Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
Mu0-CONOPEPTIDES AND THEIR USE AS LOCAL ANESTHETICS
This invention was made with Government support under Grant No. GM48677
awarded by
the National Institute of General Medical Sciences, National Institutes of
Health, Bethesda,
to Maryland. The United States Government has certain rights in the invention.
BACKGROUND OF THE INVENTION
The present invention is directed to the use of q0-conopeptides as a local
anesthetic for
treating pain. The q0-conopeptides have long-lasting anesthetic activity and
are particularly useful
for spinal anesthesia, administered either acutely for post-operative pain or
via an intrathecal pump
for severe chronic pain situations. The present invention is further directed
to new ~O-
conopeptides, their coding sequences and their propeptides.
The publications and other materials used herein to illuminate the background
of the
invention, and in particular, cases to provide additional details respecting
the practice, are
incorporated herein by reference, and for convenience, are referenced by
author and date in the
following text and respectively grouped in the appended List of References.
Conus is a genus of predatory marine gastropods (snails) which envenomate
their prey.
Venomous cone snails use a highly developed projectile apparatus to deliver
their cocktail of toxic
conotoxins into their prey. In fish-eating species such as Conus magus the
cone detects the presence
of the fish using chemosensors in its siphon and when close enough extends its
proboscis and fires
a hollow harpoon-like tooth containing venom into the fish. This immobilizes
the fish and enables
the cone snail to wind it into its mouth via an attached filament. For general
information on Conus
and their venom see the website address
http://grimwade.biochem.unimelb.edu.au/cone/
referenc.html. Prey capture is accomplished through a sophisticated arsenal of
peptides which target
3o specific ion channel and receptor subtypes. Each Conus species venom
appears to contain a unique
set of 50-200 peptides. The composition of the venom differs greatly between
species and between
individual snails within each species, each optimally evolved to paralyse it's
prey. The active
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2
components of the venom are small peptides toxins, typically 12-30 amino acid
residues in length
and are typically highly constrained peptides due to their high density of
disulphide bonds.
The venoms consist of a large number of different peptide components that when
separated
exhibit a range of biological activities: when injected into mice they elicit
a range of physiological
responses from shaking to depression. The paralytic components of the venom
that have been the
focus of recent investigation are the a-, c~,>- and q-conotoxins. All of these
conotoxins act by
preventing neuronal communication, but each targets a different aspect of the
process to achieve
this. The a-conotoxins target nicotinic ligand gated channels, the ~,-
conotoxins target the voltage-
gated sodium channels and the c~-conotoxins target the voltage-gated calcium
channels (Olivera et
al., 1985). For example a linkage has been established between a-, aA- & ~-
conotoxins and the
nicotinic ligand-gated ion channel; c.~-conotoxins and the voltage-gated
calcium channel; q
conotoxins and the voltage-gated sodium channel; 8-conotoxins and the voltage-
gated sodium
channel; K-conotoxins and the voltage-gated potassium channel; conantokins and
the ligand-gated
glutamate (NMDA) channel. For a partial list of Conus peptides and their amino
acid sequences see
the website address http://pir.georgetown.edu.
However, the structure and function of only a small minority of these peptides
have been
determined to date. For peptides where function has been determined, three
classes of targets have
been elucidated: voltage-gated ion channels; ligand-gated ion channels, and G-
protein-linked
receptors.
2o Conus peptides which target voltage-gated ion channels include those that
delay the
inactivation of sodium channels, as well as Mockers specific for sodium
channels, calcium channels
and potassium channels. Peptides that target ligand-gated ion channels include
antagonists of
NMDA and serotonin receptors, as well as competitive and noncompetitive
nicotinic receptor
antagonists. Peptides which act on G-protein receptors include neurotensin and
vasopressin receptor
agonists. The unprecedented pharmaceutical selectivity of conotoxins is at
least in part defined by
a specific disulfide bond frameworks combined with hypervariable amino acids
within disulfide
loops (for a review see McIntosh et al., 1998).
The pain response is a protective reflex system warning an individual of
hostile situations
and tissue injury. The origins of clinically significant acute and chronic
pain in a mammal are
3o different, but the biochemical and neurological pathways are similar. In
the following discussion
on pain and its management, the focus is primarily on humans, however, it
should be understood
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3
that the concepts of pain are applicable to mammalian animals and the
management of such pain
is applicable to veterinary medicine.
Acute pain is often associated with surgery and with trauma. The intensity of
acute
postoperative pain varies considerably depending on the extent of the surgical
procedure performed,
on the individual's pain sensitivity, and on the type of anesthetic management
employed during
surgery. In general, major operations on the thorax and the upper abdominal
region induce the most
intensive postoperative pain. Extensive orthopedic operations also produce
strong postoperative
pain.
Chronic pain is persistent pain which has long outlasted the onset of any
known or suspected
Io physical cause. It can occur after a known injury or disease, or it can
occur without any known
physical cause whatsoever. Moreover, it can be accompanied by known tissue
pathology, such as
chronic inflammation that occurs in some types of arthritis, or it can occur
long after the healing of
the injured tissue which is suspected or known to be the cause of chronic
pain. Chronic pain is a
very general concept and there are several varieties of chronic pain related
to the musculoskeletal
system, visceral organs, skin, and nervous system.
Neuropathic pain can occur as a form of chronic pain and can also occur under
acute
conditions such as those following surgery or accidental trauma. Neuropathic
pain can be defined
as pain that results from an abnormal functioning of the peripheral and/or
central nervous system.
A critical component of this abnormal functioning is an exaggerated response
of pain-related nerve
2o cells either in the peripheral or in the central nervous system. This
exaggerated responsiveness is
manifested behaviorally as increased sensitivity to pain, i.e., as
hyperalgesia or allodynia, both of
which can occur in chronic neuropathic and acute inflammatory pains. An
example is the pain from
causalgia wherein even a light touch to the skin is felt as an excruciating
burning pain (allodynia)
or a normally mild pain is experienced as an excruciating one (hyperalgesia).
Neuropathic pain is
thought to be a consequence of damage to peripheral nerves or to regions of
the central nervous
system. However, abnormal functioning of pain-related regions of the nervous
system call also
occur with chronic inflammatory conditions such as certain types of arthritis
and metabolic disorders
such as diabetes as well as with acute inflammatory conditions. Thus, many
types of chronic pains
that are related to inflammation as well as acute pains that are related to
inflammation can be
considered to be at least partly neuropathic pains.
The modern concept of pain treatment emphasizes the significance of
prophylactic
prevention of pain, as pain is more easily prevented than relieved.
Additionally, the hormonal stress
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4
responses associated with pain are considered harmful to the patient, impair
the healing process and
overall recovery, and generally are to be avoided.
While compounds utilized as general anesthetics reduce pain by producing a
loss of
consciousness, local anesthetics act to induce a loss of sensation in the
localized area of
administration in the body. The mechanism by which local anesthetics induce
their effect, while
not having been determined definitively, is generally thought to be based upon
the ability to
interfere with the initiation and transmission of the nerve impulse conduction
along an axon through
a reversible blockade of sodium channels. Currently used local anesthetics
have durations of action
lasting only several hours. While this length of duration meets many needs,
particularly the control
of acute pain, local anesthetic agents with longer duration of action would
have broad clinical
application for the treatment of postoperative and chronic pain (Kuzma et al.,
1997).
The duration of action of a local anesthetics is proportional to the time
during which it is in
actual contact with the nervous tissues. In an effort to increase the duration
of action, procedures or
formulations that maintain localization of the drug at the nerve greatly
prolong anesthesia. All local
anesthetics are potentially toxic, and therefore it is of great importance
that the choice of drug,
concentration, rate and site of administration, as well as other actors, be
considered in their use. On
the other hand, a local anesthetic must remain at the site long enough to
allow sufficient time for the
localized pain to subside. Different devices and formulations are known in the
art for administration
of local anesthetics. See U.S. Patent No. 5,747,060, which discloses such
devices and formulations.
2o Side effects which have been associated with the use of different drugs for
treating pain or
as local anesthetics includes include respiratory depression, reduced cough
reflex, bronchial spasms,
nausea, vomiting, release of histamine, peripheral vasodilation, orthostatic
hypotension, vagal
impact on the heart, contraction of smooth muscles (sphincters), reduced
peristaltic motility in the
gastrointestinal tract, urinary retention, stimulated release of adrenalin,
anti-diuretic hormone,
changes in the regulation of body temperature and sleep pattern, tolerance,
addiction, tachycardia,
increase in blood pressure, and agitation. Not all of these side effects are
seen with any given drug
used to treat pain.
Thus, there is a need to develop additional drugs and methods which can be
used for the
treatment of pain, which can act as local anesthetics, which have a longer
duration of action and
3o which have reduced side effects. Accordingly, an object of the invention is
to provide methods and
compositions for the treatment of acute or chronic pain which provide
effective control of pain with
longer duration of action and reduced side effects associated with traditional
analgesics.
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SUMMARY OF THE INVENTION
The present invention is directed to the new q0-conopeptides, their coding
sequences and
their propeptides and to the use of q0-conopeptides as a local anesthetic for
treating pain. The ~O-
conopeptides have long lasting anesthetic activity and are particularly useful
for spinal anesthesia,
5 either administered acutely for post-operative pain or via an intrathecal
pump for severe chronic pain
situations or for treatment of pain in epithelial tissue.
More specifically, the present invention is directed to q0-conopeptides having
the general
formula I:
Xaa,-Xaa,-Cys-Xaa3-Xaa4-Xaas-Xaab-Xaa~ XaaB-Cys-Xaaq-Xaa,o-Xaa"-Xaa,~-Xaa,3-
Xaa,4
l0 Xaa,S-Xaa,6-Xaa"-Cys-Cys-Xaa,B-Xaa,9-Xaa,o-Xaa,,-Cys-Xaa2,-Xaa23-Xaa,4-
Xaa~s-Cys-Xaa26
Xaa~,-Xaa,B-Xaa~~-Xaa;o (SEQ ID NO:1),
wherein Xaa, is des-Xaa,, Pro, hydroxy-Pro (Hyp), Arg, Lys, ornithine, homo-
Lys,
homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys
or any synthetic
basic amino acid; Xaa, is des-Xaa,, Ala, Gly, Asp, Glu, y-carboxy-glutamate
(Gla), any synthetic
acidic amino acid, Tlu, Ser, g-Thr (where g is glycosylation), g-Ser, Trp (D
or L), neo-Trp or halo-
Trp (D or L) or Xaa, may be pyroglutamate if Xaa, is des-Xaa,; Xaa3 is Arg,
Lys, ornithine, homo-
Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-
Lys, any
synthetic basic amino acid, Ser, Thr, g-Ser, g-Thr, Ala, an aliphatic amino
acids bearing linear or
branched saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-
natural derivatives
2o of the aliphatic amino acid, His, Glu, Gln, Gla, Asp, Asn or any synthetic
acidic amino acid; Xaa4
is Glu, Gla, Gln, Asp, Asn, any synthetic acidic amino acid, Lys, Arg,
ornithine, homo-Lys,
homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys,
any synthetic
basic amino acid, Ala, an aliphatic amino acids bearing linear or branched
saturated hydrocarbon
chains such as Leu (D or L), Ile and Val or non-natural derivatives of the
aliphatic amino acid, Ser,
Thr, Pro, Hyp, g-Ser, g-Thr, g-Hyp or any synthetic hydroxylated amino acid;
XaaS is Lys, Arg,
ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N"-trimethyl-
Lys, any synthetic basic amino acid, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-
halo-Tyr, di-halo-Tyr,
O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, an aliphatic amino acids bearing
linear or branched
saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural
derivatives of the
3o aliphatic amino acid. Glu, Gla, Gln, Asp, Asn, any synthetic acidic amino
acid, Pro or Hyp; Xaab
is Trp (D or L), neo-Trp, halo-Trp (D or L), Gly, Tyr, meta-Tyr, ortho-Tyr,
nor-Tyr, mono-halo-Tyr,
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6
di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, vitro-Tyr, Glu, Gla, Gln, Asp, Asn,
any synthetic acidic
amino acid; Xaa, is Glu, Gla, Gln, Asp, Asn, any synthetic acidic amino acid,
Met, norleucine (Nle),
Ala, an aliphatic amino acids bearing linear or branched saturated hydrocarbon
chains such as Leu
(D or L), Ile and Val or non-natural derivatives of the aliphatic amino acid,
Tyr, meta-Tyr, ortho-
Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, vitro-
Tyr, Lys, Arg,
omithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N"-trimethyl-
Lys or any synthetic basic amino acid; XaaB is Leu, Phe, Tyr, .meta-Tyr, ortho-
Tyr, nor-Tyr, mono-
halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, vitro-Tyr, Trp (D or L),
neo-Trp, halo-Trp
(D or L) or any synthetic aromatic amino acid; Xaag is Pro, Hyp, Gly, an
aliphatic amino acids
1o bearing linear or branched saturated hydrocarbon chains such as Leu (D or
L), Ile and Val or non-
natural derivatives of the aliphatic amino acid; Xaa,o is Thr, Ser, g-Thr, g-
Ser, Ala, an aliphatic
amino acids bearing linear or branched saturated hydrocarbon chains such as
Leu (D or L), Ile and
Val or non-natural derivatives of the aliphatic amino acid, Phe, Tyr, meta-
Tyr, ortho-Tyr, nor-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, vitro-Tyr, Trp (D or
L), neo-Trp, halo-
Trp (D or L) or any synthetic aromatic amino acid; Xaa, ~ is Pro, Hyp, Ser,
Thr, g-Hyp, g-Ser, g-Thr
or any hydroxylated amino acid; Xaa,~ is an aliphatic amino acids bearing
linear or branched
saturated hydrocarbon chains such as Leu (D or L), Ile and Val or non-natural
derivatives of the
aliphatic amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr,
di-halo-Tyr, O-
sulpho-Tyr, O-phospho-Tyr, vitro-Tyr, Lys, Arg, ornithine, homo-Lys,
homoarginine, nor-Lys, N-
methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys or any synthetic basic
amino acid; Xaa,3 is
Pro, Hyp, an aliphatic amino acids bearing linear or branched saturated
hydrocarbon chains such as
Leu (D or L), Ile and Val or non-natural derivatives of the aliphatic amino
acid, Lys, Arg, ornithine,
homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-
trimethyl-Lys or
any synthetic basic amino acid; Xaa~4 is Gly, His, Lys, Arg, ornithine, homo-
Lys, homoarginine,
nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys or any
synthetic basic amino
acid; Xaa,S is des-Xaa,S, Ser, Thr, g-Ser, g-Thr, Val, Asn, Phe, Tyr, meta-
Tyr, ortho-Tyr, nor-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, vitro-Tyr, Trp (D or
L), neo-Trp, halo-
Trp (D or L) or any synthetic aromatic amino acid; Xaa,b is Met, Nle, Leu,
Phe, Tyr, meta-Tyr,
ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr,
vitro-Tyr, Arg, Lys,
3o ornithine, homo-Lys, homoarginine, nor-Lys, N-methyl-Lys, N,N'-dimethyl-
Lys, N,N',N"-trimethyl-
Lys or any synthetic basic amino acid; Xaa" is Pro, Hyp, Ser, Thr, g-Hyp, g-
Ser, g-Thr, any
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hydroxylated amino acid, Ala, Glu, Gla, Gln, Asp, Asn, any synthetic acidic
amino acid, His or Gly;
Xaa,B is Gly, Asn or Gln; Xaa,9 is Leu, Trp (D or L), neo-Trp or halo-Trp (D
or L); Xaa,o is des-
Xaa,o, Leu or Trp (D or L), neo-Trp or halo-Trp (D or L); Xaaz, is des-Xaa,,
or an aliphatic amino
acids bearing linear or branched saturated hydrocarbon chains such as Leu (D
or L), Ile and Val or
non-natural derivatives of the aliphatic amino acid; Xaa~, is des-Xaa", Gly,
Met, Nle, Phe, Tyr,
meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-
phospho-Tyr, nitro-
Tyr, Trp (D or L), neo-Trp, halo-Trp (D or L) or any synthetic aromatic amino
acid; Xaa,; is des-
Xaa,;, Pro, Hyp, Ala, an aliphatic amino acids bearing linear or branched
saturated hydrocarbon
chains such as Leu (D or L), Ile and Val or non-natural derivatives of the
aliphatic amino acid, Phe,
Tyr, meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-
phospho-Tyr,
nitro-Tyr, Trp (D or L), neo-Trp, halo-Trp (D or L) or any synthetic aromatic
amino acid; Xaa,4 is
an aliphatic amino acids bearing linear or branched saturated hydrocarbon
chains such as Leu (D
or L), Ile and Val or non-natural derivatives of the aliphatic amino acid,
Phe, Tyr, meta-Tyr, ortho-
Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-
Tyr, Trp (D or L),
neo-Trp, halo-Trp (D or L) or any synthetic aromatic amino acid; Xaa~S is Ala,
an aliphatic amino
acids bearing linear or branched saturated hydrocarbon chains such as Leu (D
or L), Ile and Val or
non-natural derivatives of the aliphatic amino acid, Tyr, meta-Tyr, ortho-Tyr,
nor-Tyr, mono-halo-
Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa,b is an
aliphatic amino acids
bearing linear or branched saturated hydrocarbon chains such as Leu (D or L),
Ile and Val or non-
2o natural derivatives of the aliphatic amino acid; Xaa2~ is des-Xaa,~, Asp,
Glu, Gla, Pro, Hyp, Ser, Thr,
g-Hyp, g-Ser, g-Ser or any synthetic hydroxylated amino acid; Xaa,B is des-
Xaa,B, Glu, Gla, Gln,
Asp, Asn, any synthetic acidic amino acid, Lys, Arg, ornithine, homo-Lys,
homoarginine, nor-Lys,
N-methyl-Lys, N,N'-dimethyl-Lys, N,N',N"-trimethyl-Lys, any synthetic basic
amino acid, Ile, Ser,
Thr, g-Ser or g-Thr; Xaa~9 is des-Xaa,9, Pro, Hyp, Tyr, meta-Tyr, ortho-Tyr,
nor-Tyr, mono-halo-
Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa3o is des-Xaa;o
or Phe, with the
proviso that the peptide is not MrVIA/B as defined below. The Cys residues may
be in D or L
configuration and may optionally be substituted with homocysteine (D or L).
