U.S. patent application number 13/051446 was filed with the patent office on 2011-08-11 for novel x-conotoxin peptides (-ii).
This patent application is currently assigned to XENOME LTD.. Invention is credited to Dianne Alewood, Paul Francis Alewood, Richard James Lewis, Elka Palant.
Application Number | 20110195909 13/051446 |
Document ID | / |
Family ID | 32469447 |
Filed Date | 2011-08-11 |
United States Patent
Application |
20110195909 |
Kind Code |
A1 |
Lewis; Richard James ; et
al. |
August 11, 2011 |
NOVEL X-CONOTOXIN PEPTIDES (-II)
Abstract
An isolated, synthetic or recombinant .chi.-conotoxin peptide
having the ability to inhibit neuronal amine transporter comprising
the following sequence of amino acids: Cys Cys Gly Tyr Lys Leu Cys
Xaa5 Xaa6 Cys, SEQ ID NO: 3, where Xaa5 and Xaa6 are independently
absent or represent any amino acid residue except Cys; or a
sequence in which Gly, Tyr, Lys or Leu are subject to conservative
amino acid substitution or side chain modification with the proviso
that the peptide is not Mr1A, Mr1B, Mar2, CMrV1A, Bn1.5, Mr1.3 or
Au1.4; or a salt, ester, amide, prodrug or cyclised derivative
thereof.
Inventors: |
Lewis; Richard James;
(Woolloongabba, AU) ; Alewood; Paul Francis;
(Pullenvale, AU) ; Alewood; Dianne; (Pullenvale,
AU) ; Palant; Elka; (Kenmore, AU) |
Assignee: |
XENOME LTD.
Indooroopilly
AU
|
Family ID: |
32469447 |
Appl. No.: |
13/051446 |
Filed: |
March 18, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12251276 |
Oct 14, 2008 |
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13051446 |
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10537088 |
Dec 27, 2005 |
7507717 |
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PCT/AU2003/001606 |
Dec 2, 2003 |
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12251276 |
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60430307 |
Dec 2, 2002 |
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Current U.S.
Class: |
514/18.3 ;
514/21.5; 530/326; 530/327 |
Current CPC
Class: |
C07K 14/43504 20130101;
A61P 25/00 20180101; A61P 9/00 20180101; A61K 38/00 20130101; A61P
13/00 20180101; A61P 29/00 20180101; C07K 7/08 20130101 |
Class at
Publication: |
514/18.3 ;
530/327; 530/326; 514/21.5 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C07K 7/08 20060101 C07K007/08; A61P 9/00 20060101
A61P009/00; A61P 13/00 20060101 A61P013/00; A61P 25/00 20060101
A61P025/00; A61P 29/00 20060101 A61P029/00 |
Claims
1. An isolated, synthetic or recombinant .chi.-conotoxin peptide
having the ability to inhibit a neuronal amine transporter
comprising the following sequence of amino acids: TABLE-US-00019
(SEQ ID NO: 216) Xaa1Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys Leu Cys
Xaa5 Xaa6 Cys,
wherein Xaa1, Xaa2, Xaa3, and Xaa4 each represent an amino acid,
and Xaa5 and Xaa6 are independently absent or represent any amino
acid residue except Cys; or a salt, ester, amide, prodrug or
cyclised derivative thereof.
2. The .chi.-conotoxin peptide of claim 1, wherein Xaa1 is selected
from Trp, DTrp, Tyr, Phe, hPhe, Ala, MeY, Arg, Orn, pGlu, or DpGlu;
Xaa2 is selected from Arg, Ala, Asn, Lys, Phe, BHK, Orn, Lys, DArg,
Nle, DLys, DMK, DAsn, Thr, Cit, Val, Tyr, or Trp; Xaa3 is selected
from Gly, Asp, Lys, Arg, Ala, Nle, Ser or Phe; and Xaa4 is selected
from Val, Leu, Nle, Ile, Thr, Ala, Asn, Trp, Phe or Abu.
3. The .chi.-conotoxin peptide according to claim 2, wherein Xaa1
is selected from Trp, Tyr, Phe, hPhe, Ala, MeY, or Arg, Xaa2 is
selected from Arg, Asn, Lys, BHK, Orn, Lys, DArg, Nle, DLys, DMK,
DAsn, Thr, Cit or Val, Xaa3 is selected from Gly, Asp, Lys, Arg,
Ala, Nle or Ser, and Xaa4 is selected from Val, Leu, Nle, Ile, Thr,
Ala or Abu.
4. The .chi.-conotoxin peptide according to claim 3, consisting of
the following sequence of amino acids: TABLE-US-00020 (SEQ ID NO:
4) Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys Leu Cys Xaa5 Xaa6 Cys
where
Xaa1 is selected from Trp, Tyr, Phe, hPhe, Ala, MeY, or Arg, Xaa2
is selected from Arg, Asn, Lys, BHK, Orn, Lys, DArg, Nle, DLys,
DMK, DAsn, Thr, Cit or Val, Xaa3 is selected from Gly, Asp, Lys,
Arg, Ala, Nle or Ser, Xaa4 is selected from Val, Leu, Nle, Ile,
Thr, Ala or Abu, and Xaa5 and Xaa6 are independently absent or
represent any amino acid residue except Cys, or a salt, ester,
amide or prodrug thereof.
5. The .chi.-conotoxin peptide of claim 1, wherein Xaa1 is selected
from pGlu, DpGlu, Pro, Hyp or an N-acetylated amino acid residue;
Xaa2 is selected from Arg, Asn, Lys, BHK, Orn, Lys, DArg, Nle,
DLys, DMK, DAsn, Thr, Cit, or Val, Xaa3 is selected from Gly, Asp,
Lys, Arg, Ala, Nle or Ser, and Xaa4 is selected from Val, Leu, Nle,
Ile, Thr, Ala or Abu.
6. The peptide according to claim 2, wherein Xaa1 is Trp, Tyr or
hPhe.
7. The peptide according to claim 6, wherein Xaa1 is Trp.
8. The peptide according to claim 2, wherein Xaa2 is Arg, Lys or
Asn.
9. The peptide according to claim 5, wherein Xaa1 is pGlu or
DpGlu.
10. The peptide according to claim 5, wherein Xaa2 is a
deletion.
11. The peptide according to claim 5, wherein Xaa2 is BHK or
Orn.
12. The peptide according to claim 2, wherein Xaa3 is Gly or
Asp.
13. The peptide according to claim 12, wherein Xaa3 is Gly.
14. The peptide according to claim 2, wherein Xaa4 is Leu, Nle or
Val.
15. The peptide according to claim 2, wherein Xaa5 is selected from
the group consisting of His, Arg, Trp, Nal, Glu and a deletion.
16. The peptide according to claim 15, wherein Xaa5 is Arg or
His.
17. The peptide according to claim 2, wherein Xaa6 is selected from
the group consisting of Hyp, Pro, Ala, Tic, Pip, MeY, DMD, Phe,
THZ, Glu, Nle, Tyr and a deletion.
18. The peptide according to claim 17, wherein Xaa6 is Hyp or
Pro.
19. The peptide according to claim 2, wherein the Tyr of loop 1 has
been replaced with MeY and/or the Leu of loop 1 is replaced with
Hle or Nle.
20. The peptide according to claim 5, wherein the Tyr of loop 1 has
been replaced with MeY and/or the Leu of loop 1 is replaced with
Hle or Nle.
21. The peptide according to claim 2 having from 14 to 20 amino
acids.
22. The peptide according to claim 1, comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 13-18,
20-26, 28-30, 33, 34, 36-41, 43, 46, 50, 53-56, 60, 62, 64, 67-72,
78, 81-86, 88-92, 94, 95, 99, 100, 104-106, 109, 110, 114, 116,
118, 132, 140, 157, 161 and 163.
23. A composition comprising the peptide according to any one of
claims 1-5, and a pharmaceutically acceptable carrier or
diluent.
24. A method for the treatment or prophylaxis of urinary or
cardiovascular conditions or diseases or mood disorders or for the
treatment or control of pain or inflammation including the step of
administering to a mammal an effective amount of the peptide
according to any one of claims 1-5.
25. The method according to claim 24, wherein the peptide is
administered substantially simultaneously or sequentially with
other active agents useful in the treatment of the conditions,
diseases or disorders.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/251,276, filed on Oct. 14, 2008, which is a continuation of
U.S. application Ser. No. 10/537,088, filed on Dec. 27, 2005, now
U.S. Pat. No. 7,507,717 B2, issued on Mar. 24, 2009, which is the
.sctn.371 national phase of PCT/AU2003/001606, filed on Dec. 2,
2003, which claims priority from U.S. Provisional Application No.
60/430,307, filed on Dec. 2, 2002.
[0002] The present invention relates to novel .alpha.-conotoxin
peptides useful as inhibitors of neuronal amine transporters of
neurotransmitters, such as noradrenaline, serotonin, dopamine,
glutamic acid and glycine. The invention also relates to
pharmaceutical compositions comprising these peptides and the use
of these peptides in the prophylaxis or treatment of conditions,
such as but not limited to, pain, inflammation, incontinence,
cardiovascular conditions and mood disorders.
[0003] The marine snails of the genus Conus (cone snails) use a
sophisticated biochemical strategy to capture their prey. As
predators of either fish, worms or other molluscs, the cone snails
inject their prey with venom containing a cocktail of small
bioactive peptides. These toxin molecules, which are referred to as
conotoxins, interfere with neurotransmission by targeting a variety
of receptors and ion-channels. The venom from any single Conus
species may contain more than 100 different peptides. The
conotoxins are divided into classes on the basis of their
physiological targets. The .omega.-conotoxin class of peptides
target and block voltage-sensitive Ca.sup.2+-channels inhibiting
neurotransmitter release. The .alpha.-conotoxins and
.PSI.-conotoxins target and block nicotinic ACh receptors, causing
ganglionic and neuromuscular blockade. Peptides of the
.mu.-conotoxin class act to block voltage-sensitive
Na.sup.+-channels inhibiting muscle and nerve action potentials.
The .delta.-conotoxins target and delay the inactivation of
voltage-sensitive Na.sup.+-channels, enhancing neuronal
excitability. The ic-conotoxin class of peptides target and block
voltage-sensitive K.sup.+-channels, and these also cause enhanced
neuronal excitability. The conopressins are vasopressin receptor
antagonists and the conantokins are NMDA receptor antagonists. The
.gamma.-conotoxin class targets a voltage-sensitive nonspecific
cation channel. The .sigma.-conotoxin class antagonises the
5HT.sub.3 receptor and the .chi.-conotoxin class inhibits neuronal
amine transporters.
[0004] The .chi.-conotoxin class of peptides was first described in
WO00/20444 (University of Queensland), although two members of the
class were subsequently referred to in WO00/44769 (University of
Utah Research Foundation). The particular .chi.-conotoxin peptides
identified in WO 00/20444 were MrIA and MrIB which have the
following sequences:
TABLE-US-00001 .chi.-MrIA SEQ ID NO. 1 Asn Gly Val Cys Cys Gly Tyr
Lys Leu Cys His Hyp Cys .chi.-MrIB SEQ ID NO. 2 Val Gly Val Cys Cys
Gly Tyr Lys Leu Cys His Hyp Cys
[0005] In these and following sequences Hyp refers to 4-hydroxy
proline. In nature, this amino acid residue results from post
translational modification of the encoded peptide and is not
directly encoded by the nucleotide sequence.
[0006] Additional .chi.-conotoxin peptides have also now been
described by Balaji et al. (2000 J. Biol. Chem. 27539516-39522),
McIntosh J et al. (WO00/44769). These peptides, Mar2, CMrVIA and
CMRx (or UO36), have the following sequences:
TABLE-US-00002 Mar2 SEQ ID NO. 7 Gly Val Cys Cys Gly Tyr Lys Leu
Cys Cys His Hyp Cys CMrVIA SEQ ID NO. 8 Val Cys Cys Gly Tyr Lys Leu
Cys His Hyp Cys CMRx SEQ ID NO. 9 Gly Ile Cys Cys Gly Val Ser Phe
Cys Tyr Hyp Cys
[0007] Other .PI.-type conotoxin peptides have been described by
Olivera et al. (WO02/064740) although the disulphide connectivity
and activity of these peptides does not appear to be described.
Some of those peptides are as follows:
TABLE-US-00003 Bn1.5 SEQ ID NO. 10 Ala Cys Cys Gly Tyr Lys Leu Cys
Ser Pro Cys# Mr1.3 SEQ ID NO. 11 Asn Gly Val Cys Cys Gly Tyr Lys
Leu Cys Leu Pro Cys{circumflex over ( )} Au1.4 SEQ ID NO. 12 Ser
Val Cys Cys Gly Tyr Lys Leu Cys Phe Pro Cys{circumflex over (
)}
[0008] The ` ` indicates that the C-terminus is preferably free
carboxyl and `#` indicates that it is preferably amidated.
[0009] Compounds which inhibit neurotransmitter reuptake have been
found to be useful in the treatment of acute, chronic and/or
neuropathic pain, migraine and inflammation. Such compounds can
also be administered with other agents useful in these treatments
to provide improved pain/inflammation relief and/or reduce the
severity of unwanted side effects, such as nausea and stomach
upset. They have also been found to be useful in the treatment of
lower urinary tract disorders, such as urinary incontinence,
detrusor instability and interstitial cystitis. One such compound
is "imipramine" which, in addition to inhibiting noradrenaline
reuptake, has been shown to affect calcium channel blockade, and to
exhibit anticholinergic activity, local anaesthetic activity and a
number of other effects. Other compounds capable of inhibiting
noradrenaline reuptake are described in U.S. Pat. No. 5,441,985.
These compounds are said to have a reduced anticholinergic effect
relative to imipramine.
[0010] In the case of the peptides of the present invention this
inhibition of neurotransmitter reuptake is achieved by selectively
inhibiting the neuronal neurotransmitter transporter, such as the
noradrenaline transporter, which functions to rapidly clear
released noradrenaline from the synapse back into neurons.
[0011] As described in WO00/20444, the peptides .chi.-MrIA and
.chi.-MrIB are composed of a tail, residues 1-3, two loops,
residues 6-9 (loop 1) and 11-12 (loop 2), respectively and two
disulfide bonds between cysteine residues 4 and 13 and 5 and 10,
respectively. While MrIA resembles a .alpha.-conotoxin peptide in
terms of the number of cysteine residues, the disulfide
connectivity is different. In this regard the .alpha.-conotoxin
peptides are characterised by an A-C/B-D connectivity, rather than
the A-D/B-C connectivity of MrIA, where A, B, C and D represent the
first, second, third and fourth cysteine residues involved in
disulfide bond formation respectively.
[0012] It has now been found that a particular part of the MrIA
sequence is essential for the biological activity, and that the
activity of MrIA can be enhanced by making particular modifications
to its primary structure.
[0013] Accordingly in a first aspect the present invention there is
provided an isolated, synthetic or recombinant .chi.-conotoxin
peptide having the ability to inhibit neuronal amine transporter
comprising the following sequence of amino acids:
TABLE-US-00004 SEQ ID NO. 3 Cys Cys Gly Tyr Lys Leu Cys Xaa5 Xaa6
Cys
where Xaa5 and Xaa6 are independently absent or represent any amino
acid residue except Cys, or such a sequence in which Gly, Tyr, Lys
or Leu are subject to conservative amino acid substitution or side
chain modification, with the proviso that the peptide is not
.chi.-MrIA, .chi.-MrIB, Mar2, CMrVIA, Bn1.5, Mr1.3 or Au1.4; or a
salt, ester, amide, prodrug or cyclised derivative thereof.
[0014] It has also been found that the introduction of an
additional amino acid residue at the N-terminus can increase the
binding affinity of the peptide for the human noradrenaline
transporter.
[0015] In a second aspect the present invention provides an
isolated, synthetic or recombinant .chi.-conotoxin peptide having
the ability to inhibit neuronal amine transporter comprising the
following sequence of amino acids:
TABLE-US-00005 SEQ ID NO. 4 Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys
Leu Cys Xaa5 Xaa6 Cys
where [0016] Xaa1 is selected from Trp, DTrp, Tyr, Phe, hPhe, Ala,
MeY, Arg, Ben, Nap, Orn, pGlu, DpGlu and a deletion; [0017] Xaa2 is
selected from Arg, Ala, Asn, Lys, Phe, BHK, Orn, Lys, DArg, Nle,
DLys, DMK, DAsn, Thr, ABZ, Nap, Cit, Val, Tyr, Trp, pGlu, DpGlu or
a deletion; [0018] Xaa3 is selected from Gly, Asp, Lys, Arg, Ala,
Nle, Ser or Phe; [0019] Xaa4 is selected from Val, Leu, Nle, Ile,
Thr, Ala, Asn, Trp, Phe and Abu, and Xaa5 and Xaa6 are as defined
above, or such a sequence where one or more of the loop 1 residues
Gly, Tyr, Lys and Leu are subject to conservative amino acid
substitution or side chain modification, with the proviso that the
peptide is not .chi.-MrIA, .chi.-MrIB, Mar2, Mr1.3 or Au1.4; and or
a salt, ester, amide, prodrug or cyclised derivative thereof.
[0020] In a third aspect the present invention provides an
isolated, synthetic or recombinant .chi.-conotoxin peptide having
the ability to inhibit neuronal amine transporter comprising the
following sequence of amino acids:
TABLE-US-00006 SEQ ID NO. 4 Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys
Leu Cys Xaa5 Xaa6 Cys
where [0021] Xaa1 is selected from Trp, Tyr, Phe, hPhe, Ala, MeY,
Arg, Ben and Nap, [0022] Xaa2 is selected from Arg, Asn, Lys, BHK,
Orn, Lys, DArg, Nle, DLys, DMK, [0023] DAsn, Thr, ABZ, Nap, Cit and
Val, [0024] Xaa3 is selected from Gly, Asp, Lys, Arg, Ala, Nle and
Ser, [0025] Xaa4 is selected from Val, Leu, Nle, Ile, Thr, Ala and
Abu, and [0026] Xaa5 and Xaa6 are as defined above, or such a
sequence where one or more of the loop 1 residues Gly, Tyr, Lys and
Leu are subject to conservative amino acid substitution or side
chain modification, or a salt, ester, amide, prodrug or cyclised
derivative thereof.
