic agents for treating anxiety
include, but are not limited to, benzodiazepines, such as
alprazolam, brotizolam, chlordiazepoxide, clobazam, clonazepam,
clorazepate, demoxepam, diazepam, estazolam, flumazenil,
flurazepam, halazepam, lorazepam, midazolam, nitrazepam,
nordazepam, oxazepam, prazepam, quazepam, temazepam, and triazolam;
non-benzodiazepine agents, such as buspirone, gepirone, ipsapirone,
tiospirone, zolpicone, zolpidem, and zaleplon; tranquilizers, such
as barbituates, e.g., amobarbital, aprobarbital, butabarbital,
butalbital, mephobarbital, methohexital, pentobarbital,
phenobarbital, secobarbital, and thiopental; and propanediol
carbamates, such as meprobamate and tybamate.
[0198] Examples of useful therapeutic agents for treating epilepsy
or seizure include, but are not limited to, carbamazepine,
ethosuximide, gabapentin, lamotrigine, phenobarbital, phenytoin,
primidone, valproic acid, trimethadione, benzodiazepines,
gamma-vinyl GABA, acetazolamide, and felbamate.
[0199] Examples of useful therapeutic agents for treating a
pruritic condition include, but are not limited to, naltrexone;
nalmefene; danazol; tricyclics such as amitriptyline, imipramine,
and doxepin; antidepressants such as those given below; menthol;
camphor; phenol; pramoxine; capsaicin; tar; steroids; and
antihistamines.
[0200] Examples of useful therapeutic agents for treating psychosis
include, but are not limited to, phenothiazines such as
chlorpromazine hydrochloride, mesoridazine besylate, and
thoridazine hydrochloride; thioxanthenes such as chloroprothixene
and thiothixene hydrochloride; clozapine; risperidone; olanzapine;
quetiapine; quetiapine fumarate; haloperidol; haloperidol
decanoate; loxapine succinate; molindone hydrochloride; pimozide;
and ziprasidone.
[0201] Examples of useful therapeutic agents for treating cognitive
disorders include, but are not limited to, agents for treating
dementia such as tacrine; donepezil; ibuprofen; antipsychotic drugs
such as thioridazine and haloperidol; and antidepressant drugs such
as those given below.
[0202] Examples of useful therapeutic agents for treating a
migraine include, but are not limited to, sumatriptan;
methysergide; ergotamine; caffeine; and beta-blockers such as
propranolol, verapamil, and divalproex.
[0203] Examples of useful therapeutic agents for treating vomiting
include, but are not limited to, 5-HT3 receptor antagonists such as
ondansetron, dolasetron, granisetron, and tropisetron; dopamine
receptor antagonists such as prochlorperazine, thiethylperazine,
chlorpromazine, metoclopramide, and domperidone; glucocorticoids
such as dexamethasone; and benzodiazepines such as lorazepam and
alprazolam.
[0204] Examples of useful therapeutic agents for treating
dyskinesia include, but are not limited to, reserpine and
tetrabenazine.
[0205] Examples of useful therapeutic agents for treating
depression include, but are not limited to, tricyclic
antidepressants such as amitryptyline, amoxapine, bupropion,
clomipramine, desipramine, doxepin, imipramine, maprotiline,
nefazadone, nortriptyline, protriptyline, trazodone, trimipramine,
and venlafaxine; selective serotonin reuptake inhibitors such as
citalopram, (S)-citalopram, fluoxetine, fluvoxamine, paroxetine,
and setraline; monoamine oxidase inhibitors such as isocarboxazid,
pargyline, phenelzine, and tranylcypromine; and psychostimulants
such as dextroamphetamine and methylphenidate.
Sequences
[0206] The present invention provides the following specific
peptides comprising the amino acid sequences of SEQ ID NOS: 1-15,
and the pharmaceutically acceptable salts, prodrugs and solvates
thereof. If an amino acid residue is not set forth at position 30
and/or position 31, relative to SEQ ID NO: 1, then Xaa.sub.30
and/or Xaa.sub.31 are absent.