The Tyr residues may
be substituted with the 3-hydroxyl or 2-hydroxyl isomers and corresponding O-
sulpho- and O-
phospho-derivatives. The acidic amino acid residues may be substituted with
any synthetic acidic
amino acid, e.g., tetrazolyl derivatives of Gly and Ala. The nonnatural
derivatives of the aliphatic
amino acids include those synthetic derivatives bearing non-natural aliphatic
branched or linear side
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8
chains CnH~~+, up to and including n=8. The halogen is iodo, chloro, fluoro or
bromo; preferably
iodo for halogen substituted-Tyr and bromo for halogen-substituted Trp.
MrVIA/B has the sequence: Ala-Cys-Xaa3,-Lys-Lys-Trp-Glu-Tyr-Cys-Ile-Val-Xaa3,-
Ile-
Xaa;;-Gly-Phe-Xaa;4-Tyr-Cys-Cys-Xaa;,-Gly-Leu-Ile-Cys-Gly-Xaa;,-Phe-Val-Cys-
Val, wherein
Xaa3, is Arg or Ser, Xaa;~ is Pro or hydroxy-Pro, Xaa33 is Ile or Leu and
Xaa34 is Ile or Val (SEQ
ID N0:2).
The present invention is also directed to novel specific conotoxin peptides
within general
formula I having the formulas:
Ala-Cys-Arg-Gln-Xaa,-Xaa~-Xaa;-Phe-Cys-Leu-Val-Xaa4-Ile-IIe-Gly-Xaa,-Ile-Xaa,-
Cys-
1o Cys-Ala-Gly-Leu-Ile-Cys-Gly-Xaa~,-Phe-Val-Cys-Leu (SEQ ID N0:3);
Xaa4-Thr-Cys-Leu-Xaa,-Gln-Asp-Xaa,-Phe-Cys-Ile-Ile-Xaa4-Leu-Ile-Gly-Thr-Leu-
Xaa,-
Cys-Cys-Ser-Gly-Leu-Ile-Cys-Gly-Phe-Phe-Val-Cys-Val-Xaa4-Xaa,-Xaa4-Phe (SEQ ID
N0:4);
Asp-Cys-Xaa;-Ala-Asp-Gly-Ala-Phe-Cys-Gly-Ile-Xaa4-Ile-Val-Xaa,-Asn-Xaas-Met-
Cys-
Cys-Ser-Asn-Leu-Cys-Ile-Phe-Ala-Cys-Val-Xaa4-Xaa3-Xaa~ (SEQ ID NO:S);
15 Asp-Cys-His-Xaa;-Arg-XaaS-Asp-Xaas-Cys-Xaa4-Ala-Ser-Ile-Leu-Gly-Val-Ile-
Xaa,-Cys-
Cys-Xaa3-Gly-Leu-Ile-Cys-Phe-Ile-Ala-Phe-Cys-Ile (SEQ ID N0:6);
Asp-Cys-Gln-Xaa;-Xaa,-Xaas-Xaa3-Phe-Cys-Ile-Val-Xaa4-Ile-Leu-Gly-Phe-Val-Xaa,-
Cys-
Cys-Xaa4-Gly-Leu-Ile-Cys-Gly-Xaa4-Phe-Val-Cys-Val-Asp-Ile (SEQ ID N0:7);
Xaa4 Thr-Cys-Val-Ser-Xaa,-Asn-Val-Phe-Cys-Gly-Val-Xaa4-Leu-Val-Gly-Thr-Xaa,-
Leu-
2o Cys-Cys-Ser-Gly-Leu-Val-Cys-Leu-Val-Val-Cys-Ile (SEQ ID N0:8);
Cys-Arg-Xaa4-Arg-Gly-Met-Phe-Cys-Gly-Phe-Xaa4-Xaa,-Xaa4-Gly-Xaa~-Xaa,-Cys-Cys-
Asn-Gly-Xaas-Cys-Phe-Phe-Val-Cys-Ile (SEQ ID N0:9);
Arg-Xaa;-Cys-Ala-Leu-Asp-Gly-Xaa3-Leu-Cys-Ile-Ile-Xaa4-Val-Ile-Gly-Ser-Ile-Phe-
Cys-
Cys-His-Gly-Ile-Cys-Met-Ile-Xaa~-Cys-Val (SEQ ID NO:10);
25 Asp-Cys-Arg-Xaa4-Val-Gly-Gln-Xaa~-Cys-Gly-Ile-Xaa4-XaaZ-Xaa,-His-Asn-XaaS-
Arg-Cys-
Cys-Ser-Gln-Leu-Cys-Ala-Ile-Ile-Cys-Val-Ser (SEQ ID NO:11); and
Gly-Cys-Leu-Asp-Xaa4-Gly-XaarPhe-Cys-Gly-Thr-Xaa4-Phe-Leu-Gly-Ala-Xaa~-Cys-Cys-
Gly-Gly-Ile-Cys-Leu-Ile-Val-Cys-Ile-Xaa3-Thr (SEQ ID N0:12),
wherein Xaa, is Lys, N-methy-Lys, N,N-dimethyl-Lys or N,N,N-trimethyl-Lys;
Xaa~ is Tyr, mono-
3o halo-Tyr, di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr; Xaa3 is
Glu or gamma-carboxy-
Glu (Gla); Xaa4 is Pro or hydroxy-Pro; Xaas is Trp or halo-Trp; and the C-
terminus contains a
carboxyl or amide group. The halo is preferably chlorine or iodine, more
preferably iodine. In
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9
addition, the Arg residues may be substituted by Lys, ornithine, homoargine,
nor-Lys, N-methyl-
Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys or any synthetic basic amino acid;
the Xaa, residues
may be substituted by Arg, ornithine, homoargine, nor-Lys, or any synthetic
basic amino acid; the
Tyr residues may be substituted with any synthetic hydroxy containing amino
acid; the Ser residues
may be substituted with Thr or any synthetic hydroxylated amino acid; the Thr
residues may be
substituted with Ser or any synthetic hydroxylated amino acid; the Phe and Trp
residues may be
substituted with any synthetic aromatic amino acid; and the Asn, Ser, Thr or
Hyp residues may be
glycosylated. The Cys residues may be in D or L configuration and may
optionally be substituted
with homocysteine (D or L). The 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. The acidic amino acid residues may be substituted with
any synthetic acidic
amino acid, e.g., tetrazolyl derivatives of Gly and Ala. The aliphatic amino
acids may be substituted
by synthetic derivatives bearing non-natural aliphatic branched or linear side
chains C~Hz~+, up to
and including n=8.
More specifically, the present invention is directed to the following ~O-
conopeptides within
general formula I:
MrVIA: SEQ ID N0:2, whererin Xaa3o is Arg, Xaa;, is Ile and Xaa;~ is Ile;
MrVIB: SEQ ID N0:2, wherein Xaa;o is Ser, Xaa;, is Leu and Xaa;, is Val;
A657: SEQ ID N0:3, wherein Xaa, is Lys, Xaa~ is Tyr, Xaa3 is Glu and Xaa4 is
Pro;
F079: SEQ ID N0:4, wherein Xaa, is Lys, Xaa, is Tyr and Xaa4 is Pro;
Ca6.1: SEQ ID NO:S, wherein Xaa, is Lys, Xaa, is Tyr, Xaa; is Glu, Xaa4 is Pro
and
XaaS is Trp;
Tx6.12: SEQ ID N0:6, wherein Xaa, is Tyr, Xaa3 is Glu, Xaa4 is Pro and XaaS is
Trp;
Tx6.13: SEQ ID N0:7, wherein Xaa, is Lys, Xaa, is Tyr, Xaa; is Glu, Xaa4 is
Pro and
Xaas is Trp;
G28: SEQ ID N0:8, wherein Xaa, is Tyr and Xaa4 is Pro;
F763: SEQ ID N0:9, wherein Xaa, is Lys, Xaa~ is Tyr, Xaa4 is Pro and XaaS is
Trp;
F080: SEQ ID NO:10, wherein Xaa, is Tyr, Xaa3 is Glu, Xaa4 is Pro and XaaS is
Trp;
F008: SEQ ID NO:11, wherein Xaa, is Lys, Xaa, is Tyr, Xaa4 is Pro and XaaS is
Trp; and
G 18: SEQ ID N0:12, wherein Xaa, is Tyr, Xaa; is Glu and Xaa4 is Pro.
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Examples of synthetic aromatic amino acid include, but are not limited to,
such as nitro-Phe,
4-substituted-Phe wherein the substituent is C,-C3 alkyl, carboxyl,
hyrdroxymethyl, sulphomethyl,
halo, phenyl, -CHO, -CN, -S03H and -NHAc. Examples of synthetic hydroxy
containing amino
acid, include, but are not limited to, such as 4-hydroxymethyl-Phe, 4-
hydroxyphenyl-Gly, 2,6-
5 dimethyl-Tyr and 5-amino-Tyr. Examples of synthetic basic amino acids
include, but are 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. These and other synthetic basic amino
acids, synthetic hydroxy
containing amino acids or synthetic aromatic amino acids are described in
Building Block Index,
Version 3.0 (1999 Catalog, pages 4-47 for hydroxy containing amino acids and
aromatic amino
1o acids and pages 66-87 for basic amino acids; see also http://www.amino-
acids.com), incorporated
herein by reference, by and available from RSP Amino Acid Analogues, Inc.,
Worcester, MA.
Examples of synthetic acid amino acids include those derivatives bearing
acidic functionality,
including carboxyl, phosphate, sulfonate and synthetic tetrazolyl derivatives
such as described by
Ornstein et al. (1993) and in U.S. Patent No. 5,331,001, each incorporated
herein by reference.
Optionally, in the peptides of general formula I and the specific peptides
described above,
the Asn residues may be modified to contain an N-glycan and the Ser, Thr and
Hyp residues may
be modified to contain an O-glycan (e.g., g-N, g-S, g-T and g-Hyp). In
accordance with the present
invention, a glycan shall mean any N-, S- or O-linked mono-, di-, tri-, poly-
or oligosaccharide that
can be attached to any hydroxy, amino or thiol group of natural or modified
amino acids by
2o 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 (GIcNAc), D-N-acetyl-
galactosamine
(GaINAc), D-fucose or D-arabinose. These saccharides may be structurally
modified, e.g., with one
or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such as sialic
acid, including
combinations thereof. The gylcan may also include similar polyhydroxy 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-.
Core O-glycans have been described by Van de Steen et al. (1998), incorporated
herein by
3o reference. Mucin type O-linked oligosaccharides are attached to Ser or Thr
(or other hydroxylated
residues of the present peptides) by a GaINAc residue. The monosaccharide
building blocks and
the linkage attached to this first GaINAc residue define the "core glycans,"
of which eight have been
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11
identified. The type of glycosidic linkage (orientation and connectivities)
are defined for each core
glycan. Suitable glycans and glycan analogs are described further in U.S.
Serial No. 09/420,797
filed 19 October 1999 and in PCT Application No. PCT/US99/24380 filed 19
October 1999 (PCT
Published Application No. WO 00/23092), each incorporated herein by reference.
A preferred
glycan is Gal((31~3)GaINAc(al~).
Optionally, in the peptides of general formula I and the specific peptides
described above,
pairs of Cys residues may be replaced pairwise with isoteric lactam or ester-
thioether replacements,
such as Ser/(Glu or Asp), Lys/(Glu or Asp) or Cys/Ala combinations. Sequential
coupling by
known methods (Barnay et al., 2000; Hruby et al., 1994; Bitan et al., 1997)
allows replacement of
1 o native Cys bridges with lactam bridges. Thioether analogs may be readily
synthesized using halo-
Ala residues commercially available from RSP Amino Acid Analogues.
The present invention is also directed to the identification of the nucleic
acid sequences
encoding these peptides and their propeptides and the identication of nucleic
acid sequences of
additional related q0-conopeptides.
The present invention is further directed to a method of
reducing/alleviating/decreasing the
perception of pain by a subject or for inducing analgesia, particularly local
analgesia, in a subject
comprising administering to the subject an effective amount of the
pharmaceutical composition
comprising a therapeutically effective amount of a ~ O-conotoxin peptide
described herein or a
pharmaceutically acceptable salt or solvate thereof, including MrVIA and
MrVIB. The present
2o invention is also directed to a pharmaceutical composition comprising a
therapeutically effective
amount of a ~O-conotoxin peptide described herein or a pharmaceutically
acceptable salt or solvate
thereof and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows ~O-conopeptide MrVIB inhibits skin flinch sensitivity in the
Guinea pig
intracutaneous wheat assay with greater potency than lidocaine or bupivacaine.
Data represent the
number of flinches observed after 36 pin pricks in a 30 minutes test period.
Each point represents
the mean of at least three observations.
Figure 2 shows q0-conopeptide MrVIB produces a long-lasting inhibition of skin
flinch
3o sensitivity relative to either lidocaine or bupivacaine in the Guinea pig
intracutaneous wheat assay.
Data represent the percentage of flinches observed out of six total at each
time point. Each point
represents the mean of at least three observations.