[0027] In a fourth aspect the present invention provides an
isolated, synthetic or recombinant .chi.-conotoxin peptide having
the ability to inhibit neuronal amine transporter consisting of the
following sequence of amino acids:
TABLE-US-00007 SEQ ID NO. 4 Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys
Leu Cys Xaa5 Xaa6 Cys
where [0028] Xaa1 is selected from Trp, Tyr, Phe, hPhe, Ala, MeY,
Arg, Ben and Nap, [0029] Xaa2 is selected from Arg, Asn, Lys, BHK,
Orn, Lys, DArg, Nle, DLys, DMK, DAsn, Thr, ABZ, Nap, Cit and Val,
[0030] Xaa3 is selected from Gly, Asp, Lys, Arg, Ala, Nle and Ser,
[0031] Xaa4 is selected from Val, Leu, Nle, Ile, Thr, Ala and Abu,
and [0032] Xaa5 and Xaa6 are as defined above, or such a sequence
where one or more of the loop 1 residues Gly, Tyr, Lys and Leu are
subject to conservative amino acid substitution or side chain
modification or a salt, ester, amide or prodrug thereof.
[0033] It has further been found that the introduction of an
N-terminally blocked residue can provide a number of advantages
over MrIA.
[0034] Accordingly in a fifth aspect of the present invention there
is provided an isolated, synthetic or recombinant .chi.-conotoxin
peptide comprising the following sequence of amino acids:
TABLE-US-00008 SEQ ID NO. 5 Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys
Leu Cys Xaa5 Xaa6 Cys
where [0035] Xaa1 is an N-terminal residue and is selected from
pGlu, DpGlu, Pro, Hyp or an N-acetylated amino acid residue; [0036]
Xaa2 is selected from Arg, Asn, Lys, BHK, Orn, Lys, DArg, Nle,
DLys, DMK, DAsn, Thr, ABZ, Nap, Cit, Val and a deletion, [0037]
Xaa3 is selected from Gly, Asp, Lys, Arg, Ala, Nle and Ser, [0038]
Xaa4 is selected from Val, Leu, Nle, Ile, Thr, Ala and Abu, and
[0039] Xaa5 and Xaa 6 are as defined above, or such a sequence
where one or more of the loop 1 residues Gly, Tyr, Lys and Leu are
subject to conservative amino substitution or sidechain
modification, or a salt, ester, amide or prodrug thereof.
[0040] In a sixth aspect the present invention provides an
isolated, synthetic or recombinant .chi.-conotoxin peptide
consisting of the following sequence of amino acids:
TABLE-US-00009 SEQ ID NO. 5 Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys
Leu Cys Xaa5 Xaa6 Cys
where [0041] Xaa1 is an N-terminal residue and is selected from
pGlu, Pro, Hyp or an N-acetylated amino acid residue; [0042] Xaa2
is selected from Arg, Asn, Lys, BHK, Orn, Lys, DArg, Nle, DLys,
DMK, DAsn, Thr, ABZ, Nap, Cit, pGlu, Val and a deletion, [0043]
Xaa3 is selected from Gly, Asp, Lys, Arg, Ala, Nle and Ser, [0044]
Xaa4 is selected from Val, Leu, Nle, Ile, Thr, Ala and Abu, and
[0045] Xaa5 and Xaa 6 are as defined above, or such a sequence
where one or more of the loop 1 residues Gly, Tyr, Lys and Leu are
subject to conservative amino and substitution or said chain
modification, or a salt or prodrug thereof.
[0046] The peptides according to the fifth and sixth aspects of the
present invention may have a number of advantages over MrIA. A
peptide of this aspect of the invention was found to have a
duration of effect which extended beyond 24 hours following a bolus
30 nmol dose given i.t. Another peptide of the invention had an
increase in potency of over 50-fold relative to MrIA. These
peptides have also been found to be particularly stable to storage
in the pH range of 4 to 7 and 37EC, allowing long term delivery in
a device, for example an infusion pump, held at room temperature to
37EC. There are also advantages in relation to the production and
separation of the peptides from unwanted bi-products of synthesis,
allowing straightforward purification to homogeneity of >99%,
relative to MrIA using a similar procedure in which purity is
typically <93%.
[0047] It has further been found that the binding affinity of the
.chi.-peptides according to the invention can be increased by
introduction of particular residues at the N-terminus.
[0048] Accordingly a seventh aspect the present invention provides
an isolated, synthetic or recombinant .chi.-conotoxin peptide
having the ability to inhibit neuronal amine transporter comprising
the following sequence of amino acids:
TABLE-US-00010 SEQ ID NO. 6 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys Leu
Cys Xaa5 Xaa6 Cys
where Xaa2 is BHK, Orn, Arg, DArg or DMK and Xaa3, Xaa4, Xaa5 and
Xaa6 are as defined above, or such a sequence where one or more of
the loop 1 residues Gly, Tyr, Lys and Leu are subject to
conservative amino acid or side chain modification, or a salt,
ester, amide, prodrug or cyclised derivative thereof.
[0049] In an eighth aspect the present invention provides an
isolated, synthetic or recombinant .chi.-conotoxin peptide having
the ability to inhibit neuronal amine transporter consisting of the
following sequence of amino acids:
TABLE-US-00011 SEQ ID NO. 6 Xaa2 Xaa3 Xaa4 Cys Cys Gly Tyr Lys Leu
Cys Xaa5 Xaa6 Cys
where Xaa2 is BHK, Orn, Arg, DArg or DMK and Xaa3, Xaa4, Xaa5 and
Xaa6 are as defined above, or such a sequence where one or more of
the loop 1 residues Gly, Tyr, Lys and Leu are subject to
conservative amino acid or side chain modification, or a salt,
ester, amide, prodrug or cyclised derivative thereof.
[0050] In SEQ ID NO. 4 Xaa1 is preferably Trp, Tyr or hPhe. More
preferably Xaa1 is Trp.
[0051] In SEQ ID NO. 5 Xaa1 is preferably pGlu.
[0052] In SEQ ID NO. 4 Xaa2 is preferably Arg, Lys or Asn.
[0053] In SEQ ID NO. 5 Xaa2 is preferably a deletion.
[0054] In SEQ ID NO. 6 Xaa2 is preferably BHK or Orn.
[0055] In SEQ ID NOS. 4, 5 and 6 Xaa3 is preferably Gly or Asp.
More preferably Xaa3 is Gly.
[0056] In SEQ ID NOS. 4, 5 and 6 Xaa4 is preferably Leu, Nle or
Val.
[0057] In SEQ ID NOS, 3, 4, 5 and 6 the following preferred
definitions apply for Xaa5 and Xaa6:
[0058] Preferably Xaa5 is selected from His, Arg, Trp, Nal, Glu and
a deletion. More preferably Xaa5 is Arg or His.
[0059] Xaa6 is selected from Hyp, Pro, Ala, Tic, Pip, MeY, DMD,
Phe, THZ, Glu, Nle, Tyr and a deletion. More preferably Xaa6 is Hyp
or Pro.
[0060] Preferably, the neuronal amine transporter is the neuronal
noradrenaline transporter.
[0061] The .chi.-conotoxin peptide may be naturally occurring
peptides isolated from a cone snail, or derivatives or synthetic
versions thereof.
[0062] Preferably, the .chi.-conotoxin peptide is a selective
inhibitor of the neuronal noradrenaline transporter. The terms
"selective" and "selectively" as used herein mean that the activity
of the peptide as an inhibitor of neuronal noradrenaline
transporter is considerably greater than any activity at the
.alpha..sub.1-adrenoceptors. Preferably the peptide inhibitor is
10-fold more selective towards the neuronal noradrenaline
transporter, more preferably 100-fold more selective and most
preferably more than 1000-fold more selective. The peptide is also
preferably selective over .alpha..sub.2-adrenoceptors and/or
serotonin reuptake transporter (SERT) The selectivity of an
inhibitor of the neuronal noradrenaline transporter can be measured
using techniques known in the art, for example using appropriate
labelled ligand displacement assays.
[0063] U.S. Pat. No. 5,441,985 indicates that inhibitors of
noradrenaline reuptake which have a negligible anticholinergic
effect are particularly useful in the treatment of lower urinary
tract disorders. It has been found that the peptides of this
invention also have no detectable or substantially no detectable
anticholinergic effect.
[0064] Accordingly in a preferred embodiment of the invention the
.chi.-conotoxin peptide has the ability to selectively inhibit
neuronal noradrenaline transporter, and has negligible or no
substantial anticholinergic effect.
[0065] The peptides of the present invention preferably have no
activity as a sodium channel blocker or as an inhibitor of dopamine
transporter. The absence, in the peptides of the invention and in
particular the preferred peptides according to the invention, of
these additional pharmacological activities commonly associated
with other noradrenaline transporter inhibitors makes these
peptides useful pharmacological tools.
[0066] The peptides according to the present invention may be
termed derivatives of MrIA.
[0067] The term "derivative" as used herein in connection with a
naturally occurring .chi.-conotoxin peptide, such as .chi.-MrIA,
refers to a peptide which differs from the naturally occurring
peptides by one or more amino acid deletions, additions,
substitutions, or side-chain modifications. All such derivatives of
.chi.-MrIA according to the present invention have the ability to
inhibit the neuronal noradrenaline transporter.
[0068] Substitutions encompass amino acid alterations in which an
amino acid is replaced with a different naturally-occurring or a
non-conventional amino acid residue. Such substitutions may be
classified as "conservative", in which case an amino acid residue
contained in a polypeptide is replaced with another
naturally-occurring amino acid of similar character either in
relation to polarity, side chain functionality or size, for example
SerThrProHypGlyAla, ValIleLeu, HisLys AsnGlnAspGlu or PheTrpTyr. It
is to be understood that some non-conventional amino acids may also
be suitable replacements for the naturally occurring amino acids.
For example Lys residues may be substituted by ornithine,
homoarginine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys and
N,N,N-trimethyl-Lys. Lys residues can also be replaced with
synthetic basic amino acids including, but not limited to,
N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala,
2-[3-(2S)pyrrolininyl]-Gly and 2-[3-(2S)pyrrolininyl]-Ala. Tyr
residues may be substituted with 4-methoxy tyrosine (MeY),
meta-Tyr, ortho-Tyr, nor-Tyr, .sup.125I-Tyr, mono-halo-Tyr,
di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, and nitro-Tyr. Tyr
residues may also be substituted with the 3-hydroxyl or 2-hydroxyl
isomers (meta-Tyr or ortho-Tyr, respectively) and corresponding
O-sulpho- and O-phospho derivatives. Tyr residues can also be
replaced with synthetic hydroxyl containing amino acids including,
but not limited to 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly,
2,6-dimethyl-Tyr and 5-amino-Tyr. Aliphatic amino acids may be
substituted by synthetic derivatives bearing non-natural aliphatic
branched or linear side chains C.sub.nH.sub.2n+2 up to and
including n=8. Examples of suitable conservative substitutions by
non-conventional amino acids are given in WO02/064740, the entire
contents of which is incorporated herein by reference. According to
the present invention substitutions in loop 1 are restricted to
conservative substitutions.
[0069] Substitutions may also be "non-conservative", in which an
amino acid residue which is present in a peptide is substituted
with an amino acid having different properties, such as
naturally-occurring amino acid from a different group (eg.
substituting a charged or hydrophobic amino acid with alanine), or
alternatively, in which a naturally-occurring amino acid is
substituted with a non-conventional amino acid. According to the
present invention such non-conservative substitutions are
restricted to amino acid residues which are not part of loop 1 of
the peptide, and that have little or no deleterious effect on
activity.
[0070] Amino acid substitutions are typically of single residues,
but may be of multiple residues, either clustered or dispersed.
[0071] Additions encompass the addition of one or more naturally
occurring or non-conventional amino acid residues. According to the
present invention, except where an N-terminal residue is specified
or where the complete sequence is designated, additions may occur
at the N- or C-termini of the peptides according to the invention.
Deletion encompasses the deletion of one or more amino acid
residues. Many peptides according to the present invention
represent derivatives of .chi.-MrIA which have undergone one or
more amino acid deletions.
[0072] As stated above the present invention includes peptides in
which one or more of the amino acids has undergone sidechain
modifications. Examples of side chain modifications contemplated by
the present invention include but are not limited to modifications
of amino groups such as by reductive alkylation by reaction with an
aldehyde followed by reduction with NaBH.sub.4; amidination with
methylacetimidate; acylation with acetic anhydride; carbamoylation
of amino groups with cyanate; trinitrobenzylation of amino groups
with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of
amino groups with succinic anhydride and tetrahydrophthalic
anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate
followed by reduction with NaBH.sub.4; and N-acetylation.
[0073] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0074] The tyrosine residue may be altered, for example by
methoxylation at the 4-position. Tyrosine may also be altered by
nitration with tetranitromethane to form a 3-nitrotyrosine
derivative. Examples of tyrosine derivatives are given in
WO02/064740.
[0075] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivatisation, for example, to a corresponding amide.
[0076] Acidic amino acids may be substituted with tetrazolyl
derivatives of Gly and Ala, as described in WO02/600923.
[0077] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH. Any modification of cysteine residues
must not affect the ability of the peptide to form the necessary
disulphide bonds. It is also possible to replace the sulphydryl
groups of cysteine with selenium equivalents such that the peptide
forms a diselenium bond in place of one or more of the disulphide
bonds, or mixed selenium/sulfide bonds. Individual Cys residues may
also be replaced with homoCys or penicillamine so that disulfide
bridges may be formed between Cys-homoCys, Cys-penicillamine or
homoCys-penicillamine. Cys residues may also be replaced with
isosteric lactam replacements as described in detail in
WO02/600923.
[0078] Tryptophan residues may be modified by, for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
[0079] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0080] Proline residues may be modified by, for example,
hydroxylation in the 4-position.
[0081] Other derivatives contemplated by the present invention
include a range of glycosylation variants. Altered glycosylation
patterns may result from expression of recombinant molecules in
different host cells. Ser, Thr and Hyp residues may be modified to
contain an O-glycan, while Asn and Gln residues can be modified to
form a N-glycan. In accordance with the present invention, the term
"glycan" refers to an N-, S- or O-linked mono-, di-, tri, poly- or
oligosaccharide that can be attached to any hydroxy, amino or thiol
group of natural of modified amino acids by synthetic or enzymatic
methodologies known in the art. The monosaccharides making up the
glycan can include D-allose, D-altrose, D-glucose, D-mannose,
D-gulose, D-idose, D-galactose, D-talose, D-galactosamine,
D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),
D-N-acetyl-galactosamine (GalNac), D-fucose or D-arabinose. These
saccharides may be structurally modified ie., with one or more
O-sulphate, O-phosphate, O-acetyl or acidic groups such as sialic
acid, including combinations thereof. The glycan may also include
similar polyhydroxyl groups, such as D-penicillamine 2,5 and
halogenated derivatives thereof or polypropylene glycol
derivatives. The glycosidic linkage is beta and 1-4 or 1-3,
preferably 1-3. The linkage between the glycan and the amino acid
may be alpha or beta, preferably alpha and is 1-.
[0082] A list of some amino acids having modified side chains and
other unnatural amino acids is shown in Table 1.