[0207] SEQ ID NO: 1 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Xaa.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Xaa.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Xaa.sub-
.28-Xaa.sub.29-Xaa.sub.30-Xaa.sub.31.
[0208] SEQ ID NO: 2 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30-NH.sub.2.
[0209] SEQ ID NO: 3 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30.
[0210] SEQ ID NO: 4 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30.
[0211] SEQ ID NO: 5 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Ile.sub-
.28-Ile.sub.29.
[0212] SEQ ID NO: 6 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30.
[0213] SEQ ID NO: 7 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-.alpha.-Me-Leu.sub.29-Trp.sub.30, wherein "Me" is "methyl."
[0214] SEQ ID NO: 8 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-N-Me-Leu.sub.29-Trp.sub.30.
[0215] SEQ ID NO: 9 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Ile.sub.29-Leu.sub.30-Trp.sub.31.
[0216] SEQ ID NO: 10 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Ile.sub.30.
[0217] SEQ ID NO: 11 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-.alpha.-Me-Leu.sub.29-Ile.sub.30.
[0218] SEQ ID NO: 12 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-Arg.sub.22-
-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub.28-N-Me-Le-
u.sub.29-Ile.sub.30.
[0219] SEQ ID NO: 13 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.5-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21--
Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Ile.sub.-
28-Leu.sub.29-Trp.sub.30.
[0220] SEQ ID NO: 14 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Ile.sub-
.28-Ile.sub.29-Trp.sub.30.
[0221] SEQ ID NO: 15 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Leu.sub-
.28-Trp.sub.29.
[0222] Other sequences discussed herein include the following
peptide sequences. If an amino acid residue is not set forth at
position 30 and/or position 31, relative to SEQ ID NO: 1, then
Xaa.sub.30 and/or Xaa.sub.31 are absent.
[0223] SEQ ID NO: 16 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30 (ProTx II).
[0224] SEQ ID NO: 17 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Ile.sub-
.28-Ile.sub.29 (PaTx I).
[0225] SEQ ID NO: 18 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Tyr.sub.19-Val.sub.20-Cys.sub.21-
-Glu.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Tyr.sub.27-Asn.sub-
.28-Leu.sub.29 (JzTx XII).
[0226] SEQ ID NO: 19 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Leu.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Asp.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Arg.sub-
.28-Leu.sub.29 (GsAF I).
[0227] SEQ ID NO: 20 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Lys.sub.12-Arg.sub.13-Ala.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Arg.sub.20-Cys.sub.21-
-Lys.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Arg.sub.26-Lys.sub.27-Ile.sub-
.28-Ile.sub.29 (JzTx V).
[0228] SEQ ID NO: 21 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Glu.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Ile.sub.29-Glu.sub.30-Glu.sub.31-Gly.sub.32 (VsTx II).
[0229] SEQ ID NO: 22 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Glu.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Ile29-Glu.sub.30-Trp.sub.31 (GsAF II).
[0230] SEQ ID NO: 23 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Lys.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Asp.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Gln.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Arg.sub-
.28-Leu.sub.29 (GrTx I).
[0231] SEQ ID NO: 24 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Glu.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Leu.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Arg.sub.27-Ile.sub-
.28-Ile.sub.29-Asn.sub.30-Met.sub.31 (GsMtx II/PaTX II).
[0232] SEQ ID NO: 25 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Ala.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30.
[0233] SEQ ID NO: 26 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Ala.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Trp.sub.30.
[0234] SEQ ID NO: 27 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Ala.sub-
.28-Leu.sub.29-Trp.sub.30-Xaa.sub.31.
[0235] SEQ ID NO: 28 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Ala.sub.29-Trp.sub.30.
[0236] SEQ ID NO: 29 is
Tyr.sub.1-Cys.sub.2-Gln.sub.3-Lys.sub.4-Trp.sub.5-Met.sub.6-Trp.sub.7-Thr-
.sub.8-Cys.sub.9-Asp.sub.10-Ser.sub.11-Glu.sub.12-Arg.sub.13-Lys.sub.14-Cy-
s.sub.15-Cys.sub.16-Glu.sub.17-Gly.sub.18-Met.sub.19-Val.sub.20-Cys.sub.21-
-Arg.sub.22-Leu.sub.23-Trp.sub.24-Cys.sub.25-Lys.sub.26-Lys.sub.27-Lys.sub-
.28-Leu.sub.29-Ala.sub.30.