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12
SUMMARY OF THE SEQUENCE LISTING
SEQ ID NO:1 is a generic formula for ~O-conopeptides. SEQ ID N0:2 is a generic
formula
for y0-conopeptides MrVIA and MrVIB. SEQ ID N0:3 is a generic formula for q0-
conopeptide
A657. SEQ ID N0:4 is a generic formula for q0-conopeptide F079. SEQ ID NO:S is
a generic
formula for ~O-conopeptide Ca6.1. SEQ ID N0:6 is a generic formula for ~O-
conopeptide Tx6.12.
SEQ ID N0:7 is a generic formula for q0-conopeptide Tx6.13. SEQ ID N0:8 is a
generic formula
for the ~O-conopeptide G28. SEQ ID N0:9 is a generic formula for the ~O-
conopeptide F763.
SEQ ID NO:10 is a generic formula for the p0-conopeptide F080. SEQ ID NO:11 is
a generic
formula for the ~O-conopeptide F008. SEQ ID N0:12 is a generic formula for the
q0-conopeptide
G18. SEQ ID N0:13 is a primer for amplifying "O-Superfamily" conotoxins. SEQ
ID N0:14 is
a primer for amplifying "O-Superfamily" conotoxins. SEQ ID NO:15 is a
nucleotide sequence for
the gene coding for the A657 propeptide. SEQ ID N0:16 is an amino acid
sequence of the A657
propeptide. SEQ ID N0:17 is a nucleotide sequence for the gene coding for the
F079 propeptide.
SEQ ID N0:18 is an amino acid sequence of the F079 propeptide. SEQ ID N0:19 is
a nucleotide
sequence for the gene coding for the Ca6.1 propeptide. SEQ ID N0:20 is an
amino acid sequence
of the Ca6.1 propeptide. SEQ ID N0:21 is a nucleotide sequence for a portion
of the gene coding
for the Tx6.12 propeptide. SEQ ID N0:22 is an amino acid sequence of a portion
of the Tx6.12
propeptide. SEQ ID N0:23 is a nucleotide sequence for a portion of the gene
coding for the Tx6. l3
propeptide. SEQ ID N0:24 is an amino acid sequence of a portion of the Tx6.13
propeptide. SEQ
2o ID N0:25 is a nucleotide sequence for the gene coding for the G28
propeptide. SEQ ID N0:26 is
an amino acid sequence of the G28 propeptide. SEQ ID N0:27 is a nucleotide
sequence for the
gene coding for the F763 propeptide. SEQ ID N0:28 is an amino acid sequence of
the F763
propeptide. SEQ ID N0:29 is a nucleotide sequence for the gene coding for the
F080 propeptide.
SEQ ID N0:30 is an amino acid sequence of the F080 propeptide. SEQ ID N0:31 is
a nucleotide
sequence for the gene coding for the F008 propeptide. SEQ ID N0:32 is an amino
acid sequence
of the F008 propeptide. SEQ ID N0:33 is a nucleotide sequence for the gene
coding for the G18
propeptide. SEQ ID N0:34 is an amino acid sequence of the G18 propeptide.
DETAILED DESCRIPTION OF THE INVENTION
3o The present invention is directed to the new q0-conopeptides, their coding
sequences and
their propeptides and to the use of p0-conopeptides as a local anesthetic for
treating pain. The p0-
conopeptides have long lasting anesthetic activity and are particularly useful
for spinal anesthesia,
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13
either administered acutely for post-operative pain or via an intrathecal pump
for severe chronic pain
situations or for treatment of pain in epithelial tissue.
The present invention, in another aspect, relates to a pharmaceutical
composition comprising
an effective amount of a conotoxin peptide described herein or a
pharmaceutically acceptable salt
or solvate thereof. Such a pharmaceutical composition has the capability of
acting as analgesic
agents.
The present invention also provides for a method provides local anesthesia to
a patient
having pain. In one embodiment, the pain results from surgical or medical
procedures, and the
compounds are administered to the central nervous system (CNS), e.g. to the
spine for spinal
1 o analgesia. In a second embodiment, the pain is in an epithelial tissue
region associated with damage
or loss of epithelial tissue as a result of, for example, plastic surgery,
canker sores, burns, sore
throats, genital lesions, upper or lower gastrointestinal bronchoscopy or
endoscopy, intubation,
dermatologic abrasions or chemical skin peels, and the compounds are
administered to alleviate the
associated pain.
The conotoxin peptides described herein are sufficiently small to be
chemically synthesized.
General chemical syntheses for preparing the foregoing conotoxin peptides are
described
hereinafter. Various ones of the conotoxin peptides can also be obtained by
isolation and
purification from specific Conus species using the technique described in U.S.
Patent No. 4,447,356
(Olivera et al., 1984), the disclosure of which is incorporated herein by
reference.
2o Although the conotoxin peptides of the present invention can be obtained by
purification
from cone snails, because the amounts of conotoxin peptides obtainable from
individual snails are
very small, the desired substantially pure conotoxin peptides are best
practically obtained in
commercially valuable amounts by chemical synthesis using solid-phase
strategy. For example, the
yield from a single cone snail may be about 10 micrograms or less of conotoxin
peptide. By
"substantially pure" is meant that the peptide is present in the substantial
absence of other biological
molecules of the same type; it is preferably present in an amount of at least
about 85% purity and
preferably at least about 95% purity. Chemical synthesis of biologically
active conotoxin peptides
depends of course upon correct determination of the amino acid sequence.
The conotoxin peptides can also be produced by recombinant DNA techniques well
known
3o in the art. Such techniques are described by Sambrook et al. (1989). The
peptides produced in this
manner are isolated, reduced if necessary, and oxidized to form the correct
disulfide bonds.
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WO 00/76532 PCT/US00/15779
14
One method of forming disulfide bonds in the peptides of the present invention
is the air
oxidation of the linear peptides for prolonged periods under cold room
temperatures or at room
temperature. This procedure results in the creation of a substantial amount of
the bioactive,
disulfide-linked peptides. The oxidized peptides are fractionated using
reverse-phase high
performance liquid chromatography (HPLC) or the like, to separate peptides
having different linked
configurations. Thereafter, either by comparing these fractions with the
elution of the native material
or by using a simple assay, the particular fraction having the correct linkage
for maximum biological
potency is easily determined. However, because of the dilution resulting from
the presence of other
fractions of less biopotency, a somewhat higher dosage may be required.
The peptides are synthesized by a suitable method, such as by exclusively
solid-phase
techniques, by partial solid-phase techniques, by fragment condensation or by
classical solution
couplings.
In conventional solution phase peptide synthesis, the peptide chain can be
prepared by a
series of coupling reactions in which constituent amino acids are added to the
growing peptide chain
in the desired sequence. Use of various coupling reagents, e.g.,
dicyclohexylcarbodiimide or
diisopropylcarbonyldimidazole, various active esters, e.g., esters of N-
hydroxyphthalimide or N-
hydroxy-succinimide, and the various cleavage reagents, to carry out reaction
in solution, with
subsequent isolation and purification of intermediates, is well known
classical peptide methodology.
Classical solution synthesis is described in detail in the treatise, "Methoden
der Organischen Chemie
2o (Houben-Weyl): Synthese von Peptiden," (1974). Techniques of exclusively
solid-phase synthesis
are set forth in the textbook, "Solid-Phase Peptide Synthesis," (Stewart and
Young, 1969), and are
exemplified by the disclosure of U.S. Patent 4,105,603 (Vale et al., 1978).
The fragment
condensation method of synthesis is exemplified in U.S. Patent 3,972,859
(1976). Other available
syntheses are exemplified by U.S. Patents No. 3,842,067 (1974) and 3,862,925
(1975). The
synthesis of peptides containing 'y-carboxyglutamic acid residues is
exemplified by Rivier et al.
(1987), Nishiuchi et al. (1993) and Zhou et al. (1996).
Common to such chemical syntheses is the protection of the labile side chain
groups of the
various amino acid moieties with suitable protecting groups which will prevent
a chemical reaction
from occurring at that site until the group is ultimately removed. Usually
also common is the
protection of an a-amino group on an amino acid or a fragment while that
entity reacts at the
carboxyl group, followed by the selective removal of the a-amino protecting
group to allow
subsequent reaction to take place at that location. Accordingly, it is common
that, as a step in such
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
a synthesis, an intermediate compound is produced which includes each of the
amino acid residues
located in its desired sequence in the peptide chain with appropriate side-
chain protecting groups
linked to various ones of the residues having labile side chains.
As far as the selection of a side chain amino protecting group is concerned,
generally one
5 is chosen which is not removed during deprotection of the a-amino groups
during the synthesis.
However, for some amino acids, e.g., His, protection is not generally
necessary. In selecting a
particular side chain protecting group to be used in the synthesis of the
peptides, the following
general rules are followed: (a) the protecting group preferably retains its
protecting properties and
is not split off under coupling conditions, (b) the protecting group should be
stable under the
reaction conditions selected for removing the a-amino protecting group at each
step of the synthesis,
and (c) the side chain protecting group must be removable, upon the completion
of the synthesis
containing the desired amino acid sequence, under reaction conditions that
will not undesirably alter
the peptide chain.
It should be possible to prepare many, or even all, of these peptides using
recombinant DNA
15 technology. However, when peptides are not so prepared, they are preferably
prepared using the
Merrifield solid-phase synthesis, although other equivalent chemical syntheses
known in the art can
also be used as previously mentioned. Solid-phase synthesis is commenced from
the C-terminus
of the peptide by coupling a protected a-amino acid to a suitable resin. Such
a starting material can
be prepared by attaching an a-amino-protected amino acid by an ester linkage
to a chloromethylated
2o resin or a hydroxymethyl resin, or by an amide bond to a benzhydrylamine
(BHA) resin or para-
methylbenzhydrylamine (MBHA) resin. Preparation of the hydroxymethyl resin is
described by
Bodansky et al. ( 1966). Chloromethylated resins are commercially available
from Bio Rad
Laboratories (Richmond, CA) and from Lab. Systems, Inc. The preparation of
such a resin is
described by Stewart and Young (1969). BHA and MBHA resin supports are
commercially
available, and are generally used when the desired polypeptide being
synthesized has an
unsubstituted amide at the C-terminus. Thus, solid resin supports may be any
of those known in the
art, such as one having the formulae -O-CHI-resin support, -NH BHA resin
support, or -NH-MBHA
resin support. When the unsubstituted amide is desired, use of a BHA or MBHA
resin is preferred,
because cleavage directly gives the amide. In case the N-methyl amide is
desired, it can be
generated from an N-methyl BHA resin. Should other substituted amides be
desired, the teaching
of U.S. Patent No. 4,569,967 (Kornreich et al., 1986) can be used, or should
still other groups than
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
16
the free acid be desired at the C-terminus, it may be preferable to synthesize
the peptide using
classical methods as set forth in the Houben-Weyl text (1974).
The C-terminal amino acid, protected by Boc or Fmoc and by a side-chain
protecting group,
if appropriate, can be first coupled to a chloromethylated resin according to
the procedure set forth
in Horiki et al. (1978), using KF in DMF at about 60°C for 24 hours
with stirring, when a peptide
having free acid at the C-terminus is to be synthesized. Following the
coupling of the BOC-
protected amino acid to the resin support, the a-amino protecting group is
removed, as by using
trifluoroacetic acid (TFA) in methylene chloride or TFA alone. The
deprotection is carried out at
a temperature between about 0°C and room temperature. Other standard
cleaving reagents, such as
1 o HCl in dioxane, and conditions for removal of specific a-amino protecting
groups may be used as
described in Schroder & Lubke (1965).
After removal of the a-amino-protecting group, the remaining a-amino- and side
chain-
protected amino acids are coupled step-wise in the desired order to obtain the
intermediate
compound defined hereinbefore, or as an alternative to adding each amino acid
separately in the
synthesis, some of them may be coupled to one another prior to addition to the
solid phase reactor.
Selection of an appropriate coupling reagent is within the skill of the art.
Particularly suitable as
a coupling reagent is N,N'-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU,
TBTU in the
presence of HoBt or HoAt).
The activating reagents used in the solid phase synthesis of the peptides are
well known in
the peptide art. Examples of suitable activating reagents are carbodiimides,
such as N,N'-
diisopropylcarbodiimide and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide.
Other activating
reagents and their use in peptide coupling are described by Schroder & Lubke
(1965) and Kapoor
( 1970).
Each protected amino acid or amino acid sequence is introduced into the solid-
phase reactor
in about a twofold or more excess, and the coupling may be carried out in a
medium of
dimethylformamide (DMF):CHZCI, (1:1) or in DMF or CH,CI, alone. In cases where
intermediate
coupling occurs, the coupling procedure is repeated before removal of the a-
amino protecting group
prior to the coupling of the next amino acid. The success of the coupling
reaction at each stage of
the synthesis, if performed manually, is preferably monitored by the ninhydrin
reaction, as described
3o by Kaiser et al. (1970). Coupling reactions can be performed automatically,
as on a Beckman 990
automatic synthesizer, using a program such as that reported in Rivier et al.
(1978).
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17
After the desired amino acid sequence has been completed, the intermediate
peptide can be
removed from the resin support by treatment with a reagent, such as liquid
hydrogen fluoride or
TFA (if using Fmoc chemistry), which not only cleaves the peptide from the
resin but also cleaves
all remaining side chain protecting groups and also the a-amino protecting
group at the N-terminus
if it was not previously removed to obtain the peptide in the form of the free
acid. If Met is present
in the sequence, the Boc protecting group is preferably first removed using
trifluoroacetic acid
(TFA)/ethanedithiol prior to cleaving the peptide from the resin with HF to
eliminate potential S-
alkylation. When using hydrogen fluoride or TFA for cleaving, one or more
scavengers such as
anisole, cresol, dimethyl sulfide and methylethyl sulfide are included in the
reaction vessel.
Cyclization of the linear peptide is preferably affected, as opposed to
cyclizing the peptide
while a part of the peptido-resin, to create bonds between Cys residues. To
effect such a disulfide
cyclizing linkage, fully protected peptide can be cleaved from a
hydroxymethylated resin or a
chloromethylated resin support by ammonolysis, as is well known in the art, to
yield the fully
protected amide intermediate, which is thereafter suitably cyclized and
deprotected. Alternatively,
deprotection, as well as cleavage of the peptide from the above resins or a
benzhydrylamine (BHA)
resin or a methylbenzhydrylamine (MBHA), can take place at 0°C with
hydrofluoric acid (HF) or
TFA, followed by oxidation as described above.
The peptides are also synthesized using an automatic synthesizer. Amino acids
are
sequentially coupled to an MBHA Rink resin (typically 100 mg of resin)
beginning at the C-
2o terminus using an Advanced Chemtech 357 Automatic Peptide Synthesizer.
Couplings are carried
out using 1,3-diisopropylcarbodimide in N-methylpyrrolidinone (NMP) or by 2-
(1H-benzotriazole
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diethylisopro-
pylethylamine
(DIEA). The FMOC protecting group is removed by treatment with a 20% solution
of piperidine
in dimethylformamide(DMF). Resins are subsequently washed with DMF (twice),
followed by
methanol and NMP.
Additional conotoxin peptides are identified by cloning by reverse
transcription-polymerase
chain reaction (RT-PCR) from cone snail venom duct mRNA. The PCR primers are
based on the
DNA sequences coding for the precursor peptides of the "O-Superfamily" as
described herein. RT-
PCR of venom duct mRNA produces a product of about 250-300 nucleotides in
Conus species that
express conotoxin genes. The PCR product is then cloned into a plasmid vector
and individual
clones are sequenced to determine the sequence of various conotoxin genes.
Alternatively, cDNA
libraries are prepared from Conus venom duct using conventional techniques.
DNA from single
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WO 00/76532 PCT/US00/15779
18
clones is amplified by conventional techniques using primers which correspond
approximately to
the M13 universal priming site and the M13 reverse universal priming site.
Clones having a size
of approximately 250 nucleotides are sequenced and screened for similarity in
sequence to the
propeptide described herein. In this manner, conotoxins having the basic
structure and activity
described herein are cloned from many Conus species.