TABLE-US-00012 TABLE 1 Non-conventional Non-conventional amino acid
Code amino acid Code L-.alpha.-aminobutyric acid Abu
L-.alpha.-methylhistidine Mhis .alpha.-amino-.alpha.-methylbutyrate
Mgabu L-.alpha.-methylisoleucine Mile aminocyclopropane- Cpro
L-.alpha.-methylleucine Mleu carboxylate L-.alpha.-methylmethionine
Mmet aminoisobutyric acid Aib L-.alpha.-methylnorvaline Mnva
aminonorbornyl- Norb L-.alpha.-methylphenylalanine Mphe carboxylate
L-.alpha.-methylserine Mser cyclohexylalanine Chexa
L-.alpha.-methyltryptophan Mtrp cyclopentylalanine Cpen
L-.alpha.-methylvaline Mval D-alanine DAla N-(N-(2,2-diphenylethyl)
Nnbhm D-arginine DArg carbamylmethylglycine D-asparagine DAsn
1-carboxy-1-(2,2-diphenyl- Nmbc D-aspartic acid DAsp
ethylamino)cyclopropane D-cysteine DCys L-N-methylalanine Nmala
D-glutamine DGln L-N-methylarginine Nmarg D-glutamic acid DGlu
L-N-methylaspartic acid Nmasp D-histidine DHis L-N-methylcysteine
Nmcys D-isoleucine DIle L-N-methylglutamine Nmgln D-leucine DLeu
L-N-methylglutamic acid Nmglu D-lysine DLys L-N-methylhistidine
Nmhis D-methionine DMet L-N-methylisolleucine Nmile D-ornithine
DOrn L-N-methylleucine Nmleu D-phenylalanine DPhe L-N-methyllysine
Nmlys D-proline DPro L-N-methylmethionine Nmmet D-serine DSer
L-N-methylnorleucine Nmnle D-threonine DThr L-N-methylnorvaline
Nmnva D-tryptophan DTrp L-N-methylornithine Nmorn D-tyrosine DTyr
L-N-methylphenylalanine Nmphe D-valine DVal L-N-methylproline Nmpro
D-.alpha.-methylalanine DMala L-N-methylserine Nmser
D-.alpha.-methylarginine DMarg L-N-methylthreonine Nmthr
D-.alpha.-methylasparagine DMasn L-N-methyltryptophan Nmtrp
D-.alpha.-methylaspartate DMasp L-N-methyltyrosine Nmtyr
D-.alpha.-methylcysteine DMcys L-N-methylvaline Nmval
D-.alpha.-methylglutamine DMgln L-N-methylethylglycine Nmetg
D-.alpha.-methylhistidine DMhis L-N-methyl-t-butylglycine Nmtbug
D-.alpha.-methylisoleucine DMile L-norleucine Nle
D-.alpha.-methylleucine DMleu L-norvaline Nva
D-.alpha.-methyllysine DMlys .alpha.-methyl-aminoisobutyrate Maib
D-.alpha.-methylmethionine DMmet
.alpha.-methyl-.gamma.-aminobutyrate Mgabu
D-.alpha.-methylornithine DMorn .alpha.-methylcyclohexylalanine
Mchexa D-.alpha.-methylphenylalanine DMphe
.alpha.-methylcyclopentylalanine Mcpen D-.alpha.-methylproline
DMpro .alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylserine DMser .alpha.-methylpenicillamine Mpen
D-.alpha.-methylthreonine DMthr N-(4-aminobutyl)glycine Nglu
D-.alpha.-methyltryptophan DMtrp N-(2-aminoethyl)glycine Naeg
D-.alpha.-methyltyrosine DMty N-(3-aminopropyl)glycine Norn
D-.alpha.-methylvaline DMval N-amino-.alpha.-methylbutyrate Nmaabu
D-N-methylalanine DNmala .alpha.-napthylalanine Anap
D-N-methylarginine DNmarg N-benzylglycine Nphe D-N-methylasparagine
DNmasn N-(2-carbamylethyl)glycine Ngln D-N-methylaspartate DNmasp
N-(carbamylmethyl)glycine Nasn D-N-methylcysteine DNmcys
N-(2-carboxyethyl)glycine Nglu D-N-methylglutamine DNmgln
N-(carboxymethyl)glycine Nasp (-carboxyglutamate Gla
N-cyclobutylglycine Ncbut 4-hydroxyproline Hyp N-cyclodecylglycine
Ncdec 5-hydroxylysine Hlys N-cylcododecylglycine Ncdod
2-aminobenzoyl Abz N-cyclooctylglycine Ncoct (anthraniloyl)
N-cyclopropylglycine Ncpro Cyclohexylalanine Cha
N-cycloundecylglycine Ncund Phenylglycine Phg
N-(2,2-diphenylethyl)glycine Nbhm 4-phenyl-phenylalanine Bib
N-(3,3-diphenylpropyl)glycine Nbhe L-pyroglutamic acid pGlu &
Pyr N-(1-hydroxyethyl)glycine Nthr L-Citrulline Cit
N-(hydroxyethyl)glycine Nser L-1,2,3,4-tetrahydroiso- Tic
N-(imidazolylethyl))glycine Nhis quinoline-3-carboxylic acid
N-(3-indolylyethyl)glycine Nhtrp L-Pipecolic acid (homo Pip
N-methyl-.gamma.-aminobutyrate Nmgabu proline) D-N-methylmethionine
Dnmmet L-homoleucine Hle N-methylcyclopentylalanine Nmcpen L-Lysine
(dimethyl) DMK D-N-methylphenylalanine Dnmphe L-Naphthylalanine Nal
D-N-methylproline Dnmpro L-dimethyldopa or DMD D-N-methylthreonine
Dnmthr L-dimethoxyphenylalanine N-(1-methylethyl)glycine Nval
L-thiazolidine-4-carboxylic THZ N-methyla-napthylalanine Nmanap
acid N-methylpenicillamine Nmpen L-homotyrosine hTyr
N-(p-hydroxyphenyl)glycine Nhtyr L-3-pyridylalanine PYA
N-(thiomethyl)glycine Ncys L-2-furylalanine FLA penicillamine Pen
L-histidine(benzyloxymethyl) HBO L-.alpha.-methylalanine Mala
L-histidine(3-methyl) HME L-.alpha.-methylasparagine Masn
D-N-methylglutamate Dnmglu L-.alpha.-methyl-t-butylglycine Mtbug
D-N-methylhistidine Dnmhis L-methylethylglycine Metg
D-N-methylisoleucine Dnmile L-.alpha.-methylglutamate Mglu
D-N-methylleucine Dnmleu L-.alpha.-methylhomophenylalanine Mhphe
D-N-methyllysine Dnmlys N-(2-methylthioethyl)glycine Nmet
N-methylcyclohexylalanine Nmchexa L-.alpha.-methyllysine Mlys
D-N-methylornithine Dnmorn L-.alpha.-methylnorleucine Mnle
N-methylglycine Nala L-.alpha.-methylornithine Morn
N-methylaminoisobutyrate Nmaib L-.alpha.-methylproline Mpro
N-(1-methylpropyl)glycine Nile L-.alpha.-methylthreonine Mthr
N-(2-methylpropyl)glycine Nleu L-.alpha.-methyltyrosine Mtyr
D-N-methyltryptophan Dnmtrp L-N-methylhomophenylalani Nmhphe
D-N-methyltyrosine Dnmtyr N-(N-(3,3-diphenylpropyl) Nnbhe
D-N-methylvaline Dnmval carbamylmethylglycine L-t-butylglycine Tbug
O-methyl-L-serine Omser L-ethylglycine Etg O-methyl-L-homoserine
Omhser L-homophenylalanine Hphe O-methyl-L-tyrosine MeY
L-.alpha.-methylarginine Marg .gamma.-aminobutyric acid Gabu
L-.alpha.-methylaspartate Masp O-methyl-L-homotyrosine Omhtyr
L-.alpha.-methylcysteine Mcys L-.beta.-homolysine BHK
L-.alpha.-methylglutamine Mgln L-ornithine Orn N-cycloheptylglycine
Nchep N-cyclohexylglycine Nchex N-(3-guanidinopropyl)glycine Narg
D-N-methylserine Dnmser L-Diphenylalanine DPA
[0083] Particularly preferred sidechain modifications include the
replacement of Tyr with MeY and/or replacement of Pro with Hyp
and/or replacement of Leu with Hle or Nle.
[0084] These types of modifications, and others which involve more
substantive sidechain modifications, may be important to stabilise
the peptide if administered to an individual or used as a
diagnostic reagent, or to improve solubility or bioavailability, or
to provide other pharmacologies. For example it is possible to
extend or contract sidechain length, or insert or remove functional
groups to achieve these effects, eg by inserting nitroxide donor
groups.
[0085] The peptides according to the present invention may be in
the form of a salt, ester, amide, prodrug or, where appropriate, a
cyclised derivative. The .chi.-conotoxins of the present invention
are typically amidated at the C-terminal, however compounds with a
free carboxyl terminus or other modifications, such as
esterification at the C-terminal are considered to be within the
scope of the present invention. Preferably the peptides are
amidated or have a free carboxyl at the C-terminal. The peptides
according to the present invention generally have a free
N-terminus, although the N-terminus may be capped using a suitable
capping group. Examples of suitable capping groups include, but are
not limited to, acetyl (Ac), benzoyl (Ben) and Naphthyl (Nap).
[0086] Examples of suitable salts include the chloride, acetate,
lactate and glutamate salts. Conventional procedures for the
preparation of suitable salts are well known in the art.
[0087] The peptides according to the present invention may also be
in the form of prodrugs. Prodrugs are understood to include all
derivatives of peptides according to the invention which are
readily convertible in vivo into the required active peptide.
Conventional procedures for the preparation of suitable prodrugs
according to the invention are described in text books, such as
"Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985.
[0088] Certain peptides according to the present invention may also
be in cyclised form, such that there is no N- or C-termini. Such
peptide derivatives may have improved stability and bioavailability
relative to the non-cyclised peptides. Methods for cyclising
conotoxin peptides are described in WO 00/15654 (University of
Queensland), the entire contents of which is incorporated herein by
reference.
[0089] Certain peptides according to the present invention may also
be in cyclised form, such that the N- or C-termini are linked
head-to-tail either directly, or through the insertion of a linker
moiety, such moiety itself generally consisting of one or more
amino acid residues as required to join the backbone in such a
manner as to avoid altering the three-dimensional structure of the
peptide with respect to the noncyclised form. Such peptide
derivatives may have improved stability and bioavailability
relative to the non-cyclised peptides. Methods for cyclising
conotoxin peptides are described in WO 00/15654 (University of
Queensland), the entire contents of which is incorporated herein by
reference.
[0090] Other procedures known in the art for selective oxidation of
the cysteine residues may also be used such as those described in
Tam J P, Lu Y A, Yang J L. "Marked increase in membranolytic
selectivity of novel cyclic tachyplesins constrained with an
antiparallel two-beta strand cystine knot framework", Biochem
Biophys Res Commun. 2000; 267(3):783-790; Yu Q, Lehrer R I, Tam J
P. "Engineered salt-insensitive .alpha.-defensins with end-to-end
circularized structures" J Biol Chem. 2000; 275(6):3943-3949; and
Tam J P, Lu YA, Yang J L, Chiu K W. "An unusual structural motif of
antimicrobial peptides containing end-to-end macrocycle and
cystine-knot disulfides" Proc Natl Acad Sci USA. 1999;
96(16):8913-8918.
[0091] The peptides of the present invention retain the Cys
residues and characteristic disulphide bonding pattern of
.chi.-conotoxin peptides. Derivatives may include additional Cys
residues provided they are protected during formation of the
disulphide bonds.
[0092] In SEQ ID NOS. 3 and 4 the Gly residue in loop 1 may be
conservatively substituted or subjected to conservative side chain
modification. One non-limiting example of a modification is
DLys.
[0093] In SEQ ID NOS. 3 and 4 the Tyr residue in loop 1 may be
conservatively substituted or subjected to conservative side chain
modification. Examples of suitable replacements or modifications
include, but are not limited to, MeY and hTyr.
[0094] In SEQ ID NOS. 3 and 4 the Lys residue in loop 1 may be
conservatively substituted or subjected to conservative side chain
modification. Examples of suitable replacements or modifications
include, but are not limited to, DMK. Other less favoured
modifications include Ala, Leu, Arg, Phe, His, Nle and Cit.
[0095] In SEQ ID NOS. 3 and 4 the Leu residue in loop 1 may be
conservatively substituted or subjected to conservative side chain
modification. Examples of suitable replacements or modifications
include, but are not limited to, Hle and Nle.
[0096] Chimeras of the .chi.-conotoxins of the present invention,
with other conotoxins or additionally with other peptides or
proteins, can be made to engineer the activity into other
molecules, in some instances to produce a new molecule with extra
functionality. For example, amino acids that bind to the N-type
calcium channel can be combined with amino acids that inhibit NET
to produce a peptide with activity at NET (using loop 1 residues of
.chi.-conopeptides) and activity at the N-type calcium channel
(using loop 2 of CVID), as in the N-/C-cyclised CCSKLMYDCCGYKLG.
Similarly, a cyclic peptide can be contrasted with loop 1 chi
residues and a loop of amino acids having activity at opiate
receptors, as in cCCRRQICCGYKLG. These chimeric peptides may be
particularly useful as they possess pharmacologies that are
additive or even synergistic, and are expected to be beneficial in
the treatment of a wide range of pain syndromes that present in
humans.
[0097] A subset of these MrIA analogues may act at receptors in
addition to the NET allowing synergistic or additional effects.
Preferably these additional interactions synergise to enhance the
antinociceptive effects. More preferably, these additional
interactions occur at opioid receptors, opioid receptor like
receptors, GPCRs of the MRG family, the NMDA receptors, glutamate
receptors, the neurokinins, cyclooxygenase receptors, serotergenic
receptors, adrenergic receptors, vanilloid receptors,
benzodiazepines receptors, N-type calcium channel antagonists,
neuronal nicotinic receptors, muscarinic acetylcholine capsaicin
receptors, TNF-.alpha., tetrodotoxin-resistant and
tetrodotoxin-sensitive Na Channels, voltage-sensitive calcium
channel and endothelian receptors.
[0098] Preferably the .chi.-conotoxin peptides according to the
invention have 10 to 30 amino acids, more preferably 11 to 20.
[0099] The peptides according to the invention may be part of a
larger peptide. For example, the N-terminus "head" region of the
peptides of the first, second, third and seventh aspects of the
present invention may be extended to any suitable length by
introduction of additional amino acid residues. Similarly the
C-terminus may also be extended by addition of a peptide "tail". In
some cases the activity of the peptide can be improved by such
modifications.
[0100] The peptides according to the present invention may be
modified by biotinylation for use in biological assays, attachment
of antibodies for targeting the site of action, attachment of
sugars and lipids to improve permeability, and the like.