[0237] The following examples are illustrative, but not limiting,
of the peptides, compositions and methods of the present invention.
Suitable modifications and adaptations of the variety of conditions
and parameters normally encountered in clinical therapy and which
are obvious to those skilled in the art in view of this disclosure
are within the spirit and scope of the invention.
EXAMPLE 1
Synthesis of Peptides
[0238] Several peptides of SEQ ID NO: 1 set forth in Tables 2 and 3
were synthesized. Fmoc-L-amino acids and HCTU were purchased from
Protein Technologies Inc. Resins were purchased from EMD chemicals
(Fmoc-amino acid-NovaSyn resin) and Anaspec (H-amino acid-2-Cl-Trt
resin and H-Trp(Boc)-2-Cl-Trt resin). HPLC-graded solvents and
ReagentPlus.RTM.-graded reagents were purchased from Sigma-Aldrich.
HATU was purchased from Genscript.
[0239] Linear peptides were obtained by the solid-phase technique
("Fmoc Solid Phase Peptide Synthesis, A Practical Approach," W. C.
Chan and P. D. White, Oxford University Press (2000)) using an ABI
433 and a Pioneer peptide synthesizer.
[0240] Synthesis Route 1: Peptides were assembled stepwise on
0.05-0.1 mmol of Fmoc-amino acid-NovaSyn resin (0.2-0.3 mmol/g) or
Anaspec (H-amino acid-2-Cl-Trt resin (0.4-0.5 mmol/g)) using 5-10
fold excess of Fmoc amino acids. Fmoc protecting groups were
removed using 20% piperidine in DMF and the free amine was coupled
with amino acids/HCTU/NMM (1:1:2). The side-chain protecting groups
used for trifunctional residues were: trityl for Cys, His, Asn and
Gln; t-butyl for Asp, Glu, Ser, Thr and Tyr;
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg; and
t-butyloxycarbonyl for Lys and Trp.
[0241] Linear peptide was treated with TFA:TIS:EDT or
TFA:TIS:EDT:thioanisole: phenol:H.sub.2O (81.5:1:2.5:5:5:5 by
volume) for 2 hours at room temperature to cleave from the resin.
Cleaved peptides were treated with cold diethyl ether to
precipitate peptides, and precipitated peptides were washed with
diethyl ether three times. Crude linear peptides were purified by
reversed Prep-HPLC and white solids were obtained.
[0242] Purified linear peptides (1 mg/10-20 mL) were dissolved in
0.1 M Tris/HCl buffer, 2.0 M Urea, 0.15 mM GSH, 0.3 mM GSSG, pH 8
(adjusted with Saturated Aqueous NaHCO.sub.3) overnight and
purified by reversed Prep-HPLC (C18, 5 .mu.m, 250 mm.times.21 mm,
buffer; A: 0.1% v/v TFA in H.sub.2O and B: 0.1% v/v TFA in MeCN).
Purified peptides were characterized by analytical LCMS and
NMR.
[0243] Synthesis Route 2: Peptides were assembled stepwise on
0.025-0.05 mmol of H-Trp(Boc)-2-Cl-Trt resin (0.42 mmol/g) using
8-16 fold excess of Fmoc amino acids. Fmoc protecting groups were
removed using 20% piperidine in DMF and the free amine was coupled
with amino acids/HATU/2,4,6-collidine. The side-chain protecting
groups used for trifunctional residues were: trityl for Cys, His,
Asn and Gln; t-butyl for Asp, Glu, Ser, Thr and Tyr;
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg; and
t-butyloxycarbonyl for Lys and Trp.
[0244] Linear peptide was treated with
TFA:TIS:3,6-Dioxa-1,8-octane-dithiol:thioanisole:phenol:H.sub.2O
(81.5:1:2.5:5:5:5 by vol.) for 2 hours at room temperature to
cleave from the resin. Cleaved peptides were treated with cold
diethyl ether to precipitate peptides out and precipitated peptides
were washed with diethyl ether for 3 times. Crude linear peptides
were purified by reversed Prep-HPLC and white solids were
obtained.