Muteins, analogs or active fragments (collectively referred to herein as
derivatives) of ~O-
conopeptides are also contemplated for use as local anesthetics. See, e.g.,
Hammerland et al. ( 1992).
Derivative muteins, analogs or active fragments of q0-conopeptides may be
synthesized according
to known techniques, including conservative amino acid substitutions, such as
outlined in U.S.
1o Patent Nos. 5,545,723; 5,534,615 and 5,364,769. The derivative muteins,
analogs or active
fragments may be conveniently assayed for activity by using a hindlimb
paralysis test such as
described in Example 2 or a local anesthetic test such as described in Example
3.
A variety of peptides from Conus target sodium channels. q-Conopeptides (i.e.,
GVIA)
block sodium channels expressed by muscle cells (Olivera et al., 1990). 8-
Conopeptides (i.e.,
GmVIA) delay the inactivation of neuronal sodium channels (Olivera et al.,
1990). Another class
of conopeptide (i.e., ~-PnIVA and q-PnIVB; unfortunately also called q but
having a distinct
cysteine framework from that which is considered a q-conopeptide) blocks
sodium channels in
molluscan neurons, but has no effect on sodium currents in bovine chromaffin
cells or in rat brain
synaptosomes (Fainzilber et al., 1995). Finally, the q0-conopeptides (MrVIA
and MrVIB) block
mammalian sodium channels (McIntosh et al., 1995).
Since the q0-conopeptides have been shown to have a slow and incomplete
washout from
Xenopus oocytes expressing cloned rat type II sodium channels (Terlau et al.,
1996), the present
invention examined whether the q 0-conopeptides might represent a candidate
for a long-lasting
local anesthetic.
Thus, the present invention is directed to a method for inducing local
analgesia by
administering the q0-conopeptides described herein. In one embodiment,
In a second embodiment, q0-conopeptides are used to provide local anesthesia
for pain
associated with any epithelial tissue region in a subject, for example, pain
associated with epithelial
ulcers, such as a canker sore or genital lesions. Canker sores can occur alone
or in groups on the
3o inside of the cheek or lip or underneath the tongue. Severely affected
people have continuously
recurring ulcers which last for one to two weeks (Clayman). Genital ulcers are
usually caused by
sexually transmitted diseases, including herpes and syphilis. The early stages
of syphilis are
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19
characterized by a hard chancre, a painful ulcer where bacteria has penetrated
the skin. This may be
followed by shallow, elongated ulcers once the chancre has healed. Such ulcers
are painful. Genital
ulceration may also be a side effect of drugs taken orally or caused by
solutions applied to genital
warts. Pain in epithelial tissue is also caused by burns. Burns affecting the
epidermal layer are
usually associated with pain, restlessness and fever. Treatment of such a burn
in accordance with
the method of the invention can provide relief from the attendant pain. Pain
as a result of damage
to or loss of epithelial tissue is also associated with other conditions and
procedures, such as sore
throats and plastic surgery, for example carbon dioxide laser surgery to
remove for skin resurfacing
and removal of wrinkles (Rosenberg et al., 1996), burns, genital lesions,
upper or lower
1o gastrointestinal bronchoscopy or endoscopy, intubation, dermatologic
abrasions or chemical skin
peels. The ~O-conopeptides administered in accordance with the method of the
invention is
beneficial in relieving pain associated with such damaged tissues.
Pharmaceutical compositions containing a ~t0-conopeptide or pharmaceutically
acceptable
salts thereof as the active ingredient (agent) can be prepared according to
conventional
pharmaceutical compounding techniques. See, for example, Remington's
Pharmaceutical Sciences,
18th Ed. (1990, Mack Publishing Co., Easton, Pa.). Typically, a
therapeutically effective amount
of the active ingredient will be admixed with a pharmaceutically acceptable
carrier. The carrier may
take a wide variety of forms depending on the form of preparation desired for
administration, e.g.,
intravenous, oral or parenteral. The compositions may further contain
antioxidizing agents,
2o stabilizing agents, preservatives and the like.
"Pharmaceutical composition" means physically discrete coherent portions
suitable for
medical administration. "Pharmaceutical composition in dosage unit form" means
physically
discrete coherent units suitable for medical administration, each containing a
daily dose or a
multiple (up to four times) or a sub-multiple (down to a fortieth) of a daily
dose of the active
compound in association with a carrier and/or enclosed within an envelope.
Whether the
composition contains a daily dose, or for example, a half, a third or a
quarter of a daily dose, will
depend on whether the pharmaceutical composition is to be administered once
or, for example,
twice, three times or four times a day, respectively.
The term "salt", as used herein, denotes acidic and/or basic salts, formed
with inorganic or
organic acids and/or bases, preferably basic salts. While pharmaceutically
acceptable salts are
preferred, particularly when employing the compounds of the invention as
medicaments, other salts
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
find utility, for example, in processing these compounds, or where non-
medicament-type uses are
contemplated. Salts of these compounds may be prepared by art-recognized
techniques.
Examples of such pharmaceutically acceptable salts include, but are not
limited to, inorganic
and organic addition salts, such as hydrochloride, sulphates, nitrates or
phosphates and acetates,
5 trifluoroacetates, propionates, succinates, benzoates, citrates, tartrates,
fumarates, maleates,
methane-sulfonates, isothionates, theophylline acetates, salicylates,
respectively, or the like. Lower
alkyl quaternary ammonium salts and the like are suitable, as well.
As used herein, the term "pharmaceutically acceptable" carrier means a non-
toxic, inert solid,
semi-solid liquid filler, diluent, encapsulating material, formulation
auxiliary of any type, or simply
10 a sterile aqueous medium, such as saline. Some examples of the materials
that can serve as
pharmaceutically acceptable carriers are sugars, such as lactose, glucose and
sucrose, starches such
as corn starch and potato starch, cellulose and its derivatives such as sodium
carboxymethyl
cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt,
gelatin, talc; excipients
such as cocoa butter and suppository waxes; oils such as peanut oil,
cottonseed oil, safflower oil,
15 sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene
glycol, polyols such as
glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl
oleate and ethyl laurate,
agar; buffering agents such as magnesium hydroxide and aluminum hydroxide;
alginic acid;
pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and
phosphate buffer solutions,
as well as other non-toxic compatible substances used in pharmaceutical
formulations.
2o Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate
and magnesium
stearate, as well as coloring agents, releasing agents, coating agents,
sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be present in the
composition, according
to the judgment of the formulator. Examples of pharmaceutically acceptable
antioxidants include,
but are not limited to, water soluble antioxidants such as ascorbic acid,
cysteine hydrochloride,
sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil
soluble antioxidants, such
as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin,
propyl gallate, aloha-tocopherol and the like; and the metal chelating agents
such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric
acid and the like.
For oral administration, the compounds can be formulated into solid or liquid
preparations
such as capsules, pills, tablets, lozenges, melts, powders, suspensions or
emulsions. In preparing
the compositions in oral dosage form, any of the usual pharmaceutical media
may be employed,
such as, for example, water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents,
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
21
suspending agents, and the like in the case of oral liquid preparations (such
as, for example,
suspensions, elixirs and solutions); or carriers such as starches, sugars,
diluents, granulating agents,
lubricants, binders, disintegrating agents and the like in the case of oral
solid preparations (such as,
for example, powders, capsules and tablets). Because of their ease in
administration, tablets and
capsules represent the most advantageous oral dosage unit form, in which case
solid pharmaceutical
carriers are obviously employed. If desired, tablets may be sugar-coated or
enteric-coated by
standard techniques. The active agent can be encapsulated to make it stable to
passage through the
gastrointestinal tract while at the same time allowing for passage across the
blood brain barrier. See
for example, WO 96/11698.
l0 For parenteral administration, the compound may be dissolved in a
pharmaceutical carrier
and administered as either a solution or a suspension. Illustrative of
suitable carriers are water,
saline, dextrose solutions, fructose solutions, ethanol, or oils of animal,
vegetative or synthetic
origin. The carrier may also contain other ingredients, for example,
preservatives, suspending
agents, solubilizing agents, buffers and the like. When the compounds are
being administered
intrathecally, they may also be dissolved in cerebrospinal fluid.
For topical administration, the compound may be formulated as an ointment,
cream, gel or
paste comprising the compound to be administered in a pharmaceutical
acceptable carrier. One
means of topical administration is a transdermal patch containing the compound
to be administered.
A variety of administration routes are available. The particular mode selected
will depend
of course, upon the particular drug selected, the severity of the disease
state being treated and the
dosage required for therapeutic efficacy. The methods of this invention,
generally speaking, may
be practiced using any mode of administration that is medically acceptable,
meaning any mode that
produces effective levels of the active compounds without causing clinically
unacceptable adverse
effects. Such modes of administration include oral, rectal, sublingual,
topical, nasal, transdermal or
parenteral routes. The term "parenteral" includes subcutaneous, intravenous,
epidural, irrigation,
intramuscular, release pumps, or infusion.
For example, administration of the active agent according to this invention
may be achieved
using any suitable delivery means, including:
(a) pump (see, e.g., Lauer & Hatton (1993), Zimm et al. (1984) and Ettinger et
al. (1978));
(b) microencapsulation (see, e.g., U.S. Patent Nos. 4,352,883; 4,353,888; and
5,084,350);
(c) continuous release polymer implants (see, e.g., U.S. Patent No.
4,883,666);
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WO 00/76532 PCT/US00/15779
22
(d) macroencapsulation (see, e.g., U.S. Patent Nos. 5,284,761, 5,158,881,
4,976,859 and
4,968,733 and published PCT patent applications.-W092/19195, WO 95/05452);
(e) naked or unencapsulated cell grafts to the CNS (see, e.g., U.S. Patent
Nos. 5,082,670 and
5,618,531 );
(f) injection, either subcutaneously, intravenously, intra-arterially,
intramuscularly, or to
other suitable site;
(g) oral administration, in capsule, liquid, tablet, pill, or prolonged
release formulation; or
(h) topical (see, e.g., U.S. Patent Nos. 6,046,187 and 6,030,974).
In one embodiment of this invention, an active agent is delivered directly
into the CNS,
preferably to the brain ventricles, brain parenchyma, the intrathecal space or
other suitable CNS
location, most preferably intrathecally.
Alternatively, targeting therapies may be used to deliver the active agent
more specifically
to certain types of cells, by the use of targeting systems such as antibodies
or cell-specific ligands.
Targeting may be desirable for a variety of reasons, e.g. if the agent is
unacceptably toxic, if it
would otherwise require too high a dosage, or if it would not otherwise be
able to enter target cells.
The active agents, which are peptides, can also be administered in a cell
based delivery
system in which a DNA sequence encoding an active agent is introduced into
cells designed for
implantation in the body of the patient, especially in the spinal cord region.
Suitable delivery
systems are described in U.S. Patent No. 5,550,050 and published PCT
Application Nos. WO
92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO
96/40871, WO 96/40959 and WO 97/12635. Suitable DNA sequences can be prepared
synthetically
for each active agent on the basis of the developed sequences and the known
genetic code.
The active agent is preferably administered in an therapeutically effective
amount. By a
"therapeutically effective amount" or simply "effective amount" of an active
compound is meant a
sufficient amount of the compound to treat or alleviate pain or to induce
analgesia at a reasonable
benefit/risk ratio applicable to any medical treatment. The actual amount
administered, and the rate
and time-course of administration, will depend on the nature and severity of
the condition being
treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is
within the responsibility
of general practitioners or spealists, and typically takes account of the
disorder to be treated, the
3o condition of the individual patient, the site of delivery, the method of
administration and other
factors known to practitioners. Examples of techniques and protocols can be
found in Remington's
Parmaceutical Sciences.
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
23
For the treatment of pain, if the route of administration is directly to the
CNS, the dosage
contemplated is from about 1 ng to about 100 mg per day, preferably from about
100 ng to about
mg per day, more preferably from about 1 ~g to about 100 ~g per day. If
administered
5 peripherally, the dosage contemplated is somewhat higher, from about 100 ng
to about 1000 mg per
day, preferably from about 10 ~g to about 100 mg per day, more preferably from
about 100 ~g to
about 10 mg per day.
If the ~O-conopeptide is delivered by continuous infusion (e.g., by pump
delivery,
biodegradable polymer delivery or cell-based delivery), then a lower dosage is
contemplated than
to for bolus delivery.
However, it will be understood that the amount of the active compound actually
administered will be determined by a physician, in the light of the relevant
circumstances including
the condition to be treated, the chosen route of administration, the age,
weight, and response of the
individual patient, and the severity of the patient's symptoms, and therefore
the above dosage ranges
are not intended to limit the scope of the invention in any way. As used
herein the terms
"pharmaceutical compositions" and "pharmaceutically acceptable" include
compositions and
ingredients for both human and veterinary use.
The present data suggest that ~O-conopeptides are extremely potent and long-
lasting local
anesthetic agents, most likely due to their ability to block neuronal sodium
channels. Moreover,
2o since ~O-conopeptides probably act at a site on sodium channels distinct
from other local
anesthetics or guanidinium toxins like tetrodotoxin (since they are likely to
act at an extracellular
target, but do compete for [3H]saxitoxin at site I) (Terlau et al., 1996), and
probably do not affect
sodium channels in the muscles or heart (since i.p. injection of 10 nmol is
without effect in mice
(McIntosh et al., 1995), these peptides lack the untoward side effects of
clinically used local
anesthetics.
Despite the high hydrophobicity of these peptides, there is a cluster of
charged amino acid
residues at the amino terminus. This cluster of charge, combined with the size
of the peptides,
probably results in poor permeation of the nerve sheath and thus accounts for
the poor efficacy in
the tail withdrawal assay. In contrast, when the nerve sheath is not a
barrier, such as following
intrathecal injection or intracutaneous injection, ~O-conopeptides are
effective and long-lasting.
These facts establish that p0-conopeptides are novel candidates for spinal
anesthesia, either
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
24
administered acutely for post-operative pain or via an intrathecal pump for
severe chronic pain
situations.
The practice of the present invention employs, unless otherwise indicated,
conventional
techniques of chemistry, molecular biology, microbiology, recombinant DNA,
genetics,
immunology, cell biology, cell culture and transgenic biology, which are
within the skill of the art.
See, e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992;
Glover, 1985; Anand,
1992; Guthrie and Fink, 1991; Harlow and Lane, 1988; Jakoby and Pastan, 1979;
Nucleic Acid
Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And
T~°anslation (B. D.
1 o Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney,
Alan R. Liss, Inc.,
1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical
Guide To
Molecular Cloning ( 1984); the treatise, Methods In Enzymology (Academic
Press, Inc., N.Y.); Gene
Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,
Cold Spring
Harbor Laboratory); Methods In Enzymolo~, Vols. 154 and 155 (Wu et al. eds.),
Immunochemical
Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London, 1987);
Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds.,
1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific
Publications, Oxford, 1988;
Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press, Cold Spring
Harbor, N.Y., 1986).
EXAMPLES
The present invention is further detailed in the following Examples, which are
offered by
way of illustration and are not intended to limit the invention in any manner.
Standard techniques
well known in the art or the techniques specifically described below are
utilized.
EXAMPLE 1
Isolation of q0-conoPeptides A657 and F079
PCR primers designed to amplify "O Superfamily" conotoxin genes were used in
RT-PCR
amplification of venom duct cDNA from a variety of Conus species. The primers
have the
following sequences:
Forward Primer: OCon6 CAGGATCCATGAAACTGACGTGYRTGGTG (SEQ ID N0:13)
Reverse Primer: OCon7 ATCTCGAGCACAGGTATGGATGACTCAGG (SEQ ID N0:14).