[0101] Examples of .chi.-conotoxin peptides according to the
present invention include those listed in Table 2:
TABLE-US-00013 TABLE 2 SEQ ID. NO. Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly
Tyr Lys Leu Cys Xaa5 Xaa6 Cys 13 Tyr Arg Gly Leu Cys Cys Gly MeY
Lys Leu Cys Arg Hyp Cys 14 Tyr Arg Gly Nle Cys Cys Gly MeY Lys Leu
Cys Arg Hyp Cys 15 Orn Tyr Arg Gly Nle Cys Cys Gly MeY Lys Leu Cys
Arg Hyp Cys 16 Trp Arg Gly Leu Cys Cys Gly MeY Lys Leu Cys His Hyp
Cys 17 Orn Arg Gly Nle Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys 18
Lys Tyr Arg Gly Nle Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys 19 BHK
Gly Nle Cys Cys Gly MeY Lys Hle Cys His Hyp Cys 20 Trp Arg Gly Val
Cys Cys Gly MeY Lys Leu Cys His Hyp Cys 21 Trp Lys Gly Val Cys Cys
Gly MeY Lys Leu Cys His Hyp Cys 22 Phe Arg Tyr Gly Nle Cys Cys Gly
MeY Lys Leu Cys Arg Hyp Cys 23 Tyr Orn Arg Gly Nle Cys Cys Gly MeY
Lys Leu Cys Arg Hyp Cys 24 DTrp Arg Gly Leu Cys Cys Gly MeY Lys Leu
Cys Arg Ala Cys 25 Trp Arg Gly Val Cys Cys Gly MeY Lys Leu Cys His
Hyp Cys 26 Trp Arg Gly Leu Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys
27 BHK Gly Nle Cys Cys Gly Tyr Lys Hle Cys His Hyp Cys 28 Tyr Phe
Arg Gly Nle Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys 29 Trp Arg Gly
Val Cys Cys Gly MeY Lys Leu Cys Arg Ala Cys 30 Trp Arg Gly Val Cys
Cys Gly Tyr Lys Leu Cys His Hyp Cys 31 Trp Gly Leu Cys Cys Gly MeY
Lys Leu Cys Arg Tyr Cys 32 BHK Gly Nle Cys Cys Gly MeY Lys Leu Cys
His Hyp Cys 33 Trp Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp
Cys 34 Ac Tyr Arg Gly Leu Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys
35 Trp Gly Leu Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys 36 Trp Arg
Gly Leu Cys Cys Gly MeY Lys Leu Cys Arg Lys Cys 37 Trp Arg Gly Leu
Cys Cys Gly MeY Lys Leu Cys Arg Ala Cys 38 Trp Arg Gly Val Cys Cys
Gly Tyr Lys Leu Cys Arg Ala Cys 39 Trp Asn Gly Val Cys Cys Gly Tyr
Lys Leu Cys Arg Hyp Cys 40 Trp Arg Gly Val Cys Cys Gly MeY Lys Leu
Cys His Ala Cys 41 pGlu Tyr Arg Gly Nle Cys Cys Gly MeY Lys Leu Cys
Arg Hyp Cys 42 Orn Gly Nle Cys Cys Gly MeY Lys Hle Cys His Hyp Cys
43 Trp Arg Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys Tyr 44
Trp Gly Leu Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys Tyr 45 Orn Gly
Nle Cys Cys Gly MeY Lys Leu Cys His Hyp Cys 46 Trp Asn Gly Val Cys
Cys Gly MeY Lys Leu Cys His Hyp Cys 47 Asn Gly Nle Cys Cys Gly MeY
Lys Hle Cys His Hyp Cys 48 Orn Gly Nle Cys Cys Gly MeY Lys Nle Cys
His Hyp Cys 49 BHK Gly Val Cys Cys Gly Tyr Lys Hle Cys His Hyp Cys
50 Tyr Asn Gly Nle Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 51 Orn
Gly Leu Cys Cys Gly MeY Lys Leu Cys His Hyp Cys 52 Orn Gly Nle Cys
Cys Gly Tyr Lys Hle Cys His Hyp Cys 53 Trp Asn Gly Val Cys Cys Gly
MeY Lys Leu Cys His Pro Cys 54 Trp Arg Gly Val Cys Cys Gly Tyr Lys
Leu Cys His Ala Cys 55 Asp Tyr Arg Gly Nle Cys Cys Gly MeY Lys Leu
Cys Arg Hyp Cys 56 Tyr Asn Gly Val Cys Cys Gly Tyr Lys Hle Cys His
Pro Cys 57 Asn Gly Nle Cys Cys Gly Tyr Lys Nle Cys His Hyp Cys 58
BHK Gly Nle Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 59 Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Trp Pro Cys 60 Trp Asn Gly Val Cys Cys
Gly Tyr Lys Leu Cys Arg Pro Cys 61 Asn Gly Nle Cys Cys Gly MeY Lys
Nle Cys His Hyp Cys 62 Tyr Asn Gly Val Cys Cys Gly MeY Lys Leu Cys
Arg Ala Cys 63 Asn Asp Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys
64 Trp Arg Gly Leu Cys Cys Gly Tyr Lys Leu Cys Arg Gly Cys 65 pGlu
Gly Leu Cys Cys Gly MeY Lys Leu Cys Arg Hyp Cys Tyr 66 Orn Gly Nle
Cys Cys Gly Tyr Lys Nle Cys His Hyp Cys 67 hPhe Asn Gly Val Cys Cys
Gly Tyr Lys Leu Cys His Hyp Cys 68 Tyr Asn Gly Val Cys Cys Gly Tyr
Lys Leu Cys Arg Hyp Cys 69 Trp Asn Gly Val Cys Cys Gly Tyr Lys Leu
Cys Arg Ala Cys 70 Phe Gly Gly Phe Trp Cys Cys Gly MeY Lys Leu Cys
Arg Ala Cys 71 Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Trp Hyp
Cys 72 Trp Asn Gly Leu Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 73
BHK Gly Nle Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 74 Asn Gly Nle
Cys Cys Gly MeY Lys Leu Cys His Hyp Cys 75 BHK Gly Val Cys Cys Gly
MeY Lys Leu Cys His Pro Cys 76 BHK Gly Val Cys Cys Gly Tyr Lys Hle
Cys His Pro Cys 77 DArg Gly Val Cys Cys Gly MeY Lys Leu Cys His Hyp
Cys 78 Trp Arg Gly Leu Cys Cys Gly Tyr Lys Leu Cys Arg Ala Cys 79
BHK Gly Val Cys Cys Gly MeY Lys Leu Cys His Hyp Cys 80 Asn Gly Nle
Cys Cys Gly Tyr Lys Hle Cys His Hyp Cys 81 Tyr Asn Gly Val Cys Cys
Gly Tyr Lys Leu Cys Arg Ala Cys 82 Trp Asn Gly Val Cys Cys Gly Tyr
Lys Leu Cys His Pro Cys 83 Phe Gly Gly Phe Cys Cys Gly MeY Lys Leu
Cys Arg Ala Cys 84 Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Arg
Pro Cys 85 Trp Lys Asp Leu Cys Cys Gly Tyr Lys Leu Cys His Pro Cys
86 Tyr Asn Gly Val Cys Cys Gly MeY Lys Leu Cys His Pro Cys 87 BHK
Gly Val Cys Cys Gly Tyr Lys Leu Cys Arg Hyp Cys 88 Tyr Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 89 Tyr Asn Gly Val Cys Cys
Gly Tyr Lys Leu Cys Arg Pro Cys 90 Trp Lys Asp Leu Cys Cys Gly Tyr
Lys Leu Cys Trp Pro Cys 91 Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu
Cys Trp Pro Cys 92 Trp Lys Asp Val Cys Cys Gly Tyr Lys Leu Cys Trp
Pro Cys 93 BHK Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 94
Tyr Asn Gly Val Cys Cys Gly MeY Lys Leu Cys -- Pro Cys 95 Trp BHK
Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 96 Orn Gly Nle Cys
Cys Gly Tyr Lys Leu Cys His Hyp Cys 97 Asn Gly Leu Cys Cys Gly Tyr
Lys Leu Cys His Pro Cys 98 Arg Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys 99 cyclic ( Gly Tyr Lys Leu Gly Cys Cys Gly Tyr Lys Leu
Cys -- -- Cys ) 100 Trp Ala Ala Asn Gly Val Cys Cys Gly Tyr Lys Leu
Cys His Hyp Cys 101 BHK Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp
Cys 102 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Tic Cys 103
DArg Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 104 MeY Asn
Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 105 Gly Ile Leu Arg
Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 106 Trp Ala Asn
Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 107 Nle Gly Val Cys
Cys Gly MeY Lys Leu Cys His Hyp Cys 108 Orn Gly Val Cys Cys Gly Tyr
Lys Leu Cys His Pro Cys 109 Ac Trp BHK Gly Val Cys Cys Gly Tyr Lys
Leu Cys His Hyp Cys 110 Tyr Asn Lys Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys 111 BHK Gly Val Cys Cys Gly Tyr Lys Leu Cys His Tic Cys
112 Asn Gly Nle Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 113 Asn Gly
Val Cys Cys Gly MeY Lys Leu Cys His Pro Cys 114 Ac BHK Gly Val Cys
Cys Gly Tyr Lys Leu Cys His Hyp Cys 115 Asn Gly Leu Cys Cys Gly Tyr
Lys Leu Cys His Hyp Cys 116 Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu
Cys His Pro Cys 117 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Lys
Cys 118 Tyr Asn Arg Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 119
Nle Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 120 Ben Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 121 DLys Gly Val Cys
Cys Gly Tyr Lys Leu Cys His Hyp Cys 122 Asn Lys Val Cys Cys Gly Tyr
Lys Leu Cys His Pro Cys 123 Asn Gly Val Cys Cys Gly MeY Lys Leu Cys
His Hyp Cys 124 Asn Ala Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys
125 Asn Gly Ile Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 126 Asn Gly
Val Cys Cys Gly Tyr Lys Nle Cys His Hyp Cys 127 DMK Gly Val Cys Cys
Gly Tyr Lys Leu Cys His Hyp Cys 128 DAsn Gly Val Cys Cys Gly Tyr
Lys Leu Cys His Hyp Cys 129 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pip Cys 130 Ala Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys
131 Nap Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 132 Tyr
Asn Nle Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 133 Phe Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 134 Asn Gly Val Cys Cys Gly
Tyr Lys Leu Cys Nal Pro Cys
135 Thr Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 136 ABZ Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 137 Nap Gly Val Cys Cys
Gly Tyr Lys Leu Cys His Pro Cys 138 Asn Gly Thr Cys Cys Gly Tyr Lys
Leu Cys His Pro Cys 139 Cit Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Hyp Cys 140 pGlu Asn Gly Val Cys Cys Gly MeY Lys Leu Cys His Hyp
Cys 141 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His MeY Cys 142
pGlu Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 143 Ac Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 144 DpGlu Gly Val Cys
Cys Gly Tyr Lys Leu Cys His Hyp Cys 145 Asn Gly Val Cys Cys Gly Tyr
Lys Leu Cys His Ala Cys 146 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys 147 Asp Gly Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys
148 Val Cys Cys Gly Tyr Lys Leu Cys -- -- Cys 149 Asn Gly Val Cys
Cys Gly Tyr Lys Leu Cys His DMD Cys 150 Asn Gly Ala Cys Cys Gly Tyr
Lys Leu Cys His Hyp Cys 151 Asp Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys 152 Ac Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro
Cys 153 Asn Gly Ala Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 154
pGlu Asp Val Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 155 Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Phe Cys 156 Asn Ser Val Cys Cys
Gly Tyr Lys Leu Cys His Pro Cys 157 pGlu Asn Gly Val Cys Cys Gly
Tyr Lys Leu Cys His Hyp Cys 158 Asn Gly Val Cys Cys Gly Tyr Lys Leu
Cys His THZ Cys 159 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Glu
Cys 160 Asn Gly Abu Cys Cys Gly Tyr Lys Leu Cys His Hyp Cys 161 Ac
Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 162 Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Nle Cys 163 Tyr Asn Gly Val Cys
Cys Gly Tyr Lys Leu Cys Gln Pro Cys 164 DpGlu Gly Val Cys Cys Gly
Tyr Lys Leu Cys His Hyp Cys OH 165 Asn Gly Val Cys Cys Gly Tyr Lys
Leu Cys His Tyr Cys
[0102] These peptides can also be labelled and used to establish
binding assays to identify new molecules that act at the same site.
For example, a labelled peptide ligand could have tritium included
or may have radio-active iodine or similar attached through a Tyr
or other appropriate residue. A Tyr scan through each peptide will
establish a suitable location for incorporation of the Tyr. The
inhibition of binding of such labelled peptides to tissue
homogenates or expressed transporters by compounds or mixtures
would permit identification of new peptides active at this site,
including peptides present in serum and nerve and muscle tissue of
mammals, including human tissues. The assay will also allow
identification of non-peptide molecules that also act at the same
site as .chi.-conotoxin peptides, and that may have utility as
orally active forms of these peptides. Labelled peptides will
additionally permit autoradiographic studies to identify the
location of the peptide binding across various tissues.
[0103] Contrary to what was proposed in WO00/20444 the
.chi.-conotoxin peptides have been found to be non-competitive
inhibitors in relation to noradrenaline, but competitive in
relation to small molecules that also bind to the noradrenaline
transporter, such as mazindol, cocaine and tricyclic
antidepressants, such as desipramine.
[0104] Accordingly binding assays using labelled peptides of the
present invention, preferably radio isotopically labelled, can be
used to discover small molecules that could act as non-competitive
inhibitors of the noradrenaline transport through the noradrenaline
transporter. Preferably this assay would be conducted in the
presence of blocking concentrations of noradrenaline or related
small molecules which do not overlap with the chi conopeptide
binding site but which overlap with many small molecule inhibitors
of the noradrenaline transporter (e.g. tricyclic
antidepressants).
[0105] The .chi.-conotoxins of the present invention may be
prepared using standard peptide synthetic methods followed by
oxidative disulfide bond formation. For example, the linear
peptides may be synthesised by solid phase methodology using BOC
chemistry, as described by Schnoltzer et al (1992). Following
deprotection and cleavage from the solid support the reduced
peptides are purified using preparative chromatography. The
purified reduced peptides are oxidised in buffered systems, for
example as described in example 2. The oxidised peptides were
purified using preparative chromatography.
[0106] References describing the synthesis of conotoxins include
Sato et al, Lew et al and WO 91/07980.
[0107] Some of the .chi.-conotoxins according to the present
invention may also be prepared using recombinant DNA technology. A
nucleotide sequence encoding the desired peptide sequence, or its
precursor, may be inserted into a suitable vector and protein
expressed in an appropriate expression system. In some instances,
further chemical modification of the expressed peptide may be
appropriate, for example C-terminal amidation or post translational
modification of particular residues. Under some circumstances it
may be desirable to undertake oxidative bond formation of the
expressed peptide as a chemical step following peptide expression.
This may be preceded by a reductive step to provide the unfolded
peptide. Those skilled in the art may readily determine appropriate
conditions for the reduction and oxidation of the peptide.
[0108] The invention further provides an isolated nucleic acid
molecule comprising a sequence of nucleotides encoding or
complementary to sequence encoding a .chi.-conotoxin peptide as
described above.
[0109] It may also be possible to prepare antiidiotypic antibodies
using techniques known to the art. These antiidiotypic antibodies
and their use as therapeutic agents represent a further aspect of
the present invention.
[0110] The nucleic acid molecules of the present invention may be
in isolated form or they may be integrated into or ligated to or
otherwise fused or associated with other genetic molecules such as
vector molecules and in particular expression vector molecules.
Vectors and expression vectors are generally capable of replication
and, if applicable, expression in one or both of a prokaryotic cell
or a eukaryotic cell. Preferably, prokaryotic cells include E.
coli, Bacillus sp and Pseudomonas sp. Preferred eukaryotic cells
include yeast, fungal, mammalian and insect cells.
[0111] Accordingly, another aspect of the present invention
contemplates a genetic construct comprising a vector portion and a
gene capable of encoding a peptide according to the invention, or a
peptide which can be post translationally modified to provide a
peptide according to the invention.
[0112] Preferably, the gene portion of the genetic construct is
operably linked to a promoter on the vector such that said promoter
is capable of directing expression of the gene portion in an
appropriate cell.
[0113] The present invention extends to such genetic constructs and
to prokaryotic or eukaryotic cells comprising same.
[0114] It should thus be understood that the terms conotoxin
peptide or conotoxins are not limited to naturally occurring toxic
peptides obtained from the genus Conus but rather simply indicates
an initial source from which the peptides have been derived.
Conotoxin peptides are may be synthetically created, non-naturally
occurring non-toxic peptide derivatives. Conopeptides is an
alternative term interchangeable with conotoxin peptides.
[0115] The .chi.-conotoxin peptides according to the present
invention are active in inhibiting neuronal noradrenaline
transporter. Accordingly the invention provides the use of the
.chi.-conotoxin peptides as inhibitors of neuronal noradrenaline
transporter, and in the treatment or prophylaxis of diseases or
conditions in relation to which the inhibition of neuronal
noradrenaline transporter is associated with effective treatment.
Such activity in pharmacological agents is associated with activity
in the prophylaxis or treatment of diseases or conditions of the
urinary or cardiovascular systems, or mood disorders, or in the
treatment or control of acute, chronic and or neuropathic pain,
migraine or inflammation.
[0116] Examples of the formulation and use of noradrenaline
reuptake inhibitors in therapy can be found in Ardid, D et al.,
(1992) Fund. Clinical Pharmacology 6(2): 75-8; Yaksh, T. L. (1985)
Pharmacology Biochemistry and Behaviour 22:845-858; Yaksh, T. L.
& Takano, Y. (1992) J. Pharmacology & Experimental
Therapeutics 261(2): 764-772; Yaksh, T. L. & Howe, J. R. (1982)
J. Pharmacology & Experimental Therapeutics 220(2): 311-321;
Howe, J. R. et al., (1983) J. Pharmacology & Experimental
Therapeutics 224(3): 552-558; Solomon et al., (1989) J.
Pharmacology & Experimental Therapeutics 251(1): 28-38;
Fleetwood-Walker, S. M. et al., (1985) Brain Research 334:243-254;
Takagi, H & Harima, A. (1996) European Neuropsychopharmacology
6, 43-47; Eisenach, J. C. et al (1998) Anesth Analg 87, 591-6;
Dubner, R. & Hargreaves, KM (1989) Clin J Pain, 5 pS1-6; Max,
MB (1992) N Engl J Med 326, p 1287-8; Atkinson, J H et al (1998)
Pain 76, p 287-96; Mico, J. A. et al., (1997) European
Neuropsychopharmacology 7, S162.
[0117] Accordingly the present invention provides a method for the
treatment or prophylaxis of urinary or cardiovascular conditions or
diseases or mood disorders or for the treatment or control of pain
or inflammation including the step of administering to a mammal an
effective amount of an isolated, synthetic or recombinant
.chi.-conotoxin peptide having the ability to inhibit neuronal
noradrenaline transporter, wherein said .chi.-conotoxin peptide
comprises the following sequence of amino acids:
TABLE-US-00014 SEQ ID NO. 3 Cys Cys Gly Tyr Lys Leu Cys Xaa5 Xaa6
Cys
where Xaa5 and Xaa6 are independently absent or represent any amino
acid residue except Cys, or such a sequence in which Gly, Tyr, Lys
or Leu are subject to conservative amino acid substitution or side
chain modification, with the proviso that the peptide is not
.chi.-MrIA or .chi.-MrIB; or a salt, ester, amide, prodrug or
cyclised derivative thereof.
[0118] According to this embodiment of the invention the peptide
may be a peptide of SEQ ID NO. 4, 5 or 6 as described above.
[0119] In performing the method according to the present invention
the administration of the .chi.-peptide may be performed in
conjunction with other therapies useful in the treatment of the
condition, disease or disorder. Accordingly the peptide may be
administered substantially simultaneously or sequentially with
other agents useful in the treatment of the conditions, diseases or
disorders. Where the co-administration is simultaneous, the peptide
may be formulated in a composition with one or more of the other
agents. The co-administration of other agents can be performed via
the same or different route to the route of administration for the
.chi.-peptide. Where the method is for the treatment or control of
acute, chronic and/or neuropathic pain or migraine, the peptide may
be administered substantially simultaneously or sequentially with
an analgesic agent selected from the group consisting of opioid
analgesics, opioid receptor-like antagonists, GPCR antagonists of
the MRG family, NMDA antagonists, substance P antagonists, COX 1
and COX 2 inhibitors, tricyclic antidepressants (TAC), selective
serotonin reuptake inhibitors (SSRI), capsaicin receptor
antagonists, anaesthetic agents, benzodiazepines, skeletal muscle
relaxants, migraine therapeutic agents, anti-convulsants,
anti-hypertensives, anti-arrhythmics, antihistamines, steroids,
caffeine, N-type calcium channel antagonists, nicotinic receptor
partial agonists and antagonists, vanilloid receptor antagonists
and agonists, TNF-.A-inverted. antagonists and antibodies,
inhibitors of tetrodotoxin-sensitive Na Channels, P-type channel
inhibitors, endothelian antagonists and botulinum toxin. The
peptide may also be administered simultaneously with two or more
other agents, for example mixtures of SSRIs and noradrenaline
reuptake inhibitors.
[0120] Where the analgesic agent is an opioid receptor-like
analgesic agent it is preferably selected from naltrexone and
nalmefene; their pharmaceutically active salts and their optical
isomers.
[0121] Where the analgesic agent is an opioid analgesic agent it is
preferably selected from propoxyphene, meperidine, hydromorphone,
hydrocodone, morphine, codeine and tramodol; their pharmaceutically
active salts and their optical isomers.
[0122] Where the analgesic agent is an NMDA antagonist analgesic
agent it is preferably selected from 2-piperidino-1 alkanol
derivatives, dextromethorphan, eliprodil, and ifenprodil; their
pharmaceutically active salts and their optical isomers.
[0123] Where the analgesic agent is a P antagonist analgesic agent
it is preferably selected from 2-phenyl-piperidin-3-yl or
2-diphenylmethyl-1-azabicyclo[2.2.2]-octane-3-amine derivatives as
described in U.S. Patent Application No. 2001/00336943 A1 (Coe et
al.); their pharmaceutically active salts and their optical
isomers.
[0124] Where the analgesic agent is a COX 2 inhibition analgesic
agent it is preferably selected from rofecoxib and celecoxib; their
pharmaceutically active salts and their optical isomers.