[0245] Purified linear peptides (1 mg/10 mL) were dissolved in 0.1
M of Tris/HCl buffer, 2.0 M of Urea, 0.15 mM of GSH, 0.3 mM of
GSSG, pH 8 (adjusted with saturated aqueous NaHCO.sub.3) overnight
and purified by reversed Prep-HPLC (C18, 5 .mu.m, 250 mm.times.21
mm, buffer; A: 0.1% v/v TFA in H.sub.2O and B: 0.1% v/v TFA in
MeCN). Purified toxins were characterized by analytical LCMS and
NMR.
TABLE-US-00004 TABLE 2 Peptides Of SEQ ID NO: 1 IC50 IC50 SEQ
Na.sub.v1.7 Na.sub.v1.2 Sequence ID NO: (nm) (nm)
YCQKWMWTCDSERKCCEGMVCRLWCKKKLW-NH.sub.2 2 1 8 .+-. 1
YCQKWMWTCDSARKCCEGMVCRLWCKKKLW 3 0.2 .+-. 0.03 8 .+-. 2
YCQKWMWTCDSERKCCEGLVCRLWCKKKLW 4 1.7 .+-. 0.1 44.7 .+-. 15
YCQKWMWTCDSERKCCEGMVCRLWCKKII 5 552 .+-. 90 919 .+-. 130
YCQKWMWTCDSARKCCEGLVCRLWCKKKLW 6 1 .+-. 0 24 .+-. 10
YCQKWMWTCDSARKCCEGLVCRLWCKKK[aml]W 7 8 .+-. 1 28 .+-. 6
YCQKWMWTCDSARKCCEGLVCRLWCKKK[nml]W 8 18 .+-. 0 51 .+-. 10
YCQKWMWTCDSARKCCEGLVCRLWCKKKILW 9 1 .+-. 0 33 .+-. 0
YCQKWMWTCDSARKCCEGLVCRLWCKKKLI 10 4 .+-. 0 98 .+-. 10
YCQKWMWTCDSARKCCEGLVCRLWCKKK[aml]I 11 10 .+-. 1 342 .+-. 73
YCQKWMWTCDSARKCCEGLVCRLWCKKK[nml]I 12 528 + 100 138 .+-. 40
YCQKWMWTCDSARKCCEGLVCRLWCKKILW 13 3 .+-. 0 50 .+-. 10
YCQKWMWTCDSARKCCEGLVCRLWCKKIIW 14 5 .+-. 1 45 .+-. 0
YCQKWMWTCDSARKCCEGLVCRLWCKKLW 15 67 .+-. 0 63 .+-. 0
TABLE-US-00005 TABLE 3 Natural Toxins IC50 IC50 SEQ Na.sub.v1.7
Na.sub.v1.2 Sequence Name ID NO: (nm) (nm)
YCQKWMWTCDSERKCCEGMVCRLWCKKKLW ProTx II 16 1 105 .+-. 20
YCQKWMWTCDSARKCCEGLVCRLWCKKII PaTx I 17 423 .+-. 110 5000
YCQKWMWTCDSERKCCEGYVCELWCKYNL JzTx XII 18 1.527 .+-. 130 73.939
.+-. 14.440 YCQKWLWTCDSERKCCEDMVCRLWCKKRL GsAF I 19 249 .+-. 20 255
.+-. 43 YCQKWMWTCDSKRACCEGLRCKLWCRKII JzTx V 20 14 .+-. 10 157 .+-.