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Amplification products in the appropriate size range were cloned and
sequenced. A range of "O-
Superfamily" gene sequences were identified. The novel genes, A657 from C.
skinneri, F079, F080
and G28 from C. tessulatus, F763 from C. atlanticus, F008 from C. arenatus,
Tx6.12 and Tx6.13
from C. textile and G18 from C. generalis, were identified as ~O-conopeptides
on the basis of their
5 similarity to the ~-O conopeptides MrVIA and MrVIB. This similarity was much
greater than the
similarity with any of the c~-, x- or 8-conopeptides that comprise the "O
Superfamily" peptides.
The cDNA and amino acid sequence for the A657, F079, Ca6.l, Tx6.12 (portion),
Tx6.13
(portion), G28, F763, F080, F008 and G18 propeptides are set forth in Tables 1-
10, respectively.
The amino acid sequences of the mature ~O-conopeptides are as shown above.
to
TABLE 1
DNA Sequence (SEQ ID NO:15) and Protein Sequence (SEQ ID N0:16) of A657
atg etgacgtgt gtggtgatc gttgetgtg ctgttettg ace gec
aaa
Met LeuThrCys ValValI1e ValAlaVal LeuPheLeu Thr Ala
Lys
tgg ttcgteatg getgatgac eceagagat ggagcggag att aga
aca
Trp PheValMet AlaAspAsp ProArgAsp GlyAlaGlu Ile Arg
Thr
agc atg gta agg ggg gaa cct ctg tcg aag gca cgt gac gaa atg aac
Ser Met Val Arg Gly Glu Pro Leu Ser Lys Ala Arg Asp Glu Met Asn
ccc gaa gcc tct aaa ttg gag aaa agg gcg tgc cgc caa aaa tac gaa
Pro Glu Ala Ser Lys Leu Glu Lys Arg Ala Cys Arg Gln Lys Tyr Glu
ttt tgt cta gta ceg atc att gga tac ata tat tgc tge get gge tta
Phe Cys Leu Val Pro Ile Ile Gly Tyr Ile Tyr Cys Cys Ala Gly Leu
atc tgt ggt cct ttc gtc tgc ctt tgatagtgat gtcttctact gccatctgtg
Ile Cys Gly Pro Phe Val Cys Leu
ctacccctgg cttgatcttt gataggcgtt gttgcccttc actggtttat gaaccctctg
atcatactct ctggaccctt gggggtccaa catccaaata aagcgacatc ccaaaaaaaa
aaaaaaaaaa
TABLE 2
DNA Sequence (SEQ ID N0:17) and Protein Sequence (SEQ ID N0:18) of F079
gga tcc atg aaa ctg acg tgc atg gtg atc gtt gtt gtg ctg ttg ttg
Gly Ser Met Lys Leu Thr Cys Met Val Ile Val Val Val Leu Leu Leu
aac gcc tgg aca ttc gtc tcc ata aat gga aag gcg aat cgt ttt tgg
Asn Ala Trp Thr Phe Val Ser Ile Asn Gly Lys Ala Asn Arg Phe Trp
aag gca cgt gac gaa atg aag gac tcc gaa gtt tct gaa ttg gag aaa
Lys Ala Arg Asp Glu Met Lys Asp Ser Glu Val Ser Glu Leu Glu Lys
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26
agg agg aaa ccg acc tgc ctg aag cag gac aag ttt tgc ata ata ccg
Arg Arg Lys Pro Thr Cys Leu Lys Gln Asp Lys Phe Cys Ile Ile Pro
ctc att gga acc ctt tat tgc tgc agt ggg tta atc tgt ggg ttt ttt
Leu Ile Gly Thr Leu Tyr Cys Cys Ser Gly Leu Ile Cys Gly Phe Phe
gtc tgc gtc cca aag ccg ttc tgatgtcttc tactgccatc tgtgctaccc
Val Cys Val Pro Lys Pro Phe
ctggcttgat ctttgattgg cgtgtgccct tcactggtta tgaacccctc tgatcctact
gtctggacgc ctcgggcgtc caacgtccaa ataaagcgac atcccaataa aaaaaaaaaa
aaaaaaa
TABLE 3
DNA Sequence (SEQ ID N0:19) and Protein Sequence (SEQ ID N0:20) of Ca6.1
atg aaa ctg acg tgc gtg atg atc gtt get gtg ctg ttc ttg acc gcc
Met Lys Leu Thr Cys Val Met Ile Val Ala Val Leu Phe Leu Thr Ala
tgg aca ttc gtc acg get gat gac tcc att aat gca ctg gag gat ctt
Trp Thr Phe Val Thr Ala Asp Asp Ser Ile Asn Ala Leu Glu Asp Leu
ttt tcg aag gca cgt gac gaa atg gaa aac ggc gaa get tct aca ttg
Phe Ser Lys Ala Arg Asp Glu Met Glu Asn Gly Glu Ala Ser Thr Leu
aac gag aga gac tgc gaa gca gat ggt gca ttt tgt ggt atc cca att
Asn Glu Arg Asp Cys Glu Ala Asp Gly Ala Phe Cys Gly Ile Pro Ile
gtg aag aac tgg atg tgc tgc agt aac ttg tgt att ttt gcc tgc gta
Val Lys Asn Trp Met Cys Cys Ser Asn Leu Cys Ile Phe Ala Cys Val
ccc gag tat taagactgcc gtgatgtctt ctcctcccct c
Pro Glu Tyr
TABLE 4
DNA Sequence (SEQ ID N0:21) and Protein Sequence (SEQ ID N0:22) of Tx6.12
a ttg gag aaa agg gat tgc cac gaa agg tgg gat tgg tgt cca gca tca
Leu Glu Lys Arg Asp Cys His Glu Arg Trp Asp Trp Cys Pro Ala Ser
atc ctt gga gtg ata tat tgc tgc gag gga tta att tgt ttt att gcc
Ile Leu Gly Val Ile Tyr Cys Cys Glu Gly Leu Ile Cys Phe Ile Ala
ttc tgc att tgatagtgat gtcttctcct cccctc
Phe Cys Ile
5o TABLE 5
DNA Sequence (SEQ ID N0:23) and Protein Sequence (SEQ ID N0:24) of Tx6.13
a ttg gag aaa agg gat tgc caa gag aaa tgg gag ttt tgt ata gta ccg
Leu Glu Lys Arg Asp Cys Gln Glu Lys Trp Glu Phe Cys Ile Val Pro
atc ctt gga ttt gta tat tgc tgc cct ggc tta atc tgt ggc cct ttt
Ile Leu Gly Phe Val Tyr Cys Cys Pro Gly Leu Ile Cys Gly Pro Phe
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27
gtc tgc gtt gat atc tgatgtcttc tcctcccatc
Val Cys Val Asp Ile
s TABLE 6
DNA Sequence (SEQ ID N0:25) and Protein Sequence (SEQ ID N0:26) of G28
ggatcc atg aaa ctg acg tgt gtg gtg atc gtt gtt gtg ctg ttg ttg
Met Lys Leu Thr Cys Val Val Ile Val Val Val Leu Leu Leu
aac gcc tgg aca ttc gtc tcc ata aat gga aag gcg aat cct ttt tgg
Asn Ala Trp Thr Phe Val Ser Ile Asn Gly Lys Ala Asn Pro Phe Trp
aag gca cgt gac gaa atg aag gac tcc gaa gtt tct gag ttg gag aaa
Lys Ala Arg Asp Glu Met Lys Asp Ser Glu Val Ser Glu Leu Glu Lys
agg agg aaa ccg acc tgc gtg tcg tat aac gtg ttt tgc gga gta ccg
Arg Arg Lys Pro Thr Cys Val Ser Tyr Asn Val Phe Cys,Gly Val Pro
ctc gtt gga acc tac ctt tgc tgc agt ggc tta gtc tgt ctc gta gtc
Leu Val Gly Thr Tyr Leu Cys Cys Ser Gly Leu Val Cys Leu Val Val
tgc atc tagtactgat gtcttctact cccatctgtg ctacccctcg ag
Cys Ile
30
TABLE 7
DNA Sequence (SEQ ID N0:27) and Protein Sequence (SEQ ID N0:28) of F763
ggatcc atg aaa ctg acg tgc gtg gtg atc gtt get gtg ctg ttc ttg
Met Lys Leu Thr Cys Val Va1 Ile Val Ala Val Leu Phe Leu
acc gcc tgg aca ttc gtc acg get gat gac tcc ata aat ggg ttg gag
Thr Ala Trp Thr Phe Val Thr Ala Asp Asp Ser Ile Asn Gly Leu Glu
aat ctt ttt ccg aag gca cgt cac gaa atg agg aaa ccc gaa gcc tct
Asn Leu Phe Pro Lys Ala Arg His Glu Met Arg Lys Pro Glu Ala Ser
aga tcg aga ggg agg tgc cgt cct cgt ggt atg ttc tgt ggc ttt ccg
Arg Ser Arg Gly Arg Cys Arg Pro Arg Gly Met Phe Cys Gly Phe Pro
aaa cct gga cca tac tgc tgc aat ggc tgg tgc ttt ttc gtc tgc atc
Lys Pro Gly Pro Tyr Cys Cys Asn Gly Trp Cys Phe Phe Val Cys Ile
taaaactgcc gtgatgtgtt ctactcccat ctgtgctacc cctcgag
50
TABLE 8
DNA Sequence (SEQ ID N0:29) and Protein Sequence (SEQ ID N0:30) of F080
ggatcc atg aaa ctg acg tgc gtg gtg gtc gtt get gtg ctg ttc ttg
Met Lys Leu Thr Cys Val Val Val Val Ala Val Leu Phe Leu
aac gcc tgg aca ttc gcc acg get gtt gac tcc aaa cat gca ctg gcg
Asn Ala Trp Thr Phe Ala Thr Ala Val Asp Ser Lys His Ala Leu Ala
aaa ctt ttt atg aag gca cgt gac gaa atg tat aac ccc gat gcc act
Lys Leu Phe Met Lys Ala Arg Asp Glu Met Tyr Asn Pro Asp Ala Thr
aaa ttg gac gat aag aga tgg tgc get tta gat ggt gaa ctt tgt atc
Lys Leu Asp Asp Lys Arg Trp Cys Ala Leu Asp Gly Glu Leu Cys Ile
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ata ccg gtc att ggg tcc ata ttt tgc tgc cat ggc ata tgt atg atc
Ile Pro Val Ile Gly Ser Ile Phe Cys Cys His Gly Ile Cys Met Ile
tac tgc gtc tagttgaact gccgtgatgt cttctactcc cctctgtgct
Tyr Cys Val
acccctggtt tgatctttga ttgccctgtg cccttcactg attatgaatc cctctgatcc
tactctctga agacctcttg gggtccaaca tccaaataaa gcgacatccc aaaaaaaaaa
aaaaaaaaaa
TABLE 9
DNA Sequence (SEQ ID N0:31) and Protein Sequence (SEQ ID N0:32) of F008
ggatcc atg aaa ctg acg tgt gtg gtg atc gtt get gtg ctg ttc ttg
Met Lys Leu Thr Cys Val Val Ile Val Ala Val Leu Phe Leu
acc gcc tgg aca ttc gtc acg get gac tcc ata cgt gca ctg gag gat
Thr Ala Trp Thr Phe Val Thr Ala Asp Ser Ile Arg Ala Leu Glu Asp
ttt ttt gcg aag gca cgt gac gaa atg gaa aac agc gga get tct cca
Phe Phe Ala Lys Ala Arg Asp Glu Met Glu Asn Ser Gly Ala Ser Pro
ttg aac gag aga gac tgc cga cct gta ggt caa tat tgt ggc ata ccg
Leu Asn Glu Arg Asp Cys Arg Pro Val Gly Gln Tyr Cys Gly Ile Pro
3O
tat aag cac aac tgg cga tgc tgc agt cag ctt tgt gca att atc tgt
Tyr Lys His Asn Trp Arg Cys Cys Ser Gln Leu Cys Ala Ile Ile Cys
gtt tcc taacccctct gatcctactc tctgaagacc tccgggattc aacatccaaa
Val Ser
taaagcgaca tcccgatnaa aaaaaangaa aaaaaaaaaa aaaa
TABLE 10
DNA Sequence (SEQ ID N0:33) and Protein Sequence (SEQ ID N0:34) of G18
ggatcc atg aaa ctg acg tgt gtg gtg atc gtt get gtg cta ttc ttg
Met Lys Leu Thr Cys Val Val Ile Val Ala Val Leu Phe Leu
acc gcc tgg aca ttc gtc acg get gat gac acc aga tat aaa ctg gag
Thr A1a Trp Thr Phe Val Thr Ala Asp Asp Thr Arg Tyr Lys Leu Glu
aat cct ttt ctg aag gca cgc aac gaa ctg cag aaa cac gaa gcc tct
Asn Pro Phe Leu Lys Ala Arg Asn Glu Leu Gln Lys His Glu Ala Ser
caa ctg aac gag aga ggc tgc ctt gac cca ggt tac ttc tgt ggg acg
Gln Leu Asn Glu Arg Gly Cys Leu Asp Pro Gly Tyr Phe Cys Gly Thr
ccg ttt ctt gga gca tac tgc tgc ggt ggc att tgc ctt att gtc tgc
Pro Phe Leu Gly Ala Tyr Cys Cys Gly Gly Ile Cys Leu Ile Val Cys
ata gaa acg taaaggcttg atgtcttcta ctcccatctg tgctacccct cgag
Ile Glu Thr
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EXAMPLE 2
Effect of Intrathecal Administration of MrVIB
Male C57 black mice (20-25g) were obtained from Charles River Laboratories.
These mice
and the animals used in the other examples were housed in a temperature
controlled (23 ° ~ 3 ° C)
room with a 12 hour light-dark cycle with free access to food and water. All
animals were
euthanized in accordance with Public Health Service policies on the humane
care of laboratory
animals.
Intrathecal (it) drug injections were performed as described (Hylden and
Wilcox, 1980).
MrVIB (10 runol) or vehicle was administered in a volume of 5 ~l. Duration of
hind-limb paralysis
was assessed. This experiment revealed that injection of 10 nmols of MrVIB
into the intrathecal
space of C57 black mice produced a long-lasting paralysis (>20 hrs) of the
animal. The injection
initially produced a paralysis of the hind-limbs, but over the following 30
minutes resolved into
paralysis of the entire animal. Despite the long duration of anesthesia, the
animals in this
experiment recovered fully. Similar results were obtained with MrVIA. Similar
results are also
obtained with A657, F079, Ca6.l, Tx6.12, Tx6.13, G28, F763 and F080.
EXAMPLE 3
Effect of MrVIB as a Local Anesthetic
Male Hartley guinea pigs (retired breeders) were obtained form Charles River
Laboratories.
2o The local anesthetic test was performed essentially as described (Bulbring
and Wajda, 1945). On
the day prior to test day, a patch on the back of the guinea pig was denuded
of hair, first by shaving
with electric clippers and subsequently with depilatory cream (Nair~).
Depilatory cream was
applied for five minutes and removed with a warm washcloth. The guinea pigs
were dried and
returned to their cages. On the following day, intradermal injections (0.1 ml
vols) of lidocaine,
bupivacaine, MrVIB or vehicle (0.5% cyclodextran) were made into the denuded
patch. The
injection produced a raised wheal on the surface of the skin which was circled
with a felt-tipped pen.
Typically, four injections were made on the back of each guinea pig. In some
cases, guinea pigs
were reused following at least one week of recovery and injecting into an
unused portion of the skin.
The stimulus consisted of mild pin pricks (not hard enough to break the skin)
with a 26G
3o needle. The response is a localized skin twitch caused by contraction of
cutaneous muscles. A unit
test consisted of six uniform pin pricks, 3-5 seconds apart, within the
injected area. Unit scores
ranged from 0 (complete anesthesia) to 6 (no anesthesia). For potency
experiments, the unit test was
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
repeated at each site at five minute intervals for 30 minutes, and unit test
scores summed (with 36
representing no anesthesia to 0 representing complete anesthesia. For duration
experiments, unit
tests were performed as described over the course of several hours to days.