[0125] Where the analgesic agent is an anaesthetic analgesic agent
it is preferably selected from nitrous oxide, halothane, lidocaine,
etidocaine, ropivacaine, chloroprocaine, sarapin and bupivacaine;
their pharmaceutically active salts and their optical isomers.
[0126] Where the analgesic agent is a benzodiazepine analgesic
agent it is preferably selected from diazepam, chlordiazepoxide,
alprazolam, lorazepam, midazolam, L-365260; their pharmaceutically
active salts and their optical isomers.
[0127] Where the analgesic agent is a skeletal muscle relaxant
analgesic agent it is preferably selected from flexeril,
carisoprodol, robaxisal, norgesic and dantrium their
pharmaceutically active salts and their optical isomers.
[0128] Where the analgesic agent is a migraine therapeutic agent it
is preferably selected from elitriptan, sumatriptan, rizatriptan,
zolmitriptan, and naratriptan their pharmaceutically active salts
and their optical isomers.
[0129] Where the analgesic agent is an anticonvulsant analgesic
agent it is preferably selected from gabapentin, pregabalin,
carbamazepine, and topiramate and valproic acid their
pharmaceutically active salts and their optical isomers.
[0130] Where the analgesic agent is a COX 1 inhibitor analgesic
agent it is preferably selected from salycylic acid, acetominophen,
diclofenac, piroxican indomethacin, ibuprofen, and naproxen their
pharmaceutically active salts and their optical isomers.
[0131] Where the analgesic agent is a tricyclic antidepressant
analgesic agent it is preferably selected from amitriptyline,
desipramine, perphenazine, protriptyline, and tranylcypromine their
pharmaceutically active salts and their optical isomers.
[0132] Where the analgesic agent is a SSRI analgesic agent it is
preferably selected from tramadol and milnacipran; their
pharmaceutically active salts and their optical isomers.
[0133] Where the analgesic agent is a mixture of SSRI and
Noradrenaline reuptake inhibitors, the latter is preferably
selected from reboxetine and atomoxetine; their pharmaceutically
active salts and their optical isomers.
[0134] The analgesic agent may also be selected from adenosine,
baclofen, clonidine, mexilitene, diphenyl-hydramine, hydroxysine,
caffeine, prednisone, methylprednisone, decadron, paroxetine,
sertraline, fluoxetine, Ziconotide.RTM. and levodopa their
pharmaceutically active salts and their optical isomers.
[0135] Where the analgesic agent is a TNF-.alpha. antagonist or
antibody, the agent is preferably selected from etanercept,
infliximab and thalidomide; their pharmaceutically active salts and
their optical isomers.
[0136] Where the analgesic agent is an endothelian antagonist, the
agent is preferably selected from bosentan and tesosentan; their
pharmaceutically active salts and their optical isomers.
[0137] Where the analgesic agent is a vanilloid antagonist, the
analgesic agent is preferably selected from ananamide, capsazepine,
thiocarbamic acid derivatives (as described in WO02/16317 A1) and
thiourea derivatives (as described in WO02/16318 A1); their
pharmaceutically active salts and their optical isomers.
[0138] Where the analgesic agent is selected from nicotine receptor
partial agonist it is preferably selected from
1,2,3,4,5,6-hexahydro-1,5-methano-pyrido[1,2-a][1,5]diazocin-8-one
derivatives,
diazatetracyclo[9.3.1.0.sup.2,10.0.sup.4,8]pentadeca-2(10),3,8-triene
derivatives, 10-aza-tricyclo[6.3.1.0.sup.2,7]dodeca-2(7),3,5-triene
derivatives,
triazatetracyclo[9.3.1.0.sup.2,10.0.sup.4,8]pentadeca-2(10),3,5,8-tetraen-
e derivatives,
5,8,14-triazatetracyclo[10.3.1.0.sup.2,11.0.sup.4,9]hexadeca-2(11),3,5,7,-
9-pentaene derivatives,
diazatetracyclo[9.3.1.0.sup.2,10.0.sup.4,8]pentadeca-2(10),3,6,8-tetraene
derivatives, 10-azatricyclo[6.3.1.0.sup.2,7]dodeca-2(7),3,5-triene
derivatives,
5,7,14-triazatetracyclo[10.3.1.0.sup.2,10.0.sup.4,8]hexadeca-2(10),3,5,8--
tetraene derivatives,
5,8,15-triazatetracyclo[11.3.1.0.sup.2,11.0.sup.4,9]heptadeca-2(11),3,5,7-
,9-pentaene derivatives,
5,14-diazatetracyclo[10.3.1.0.sup.2,10.0.sup.4,8]hexadeca-2(10),3,5,8-tet-
raene derivatives,
11-azatricyclo[7.3.1.0.sup.2,7]trideca-2(7),3,5-triene derivatives,
all of which are described in U.S. Patent Application No.
2001/00336943 A1 and their pharmaceutically acceptable salts and
their optical isomers.
[0139] Examples of conditions associated with acute, chronic and/or
neuropathic pain and inflammatory pain include soft tissue and
peripheral damage, such as acute trauma, osteoarthritis, rheumatoid
arthritis, musculo-skeletal pain, particularly after trauma, spinal
pain, dental pain, myofascial pain syndromes, headache, episiotomy
pain, and burns; deep and visceral pain, such as heart pain, muscle
pain, eye pain, orofacial pain, for example, odontalgia, abdominal
pain, gynaecological pain, for example, dysmenorrhea, and labour
pain; pain associated with nerve and root damage, such as pain
associated with peripheral nerve disorders, for example, nerve
entrapment and brachial plexus avulsions, amputation, peripheral
neuropathies, neuralgia, tic douloureux, atypical facial pain,
nerve root damage, pain and/or chronic nerve compression, and
arachnoiditis; pain associated with carcinoma, often referred to as
cancer pain; pain associated with AIDS, central nervous system
pain, such as pain due to spinal cord or brain stem damage; low
back pain; sciatica; headache, including migraine, acute or chronic
tension headache, cluster headache, temporomandibular pain and
maxillary sinus pain; ankylosing spondylitis, gout; post operative
pain; phantom pains; diabetic neuropathy; shingles; and scar
pain.
[0140] Examples of the formulation and use of conotoxin peptides in
the treatment of pain can be found in WO9107980; U.S. Pat. No.
5,587,454 and WO9701351. These documents relate to omega
conotoxins. Also see Bowersox S S, Gadbois T, Singh T, Pettus M,
Wang Y X & Luther R R (1996) J Pharmacol Exp Ther, 279(3) pages
1243-9 which relates to conotoxin peptides that are selective
N-type Voltage-sensitive calcium channel blockers and their use in
the treatment of acute, persistent and neuropathic pain in
rats.
[0141] Examples of diseases or conditions of the urinary system
include urinary and fecal incontinence. Examples of cardiovascular
diseases or conditions include arrhythmias of various origins and
coronary heart failure. Examples of mood disorders include
depression, anxiety, cravings, an addictive disorder and withdrawal
syndrome, an adjustment disorder, age-associated learning and
mental disorders, anorexia nervosa, apathy, attention-deficit
disorders due to general medical conditions, attention-deficit
hyperactivity disorder, bipolar disorder, bulimia nervosa, chronic
fatigue syndrome, chronic or acute stress, conduct disorder,
cyclothymic disorder, depression, dysthymic disorder, fibromyalgia
and other somatoform disorders, generalised anxiety disorder,
incontinence, inhalation disorders, intoxication disorders, mania,
obesity, obsessive compulsive disorders and related spectrum
disorders, oppositional defiant disorder, panic disorder,
peripheral neuropathy, post-traumatic stress disorder, premenstrual
dysphoric disorder, psychotic disorders, seasonal affective
disorder, sleep disorders, social phobia, specific developmental
disorders, selective serotonin reuptake inhibition (SSRI) "poop
out" syndrome, and TIC disorders.
[0142] Examples of the use of selective noreprinephrine reuptake
inhibitors in the treatment of diseases or conditions of the
urinary system include Springer, J P., Kropp, B P & Thor K B
(1994) J Urol 152(2), p 515-9 (relates to lower urinary tract);
Penttila, O. et al (1975) Ann Clin Res (7), 32-6 (relates to
treatment of ulcerative colitis) and Dinan, T G et al (1990) J
Psychosom Res 34, p 575-80 (relates to treatment of irritable bowel
syndrome).
[0143] Preferably the mammal is in need of such treatment although
the peptide may be administered in a prophylactic sense.
[0144] The invention also provides a composition comprising an
isolated, synthetic or recombinant .chi.-conotoxin peptide having
the ability to inhibit neuronal noradrenaline transporter, wherein
said .chi.-conotoxin peptide comprises the following sequence of
amino acids:
TABLE-US-00015 SEQ ID NO. 3 Cys Cys Gly Tyr Lys Leu Cys Xaa5 Xaa6
Cys
where Xaa5 and Xaa6 are independently absent or represent any amino
acid residue except Cys, or such a sequence in which loop1 residues
Gly, Tyr, Lys or Leu are subject to conservative amino acid
substitution or side chain modification, with the proviso that the
peptide is not .chi.-MrIA or .chi.-MrIB; or a salt, ester, amide,
prodrug or cyclised derivative thereof, and a pharmaceutically
acceptable carrier or diluent.
[0145] According to this embodiment of the invention the peptide
may be a peptide of SEQ ID NO. 4, 5 or 6 as described above.
[0146] Preferably the composition is in the form of a
pharmaceutical composition.
[0147] There is also provided the use of an isolated, synthetic or
recombinant .chi.-conotoxin peptide having the ability to inhibit
neuronal noradrenaline transporter, wherein said .chi.-conotoxin
peptide comprises the following sequence of amino acids:
TABLE-US-00016 SEQ ID NO. 3 Cys Cys Gly Tyr Lys Leu Cys Xaa5 Xaa6
Cys
where Xaa5 and Xaa6 are independently absent or represent any amino
acid residue except Cys, or such a sequence in which loop 1
residues Gly, Tyr, Lys or Leu are subject to conservative amino
acid substitution or side chain modification, with the proviso that
the peptide is not .chi.-MrIA or .chi.-MrIB; or a salt, ester,
amide, prodrug or cyclised derivative thereof, in the manufacture
of a medicament for the treatment or prophylaxis of urinary or
cardiovascular conditions or diseases, or mood disorders, or for
the treatment or control of pain or inflammation.
[0148] According to this embodiment of the invention the peptide
may be a peptide of SEQ ID NO. 4, 5 or 6 as described above.
[0149] It is also noted that noradrenaline transporter is expressed
not only by nerve cells, but also by other tissues including the
placenta, pulmonary endothelial cells and the uterus. The peptides
according to the present invention may also be effective in
inhibiting these noradrenaline transporters, and may be useful in
treating conditions in which these transporters are implicated.
[0150] As will be readily appreciated by those skilled in the art,
the route of administration and the nature of the pharmaceutically
acceptable carrier will depend on the nature of the condition and
the mammal to be treated. It is believed that the choice of a
particular carrier or delivery system, and route of administration
could be readily determined by a person skilled in the art. In the
preparation of any formulation containing the peptide actives care
should be taken to ensure that the activity of the peptide is not
destroyed in the process and that the peptide is able to reach its
site of action without being destroyed. In some circumstances it
may be necessary to protect the peptide by means known in the art,
such as, for example, micro encapsulation. Similarly the route of
administration chosen should be such that the peptide reaches its
site of action.
[0151] For example the preferred route of administration for the
treatment of urinary diseases is oral, topical, intranasal,
intrarectal, intramucosal and intravenous. The same may be used for
the treatment of pain and mode disorders, in addition to
intrathecal administration. A method and formulations for use with
conotoxin peptides in intrathecal administration is described in WO
9701351, the contents of which are incorporated by
cross-reference.
[0152] The pharmaceutical forms suitable for injectable use include
sterile injectable solutions or dispersions, and sterile powders
for the extemporaneous preparation of sterile injectable solutions.
They should be stable under the conditions of manufacture and
storage and may be preserved against oxidation and the
contaminating action of microorganisms such as bacteria or
fungi.
[0153] Those skilled in the art may readily determine appropriate
formulations for the peptides or modified peptides of the present
invention using conventional approaches. Identification of
preferred pH ranges and suitable excipients, for example
antioxidants, is routine in the art (see for example Cleland et al,
1993). Buffer systems are routinely used to provide pH values of a
desired range and include carboxylic acid buffers for example
acetate, citrate, lactate and succinate. A variety of antioxidants
are available for such formulations including phenolic compounds
such as BHT or vitamin E, reducing agents such as methionine or
sulphite, and metal chelators such as EDTA.
[0154] Conventional approaches for the formulation of
pharmaceutically active peptides are described in the following
articles, the methodology of which are incorporated by reference:
Ryan, J et al., (1986) Clin Pharmacol Ther (39), 40-2. (a clinical
trial detailing the oral administration of the peptide nifalatide);
Krames E. S. et al. (1986) Pain 24, 205-9 (describes the
intrathecal delivery of a peptide); WO9614079A1 (which describes
oral and rectal administration of formulations of the peptide
cyclosporin); WO9640064A1 (which describes formulations for peptide
stability); WO9805309A1 (describes peptide formulations--a
pharmaceutical composition of cyclosporin for internal use and
WO9802148A2 (which describes sustained release rectal and oral
peptide formulations).
[0155] The solvent or dispersion medium for the injectable solution
or dispersion may contain any of the conventional solvent or
carrier systems for peptide actives, and may contain, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about where necessary by the
inclusion of various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal
and the like. In many cases, it will be preferable to include
agents to adjust osmolality, for example, sugars or sodium
chloride. Preferably, the formulation for injection will be
isotonic with blood. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin. Pharmaceutical forms suitable for injectable use may be
delivered by any appropriate route including intravenous,
intramuscular, intracerebral, intrathecal, epidural injection or
infusion.
[0156] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients such as these
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredient into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, preferred methods of
preparation are vacuum drying or freeze-drying a of a previously
sterile-filtered solution of the active ingredient plus any
additional desired ingredients.
[0157] When the active ingredients are suitably protected they may
be orally administered, for example, with an inert diluent or with
an assimilable edible carrier, or it may be enclosed in hard or
soft shell gelatin capsule, or it may be compressed into tablets,
or it may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compound may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
preferably contain at least 1% by weight of active compound. The
percentage of the compositions and preparations may, of course, be
varied and may conveniently be between about 5 to about 80% of the
weight of the unit. The amount of active compound in such
therapeutically useful compositions in such that a suitable dosage
will be obtained.
[0158] The tablets, troches, pills, capsules and the like may also
contain the components as listed hereafter: a binder such as gum,
acacia, corn starch or gelatin; excipients such as dicalcium
phosphate; a disintegrating agent such as corn starch, potato
starch, alginic acid and the like; a lubricant such as magnesium
stearate; and a sweetening agent such a sucrose, lactose or
saccharin may be added or a flavouring agent such as peppermint,
oil of wintergreen, or cherry flavouring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills, or capsules may be coated with
shellac, sugar or both. A syrup or elixir may contain the active
compound, sucrose as a sweetening agent, methyl and propylparabens
as preservatives, a dye and flavouring such as cherry or orange
flavour. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compound(s) may be
incorporated into sustained-release preparations and
formulations.
[0159] The present invention also extends to any other forms
suitable for administration, for example topical application such
as creams, lotions, transdermal patches, sprays and gels, or
compositions suitable for inhalation or intranasal delivery, for
example solutions or dry powders.
[0160] Parenteral dosage forms are preferred, including those
suitable for intravenous, subcutaneous, intrathecal, intracerebral
or epidural delivery.
[0161] The composition may also be formulated for delivery via slow
release implants, including implantable pumps, such as osmotic
pumps.
[0162] Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the
like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, use thereof in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0163] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the novel dosage unit
forms of the invention are dictated by and directly dependent on
(a) the unique characteristics of the active material and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
material for the treatment of disease in living subjects having a
diseased condition in which bodily health is impaired as herein
disclosed in detail.
[0164] The principal active ingredient is compounded for convenient
and effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form. A unit
dosage form can, for example, contain the principal active compound
in amounts ranging from 0.25 .mu.g to about 2000 mg. Expressed in
proportions, the active compound is generally present in from about
0.25 .mu.g to about 200 mg/ml of carrier. In the case of
compositions containing supplementary active ingredients, the
dosages are determined by reference to the usual dose and manner of
administration of the said ingredients.
[0165] The invention will now be described with reference to the
accompanying drawings and examples, however it is to be understood
that the particularity of the following description is not to
supersede the generality of the preceding description of the
invention.
[0166] Referring to the FIGURE:
[0167] FIG. 1: Anti-allodynic effects of (A) i.t. SEQ ID NO. 20 and
(B) MrIA in Chronic Constriction Injury (CCI) of the rat sciatic
nerve.
EXAMPLES
Example 1
Synthesis
a) Assembly
[0168] The peptides described herein were prepared according to the
following methods: [0169] (i) Assembly of MrIA and some MrIA
derivatives was carried out using Fmoc-chemistry methods adapted
from that described by Schnolzer et al., (1992) on a Polymer Labs
Rink amide resin. Conventional Trt/t-Bu side chain protection was
used throughout. The coupling efficiency was monitored using the
ninhydrin test (Sarin et al., 1981) until a coupling efficiency of
99.5% or better was achieved. In some cases a second coupling step
was required to achieve this level of coupling efficiency. [0170]
Cleavage was carried out using a mixture of
TFA:water:triisopropylsilane:EDT (90:5:2.2:2.5) over 5 h at room
temperature, then the product obtained by precipitation from cold
diethyl ether. Purification of the crude reduced product was
carried out by RP-HPLC on a Vydac C-8 column using a 1%/min
gradient from 0% B to 45% B where A=0.1% TFA/water, B=90%
Acetonitrile/water plus 0.043% TFA. Eluent was delivered to a mass
spectrometer and samples collected on the basis of mass directed
fractionation (MDF). [0171] (ii) Other MrIA derivatives were
prepared using Boc-chemistry and conventional side chain protecting
groups on a MBHA resin using (Schnolzer et al, 1992). Cleavage is
carried out using HF:scavengers (9:1) for 1 h at 0 to -10EC.
b) Oxidation
[0172] Oxidation of the pure reduced peptides was carried out using
the following optimised buffer systems: [0173] (i) 30% DMSO/0.1M
NH.sub.4HCO.sub.3 at pH 6 for 12 h purified by RP-HPLC on a C-8
column as above; [0174] (ii) 30% isopropanol/0.1M NH.sub.4HCO.sub.3
at pH 8.0; and [0175] (iii) Mixture of isopropanol/DMSO/0.1M
NH.sub.4HCO.sub.3 pH 8.0.