20 YCQKWMWTCDEERKCCEGLVCRLWCKKKIEEG VsTx II 21 9.261 .+-. 2,210
42.409 .+-. 10.010 YCQKWMWTCDEERKCCEGLVCRLWCKKKIEW GsAF II 22 70
.+-. 10 410 .+-. 20 YCQKWMWTCDSKRKCCEDMVCQLWCKKRL GrTx I 23 1.007
.+-. 600 2.690 .+-. 460 YCQKWMWTCDEERKCCEGLVCRLWCKRIINM GsMTx
II/PaTX II 24 260 .+-. 50 2.699 .+-. 790
EXAMPLE 2
Sodium Channel Assays
[0246] The peptides of SEQ ID NO: 1 set forth in Table 2 and the
natural toxins set forth in Table 3 were assayed for sodium
channel-blocking activity. As shown in Table 2, peptides of the
present invention are potent antagonists of the Nav1.7 sodium
channel. In addition, the peptides show selectivity as Na.sub.v1.7
channel blockers, relative to Na.sub.v1.2.
[0247] Having now fully described this invention, it will be
understood by those of ordinary skill in the art that the same can
be performed within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any embodiment thereof.
[0248] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
[0249] All patents and publications cited herein are fully
incorporated by reference herein in their entirety.
Sequence CWU 1
1
29131PRTArtificial sequenceIsolated
peptideMISC_FEATURE(12)..(12)any natural or modified amino acid
residueMISC_FEATURE(19)..(19)any natural or modified amino acid
residueMISC_FEATURE(28)..(28)any natural or modified amino acid
residueMISC_FEATURE(29)..(29)any natural or modified amino acid
residueMISC_FEATURE(30)..(30)any natural or modified amino acid
residue or is absentMISC_FEATURE(31)..(31)any natural or modified
amino acid residue or is absent 1Tyr Cys Gln Lys Trp Met Trp Thr
Cys Asp Ser Xaa Arg Lys Cys Cys 1 5 10 15 Glu Gly Xaa Val Cys Arg
Leu Trp Cys Lys Lys Xaa Xaa Xaa Xaa 20 25 30 230PRTArtificial
sequenceAn embodiment of the present
inventionMISC_FEATURE(30)..(30)tryptophan modified with an amino
group 2Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu Arg Lys Cys
Cys 1 5 10 15 Glu Gly Met Val Cys Arg Leu Trp Cys Lys Lys Lys Leu
Trp 20 25 30 330PRTArtificial sequenceAn embodiment of the present
invention 3Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys
Cys Cys 1 5 10 15 Glu Gly Met Val Cys Arg Leu Trp Cys Lys Lys Lys
Leu Trp 20 25 30 430PRTArtificial sequenceAn embodiment of the
present invention 4Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu
Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys
Lys Lys Leu Trp 20 25 30 529PRTArtificial sequenceAn embodiment of
the present invention 5Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser
Glu Arg Lys Cys Cys 1 5 10 15 Glu Gly Met Val Cys Arg Leu Trp Cys
Lys Lys Ile Ile 20 25 630PRTArtificial sequenceAn embodiment of the
present invention 6Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala
Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys
Lys Lys Leu Trp 20 25 30 730PRTArtificial sequenceAn embodiment of
the present inventionMISC_FEATURE(29)..(29)alpha-methylated leucine
residue 7Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys
Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys
Leu Trp 20 25 30 830PRTArtificial sequenceAn embodiment of the
present inventionMISC_FEATURE(29)..(29)N-methylated leucine residue
8Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys Cys Cys 1
5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys Leu Trp 20
25 30 931PRTArtificial sequenceAn embodiment of the present
invention 9Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys
Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys
Ile Leu Trp 20 25 30 1030PRTArtificial sequenceAn embodiment of the
present invention 10Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala
Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys
Lys Lys Leu Ile 20 25 30 1130PRTArtificial sequenceAn embodiment of
the present inventionMISC_FEATURE(29)..(29)alpha-methylated leucine