MrVIB produced a potent (Figure 1) and long lasting (Figure 2) local
anesthetic effect in the
5 intracutaneous wheat test in the guinea pig. The EDso for this response ( ~
100 pmol) was at least
two orders of magnitude greater than the EDSO's for lidocaine and bupivacaine.
Moreover, the
duration of roughly equieffective doses of MrVIB (roughly 24 and 48 hours for
full recovery
following 1 and 10 nmol, respectively) was much longer than that of lidocaine
and bupivacaine (~
30 and 90 minutes for full recovery, respectively). As expected, bupivacaine
ahd a slightly longer
10 duration thatn lidocaine, consistent with clinical observations. It was
seen during the experiment
that the intracutaneous wheat consistently turned red several hours following
injection of MrVIB,
possibly suggesting an antigenic action. Similar results are obtained with
MrVIA, A657, F079,
Ca6.l, Tx6.12, Tx6.13, G28, F763 and F080.
15 While the invention has been disclosed in this patent application by
reference to the details
of preferred embodiments of the invention, it is to be understood that the
disclosure is intended in
an illustrative rather than in a limiting sense, as it is contemplated that
modifications will readily
occur to those skilled in the art, within the spirit of the invention and the
scope of the appended
claims.
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Bitan, G. et al. (1997). J. Peptide Res. 49:421-426.
Bodansky et al. (1966). Chem. Ind. 38:1597-98.
Bulbring, W. and Wajda, J. (1945). J. Pharmacol. Exp. Ther. 85:78-84.
Ettinger, L.J. et al. (1978). Cancer 41:1270-1273.
Fainzilber, M. et al. (1995). Biochemistry 34:8649-8656.
Hammerland et al. (1992). Eur. J. Pharmacol. 226:239-242.
Horiki, K. et al. (1978). Chemistry Letters 165-68.
3o Hubry, V. et al. (1994). Reactive Polymers 22:231-241.
Hylden, J.L.K.and Wilcox, G. (1980). Eur. J. Pharmacol. 67:313-316.
Kaiser et al. (1970). Anal. Biochem. 34:595.
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
31
Kapoor (1970). J. Pharm. Sci. 59:1-27.
Kornreich, W.D. et al. (1986). U.S. Patent No. 4,569,967.
Kuzma, P.K. et al. (1997). Regional Anesthesia 22:543-551.
Luer, M.S. et al. (1993). Annals ofPharmacotherapy, 27:912-921.
McIntosh, J.M. et al. (1995). J. Biol. Chem. 270:16796-16802.
McIntosh, J. M. et al. (1998). Methods Enzymol. 294:605-624.
Methoden der Organischen Chemie (Houben-Weyl): Synthese von Peptiden, E.
Wunsch (Ed.),
Georg Thieme Verlag, Stuttgart, Ger. (1974).
Nishiuchi, Y. et al. (1993). Int. J. Pept. Protein Res. 42:533-538.
1 o Nowak, L. et al. ( 1984). Nature 307:462-465.
Olivera, B.M. et al. (1984). U.S. Patent 4,447,356.
Olivera, B.M. et al. (1985). Science 230:1338-1343.
Olivera, B.M. et al. (1990). Science 249:257-263.
Ornstein, et al. (1993). Biorganic Medicinal Chemistry Letters 3:43-48.
Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA
(1990).
Rivier, J.R. et al. (1978). Biopolymers 17:1927-38.
Rivier, J.R. et al. (1987). Biochem. 26:8508-8512.
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Cold Spring Harbor
2o Laboratory, Cold Spring Harbor, NY.
Schroder & Lubke (1965). The Peptides 1:72-75, Academic Press, NY.
Stewart and Young, Solid-Phase Peptide Synthesis, Freeman & Co., San
Francisco, CA (1969).
Terlau et al. (1996). J. Neurophysiol. 76:1423-1429.
Vale et al. (1978). U.S. Patent 4,105,603.
Van de Steen, P. et al. (1998). Critical Rev. in Biochem. and Mol. Biol.
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Zhou L.M., et al. (1996). J. Neurochem. 66:620-628.
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CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
32
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CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
1
SEQUENCE LISTING
<110> Olivera, Baldomero M.
McIntosh, J. Michael
McCabe, R. Tyler
Garrett, James E.
Layer, Richard T.
Wagstaff, John D.
Jones, Robert M.
Cognetix, Inc.
University of Utah Research Foundation
<120> Mu0-Conopeptides and Their Use as Local Anesthetics
<130> Mu0-Conotoxins
<140>
<141>
<150> US 60/138,507
<151> 1999-06-10
<160> 34
<170> PatentIn Ver. 2.0
<210> 1
<211> 36
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: generic
Mu0-conopeptide sequence
<220>
<221> PEPTIDE
<222> (1)
<223> Xaa at residue 1 is des-Xaa, Pro, hydroxy-Pro
(Hyp), Arg, Lys, ornithine, homo-Lys,
homoarginine, nor-Lys, N-methyl-Lys,
N,N'-dimethyl-Lys, N,N',N " -trimethyl-Lys or any
synthetic basic amino acid
<220>
<221> PEPTIDE
<222> (2)
<223> Xaa at residue 2 is des-Xaa, Ala, Gly, Asp, Glu,
gamma-carboxy-glutamate (Gla), any synthetic
acidic amino acid, Thr, Ser, g-Thr (where g is
glycosylation), g-Ser, Trp (D or L), neo-Trp or
<220>
<221> PEPTIDE
<222> (2)..(4)
<223> or halo-Trp (D or L) or Xaa2 may be pyroglutamate
if Xaa at residue 1 is des-Xaa; Xaa at residue 4
is Arg, Lys, ornithine, homo-Lys, homoarginine,
nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
<220>
<221> PEPTIDE
<222> (4)
<223> N,N',N " -trimethyl-Lys, any synthetic basic amino
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
2
acid, Ser, Thr, g-Ser, g-Thr, Ala, an aliphatic
amino acids bearing linear or branched saturated
hydrocarbon chains such as Leu (D or L), Ile and
Val
<220>
<221> PEPTIDE
<222> (4) .. (5)
<223> or non-natural derivatives of the aliphatic
amino
acid, His, Glu, Gln, Gla, Asp, Asn or any
synthetic acidic amino acid; Xaa at residue
5 is
Glu, Gla, Gln, Asp, Asn, any synthetic
acidic
amino acid,
<220>
<221> PEPTIDE
<222> (5)
<223> Lys, Arg, ornithine, homo-Lys, homoarginine,
nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N " -trimethyl-Lys, any synthetic
basic amino
acid, Ala, an aliphatic amino acids bearing
linear
or branched
<220>
<221> PEPTIDE
<222> (5)
<223> saturated hydrocarbon chains such as Leu
(D or L),
Ile and Val or non-natural derivatives
of the
aliphatic amino acid, Ser, Thr, Pro, Hyp,
g-Ser,
g-Thr, g-Hyp or any synthetic hydroxylated
amino
acid;
<220>
<221> PEPTIDE
<222> (6)
<223> Lys, Arg, ornithine, homo-Lys, homoarginine,
nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N " -trimethyl-Lys, any synthetic
basic amino
acid, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr,
mono-halo-Tyr,
<220>
<221> PEPTIDE
<222> (6)
<223> di-halo-Tyr, O-sulpho-Tyr, 0-phospho-Tyr,
nitro-Tyr, an aliphatic amino acids bearing
linear
or branched saturated hydrocarbon chains
such as
Leu (D or L), I1e and Val or non-natural
derivatives of
<220>
<221> PEPTIDE
<222> (6)..(7)
<223> of the aliphatic amino acid, Glu, Gla,
Gln, Asp,
Asn, any synthetic acidic amino acid, Pro
or Hyp;
Xaa at residue 7 is Trp (D or L), neo-Trp,
halo-Trp (D or L), Gly, Tyr, meta-Tyr,
ortho-Tyr,
nor-Tyr,
<220>
<221> PEPTIDE
<222> (7)..(8)
<223> mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
O-phospho-Tyr, nitro-Tyr, Glu, Gla, Gln,
Asp, Asn,
CA 02376357 2001-12-06
WO 00/76532 PCT/US00/15779
J
any synthetic acidic amino acid; Xaa at residue 8
is Glu, Gla, Gln, Asp, Asn, any
<220>
<221> PEPTIDE
<222> (8)
<223> synthetic acidic amino acid, Met, norleucine
(Nle), Ala, an aliphatic amino acids bearing
linear or branched saturated hydrocarbon
chains
such as Leu (D or L), Ile and Val or
<220>
<221> PEPTIDE
<222> (8)
<223> non-natural derivatives of the aliphatic
amino
acid, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
O-phospho-Tyr, nitro-Tyr, Lys, Arg, ornithine,
homo-Lys, homoarginine,
<220>
<221> PEPTIDE
<222> (8)..(9)
<223> nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N " -trimethyl-Lys or any synthetic
basic
amino acid; Xaa at residue 9 is Leu, Phe,
Tyr,
meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr,
di-halo-Tyr,
<220>
<221> PEPTIDE
<222> (9)..(11)
<223> 0-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr,
Trp (D or
L), neo-Trp, halo-Trp (D or L) or any synthetic
aromatic amino acid; Xaa at residue 11
is Pro,
Hyp, Gly, an aliphatic amino acids bearing
linear
or
<220>
<221> PEPTIDE
<222> (11)..(12)
<223> branched saturated hydrocarbon chains such
as Leu
(D or L), Ile and Val or non-natural derivatives
of the aliphatic amino acid; Xaa at residue
12 is
Thr, Ser, g-Thr, g-Ser, Ala, an aliphatic
amino
<220>
<221> PEPTIDE
<222> (12)
<223> acids bearing linear or branched saturated
hydrocarbon chains such as Leu (D or L),
Ile and
Val or non-natural derivatives of the aliphatic
amino acid, Phe, Tyr, meta-Tyr, ortho-Tyr,
nor-Tyr,
<220>
<221> PEPTIDE
<222> (12)..(13)
<223> mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
O-phospho-Tyr, nitro-Tyr, Trp (D or L),
neo-Trp,
halo-Trp (D or L) or any synthetic aromatic
amino
acid; Xaa at residue 13 is Pre, Hyp, Ser,
Thr,
g-HYP
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4
<220>
<221> PEPTIDE
<222> (13)..(14)
<223> g-Ser, g-Thr or any hydroxylated amino
acid; Xaa
at residue 14 is an al,~phatic amino acids
bearing
linear or branched sat~~rated hydrocarbon
chains
such as Leu (D or L), Ile and Val or non-natural
<220>
<221> PEPTIDE
<222> (14)
<223> derivatives of the aliphatic amino acid,
Phe, Tyr,
meta-Tyr, ortho-Tyr, ncr-Tyr, mono-halo-Tyr,
di-halo-Tyr, 0-sulpho-Tyr, 0-phospho-Tyr,
nitro-Tyr, Lys, Arg, ornithine, homo-Lys,
homoarginine,
<220>
<221> PEPTIDE
<222> (14)..(15)
<223> nor-Lys, N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N " -trimethyl-Lys or any synthetic
basic
amino acid; Xaa at resv_due 15 is Pro,
Hyp, an
aliphatic amino acids bearing linear or
branched
saturated
<220>
<221> PEPTIDE
<222> (15)
<223> hydrocarbon chains such as Leu (D or L),
Ile and
Val or non-natural derivatives of the aliphatic
amino acid, Lys, Arg, ornithine, homo-Lys,
homoarginine, nor-Lys, N-methyl-Lys,
N,N'-dimethyl-Lys,
<220>
<221> PEPTIDE
<222> (15)..(16)
<223> N,N',N " -trimethyl-Lys or any synthetic
basic
amino acid; Xaa at residue 16 is Gly, His,
Lys,
Arg, ornithine, homo-Lys, homoarginine,
nor-Lys,
N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N " -trimethyl-Lys
<220>
<221> PEPTIDE
<222> (16)..(17)
<223> or any synthetic basic amino acid; Xaa
at residue
17 is des-Xaa, Ser, Thr, g-Ser, g-Thr,
Val, Asn,
Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
0-phospho-Tyr,
<220>
<221> PEPTIDE
<222> (17)..(18)
<223> nitro-Tyr, Trp (D or L;, neo-Trp, halo-Trp
(D or
L) or any synthetic aromatic amino acid;
Xaa at
residue 18 is Met, Nle, Leu, Phe, Tyr,
meta-Tyr,
ortho-Tyr, nor-Tyr, mono-halo-Tyr, di-halo-Tyr,
<220>
<221> PEPTIDE
<222> (18)
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WO 00/76532 PCT/US00/15779
<223> O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr,
Arg, Lys,
ornithine, homo-Lys, homoarginine, nor-Lys,
N-methyl-Lys, N,N'-dimethyl-Lys,
N,N',N " -trimethyl-Lys or any synthetic
basic
5 amino acid
<220>
<221> PEPTIDE
<222> (19)..(22)
<223> Pro, Hyp, Ser, Thr, g-Hyp, g-Ser, g-Thr,
any
hydroxylated amino acid, Ala, Glu, Gla,
Gln, Asp,
Asn, any synthetic acidic amino acid, His
or Gly;
Xaa at residue 22 is Gly, Asn or Gln
<220>
<221> PEPTIDE
<222> (23)..(24)
<223> Xaa at residue 23 is Leu, Trp (D or L),
neo-Trp
or halo-Trp (D or L); Xaa at residue 24
is
des-Xaa, Leu or Trp (D or L), neo-Trp or
halo-Trp
(D or L)
<220>
<221> PEPTIDE
<222> (25)
<223> Xaa at residue 25 is des-Xaa or an aliphatic
amino acids bearing linear or branched
saturated
hydrocarbon chains such as Leu (D or L),
Ile and
Val or non-natural derivatives of the aliphatic
amino
<220>
<221> PEPTIDE
<222> (25)..(27)
<223> acid; Xaa at residue 27 is des-Xaa, Gly,
Met, Nle,
Phe, Tyr, meta-Tyr, ortho-Tyr, nor-Tyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
0-phospho-Tyr, nitro-Tyr, Trp (D or L),
neo-Trp,
<220>
<221> PEPTIDE
<222> (27)..(28)
<223> halo-Trp (D or L) or any synthetic aromatic
amino
acid; Xaa at residue 28 is des-Xaa, Pro,
Hyp, Ala,
an aliphatic amino acids bearing linear
or
branched saturated hydrocarbon chains such
as
<220>
<221> PEPTIDE
<222> (28)
<223> Leu (D or L), Ile and Val or non-natural
derivatives of the aliphatic amino acid,
Phe, Tyr,
meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr,
di-halo-Tyr, 0-sulpho-Tyr, 0-phospho-Tyr,
nitro-Tyr,
<220>
<221> PEPTIDE
<2?2> (28)..(29)
<223> Trp (D or L), neo-Trp, halo-Trp (D or L)
or any
synthetic aromatic amino acid; Xaa at residue
29
is an aliphatic amino acids bearing linear
or
branched saturated hydrocarbon chains such
as
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6
<220>
<221> PEPTIDE
<222> (29)
<223> Leu (D cr L), Ile and Val or non-natural
derivatives of the aliphatic amino acid,
Phe, Tyr,
meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr,
di-halo-Tyr, O-sulpho-Tyr, 0-phospho-Tyr,
nitro-Tyr,
<220>
<221> PEPTIDE
<222> (29)..(30)
<223> Trp (D cr L), neo-Trp, halo-Trp (D or L)
or any
synthetic aromatic amino acid; Xaa at residue
30
is Ala, an aliphatic amino acids bearing
linear or
branched saturated hydrocarbon chains such
as
<220>
<221> PEPTIDE
<222> (30)
<223> Leu (D or L), Ile and Val or non-natural
derivati<.les of the aliphatic amino acid,
Tyr,
meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr,
di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr
or
nitro-Tyr
<220>
<221> PEPTIDE
<222> (32)
<223> Xaa at residue 32 is an aliphatic amino
acids
bearing linear or branched saturated hydrocarbon
chains such as Leu (D or L), Ile and Val
or
non-natural derivatives of the aliphatic
amino
acid;
<220>
<221> PEPTIDE
<222> (33)..(34)
<223> Xaa at residue 33 is des-Xaa, Asp, Glu,
Gla, Pro,
Hyp, Ser, Thr, g-Hyp, g-Ser, g-Ser or any
synthetic hydroxylated amino acid; Xaa
at residue
34 is des-Xaa, Glu, Gla, Gln, Asp, Asn,
any
synthetic
<220>
<221> PEPTIDE
<222> (34)
<223> acidic amino acid, Lys, Arg, ornithine,
homo-Lys,
homoarginine, nor-Lys, N-methyl-Lys,
N,N'-dimethyl-Lys, N,N',N " -trimethyl-Lys,
any
synthetic basic amino acid, Ile, Ser, Thr,
g-Ser
or g-Thr
<220>
<221> PEPTIDE
<222> (35)..(36)
<223> Xaa at residue 35 is des-Xaa, Pro, Hyp,
Tyr,
meta-Tyr, ortho-Tyr, nor-Tyr, mono-halo-Tyr,
di-halo-Tyr, 0-sulpho-Tyr, 0-phospho-Tyr
or
nitro-Tyr; Xaa at residue 36 is des-Xaa
or Phe
<400> 1
Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
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7
Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa
20 25 30
Xaa Xaa Xaa Xaa
35
<210> 2
<211> 31
<212> PRT
<213> Conus magus
<220>
<221> PEPTIDE
<222> (3)..(27)
<223> Xaa at residue 3 may be Arg or Ser; Xaa at
residues 12, 21 and 27 5 may be Pro or
hydroxy-Pro; Xaa at residue 14 may be Ile or Leu ;
Xaa at residue 17 may be Ile or Val
<900> 2
Ala Cys Xaa Lys Lys Trp Glu Tyr Cys Ile Val Xaa Ile Xaa Gly Phe
1 5 10 15
Xaa Tyr Cys Cys Xaa Gly Leu Ile Cys Gly Xaa Phe Val Cys Val
20 25 30
<210> 3
<211> 31
<212> PRT
<213> Conus skinneri
<220>
<221> PEPTIDE
<222> (5)..(18)
<223> Xaa at residue 5 is Lys, N-methyl-Lys,
N,N-dimethyl-Lys or N,N,N-trimethyl-Lys; Xaa at
residue 6, 16 and 18 may be Tyr, mono-halo-Tyr,
di-halo-Tyr, 0-sulpho-Tyr, O-phospho-Tyr or
nitro-Tyr.