[0176] In each case the desired product was purified by RP-HPLC on
a C-8 column as above.
c) In addition to preparing peptides according to the invention,
the peptides listed in Table 3 were also prepared:
TABLE-US-00017 TABLE 3 SEQ ID. NO. Xaa1 Xaa2 Xaa3 Xaa4 Cys Cys Gly
Tyr Lys Leu Cys Xaa5 Xaa6 Cys 166 Asn Gly Val Cys Cys DLys Tyr Lys
Leu Cys His Pro Cys 167 Asn Gly Val Cys Cys Gly Tyr DMK Leu Cys His
Hyp Cys 168 Asn Gly Val Cys Cys Gly hTyr Lys Leu Cys His Hyp Cys
169 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys PYA Pro Cys 170 Tyr Asn
Gly Val Cys Cys Gly Tyr Lys Leu Cys Lys Pro Cys 171 Tyr Asn Gly Val
Cys Cys Gly Leu Lys Leu Cys His Pro Cys 172 Asn Gly Val Cys Cys Gly
Tyr Ala Leu Cys His Pro Cys 173 Cys Cys Gly Tyr Lys Leu Cys His Hyp
Cys 174 Tyr Asn Gly Val Cys Cys Gly Tyr Leu Leu Cys His Pro Cys 175
Tyr Asn Gly Val Cys Cys Gly Tyr Lys Asn Cys His Pro Cys 176 Asn Gly
Val Cys Cys Gly FLA Lys Leu Cys His Pro Cys 177 Asn Gly Val Cys Cys
Gly Tyr Arg Leu Cys His Hyp Cys 178 Asn Gly Val Cys Cys Gly Tyr Lys
Leu Cys HBO Pro Cys 179 Tyr Asn Gly Val Cys Cys Gly Tyr Phe Leu Cys
His Pro Cys 180 Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys HME Pro Cys
181 Asn Gly Val Cys Cys Gly Tyr His Leu Cys His Pro Cys 182 pGlu
Gly Val Cys Cys Gly Tyr Nle Leu Cys His Hyp Cys 183 Asn Gly Val Cys
Cys DGlu Tyr Lys Leu Cys His Pro Cys 184 Tyr Asn Gly Val Cys Cys
Gly Asn Lys Leu Cys His Pro Cys 185 Asn Gly Val Cys Cys Gly Tyr Nle
Leu Cys His Pro Cys 186 Asn Gly Val Cys Cys Ser Tyr Lys Leu Cys His
Pro Cys 187 pGlu Gly Val Cys Cys Gly Trp Lys Leu Cys His Hyp Cys
188 Asn Gly Val Cys Cys DSer Tyr Lys Leu Cys His Pro Cys 189 pGlu
Gly Val Cys Cys Gly Tyr Cit Leu Cys His Hyp Cys 190 Asn Gly Val Cys
Cys Gly Tyr Lys Leu Cys Ala Hyp Cys 191 Asn Gly Val Cys Cys Gly Tic
Lys Leu Cys His Pro Cys 192 Asn Gly Val Cys Cys DPhe Tyr Lys Leu
Cys His Pro Cys 193 Gly Ile Cys Cys Gly Val Ser Phe Cys Tyr Hyp Cys
194 Asn Gly Val Cys Cys Gly Tyr Gln Leu Cys His Pro Cys 195 Tyr Asn
Gly Val Cys Cys Gly Glu Lys Leu Cys His Pro Cys 196 Asn Gly Val Cys
Cys Gly Tyr Lys Lys Cys His Pro Cys 197 pGlu Gly Val Cys Cys Gly
Glu Lys Leu Cys His Hyp Cys 198 pGlu Gly Val Cys Cys Gly Ile Lys
Leu Cys His Hyp Cys 199 Arg Asn Cys Cys Arg Leu Gln Val Cys -- --
Cys Gly 200 Val Gly Val Asp Asp Gly Tyr Lys Leu Cys His Hyp Cys 201
Tyr Asn Gly Val Cys Cys Gly Lys Lys Leu Cys His Pro Cys 202 Asn Gly
Val Cys Cys Gly Tyr Lys Ala Cys His Hyp Cys 203 Asn Gly Val Cys Cys
Gly Tyr Ala Leu Cys His Hyp Cys 204 Asn Gly Val Cys Cys Gly Ala Lys
Leu Cys His Hyp Cys 205 Asn Gly Val Cys Cys Ala Tyr Lys Leu Cys His
Hyp Cys 206 Asn Gly Val Cys Cys Gly DMD Lys Leu Cys His Pro Cys 207
Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Arg Pro Cys 208 Tyr Asn
Gly Val Cys Cys Gly Tyr Ile Leu Cys His Pro Cys 209 Tyr Asn Gly Val
Cys Cys Gly Tyr Lys Asp Cys His Pro Cys 210 Tyr Asn Gly Val Cys Cys
Gly Tyr Lys Leu Cys Glu Pro Cys 211 Tyr Asn Gly Val Cys Cys Gly Tyr
Trp Leu Cys His Pro Cys 212 Tyr Asn Gly Val Cys Cys Gly Tyr Tyr Leu
Cys His Pro Cys 213 Asn Gly Val Cys Cys Gly DMD Lys Leu Cys His Hyp
Cys 214 Asn Gly Val Cys Cys Gly DPA Lys Leu Cys His Hyp Cys 215 Asn
Gly Val Cys Cys Gly DMK Lys Leu Cys His Hyp Cys
Example 2
Binding Studies
[0177] The binding activity at the human noradrenaline transporter
(hNET) and noradrenaline (NA) uptake were measured for several
peptides according to the invention, as well as for MrIA and other
peptides not according to the invention.
(i) hNET Radioligand Assay [0178] The ability of .chi.-conotoxins
to act as inhibitors of the human noradrenaline transporter (hNET)
can be measured by competitive inhibition of .sup.3H-nisoxetine
from membrane prepared from COS-7 mammalian cells expressing hNET.
Similar results were obtained with other .sup.3H-small molecules,
such as maxindole. [0179] COS-7 cells (ATCC) grown in 150 mm dishes
containing DMEM and 10% serum were transiently transfected with
plasmid DNA encoding mammalian (human) NET (Percy et al 1999, Br J
Pharmacol 128: 774-780) using metafectene reagent (Biontex). Cells
were harvested 48 hrs post transfection, cells were scraped,
washed, homogenised and centrifuged using TEM buffer. For each 150
mm dish membrane was resuspended in 500 .mu.L TEM with 10%
glycerol. BCA protein estimates were performed giving .apprxeq.6
.mu.g/.mu.L. 1 .mu.L membrane+49 .mu.L assay buffer was used per
well in the assay (assay buffer is 20 mM TrisHCl pH 7.4, 75 mM
NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% BSA). Total assay volume was
150 .mu.L and each data point performed in triplicate. Peptides at
various concentrations (10.sup.-4 to 10.sup.-11M) or control ligand
(nisoxetine) were added to the assay plate followed by 4.3 nM
.sup.3H-nisoxetine (Perkin Elmer cat # NET1084). Finally the
membrane was added and the assay was incubated for 1 hr at RT after
which the reaction was filtered onto GF filtermats B (Perkin Elmer
cat #: 1450-521) pretreated with 0.6% PEI using a Tomtec cell
harvester and washed 3 times using wash buffer (20 mM HEPES pH 7.4,
125 mM NaCl @ 4.degree. C.). Filtermats were then dried, placed in
a filter bag, 9 mLs betaplate scintillant (Perkin Elmer cat
#1205-440) added and filtermats counted on a Wallac MicroBeta
instrument. Each data point was performed in triplicate and the
results summarised in Table 4 are from n.gtoreq.3 experiments.
(ii) NA Uptake Assay
[0179] [0180] The ability of .chi.-conotoxins to act as inhibitors
of the human noradrenaline transporter (hNET) was also measured by
non-competitive inhibition of the function of noradrenaline
transporter to transport .sup.3H-noradrenaline into COS-7 mammalian
cells expressing hNET. [0181] COS-7 cells (ATCC) grown in 24 well
plates containing DMEM and 10% serum were transiently transfected
with plasmid DNA encoding mammalian (human) NET using metafectene
reagent (Biontex). Uptake assays were performed at RT 48 hrs post
transfection in transport buffer containing 125 mM NaCl, 4.8 mM
KCl, 1.2 mM MgSO.sub.4, 1.2 mM KH.sub.2PO.sub.4, 1.3 mM CaCl.sub.2,
25 mM HEPES pH7.4, 5.55 mM glucose, 1.02 mM ascorbic acid, 10 .mu.M
U-0521 and 100 .mu.M pargyline. Total assay volume was 250 .mu.L.
Cells was 3 times with warm PBS followed by the addition of assay
buffer. To which was added control or competing ligand at various
concentrations (10.sup.-4 to 10.sup.-11M). Assay was incubated for
20 mins after which 100 nM 3H-noradrenaline was added and allowed
to incubate for 10 mins. Assay stopped by removal and washing with
cold PBS. Cells lysed with 500 .mu.L 0.1% SDS, 0.1N NaCl. 100 .mu.L
aliquots taken and added to flexible 96 well plate (for the
counter) to which supermix scintillant was added (100 .mu.L), mixed
well and counted for 3 mins per well. Each data point was performed
in triplicate and the results, summarised in Table 4, are from
n.gtoreq.3 experiments. NT=not tested.
TABLE-US-00018 [0181] TABLE 4 Av IC50 Displacement of 3H- AvIC50
Inhibition of SEQ ID. NO. nisoxetine from hNET NA uptake via hNET
13 -7.85 -7.60 14 -7.80 -7.48 15 -7.66 -7.33 16 -7.54 -7.44 17
-7.55 -7.21 18 -7.46 -7.29 19 -7.45 -7.35 20 -7.42 -7.59 21 -7.40
NT 22 -7.37 -7.09 23 -7.35 -7.21 24 -7.35 NT 25 -7.32 NT 26 -7.30
NT 27 -7.25 -7.24 28 -7.14 -7.16 29 -7.13 NT 30 -7.07 NT 31 -7.06
NT 32 -7.06 -7.16 33 -7.06 NT 34 -7.05 NT 35 -7.02 NT 36 -7.01
-7.07 37 -7.00 NT 38 -6.99 NT 39 -6.99 NT 40 -6.98 NT 41 -6.96
-6.88 42 -6.93 -7.33 43 -6.91 NT 44 -6.89 NT 45 -6.88 -6.93 46
-6.88 NT 47 -6.88 -6.78 48 -6.88 -7.17 49 -6.87 NT 50 -6.86 NT 51
-6.84 -7.11 52 -6.83 -7.09 53 -6.81 -6.84 54 -6.80 NT 55 -6.78
-6.77 56 -6.78 -7.10 57 -6.77 -6.94 58 -6.76 NT 59 -6.76 NT 60
-6.75 NT 61 -6.75 -6.90 62 -6.74 NT 63 -6.72 NT 64 -6.72 NT 65
-6.71 NT 66 -6.70 -7.04 67 -6.69 -7.12 68 -6.66 NT 69 -6.66 NT 70
-6.65 NT 71 -6.64 -7.42 72 -6.62 -7.22 73 -6.60 NT 74 -6.60 -6.67
75 -6.60 NT 76 -6.58 NT 77 -6.56 -6.91 78 -6.56 NT 79 -6.56 -7.11
80 -6.55 -6.87 81 -6.53 NT 82 -6.53 -5.50 83 -6.52 NT 84 -6.52 NT
85 -6.51 -7.05 86 -6.50 -6.96 87 -6.48 NT 88 -6.48 -6.87 89 -6.47
-6.95 90 -6.45 NT 91 -6.41 NT 92 -6.39 -7.44 93 -6.39 NT 94 -6.37
NT 95 -6.36 NT 96 -6.33 -6.54 97 -6.30 NT 98 -6.29 NT 99 -6.29
-6.99 100 -6.19 NT 101 -6.16 NT 102 -6.15 NT 103 -6.15 -6.50 104
-6.14 NT 105 -6.12 NT 106 -6.09 NT 107 -6.08 -6.66 108 -6.06 NT 109
-6.03 NT 110 -6.01 NT 111 -6.01 NT 112 -5.99 NT 113 -5.96 NT 114
-5.96 NT 115 -5.95 -6.61 116 -5.95 NT 117 -5.94 NT 118 -5.93 NT 119
-5.93 -6.32 120 -5.91 NT 121 -5.88 -6.34 122 -5.88 NT 123 -5.87
-6.45 124 -5.87 NT 125 -5.85 NT 126 -5.81 -6.32 127 -5.81 -6.46 128
-5.79 -6.28 129 -5.79 NT 130 -5.78 NT 131 -5.75 NT 1 (MrlA) -5.74
-6.30 132 -5.74 NT 133 -5.74 -6.30 134 -5.74 NT 135 -5.71 -6.31 7
(Mar2) -5.69 NT 136 -5.68 -6.34 137 -5.67 NT 138 -5.64 NT 139 -5.64
-6.36 140 -5.64 -6.58 141 -5.61 NT 142 -5.60 -6.20 143 -5.59 NT 144
-5.56 NT 145 -5.53 NT 146 -5.51 -6.13 147 -5.50 NT 148 -5.50 -6.46
149 -5.48 NT 150 -5.46 -6.01 151 -5.45 NT 152 -5.44 NT 153 -5.43 NT
154 -5.40 NT 155 -5.39 NT 156 -5.37 NT 157 -5.33 -6.12 158 -5.33 NT
159 -5.31 NT 160 -5.30 -6.04 161 -5.21 NT 162 -5.19 NT 163 -5.16 NT
164 -5.16 NT 165 -5.15 NT 166 -5.35 NT 167 -5.07 -5.84 168 -5.04
-5.58 169 -5.03 NT 170 -5.00 NT 171 -4.97 NT 172 -4.97 NT 173 -4.89
-5.38 174 -4.76 NT 175 -4.74 NT 176 -4.71 NT 177 -4.64 NT 178 -4.63
NT 179 -4.60 NT 180 -4.53 NT 181 -4.32 NT 182 -4.24 NT 183 -4.21 NT
184 -4.10 NT 185 -4.09 NT 186 -4.04 NT 187 -4.04 NT 188 -4.02 NT
189 -4.02 NT 190 -3.99 -5.10 191 -3.96 NT 192 -3.95 NT 193 -3.92 NT
194 -3.91 NT 195 -3.77 NT 196 -3.74 NT 197 -3.72 NT 198 -3.67 NT
199 -3.62 NT 200 -3.55 NT 201 -3.36 NT 202 -2.00 NT 203 -2.00 -4.86
204 -2.00 NT 205 -2.00 NT 206 -2.00 NT 207 -2.00 NT 208 -2.00 NT
209 -2.00 NT 210 -2.00 NT 211 -2.00 NT 212 -2.00 NT 213 -2.00 -5.40
214 -2.00 -5.41 215 -2.00 -6.77
Example 3
Antinociceptive Efficacy of SEQ ID NO. 20 in Rats with Neuropathic
Pain secondary to a chronic constriction injury of the sciatic
nerve
Method
Animals
[0182] Adult male Sprague-Dawley rats were purchased from the
Animal Resources Centre (ARC), Perth, Australia, and the Herston
Medical Research Centre, The University of Queensland. Rats were
housed in a temperature controlled environment (21.+-.2EC) with a
12 h/12 h light/dark cycle. Food and water were available ad
libitum. Ethical approval for this study was obtained from the
Animal Experimentation Ethics Committee of The University of
Queensland.
Reagents and Materials
[0183] Isoflurane (Forthane) was obtained from Abbott Australasia
Pty Ltd (Sydney, Australia). Sodium benzylpenicillin vials (600 mg)
were purchased from CSL Ltd (Melbourne, Australia). Normal saline
ampoules were obtained from Delta West Pty Ltd (Perth, Australia)
and heparinised saline (50 IU/5 ml) was purchased from Astra
Pharmaceuticals Pty Ltd (Sydney, Australia). Single lumen
polyethylene tubing (La 0.2 mm, O.D. 0.6 mm) was purchased from
Auburn Plastics and Engineering Pty Ltd (Sydney, Australia).
Sterile siliconized silk sutures (Dysilk.TM.) were obtained from
Dynek Pty Ltd (Adelaide, South Australia) and Michel clips were
purchased from Medical and Surgical Requisites Pty Ltd (Brisbane,
Australia).
Chronic Constriction Injury (CCI) of the Sciatic Nerve
[0184] Rats were anaesthetised with ketamine (80 mg/kg) and
xylazine (8 mg/kg) administered by intraperitoneal injection, and a
chronic constriction injury (CCI) of the sciatic nerve was produced
according to the method of Bennett and Xie (1988). Briefly, the
left common sciatic nerve was exposed at mid-thigh level by blunt
dissection through the biceps femoris. Proximal to the
trifurcation, .about.10 mm of nerve was freed of adhering tissue
and four loose ligatures (3.0 silk) were tied around the sciatic
nerve (.about.1 mm apart). The incision was closed in layers. After
surgery, rats received benzylpenicillin (60 mg s.c.) to prevent
infection and were kept warm during surgical recovery. Rats were
housed singly for 14 days prior to opioid or vehicle
administration. Rats were inspected daily from the time of
CCI-surgery with regard to posture of the affected hindpaw,
exploring behaviour, body weight and water intake, and any signs of
autotomy.