residue 11Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys
Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys
Leu Ile 20 25 30 1230PRTArtificial sequenceAn embodiment of the
present inventionMISC_FEATURE(29)..(29)N-methylated leucine residue
12Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys Cys Cys 1
5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys Leu Ile 20
25 30 1330PRTArtificial sequenceAn embodiment of the present
invention 13Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala Arg Lys
Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Ile
Leu Trp 20 25 30 1430PRTArtificial sequenceAn embodiment of the
present invention 14Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Ala
Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys
Lys Ile Ile Trp 20 25 30 1529PRTArtificial sequenceAn embodiment of
the present invention 15Thr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser
Ala Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys
Lys Lys Leu Trp 20 25 1630PRTArtificial sequenceProtoxin II 16Tyr
Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu Arg Lys Cys Cys 1 5 10
15 Glu Gly Met Val Cys Arg Leu Trp Cys Lys Lys Lys Leu Trp 20 25 30
1729PRTGrammostola spatulataPhrixotoxin I 17Tyr Cys Gln Lys Trp Met
Trp Thr Cys Asp Ser Ala Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val
Cys Arg Leu Trp Cys Lys Lys Ile Ile 20 25 1829PRTChilobrachys
jingzhaoJzTx XII 18Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu
Arg Leu Cys Cys 1 5 10 15 Glu Gly Tyr Val Cys Glu Leu Trp Cys Lys
Tyr Asn Leu 20 25 1929PRTGrammostola spatulataGsAF I 19Tyr Cys Gln
Lys Trp Leu Trp Thr Cys Asp Ser Glu Arg Lys Cys Cys 1 5 10 15 Glu
Asp Met Val Cys Arg Leu Trp Cys Lys Lys Arg Leu 20 25
2029PRTChilobrachys jingzhaoJzTx V 20Tyr Cys Gln Lys Trp Met Trp
Thr Cys Asp Ser Lys Arg Ala Cys Cys 1 5 10 15 Glu Gly Leu Arg Cys
Lys Leu Trp Cys Arg Lys Ile Ile 20 25 2132PRTGrammostola roseaVsTx
II 21Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Glu Glu Arg Lys Cys
Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys Ile
Glu Glu Gly 20 25 30 2231PRTGrammostola spatulataGsAF II 22Tyr Cys
Gln Lys Trp Met Trp Thr Cys Asp Glu Glu Arg Lys Cys Cys 1 5 10 15
Glu Gly Leu Val Cys Arg Leu Trp Cys Lys Lys Lys Ile Glu Trp 20 25
30 2329PRTGrammostola spatulataGrTx I 23Tyr Cys Gln Lys Trp Met Trp
Thr Cys Asp Ser Lys Arg Lys Cys Cys 1 5 10 15 Glu Asp Met Val Cys
Gln Leu Trp Cys Lys Lys Arg Leu 20 25 2431PRTGrammostola
spatulataGsMtx II / PaTx II 24Tyr Cys Gln Lys Trp Met Trp Thr Cys
Asp Glu Glu Arg Lys Cys Cys 1 5 10 15 Glu Gly Leu Val Cys Arg Leu
Trp Cys Lys Arg Ile Ile Asn Met 20 25 30 2530PRTArtificial
sequenceAn embodiment of the present invention 25Tyr Cys Gln Lys
Trp Met Trp Thr Cys Asp Ser Ala Arg Lys Cys Cys 1 5 10 15 Glu Gly
Met Val Cys Arg Leu Trp Cys Lys Lys Lys Leu Trp 20 25 30
2630PRTArtificial sequenceAn embodiment of the present invention
26Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu Arg Lys Cys Cys 1
5 10 15 Glu Gly Ala Val Cys Arg Leu Trp Cys Lys Lys Lys Leu Trp 20
25 30 2730PRTArtificial sequenceAn embodiment of the present
invention 27Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu Arg Lys
Cys Cys 1 5 10 15 Glu Gly Met Val Cys Arg Leu Trp Cys Lys Lys Ala
Leu Trp 20 25 30 2830PRTArtificial sequenceAn embodiment of the
present invention 28Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser Glu
Arg Lys Cys Cys 1 5 10 15 Glu Gly Met Val Cys Arg Leu Trp Cys Lys
Lys Lys Ala Trp 20 25 30 2930PRTArtificial sequenceAn embodiment of
the present invention 29Tyr Cys Gln Lys Trp Met Trp Thr Cys Asp Ser
Glu Arg Lys Cys Cys 1 5 10 15 Glu Gly Met Val Cys Arg Leu Trp Cys
Lys Lys Lys Leu Ala 20 25 30
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