<220>
<221> PEPTIDE
<222> (7)..(27)
<223> Xaa at residue 7 may be Glu or gamma-carboxy-Glu;
Xaa at residues 12 and 27 may be Pro or
hydroxy-Pro.
<400> 3
Ala Cys Arg Gln Xaa Xaa Xaa Phe Cys Leu Val Xaa Ile Ile Gly Xaa
1 5 10 15
Ile Xaa Cys Cys Ala Gly Leu Ile Cys Gly Xaa Phe Val Cys Leu
20 25 30
<210> 4
<211> 36
<212> PRT
<213> Conus tessulatus
<220>
<221> PEPTIDE
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WO 00/76532 PCT/US00/15779
8
<222> (1)..(34)
<223> Xaa at residue 1 may be Glu or gamma-carboxy-Glu;
Xaa at residues 5, 8 and 34 may be Lys,
N-methyl-Lys, N,N-dimethyl-Lys or
N,N,N-trimethyl-Lys.
<220>
<221> PEPTIDE
<222> (13)..(35)
<223> Xaa at residues 1, 33 and 35 may be Pro or
hydroxy-Pro ; Xaa at residue 19 may be Tyr,
mono-halo-Tyr, di-halo-Tyr, 0-sulpho-Tyr,
0-phospho-Tyr or nitro-Tyr.
<400> 4
Xaa Thr Cys Leu Xaa Gln Asp Xaa Phe Cys Ile Ile Xaa Leu Ile Gly
1 5 10 15
Thr Leu Xaa Cys Cys Ser Gly Leu Ile Cys Gly Phe Phe Val Cys Val
20 25 30
Xaa Xaa Xaa Phe
25
<210> 5
<211> 32
<212> PRT
<213> Conus caracteristicus
<220>
<221> PEPTIDE
<222> (3)..(31)
<223> Xaa at residues 3 and 31 may be Glu or
gamma-carboxy-Glu; Xaa at residues 12 and 30 may
be Pro or hydroxy-Pro; Xaa at residue i5 may be
Lys, N-methyl-Lys, N,N-dimethyl-Lys or
N,N,N-trimethyl-Lys.
<220>
<221> PEPTIDE
<222> (14)..(32)
<223> Xaa at residue 14 may be Trp or bromo-Trp; Xaa at
residue 32 may be Tyr, mono-halo-Tyr, di-halo-Tyr,
0-sulpho-Tyr, 0-phospho-Tyr or nitro-Tyr.
<400> 5
Asp Cys Xaa Ala Asp Gly Ala Phe Cys Gly Ile Xaa Ile Val Xaa Asn
1 5 10 15
Xaa Met Cys Cys Ser Asn Leu Cys Ile Phe Ala Cys Val Xaa Xaa Xaa
20 25 30
<210> 6
<211> 31
<212> PRT
<213> Conus textile
<220>
<221> PEPTIDE
<222> (4)..(21)
<223> Xaa at residues 4 and 21 is Glu or
gamma-carboxy-Glu; Xaa at residues 6 and 8 is Trp
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9
or halo-Trp; Xaa at residue 10 is Pro or
hydroxy-Pro; Xaa at residue 18 is Tyr,
mono-halo-Tyr, di-halo-Tyr,
<220>
<221> PEPTIDE
<222> (4)..(21)
<223> 0-sulpho-Tyr, O-phospho-Tyr or nitro-Tyr
<400> 6
Asp Cys His Xaa Arg Xaa Asp Xaa Cys Xaa Ala Ser Ile Leu Gly Val
1 5 10 15
Ile Xaa Cys Cys Xaa Gly Leu Ile Cys Phe Ile Ala Phe Cys Ile
20 25 30
<210> 7
<211> 33
<212> PRT
<213> Conus textile
<220>
<221> PEPTIDE
<222> (4)..(27)
<223> Xaa at residues 4 and 7 is Glu or
gamma-carboxy-Glu; Xaa at residue 5 isLys,
N-methy-Lys, N,N-dimethyl-Lys or
N,N,N-trimethyl-Lys; Xaa at residue 6 is Trp
or
halo-Trp; Xaa at residues 12, 21 and 27
<220>
<221> PEPTIDE
<222> (4)..(27)
<223> is Pro or hydroxy-Pro; Xaa at residue 18 isTyr,
mono-halo-Tyr, di-halo-Tyr, O-sulpho-Tyr,
O-phospho-Tyr or nitro-Tyr
<400> 7
Asp
Cys
Gln
Xaa
Xaa
Xaa
Xaa
Phe
Cys
Ile
Val
Xaa
Ile
Leu
G1y
Phe
1 .. 10 15
Val Xaa Cys Cys Xaa Gly Leu Ile Cys Gly Xaa Phe Val Cys Val Asp
20 25 30
Ile
<210> 8
<211> 31
<212> PRT
<213> Conus tessula~us
<220>
<221> PEPTIDE
<:222> (1) . . (18)
<223> Xaa at residues 1 and 13 is Pro or hydroxy-Pro;
Xaa at residues 6 and 18 is Tyr, mono-halo-Tyr,
di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or
nitro-Tyr
<400> 8
Xaa Thr Cys Val Ser Xaa Asn Val Phe Cys Gly Val Xaa Leu Val G1y
1 ., 10 15
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Thr Ile
Xaa
Leu
Cys
Cys
Ser
Gly
Leu
Val
Cys
Leu
Val
Val
Cys
20 25 30
5 <210> 9
<211> 27
<212> PRT
<213> Conus atlanticus
10 <220>
<221> PEPTIDE
<222> (3)..(21)
<223> Xaa at residues 3, 11, 13 and 15 is Pro
or
hydroxy-Pro; Xaa at residue 12 is Lys,
N-methy-Lys, N,N-dimethyl-Lys or
N,N,N-trimethyl-Lys; Xaa at residue 16
is Tyr,
mono-halo-Tyr, di-halo-Tyr,
<220>
<221> PEPTIDE
<222> (3)..(21)
<223> 0-sulpho-Tyr, 0-phospho-Tyr or nitro-Tyr;
Xaa at
residue 21 is Trp or halo-Trp
<400> 9
Cys Xaa
Arg Xaa
Xaa
Arg
Gly
Met
Phe
Cys
Gly
Phe
Xaa
Xaa
Xaa
Gly
1 5 10 15
Cys
Cys
Asn
Gly
Xaa
Cys
Phe
Phe
Val
Cys
Ile
20 25
<210> 10
<211> 30
<212> PRT
<213> Conus tessulatus
<220>
<221> PEPTIDE
<222> (2) . . (13)
<223> Xaa at residue 2 is Trp or halo-Trp; Xaa
at resude
8 is Glu or gamma-carboxy-Glu; Xaa 13 is
Pro or
hydroxy-Pro
<400> 10
Arg Ile
Xaa Gly
Cys
Ala
Leu
Asp
Gly
Xaa
Leu
Cys
Ile
Ile
Xaa
Val
1 5 10 15
Ser e Phe Cys Cys His Gly Ile Cys Met Ile Xaa
Il Cys Val
20 25 30
<210> 11
<211> 30
<212> PRT
<213> Conus arenatus
<220>
<221> PEPTIDE
<222> (4)..(17)
<223> Xaa at residues 4 and 12 is Pro or hydroxy-Pro;
Xaa at residues 8 and 13 is Tyr, mono-halo-Tyr,
di-halo-Tyr, O-sulpho-Tyr, 0-phospho-Tyr
or
nitro-Tyr; Xaa at residue i4 is Lys, N-methy-Lys,
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<220>
<221> PEPTIDE
<222> (4)..(17)
<223> N,N-dimethyl-Lys or N,N,N-trimethyl-Lys; Xaa at
residue 17 is Trp or halo-Trp
<400> 11
Asp Cys Arg Xaa Val Gly Gln Xaa Cys Gly Ile Xaa Xaa Xaa His Asn
1 5 10 15
Xaa Arg Cys Cys Ser Gln Leu Cys Ala Ile Ile Cys Val Ser
25 30
15 <210> 12
<211> 30
<212> PRT
<213> Conus generalis
20 <220>
<221> PEPTIDE
<222> (5)..(29)
<223> Xaa at residues 5 is Pro or hydroxy-Pro;
and 12
Xaa at residues 7 isTyr, mono-halo-Tyr,
and 17
di-halo-Tyr, O-sulpho-Tyr, O-phospho-Tyr or
nitro-Tyr; Xaa at 29 is Glu or
residue
gamma-carboxy-Glu
<400> 12
Gly Cys Gly Thr Xaa Phe Leu Gly
Cys Ala
Leu
Asp
Xaa
Gly
Xaa
Phe
1 5 10 15
Xaa Ile Val Cys Ile Xaa Thr
Cys
Cys
Gly
Gly
Ile
Cys
Leu
20 25 30
<210> 13
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of ArtificialSequence: amplification
primer
<400> 13
caggatccat 29
gaaactgacg
tgyrtggtg
<210> 14
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: amplification
primer
<400> 14
atctcgagca caggtatgga tgactcagg 29
<210> 15
<211> 424
<212> DNA
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<213> Conusskinneri
<220>
<221> CDS
<222> (1)..(264)
<400> 15
atg aaa acg tgt gtggtgatc gttgetgtgctg ttcttgacc gcc 48
ctg
Met Lys Thr Cys ValValIle ValAlaValLeu PheLeuThr Ala
Leu
1 5 10 15
tgg aca gtc atg getgatgac cccagagatgga gcggagatt aga 96
ttc
Trp Thr Val Met AlaAspAsp ProArgAspGly AlaGluIle Arg
Phe
20 25 30
agc atg agg ggg gaacctctg tcgaaggcacgt gacgaaatg aac 144
gta
Ser Met Arg Gly GluProLeu SerLysAlaArg AspGluMet Asn
Val
35 40 45
ccc gaa tct aaa ttggagaaa agggcgtgccgc caaaaatac gaa 192
gcc
Pro Glu Ser Lys LeuGluLys ArgAlaCysArg GlnLysTyr Glu
Ala
50 55 60
ttt tgt gta ccg atcattgga tacatatattgc tgcgetggc tta 240
cta
Phe Cys Val Pro IleIleGly TyrIleTyrCys CysAlaGly Leu
Leu
65 70 75 80
atc tgt cct ttc gtctgcctt tgatagtgat gtcttctact ccatctgtg 294
ggt g
Ile Cys Pro Phe ValCysLeu
Gly
8
5
ctacccctgg cttgatcttt gttgcccttc actggtttat gaaccctctg
354
gataggcgtt
atcatactct ctggaccctt catccaaata aagcgacatc ccaaaaaaaa
414
gggggtccaa
aaaaaaaaaa 424
<210>
16
<211>
88
<212> T
PR
<213> nusskinneri
Co
<400>
16
Met Lys LeuThrCys ValValI1e ValAlaVal LeuPheLeu ThrAla
1 5 10 15
Trp Thr PheValMet AlaAspAsp ProArgAsp GlyAlaGlu IleArg
20 25 30
Ser Met ValArgGly GluProLeu SerLysAla ArgAspGlu MetAsn
35 40 45
Pro G1u AlaSerLys LeuGluLys ArgA1aCys ArgGlnLys TyrGlu
50 55 60
Phe Cys LeuValPro IleIleGly TyrIleTyr CysCysAla GlyLeu
70 75 80
60 Ile Cys GlyProPhe ValCysLeu
85
<210> 17
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<211> 418
<212> DNA
<213> Conus tessulatus
<220>
<221> CDS
<222> (1)..(261)
<400> 17
gga tcc atg aaa ctg acg tgc atg gtg atc gtt gtt gtg ctg ttg ttg 48
Gly Ser Met Lys Leu Thr Cys Met Val Ile Val Val Val Leu Leu Leu
1 5 10 15
aac gcc tgg aca ttc gtc tcc ata aat gga aag gcg aat cgt ttt tgg 96
Asn Ala Trp Thr Phe Val Ser Ile Asn Gly Lys Ala Asn Arg Phe Trp
25 30
aag gca cgt gac gaa atg aag gac tcc gaa gtt tct gaa ttg gag aaa 144
Lys Ala Arg Asp Glu Met Lys Asp Ser Glu Val Ser Glu Leu Glu Lys
20 35 40 45
agg agg aaa ccg acc tgc ctg aag cag gac aag ttt tgc ata ata ccg 192
Arg Arg Lys Pro Thr Cys Leu Lys Gln Asp Lys Phe Cys Ile Ile Pro
50 55 60
ctc att gga acc ctt tat tgc tgc agt ggg tta atc tgt ggg ttt ttt 240
Leu Ile Gly Thr Leu Tyr Cys Cys Ser Gly Leu Ile Cys Gly Phe Phe
65 70 75 80
gtc tgc cca aag ccg ttc 291
gtc tgatgtcttc tactgccatc
tgtgctaccc
Val Cys Pro Lys Pro Phe
Val
85
ctggcttgat ctttgattgg cgtgtgcccttcactggttatgaacccctc tgatcctact351
gtctggacgc ctcgggcgtc caacgtccaaataaagcgacatcccaataa aaaaaaaaaa411
aaaaaaa 418
<210> 18
<211> 87
<212> PRT
<213> Conustessulatus
<400> 18
Gly Ser Lys Leu Thr Cys Val Ile Val Val Leu Leu
Met Met Val Leu
1 5 10 15
Asn Ala Thr Phe Val Ser Asn Gly Ala Asn Arg Phe
Trp Ile Lys Trp
20 25 30
Lys Ala Arg Asp Glu Met Lys Asp Ser Glu Val Ser Glu Leu Glu Lys
35 40 45
Arg Arg Lys Pro Thr Cys Leu Lys Gln Asp Lys Phe Cys Ile Ile Pro
50 55 60
Leu Ile Gly Thr Leu Tyr Cys Cys Ser Gly Leu Ile Cys Gly Phe Phe
65 70 75 80
Val Cys Val Pro Lys Pro Phe
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<210> 19
<211> 280
<212> DNA
<213> Conus caracterist icus
<220>
<221> CDS
<222> (1).. (249)
<400> 19
atg aaa ctg acg tgcgtgatgatc gttgetgtg ctgttcttgacc gcc 48
Met Lys Leu Thr CysValMetIle ValAlaVal LeuPheLeuThr Ala
1 5 10 15