Intrathecal Catheter Insertion
[0185] Ten to eleven days post CCI-surgery or in untreated
controls, rats were deeply anaesthetised with a mixture of ketamine
(80 mg kg.sup.-1) and xylazine (8 mg/kg) administered as a single
intraperitoneal (i.p.) injection. Prior to surgery, the back and
neck regions of the rat were shaved and the skin cleansed with
Betadine surgical scrub. The rat was then placed in a prone
position and the L6 lumbar vertebra was located by palpation of the
tuber sacrales of the os ileum (Hebel & Stromberg 1976). A 6 cm
incision was made in the midline of the back, 3 cm caudal and 3 cm
cephalad to L6. A subcutaneous pocket (for the intrathecal
catheter) was formed by blunt dissection with scissors on both
sides of the incision. The fascia covering the superficial muscles
of the back were cut in a 5 mm V-shaped incision that encompassed
L5. Additional 5 mm caudal incisions were made parallel to L6. The
fascia was then retracted and the lumbar muscles surrounding the
base of L5 and L6 were removed, as was the m. interspinalis between
the spinous processes of L5-L6.
[0186] Following removal of the L6 spinous processes with rongeurs,
the soft tissue beneath the L5 iliac arch was removed, exposing the
dura mater. The dural membrane was pierced with a 23 G needle,
releasing clear CSF. A polyethylene catheter (O.D. 0.6 mm, I.D. 0.2
mm; 20 cm in length) pre-filled with saline, was carefully advanced
a distance of 1 cm into the intrathecal space and a small volume of
saline (20 mL) was administered through the catheter. If leakage of
saline around the catheter was observed, the rat was excluded from
further experimentation. After successful completion of the `leak
test`, the intrathecal (i.t.) catheter was fixed with dental cement
onto the surrounding muscle .about.2 cm from L5, exteriorised
through a subcutaneous (s.c.) tunnel to a small incision at the
base of the neck and sutured in position. After suturing of the
lumbar muscles and skin, rats received benzylpenicillin (50000 IU
i.p.) and enrofloxacin (5 mgkg.sup.-1 s.c.) to prevent infection
and were kept warm during recovery from anaesthesia. Following
completion of the surgery, rats were housed singly for a recovery
period of 3-4 days prior to i.t. drug administration. On the day
following surgery, the local anaesthetic, lignocaine (2%, 20 mL)
was administered via the i.t. catheter. If complete paralysis of
both hind legs was not observed, rats were excluded from further
experimentation.
Drugs Administered
[0187] SEQ ID NO. 20 was prepared in 5 mM sodium acetate buffer at
pH 5.5 at delivered to rats in a single bolus dose of 0.2-30
nmoles. Stock solutions of the peptides were quantified relative to
an amino acid analysed stock solution by reversed phase HPLC with
u.v. detection at Xenome Ltd. The effects of SEQ ID NO. 20 were
compared to the effects of MrIA.
Storage of Stock Solutions
[0188] Aliquots (10 .mu.L) of stock solutions were stored at
-20.degree. C. prior to use for animal experimentation. Immediately
prior to experimentation, aliquots of the relevant compound were
thawed at room temperature and then diluted to the required
concentration with sterile saline to achieve the desired final
concentration for subsequent i.t. Unused portions were discarded to
waste to ensure that compounds only underwent one freeze-thaw
cycle.
Intrathecal Drug Dosing
[0189] On day 14 post-CCI surgery, individual groups of
drug-naive-CCI rats received an i.t. bolus injection of SEQ ID NO.
20, morphine or saline in a volume of 10-15 .mu.L. Antinociception
was assessed using von Frey filaments until responses retuned to
baseline (see below for details).
Assessment of Antinociception: CCI Rats Using Von Frey
Filaments
[0190] Tactile allodynia, the distinguishing feature of neuropathic
pain, was quantified using von Frey filaments which were used to
apply a non-noxious mechanical stimulus (light pressure) to the
hindpaw. Rats were transferred to wire mesh testing cages (20
cm.times.20 cm.times.20 cm) and allowed to acclimatise for 10 min.
Von Frey filaments were used to determine the lowest mechanical
threshold required for a brisk paw withdrawal reflex. Briefly,
starting with the von Frey filament that produced the lowest force,
the filament was applied to the plantar surface of the hindpaw
until the filament buckled slightly. Absence of a response after 5
s prompted use of the next filament of increasing weight. Filaments
used produced a buckling weight of 2, 4, 6, 8, 10, 12, 14, 16, 18
and 20 g and these were calibrated regularly. A score of 20 g was
given to animals that did not respond to any of the von Frey
filaments. Paw withdrawal thresholds (g) were converted to area
under the curve (AUCh). The maximum response on the ipsilateral
side was 45 AUCh
Verification of Correct I.T. Catheter Placement
[0191] At the completion of each experiment, malachite green dye
(30 .mu.L) was injected via the i.t. catheter whilst rats were
lightly anaesthetised with O.sub.2:CO.sub.2 (50%:50%). Thirty
seconds later, rats were decapitated and the spinal column was
exposed surgically. Data from rats where there was evidence of
subcutaneous dye leakage at the site where the catheter entered the
back muscles above L6 or failure of the dye to distribute at least
3-4 cm along the spinal cord, were excluded from the analysis.
Data Analysis
[0192] The area under the degree of antinociception versus time
curve (AUC values) for each peptide was calculated from time=0 to 3
h. Dose-response curves for each peptide was constructed by
plotting AUC values versus the i.t. peptide dose (expressed in nmol
per rat).
Results
[0193] Seq Id No. 20 (0.1 and 0.2 nmol, n=3 per dose) given by the
i.t. route produced dose-dependent relief of tactile allodynia
(defining symptom of neuropathic pain) in rats with a chronic
constriction injury of the sciatic nerve. The mean (.+-.SEM) paw
withdrawal threshold versus time curves evoked by i.t. Seq Id No.
20 (0.1 and 0.2 nmol) for the relief of tactile allodynia
(hypersensitivity to the non-noxious stimulus of light pressure) in
the ipsilateral hindpaw of rats with a chronic constriction injury
(CCI) of the sciatic nerve are shown in FIG. 1.
[0194] Seq Id No. 20 produced robust antinociception in CCI-rats
that appeared to be dose-dependent in the ipsilateral hindpaw and
which peaked at 0.75 h post-dosing. Moreover, the anti-allodynic
effect was long-lasting (>3 h) and 50-fold more potent than
MrIA. This is consistent with results from the NET uptake assay,
where Seq Id No. 20 was 20-fold more potent than MrIA at inhibiting
NA uptake. Similarly, dose-dependent antinociception was observed
in the contralateral (non-injured) paw, however, the paw withdrawal
threshold in the ipsilateral paw are approximately half those in
the contralateral paw. Due to the fact that the baseline paw
withdrawal thresholds are approximately 13 g in the contralateral
hindpaw (non-injured side) versus approximately 5.5 g in the
ipsilateral hindpaw, Seq Id No. 20 increased paw withdrawal
thresholds in the contralateral hindpaw to the maximum values (20
g) in this nociceptive test for approximately 1.5 h
post-dosing.
[0195] Importantly, close inspection of the paw withdrawal
threshold versus time curves for the ipsilateral and contralateral
hindpaws following intrathecal administration of Seq Id No. 20 in
the low dose (0.1 mmol), suggests that this compound has a more
pronounced antinociceptive effect in the ipsilateral hindpaw.
[0196] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0197] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications
which fall within the spirit and scope. The invention also includes
all of the steps, features, compositions and compounds referred to
or indicated in this specification, individually or collectively,
and any and all combinations of any two or more of said steps or
features.
Sequence CWU 1
1
216113PRTConus marmoreusMISC_FEATURE(12)..(12)Xaa is
4-hydroxyproline 1Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa
Cys1 5 10213PRTConus marmoreusMISC_FEATURE(12)..(12)Xaa is
4-hydroxyproline 2Val Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa
Cys1 5 10310PRTArtificial Sequencesynthetic 3Cys Cys Gly Tyr Lys
Leu Cys Xaa Xaa Cys1 5 10414PRTArtificial Sequencesynthetic 4Xaa
Xaa Xaa Xaa Cys Cys Gly Tyr Lys Leu Cys Xaa Xaa Cys1 5
10514PRTArtificial Sequencesynthetic 5Xaa Xaa Xaa Xaa Cys Cys Gly
Tyr Lys Leu Cys Xaa Xaa Cys1 5 10613PRTArtificial Sequencesynthetic
6Xaa Xaa Xaa Cys Cys Gly Tyr Lys Leu Cys Xaa Xaa Cys1 5
10713PRTConus marmoreusMISC_FEATURE(12)..(12)Xaa is
4-hydroxyproline 7Gly Val Cys Cys Gly Tyr Lys Leu Cys Cys His Xaa
Cys1 5 10811PRTConus marmoreusMISC_FEATURE(10)..(10)Xaa is
4-hydroxyproline 8Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
10912PRTConus marmoreusMISC_FEATURE(11)..(11)Xaa is
4-hydroxyproline 9Gly Ile Cys Cys Gly Val Ser Phe Cys Tyr Xaa Cys1
5 101011PRTArtificial Sequencesynthetic 10Ala Cys Cys Gly Tyr Lys
Leu Cys Ser Pro Cys1 5 101113PRTArtificial Sequencesynthetic 11Asn
Gly Val Cys Cys Gly Tyr Lys Leu Cys Leu Pro Cys1 5
101212PRTArtificial Sequencesynthetic 12Ser Val Cys Cys Gly Tyr Lys
Leu Cys Phe Pro Cys1 5 101314PRTArtificial Sequencesynthetic 13Tyr
Arg Gly Leu Cys Cys Gly Xaa Lys Leu Cys Arg Xaa Cys1 5
101414PRTArtificial Sequencesynthetic 14Tyr Arg Gly Xaa Cys Cys Gly
Xaa Lys Leu Cys Arg Xaa Cys1 5 101515PRTArtificial
Sequencesynthetic 15Xaa Tyr Arg Gly Xaa Cys Cys Gly Xaa Lys Leu Cys
Arg Xaa Cys1 5 10 151614PRTArtificial Sequencesynthetic 16Trp Arg
Gly Leu Cys Cys Gly Xaa Lys Leu Cys His Xaa Cys1 5
101714PRTArtificial Sequencesynthetic 17Xaa Arg Gly Xaa Cys Cys Gly
Xaa Lys Leu Cys Arg Xaa Cys1 5 101815PRTArtificial
Sequencesynthetic 18Lys Tyr Arg Gly Xaa Cys Cys Gly Xaa Lys Leu Cys
Arg Xaa Cys1 5 10 151913PRTArtificial Sequencesynthetic 19Xaa Gly
Xaa Cys Cys Gly Xaa Lys Xaa Cys His Xaa Cys1 5 102014PRTArtificial
Sequencesynthetic 20Trp Arg Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Xaa Cys1 5 102114PRTArtificial Sequencesynthetic 21Trp Lys Gly Val
Cys Cys Gly Xaa Lys Leu Cys His Xaa Cys1 5 102215PRTArtificial
Sequencesynthetic 22Phe Arg Tyr Gly Xaa Cys Cys Gly Xaa Lys Leu Cys
Arg Xaa Cys1 5 10 152315PRTArtificial Sequencesynthetic 23Tyr Xaa
Arg Gly Xaa Cys Cys Gly Xaa Lys Leu Cys Arg Xaa Cys1 5 10
152414PRTArtificial Sequencesynthetic 24Trp Arg Gly Leu Cys Cys Gly
Xaa Lys Leu Cys Arg Ala Cys1 5 102514PRTArtificial
Sequencesynthetic 25Trp Arg Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Xaa Cys1 5 102614PRTArtificial Sequencesynthetic 26Trp Arg Gly Leu
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 102713PRTArtificial
Sequencesynthetic 27Xaa Gly Xaa Cys Cys Gly Tyr Lys Xaa Cys His Xaa
Cys1 5 102815PRTArtificial Sequencesynthetic 28Tyr Phe Arg Gly Xaa
Cys Cys Gly Xaa Lys Leu Cys Arg Xaa Cys1 5 10 152914PRTArtificial
Sequencesynthetic 29Trp Arg Gly Val Cys Cys Gly Xaa Lys Leu Cys Arg
Ala Cys1 5 103014PRTArtificial Sequencesynthetic 30Trp Arg Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 103113PRTArtificial
Sequencesynthetic 31Trp Gly Leu Cys Cys Gly Xaa Lys Leu Cys Arg Tyr
Cys1 5 103213PRTArtificial Sequencesynthetic 32Xaa Gly Xaa Cys Cys
Gly Xaa Lys Leu Cys His Xaa Cys1 5 103314PRTArtificial
Sequencesynthetic 33Trp Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 103414PRTArtificial Sequencesynthetic 34Tyr Arg Gly Leu
Cys Cys Gly Xaa Lys Leu Cys Arg Xaa Cys1 5 103513PRTArtificial
Sequencesynthetic 35Trp Gly Leu Cys Cys Gly Xaa Lys Leu Cys Arg Xaa
Cys1 5 103614PRTArtificial Sequencesynthetic 36Trp Arg Gly Leu Cys
Cys Gly Xaa Lys Leu Cys Arg Lys Cys1 5 103714PRTArtificial
Sequencesynthetic 37Trp Arg Gly Leu Cys Cys Gly Xaa Lys Leu Cys Arg
Ala Cys1 5 103814PRTArtificial Sequencesynthetic 38Trp Arg Gly Val
Cys Cys Gly Tyr Lys Leu Cys Arg Ala Cys1 5 103914PRTArtificial
Sequencesynthetic 39Trp Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Arg
Xaa Cys1 5 104014PRTArtificial Sequencesynthetic 40Trp Arg Gly Val
Cys Cys Gly Xaa Lys Leu Cys His Ala Cys1 5 104115PRTArtificial
Sequencesynthetic 41Xaa Tyr Arg Gly Xaa Cys Cys Gly Xaa Lys Leu Cys
Arg Xaa Cys1 5 10 154213PRTArtificial Sequencesynthetic 42Xaa Gly
Xaa Cys Cys Gly Xaa Lys Xaa Cys His Xaa Cys1 5 104315PRTArtificial
Sequencesynthetic 43Trp Arg Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys Tyr1 5 10 154414PRTArtificial Sequencesynthetic 44Trp Gly
Leu Cys Cys Gly Xaa Lys Leu Cys Arg Xaa Cys Tyr1 5
104513PRTArtificial Sequencesynthetic 45Xaa Gly Xaa Cys Cys Gly Xaa
Lys Leu Cys His Xaa Cys1 5 104614PRTArtificial Sequencesynthetic
46Trp Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys His Xaa Cys1 5
104713PRTArtificial Sequencesynthetic 47Asn Gly Xaa Cys Cys Gly Xaa
Lys Xaa Cys His Xaa Cys1 5 104813PRTArtificial Sequencesynthetic
48Xaa Gly Xaa Cys Cys Gly Xaa Lys Xaa Cys His Xaa Cys1 5
104913PRTArtificial Sequencesynthetic 49Xaa Gly Val Cys Cys Gly Tyr
Lys Xaa Cys His Xaa Cys1 5 105014PRTArtificial Sequencesynthetic
50Tyr Asn Gly Xaa Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5
105113PRTArtificial Sequencesynthetic 51Xaa Gly Leu Cys Cys Gly Xaa
Lys Leu Cys His Xaa Cys1 5 105213PRTArtificial Sequencesynthetic
52Xaa Gly Xaa Cys Cys Gly Tyr Lys Xaa Cys His Xaa Cys1 5
105314PRTArtificial Sequencesynthetic 53Trp Asn Gly Val Cys Cys Gly
Xaa Lys Leu Cys His Pro Cys1 5 105414PRTArtificial
Sequencesynthetic 54Trp Arg Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Ala Cys1 5 105515PRTArtificial Sequencesynthetic 55Asp Tyr Arg Gly
Xaa Cys Cys Gly Xaa Lys Leu Cys Arg Xaa Cys1 5 10
155614PRTArtificial Sequencesynthetic 56Tyr Asn Gly Val Cys Cys Gly
Tyr Lys Xaa Cys His Pro Cys1 5 105713PRTArtificial
Sequencesynthetic 57Asn Gly Xaa Cys Cys Gly Tyr Lys Xaa Cys His Xaa
Cys1 5 105813PRTArtificial Sequencesynthetic 58Xaa Gly Xaa Cys Cys
Gly Tyr Lys Leu Cys His Xaa Cys1 5 105913PRTArtificial
Sequencesynthetic 59Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Trp Pro
Cys1 5 106014PRTArtificial Sequencesynthetic 60Trp Asn Gly Val Cys
Cys Gly Tyr Lys Leu Cys Arg Pro Cys1 5 106113PRTArtificial
Sequencesynthetic 61Asn Gly Xaa Cys Cys Gly Xaa Lys Xaa Cys His Xaa
Cys1 5 106214PRTArtificial Sequencesynthetic 62Tyr Asn Gly Val Cys
Cys Gly Xaa Lys Leu Cys Arg Ala Cys1 5 106313PRTArtificial
Sequencesynthetic 63Asn Asp Val Cys Cys Gly Tyr Lys Leu Cys His Pro
Cys1 5 106414PRTArtificial Sequencesynthetic 64Trp Arg Gly Leu Cys
Cys Gly Tyr Lys Leu Cys Arg Gly Cys1 5 106514PRTArtificial
Sequencesynthetic 65Xaa Gly Leu Cys Cys Gly Xaa Lys Leu Cys Arg Xaa
Cys Tyr1 5 106613PRTArtificial Sequencesynthetic 66Xaa Gly Xaa Cys
Cys Gly Tyr Lys Xaa Cys His Xaa Cys1 5 106714PRTArtificial
Sequencesynthetic 67Xaa Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 106814PRTArtificial Sequencesynthetic 68Tyr Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Arg Xaa Cys1 5 106914PRTArtificial
Sequencesynthetic 69Trp Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Arg
Ala Cys1 5 107015PRTArtificial Sequencesynthetic 70Phe Gly Gly Phe
Trp Cys Cys Gly Xaa Lys Leu Cys Arg Ala Cys1 5 10
157114PRTArtificial Sequencesynthetic 71Tyr Asn Gly Val Cys Cys Gly
Tyr Lys Leu Cys Trp Xaa Cys1 5 107214PRTArtificial
Sequencesynthetic 72Trp Asn Gly Leu Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 107313PRTArtificial Sequencesynthetic 73Xaa Gly Xaa Cys
Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 107413PRTArtificial
Sequencesynthetic 74Asn Gly Xaa Cys Cys Gly Xaa Lys Leu Cys His Xaa
Cys1 5 107513PRTArtificial Sequencesynthetic 75Xaa Gly Val Cys Cys
Gly Xaa Lys Leu Cys His Pro Cys1 5 107613PRTArtificial
Sequencesynthetic 76Xaa Gly Val Cys Cys Gly Tyr Lys Xaa Cys His Pro
Cys1 5 107713PRTArtificial Sequencesynthetic 77Xaa Gly Val Cys Cys
Gly Xaa Lys Leu Cys His Xaa Cys1 5 107814PRTArtificial
Sequencesynthetic 78Trp Arg Gly Leu Cys Cys Gly Tyr Lys Leu Cys Arg
Ala Cys1 5 107913PRTArtificial Sequencesynthetic 79Xaa Gly Val Cys
Cys Gly Xaa Lys Leu Cys His Xaa Cys1 5 108013PRTArtificial
Sequencesynthetic 80Asn Gly Xaa Cys Cys Gly Tyr Lys Xaa Cys His Xaa
Cys1 5 108114PRTArtificial Sequencesynthetic 81Tyr Asn Gly Val Cys
Cys Gly Tyr Lys Leu Cys Arg Ala Cys1 5 108214PRTArtificial
Sequencesynthetic 82Trp Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 108314PRTArtificial Sequencesynthetic 83Phe Gly Gly Phe
Cys Cys Gly Xaa Lys Leu Cys Arg Ala Cys1 5 108414PRTArtificial
Sequencesynthetic 84Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Arg
Pro Cys1 5 108514PRTArtificial Sequencesynthetic 85Trp Lys Asp Leu
Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 108614PRTArtificial
Sequencesynthetic 86Tyr Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Pro Cys1 5 108713PRTArtificial Sequencesynthetic 87Xaa Gly Val Cys
Cys Gly Tyr Lys Leu Cys Arg Xaa Cys1 5 108814PRTArtificial
Sequencesynthetic 88Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 108914PRTArtificial Sequencesynthetic 89Tyr Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Arg Pro Cys1 5 109014PRTArtificial
Sequencesynthetic 90Trp Lys Asp Leu Cys Cys Gly Tyr Lys Leu Cys Trp
Pro Cys1 5 109114PRTArtificial Sequencesynthetic 91Tyr Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Trp Pro Cys1 5 109214PRTArtificial
Sequencesynthetic 92Trp Lys Asp Val Cys Cys Gly Tyr Lys Leu Cys Trp
Pro Cys1 5 109313PRTArtificial Sequencesynthetic 93Xaa Gly Val Cys
Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 109413PRTArtificial
Sequencesynthetic 94Tyr Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys Pro
Cys1 5 109514PRTArtificial Sequencesynthetic 95Trp Xaa Gly Val Cys
Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 109613PRTArtificial
Sequencesynthetic 96Xaa Gly Xaa Cys Cys Gly Tyr Lys Leu Cys His Xaa
Cys1 5 109713PRTArtificial Sequencesynthetic 97Asn Gly Leu Cys Cys
Gly Tyr Lys Leu Cys His Pro Cys1 5 109813PRTArtificial
Sequencesynthetic 98Arg Gly Val Cys Cys Gly Tyr Lys Leu Cys His Pro
Cys1 5 109913PRTArtificial Sequencesynthetic 99Gly Tyr Lys Leu Gly
Cys Cys Gly Tyr Lys Leu Cys Cys1 5 1010016PRTArtificial
Sequencesynthetic 100Trp Ala Ala Asn Gly Val Cys Cys Gly Tyr Lys
Leu Cys His Xaa Cys1 5 10 1510113PRTArtificial Sequencesynthetic
101Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
1010213PRTArtificial Sequencesynthetic 102Asn Gly Val Cys Cys Gly
Tyr Lys Leu Cys His Xaa Cys1 5 1010313PRTArtificial
Sequencesynthetic 103Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1010414PRTArtificial Sequencesynthetic 104Xaa Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 1010517PRTArtificial
Sequencesynthetic 105Gly Ile Leu Arg Asn Gly Val Cys Cys Gly Tyr
Lys Leu Cys His Pro1 5 10 15Cys10615PRTArtificial Sequencesynthetic
106Trp Ala Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
10 1510713PRTArtificial Sequencesynthetic 107Xaa Gly Val Cys Cys
Gly Xaa Lys Leu Cys His Xaa Cys1 5 1010813PRTArtificial
Sequencesynthetic 108Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 1010914PRTArtificial Sequencesynthetic 109Trp Xaa Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1011014PRTArtificial
Sequencesynthetic 110Tyr Asn Lys Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys1 5 1011113PRTArtificial Sequencesynthetic 111Xaa Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1011213PRTArtificial
Sequencesynthetic 112Asn Gly Xaa Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1011313PRTArtificial Sequencesynthetic 113Asn Gly Val
Cys Cys Gly Xaa Lys Leu Cys His Pro Cys1 5 1011413PRTArtificial
Sequencesynthetic 114Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1011513PRTArtificial Sequencesynthetic 115Asn Gly Leu
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1011614PRTArtificial
Sequencesynthetic 116Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys1 5 1011713PRTArtificial Sequencesynthetic 117Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Lys Cys1 5 1011814PRTArtificial
Sequencesynthetic 118Tyr Asn Arg Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys1 5 1011913PRTArtificial Sequencesynthetic 119Xaa Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1012014PRTArtificial
Sequencesynthetic 120Xaa Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys1 5 1012113PRTArtificial Sequencesynthetic 121Xaa Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1012213PRTArtificial
Sequencesynthetic 122Asn Lys Val Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 1012313PRTArtificial Sequencesynthetic 123Asn Gly Val
Cys Cys Gly Xaa Lys Leu Cys His Xaa Cys1 5 1012413PRTArtificial
Sequencesynthetic 124Asn Ala Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1012513PRTArtificial Sequencesynthetic 125Asn Gly Ile
Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 1012613PRTArtificial
Sequencesynthetic 126Asn Gly Val Cys Cys Gly Tyr Lys
Xaa Cys His Xaa Cys1 5 1012713PRTArtificial Sequencesynthetic
127Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
1012813PRTArtificial Sequencesynthetic 128Xaa Gly Val Cys Cys Gly
Tyr Lys Leu Cys His Xaa Cys1 5 1012913PRTArtificial
Sequencesynthetic 129Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1013013PRTArtificial Sequencesynthetic 130Ala Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1013114PRTArtificial
Sequencesynthetic 131Xaa Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys1 5 1013214PRTArtificial Sequencesynthetic 132Tyr Asn
Xaa Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5
1013313PRTArtificial Sequencesynthetic 133Phe Gly Val Cys Cys Gly
Tyr Lys Leu Cys His Xaa Cys1 5 1013413PRTArtificial
Sequencesynthetic 134Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys Xaa
Pro Cys1 5 1013513PRTArtificial Sequencesynthetic 135Thr Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1013613PRTArtificial
Sequencesynthetic 136Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1013713PRTArtificial Sequencesynthetic 137Xaa Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 1013813PRTArtificial
Sequencesynthetic 138Asn Gly Thr Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 1013913PRTArtificial Sequencesynthetic 139Xaa Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1014014PRTArtificial
Sequencesynthetic 140Xaa Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys
His Xaa Cys1 5 1014113PRTArtificial Sequencesynthetic 141Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1014213PRTArtificial
Sequencesynthetic 142Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1014313PRTArtificial Sequencesynthetic 143Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1014413PRTArtificial
Sequencesynthetic 144Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1014513PRTArtificial Sequencesynthetic 145Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Ala Cys1 5 1014613PRTArtificial
Sequencesynthetic 146Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 1014713PRTArtificial Sequencesynthetic 147Asp Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 101489PRTArtificial
Sequencesynthetic 148Val Cys Cys Gly Tyr Lys Leu Cys Cys1
514913PRTArtificial Sequencesynthetic 149Asn Gly Val Cys Cys Gly
Tyr Lys Leu Cys His Xaa Cys1 5 1015013PRTArtificial
Sequencesynthetic 150Asn Gly Ala Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1015113PRTArtificial Sequencesynthetic 151Asp Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 1015213PRTArtificial
Sequencesynthetic 152Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 1015313PRTArtificial Sequencesynthetic 153Asn Gly Ala
Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1015413PRTArtificial
Sequencesynthetic 154Xaa Asp Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1015513PRTArtificial Sequencesynthetic 155Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Phe Cys1 5 1015613PRTArtificial
Sequencesynthetic 156Asn Ser Val Cys Cys Gly Tyr Lys Leu Cys His
Pro Cys1 5 1015714PRTArtificial Sequencesynthetic 157Xaa Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5 1015813PRTArtificial
Sequencesynthetic 158Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1015913PRTArtificial Sequencesynthetic 159Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Glu Cys1 5 1016013PRTArtificial
Sequencesynthetic 160Asn Gly Xaa Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1016114PRTArtificial Sequencesynthetic 161Tyr Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys1 5 1016213PRTArtificial
Sequencesynthetic 162Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1016314PRTArtificial Sequencesynthetic 163Tyr Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys Gln Pro Cys1 5 1016413PRTArtificial
Sequencesynthetic 164Xaa Gly Val Cys Cys Gly Tyr Lys Leu Cys His
Xaa Cys1 5 1016513PRTArtificial Sequencesynthetic 165Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys His Tyr Cys1 5 1016613PRTArtificial
Sequencesynthetic 166Asn Gly Val Cys Cys Xaa Tyr Lys Leu Cys His
Pro Cys1 5 1016713PRTArtificial Sequencesynthetic 167Asn Gly Val
Cys Cys Gly Tyr Xaa Leu Cys His Xaa Cys1 5 1016813PRTArtificial
Sequencesynthetic 168Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Xaa Cys1 5 1016913PRTArtificial Sequencesynthetic 169Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Xaa Pro Cys1 5 1017014PRTArtificial
Sequencesynthetic 170Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
Lys Pro Cys1 5 1017114PRTArtificial Sequencesynthetic 171Tyr Asn
Gly Val Cys Cys Gly Leu Lys Leu Cys His Pro Cys1 5
1017213PRTArtificial Sequencesynthetic 172Asn Gly Val Cys Cys Gly
Tyr Ala Leu Cys His Pro Cys1 5 1017310PRTArtificial
Sequencesynthetic 173Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
1017414PRTArtificial Sequencesynthetic 174Tyr Asn Gly Val Cys Cys
Gly Tyr Leu Leu Cys His Pro Cys1 5 1017514PRTArtificial
Sequencesynthetic 175Tyr Asn Gly Val Cys Cys Gly Tyr Lys Asn Cys
His Pro Cys1 5 1017613PRTArtificial Sequencesynthetic 176Asn Gly
Val Cys Cys Gly Xaa Lys Leu Cys His Pro Cys1 5 1017713PRTArtificial
Sequencesynthetic 177Asn Gly Val Cys Cys Gly Tyr Arg Leu Cys His
Xaa Cys1 5 1017813PRTArtificial Sequencesynthetic 178Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Xaa Pro Cys1 5 1017914PRTArtificial
Sequencesynthetic 179Tyr Asn Gly Val Cys Cys Gly Tyr Phe Leu Cys
His Pro Cys1 5 1018013PRTArtificial Sequencesynthetic 180Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys Xaa Pro Cys1 5 1018113PRTArtificial
Sequencesynthetic 181Asn Gly Val Cys Cys Gly Tyr His Leu Cys His
Pro Cys1 5 1018213PRTArtificial Sequencesynthetic 182Xaa Gly Val
Cys Cys Gly Tyr Xaa Leu Cys His Xaa Cys1 5 1018313PRTArtificial
Sequencesynthetic 183Asn Gly Val Cys Cys Glu Tyr Lys Leu Cys His
Pro Cys1 5 1018414PRTArtificial Sequencesynthetic 184Tyr Asn Gly
Val Cys Cys Gly Asn Lys Leu Cys His Pro Cys1 5 1018513PRTArtificial
Sequencesynthetic 185Asn Gly Val Cys Cys Gly Tyr Xaa Leu Cys His
Pro Cys1 5 1018613PRTArtificial Sequencesynthetic 186Asn Gly Val
Cys Cys Ser Tyr Lys Leu Cys His Pro Cys1 5 1018713PRTArtificial
Sequencesynthetic 187Xaa Gly Val Cys Cys Gly Trp Lys Leu Cys His
Xaa Cys1 5 1018813PRTArtificial Sequencesynthetic 188Asn Gly Val
Cys Cys Xaa Tyr Lys Leu Cys His Pro Cys1 5 1018913PRTArtificial
Sequencesynthetic 189Xaa Gly Val Cys Cys Gly Tyr Xaa Leu Cys His
Xaa Cys1 5 1019013PRTArtificial Sequencesynthetic 190Asn Gly Val
Cys Cys Gly Tyr Lys Leu Cys Ala Xaa Cys1 5 1019113PRTArtificial
Sequencesynthetic 191Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Pro Cys1 5 1019213PRTArtificial Sequencesynthetic 192Asn Gly Val
Cys Cys Xaa Tyr Lys Leu Cys His Pro Cys1 5 1019312PRTArtificial
Sequencesynthetic 193Gly Ile Cys Cys Gly Val Ser Phe Cys Tyr Xaa
Cys1 5 1019413PRTArtificial Sequencesynthetic 194Asn Gly Val Cys
Cys Gly Tyr Gln Leu Cys His Pro Cys1 5 1019514PRTArtificial
Sequencesynthetic 195Tyr Asn Gly Val Cys Cys Gly Glu Lys Leu Cys
His Pro Cys1 5 1019613PRTArtificial Sequencesynthetic 196Asn Gly
Val Cys Cys Gly Tyr Lys Lys Cys His Pro Cys1 5 1019713PRTArtificial
Sequencesynthetic 197Xaa Gly Val Cys Cys Gly Glu Lys Leu Cys His
Xaa Cys1 5 1019813PRTArtificial Sequencesynthetic 198Xaa Gly Val
Cys Cys Gly Ile Lys Leu Cys His Xaa Cys1 5 1019911PRTArtificial
Sequencesynthetic 199Arg Asn Cys Cys Arg Leu Gln Val Cys Cys Gly1 5
1020013PRTArtificial Sequencesynthetic 200Val Gly Val Asp Asp Gly
Tyr Lys Leu Cys His Xaa Cys1 5 1020114PRTArtificial
Sequencesynthetic 201Tyr Asn Gly Val Cys Cys Gly Lys Lys Leu Cys
His Pro Cys1 5 1020213PRTArtificial Sequencesynthetic 202Asn Gly
Val Cys Cys Gly Tyr Lys Ala Cys His Xaa Cys1 5 1020313PRTArtificial
Sequencesynthetic 203Asn Gly Val Cys Cys Gly Tyr Ala Leu Cys His
Xaa Cys1 5 1020413PRTArtificial Sequencesynthetic 204Asn Gly Val
Cys Cys Gly Ala Lys Leu Cys His Xaa Cys1 5 1020513PRTArtificial
Sequencesynthetic 205Asn Gly Val Cys Cys Ala Tyr Lys Leu Cys His
Xaa Cys1 5 1020613PRTArtificial Sequencesynthetic 206Asn Gly Val
Cys Cys Gly Xaa Lys Leu Cys His Pro Cys1 5 1020714PRTArtificial
Sequencesynthetic 207Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
Arg Pro Cys1 5 1020814PRTArtificial Sequencesynthetic 208Tyr Asn
Gly Val Cys Cys Gly Tyr Ile Leu Cys His Pro Cys1 5
1020914PRTArtificial Sequencesynthetic 209Tyr Asn Gly Val Cys Cys
Gly Tyr Lys Asp Cys His Pro Cys1 5 1021014PRTArtificial
Sequencesynthetic 210Tyr Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
Glu Pro Cys1 5 1021114PRTArtificial Sequencesynthetic 211Tyr Asn
Gly Val Cys Cys Gly Tyr Trp Leu Cys His Pro Cys1 5
1021214PRTArtificial Sequencesynthetic 212Tyr Asn Gly Val Cys Cys
Gly Tyr Tyr Leu Cys His Pro Cys1 5 1021313PRTArtificial
Sequencesynthetic 213Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Xaa Cys1 5 1021413PRTArtificial Sequencesynthetic 214Asn Gly Val
Cys Cys Gly Xaa Lys Leu Cys His Xaa Cys1 5 1021513PRTArtificial
Sequencesynthetic 215Asn Gly Val Cys Cys Gly Xaa Lys Leu Cys His
Xaa Cys1 5 1021614PRTArtificial Sequencesynthetic 216Xaa Xaa Xaa
Xaa Cys Cys Gly Tyr Lys Leu Cys Xaa Xaa Cys1 5 10
* * * * *