tgg aca ttc gtc acggetgatgac tccattaat gcactggaggat ctt 96
Trp Thr Phe Val ThrAlaAspAsp SerIleAsn AlaLeuGluAsp Leu
20 25 30
ttt tcg aag gca cgtgacgaaatg gaaaacggc gaagettctaca ttg 144
Phe Ser Lys Ala ArgAspGluMet GluAsnGly GluAlaSerThr Leu
35 40 45
aac gag aga gac tgcgaagcagat ggtgcattt tgtggtatccca att 192
Asn Glu Arg Asp CysGluAlaAsp GlyAlaPhe CysGlyIlePro Ile
50 55 60
gtg aag aac tgg atgtgctgcagt aacttgtgt atttttgcctgc gta 240
Val Lys Asn Trp MetCysCysSer AsnLeuCys IlePheAlaCys Val
65 70 75 80
ccc gag tat taagactgcc gtgatgtctt c 280
ctcctcccct
Pro Glu Tyr
<210> 20
<211> 83
<212> PRT
<213> Conus caracteristi cus
<400> 20
Met Lys Leu ThrCys ValMetIle ValAlaVal LeuPheLeu ThrAla
1 5 10 15
Trp Thr Phe ValThr AlaAspAsp SerIleAsn AlaLeuGlu AspLeu
20 25 30
Phe Ser Lys AlaArg AspGluMet GluAsnGly GluAlaSer ThrLeu
35 40 45
Asn Glu Ara AspCys GluAlaAsp GlyA1aPhe CysGlyIle ProIle
50 55 60
Val Lys Asn TrpMet CysCysSer AsnLeuCys IlePheAla CysVal
65 70 75 80
Pro Glu Tyr
<210> 21
<211> 132
<212> DNA
<213> Conus textile
CA 02376357 2001-12-06
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<220>
<221> CDS
<222> (2)..(106)
5 <400> 21
a ttg gag aaa agg gat tgc cac gaa agg tgg gat tgg tgt cca gca tca 49
Leu Glu Lys Arg Asp Cys His Glu Arg Trp Asp Trp Cys Pro Ala Ser
1 5 10 15
10 atc ctt gga gtg ata tat tgc tgc gag gga tta att tgt ttt att gcc 97
Ile Leu Gly Val Ile Tyr Cys Cys Glu Gly Leu Ile Cys Phe Ile Ala
25 30
ttc tgc att tgatagtgat gtcttctcct cccctc 132
15 Phe Cys Ile
<210> 22
20 <211> 35
<212> PRT
<213> Conus textile
<400> 22
25 Leu Glu Lys Arg Asp Cys His Glu Arg Trp Asp Trp Cys Pro Ala Ser
1 5 10 15
Ile Leu Gly Val Ile Tyr Cys Cys Glu Gly Leu Ile Cys Phe Ile Ala
20 25 30
Phe Cys Ile
35 <210> 23
<211> 132
<212> DNA
<213> Conus textile
<220>
<221> CDS
<222> (2)..(112)
<40C> 23
a ttg gag aaa agg gat tgc caa gag aaa tgg gag ttt tgt ata gta ccg 49
Leu Glu Lys Arg Asp Cys Gln Glu Lys Trp Glu Phe Cys Ile Val Pro
1 5 10 15
atc ctt gga ttt gta tat tgc tgc cct ggc tta atc tgt ggc cct ttt 97
Ile Leu Gly Phe Val Tyr Cys Cys Pro Gly Leu Ile Cys Gly Pro Phe
20 25 30
gtc tgc gtt gat atc tgatgtcttc tcctcccatc 132
Val Cys Val Asp Ile
35
<210> 24
<211> 37
<212> PRT
<213> Conus textile
<400> 24
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Leu Glu Lys Arg Asp Cys Gln Glu Lys Trp Glu Phe Cys Ile Val Pro
1 5 10 15
Ile Leu Phe ValTyrCys CysProGly LeuIleCys GlyProPhe
Gly
20 25 30
Val Cys Asp Ile
Val
35
<210> 25
<211> 288
<212> DNA
<213> Conustessula tus
<220>
<221> CDS
<222> (7)..(246)
<400> 25
ggatcc atg aaa acgtgt gtg atc gttgttgtg ctgttgttg 48
ctg gtg
Met Lys eu ThrCys Val Ile ValValVal LeuLeu
L Val Leu
1 5 10
aac gcc aca ttcgtctcc ataaatgga aaggcgaat cctttttgg 96
tgg
Asn Ala Thr PheValSer IleAsnGly LysAlaAsn ProPheTrp
Trp
15 20 25 30
aag gca gac gaaatgaag gactccgaa gtttctgag ttggagaaa 144
cgt
Lys Ala Asp GluMetLys AspSerGlu ValSerGlu LeuGluLys
Arg
35 40 45
agg agg ccg acctgcgtg tcgtataac gtgttttgc ggagtaccg 192
aaa
Arg Arg Pro ThrCysVal SerTyrAsn ValPheCys GlyValPro
Lys
50 55 60
ctc gtt acc tacctttgc tgcagtggc ttagtctgt ctcgtagtc 240
gga
Leu Val Thr TyrLeuCys CysSerGly LeuValCys LeuValVal
Gly
65 70 75
tgc atc tactgat gtcttctact ctgtg acccctcgag 288
tag cccat ct
Cys Ile
80
<210> 26
<211> 80
<212> PRT
<213> Conus tessulatus
<4C0> 26
Met Lys Leu ThrCysVal ValIleVal ValValLeu LeuLeuAsn A1a
1 5 10 15
Trp Thr Phe ValSerI1e AsnGlyLys AlaAsnPro PheTrpLys Ala
20 25 30
Arg Asp Glu MetLysAsp SerGluVal SerGluLeu GluLysArg Arg
35 40 45
Lys Pro Thr CysValSer TyrAsnVal PheCysGly ValProLeu Val
50 55 60
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Gly Thr Tyr Leu Cys Cys Ser Gly Leu Val Cys Leu Val Val Cys Ile
65 70 75 80
<210> 27
<211> 287
<212> DNA
<213> Conus atlanticus
<220>
<221> CDS
<222> (7).. (240)
<400> 27
ggatcc atg gc 48
aaa ctg gtg
acg t gtg
atc
gtt
get
gtg
ctg
ttc
ttg
Met Lys L eu eu eu
Thr Phe
Cys L
Val
Val
Ile
Val
Ala
Val
L
1 5 10
acc gcc tgg acattc gtcacgget gatgactcc ataaatggg ttggag 96
Thr Ala Trp ThrPhe ValThrAla AspAspSer IleAsnGly LeuGlu
15 20 25 30
aat ctt ttt ccgaag gcacgtcac gaaatgagg aaacccgaa gcctct 144
Asn Leu Phe ProLys AlaArgHis GluMetArg LysProGlu AlaSer
35 40 45
aga tcg aga gggagg tgccgtcct cgtggtatg ttctgtggc tttccg 192
Arg Ser Arg GlyArg CysArgPro ArgGlyMet PheCysGly PhePro
50 55 60
aaa cct gga ccatac tgctgcaat ggctggtgc tttttcgtc tgcatc 240
Lys Pro Gly ProTyr CysCysAsn GlyTrpCys PhePheVal CysIle
65 70 75
taaaactgcc cctcgag 287
gtgatgtgtt
ctactcccat
ctgtgctacc
<210> 28
<211> 78
<212> PRT
<213> Conus atlanticus
<400> 28
Met Lys Leu ThrCys ValValIle ValAlaVal LeuPheLeu ThrAla
1 5 10 15
Trp Thr Phe ValThr AlaAspAsp SerIleAsn GlyLeuGlu AsnLeu
2C 25 30
Phe Pro Lys AlaArg HisGluMet ArgLysPro GluAlaSer ArgSer
35 40 45
Arg Gly Arg CysArg ProArgGly MetPheCys GlyPhePro LysPro
50 55 60
Gly Pro Tyr CysCys AsnGlyTrp CysPhePhe ValCysIle
70 75
60 <210> 29
<211> 419
<212> DNA
<213> Conus tessulatus
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18
<220>
<221> CDS
<222> (7)..(249)
<400> 29
ggatcc atg aaa acg gtggtg gttget gtg ttc ttg 48
ctg tgc gtc ctg
Met Lys Thr ValVal ValAla Val Phe Leu
Leu Cys Val Leu
1 5 10
aac gcc aca gcc getgtt tccaaa cat ctg gcg 96
tgg ttc acg gac gca
Asn Ala Thr Ala AlaVal SerLys His Leu Ala
Trp Phe Thr Asp Ala
20 25 30
aaa ctt atg gca gacgaa tataac ccc gcc act 144
ttt aag cgt atg gat
15 Lys Leu Met Ala AspGlu TyrAsn Pro Ala Thr
Phe Lys Arg Met Asp
35 40 45
aaa ttg gat aga tgcget gatggt gaa tgt atc 192
gac aag tgg tta ctt
Lys Leu Asp Arg CysAla AspGly Glu Cys Ile
Asp Lys Trp Leu Leu
50 55 60
ata ccg att tcc ttttgc catggc ata atg atc 240
gtc ggg ata tgc tgt
Ile Pro Ile Ser PheCys HisGly Ile Met Ile
Val Gly Ile Cys Cys
65 70 75
tac tgc tagttgaact tgatgt cttctactcc cctctgtgct 289
gtc gccg
Tyr Cys
Val
80
acccctggtt tgatctttga ttgccctgtg cccttcactgattatgaatccctctgatcc349
tactctctga agacctcttg gggtccaaca tccaaataaagcgacatcccaaaaaaaaaa409
aaaaaaaaaa 419
<210> 30
<211> 81
<212> PRT
<213> Conus tessulatus
<400> 30
Met Lys Leu ThrCys ValValValVal AlaValLeu PheLeu AsnAla
1 5 10 15
Trp Thr Phe AlaThr AlaValAspSer LysHisAla LeuAla LysLeu
20 25 30
Phe Met Lys AlaArg AspGluMetTyr AsnProAsp AlaThr LysLeu
35 40 45
Asp Asp Lys ArgTrp CysAlaLeuAsp GlyGluLeu CysIle IlePro
50 55 60
Val Ile Gly SerIle PheCysCysHis GlyIleCys MetIle TyrCys
70 75 80
Val
<210> 31
<211> 340
<212> DNA
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<213> Conusarenatus
<220>
<221> CDS
<222> (7)..(246)
<400> 31
ggatcc atg aaa ctg tgtgtg tg gtt tg ttc ttg 48
acg g atc get ctg
g
Met Lys Leu CysVal al Ile al eu Phe Leu
Thr V Val L
Ala
V
1 5 10
acc gcc aca ttc acgget gactccata cgtgcactggag gat 96
tgg gtc
Thr Ala Thr Phe ThrAla AspSerIle ArgAlaLeuGlu Asp
Trp Val
20 25 30
15'
ttt ttt aag gca gacgaa atggaaaac agcggagettct cca 144
gcg cgt
Phe Phe Lys Ala AspGlu MetGluAsn SerGlyAlaSer Pro
Ala Arg
35 40 45
ttg aac aga gac cgacct gtaggtcaa tattgtggcata ccg 192
gag tgc
Leu Asn Arg Asp ArgPro ValGlyGln TyrCysGlyIle Pro
Glu Cys
50 55 60
tat aag aac tgg tgctgc agtcagctt tgtgcaattatc tgt 240
cac cga
Tyr Lys Asn Trp CysCys SerGlnLeu CysAlaIleIle Cys
His Arg
65 70 75
gtt tcc cccctct ctactc tgaagac c ttcaacatccaaa 296
taa gatc tc tccggga
Val Ser
80
taaagcgaca tcccgatnaaaaaaangaaaaaaaa aaaaaaaa 340
a
<210> 32
<211> 80
<212> PRT
<213> Conusarenatus
<400> 32
Met Lys Thr Cys ValIle ValAlaVal LeuPheLeuThr Ala
Leu Val
1 5 10 15
Trp Thr Val Thr AspSer IleArgAla LeuGluAspPhe Phe
Phe Ala
20 25 30
Ala Lys Arg Asp MetGlu AsnSerGly AlaSerProLeu Asn
Ala Glu
35 40 45
Glu Arg Cys Arg ValGly GlnTyrCys GlyIleProTyr Lys
Asp Pro
50 55 60
His Asn Arg Cys SerGln LeuCysAla IleIleCysVal Ser
Trp Cys
65 70 75 80
<210> 33
<211> 293
<212> DNA
<213> Conusgeneralis
<220>
<221> CDS
<222> (7) (249)
. .
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<400>
33
ggatcc tg aaactg acgtgtgtg gtgatc ctattcttg 48
a gtt
get
gtg
Met LysLeu ThrCysVal I1e LeuPheLeu
Val Val
Ala
Val
1 5 10
5
acc gcctggacattc gtcacgget gatgac accaga tataaactggag 96
Thr AlaTrpThrPhe ValThrAla AspAsp ThrArg TyrLysLeuG1u
15 20 25 30
10 aat ccttttctgaag gcacgcaac gaactg cagaaa cacgaagcctct 144
Asn ProPheLeuLys AlaArgAsn GluLeu GlnLys HisGluAlaSer
35 40 45
caa ctgaacgagaga ggctgcctt gaccca ggttac ttctgtgggacg 192
15 Gln LeuAsnGluArg GlyCysLeu AspPro GlyTyr PheCysGlyThr
50 55 60
ccg tttcttggagca tactgctgc ggtggc atttgc cttattgtctgc 290
Pro PheLeuGlyAla TyrCysCys GlyGly IleCys LeuIleValCys
20 65 70 75
ata gaaacgtaaaggcttgatgtcttcta tcccatctg ccctcgag 293
c tgctac
Ile GluThr
80
<210>
34
<211>
81
<212> T
PR
<213> nus generalis
Co
<400>
34
Met Lys Leu ThrCysVal ValIleVal AlaValLeuPhe LeuThrAla
1 5 10 15
Trp Thr Phe ValThrAla AspAspThr ArgTyrLysLeu GluAsnPro
20 25 30
Phe Leu Lys AlaArgAsn GluLeuGln LysHisGluAla SerGlnLeu
35 40 45
Asn Glu Arg GlyCysLeu AspProGly TyrPheCysGly ThrProPhe
55 60
45 Leu Gly Ala TyrCysCys GlyGlyIle CysLeuIleVal CysIleGlu
65 70 75 80
Thr
