U.S. patent application number 15/437890 was filed with the patent office on 2018-05-31 for mu-conotoxin peptides and use thereof as a local anesthetic.
This patent application is currently assigned to CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE (C.N.R.S.). The applicant listed for this patent is Evelyne BENOIT, Philippe FAVREAU, Jordi MOLGO, Reto STOCKLIN. Invention is credited to Evelyne BENOIT, Philippe FAVREAU, Jordi MOLGO, Reto STOCKLIN.
Application Number | 20180148483 15/437890 |
Document ID | / |
Family ID | 37685889 |
Filed Date | 2018-05-31 |
United States Patent
Application |
20180148483 |
Kind Code |
A2 |
FAVREAU; Philippe ; et
al. |
May 31, 2018 |
Mu-Conotoxin Peptides And Use Thereof As A Local Anesthetic
Abstract
The present invention relates to novel mu-conotoxin peptides,
biologically active fragments thereof, combinations thereof and/or
variants thereof. The invention also relates to their use in
pharmaceutical composition for the treatment or prevention of pain,
and their use in the preparation of an anesthetic.
Inventors: |
FAVREAU; Philippe; (Amancy,
FR) ; BENOIT; Evelyne; (Gif-sur-Yvette, FR) ;
MOLGO; Jordi; (Gif-sur-Yvette, FR) ; STOCKLIN;
Reto; (Bernex/Geneve, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FAVREAU; Philippe
BENOIT; Evelyne
MOLGO; Jordi
STOCKLIN; Reto |
Amancy
Gif-sur-Yvette
Gif-sur-Yvette
Bernex/Geneve |
|
FR
FR
FR
CH |
|
|
Assignee: |
CENTRE NATIONAL DE LA RECHERCHE
SCIENTIFIQUE (C.N.R.S.)
PARIS CEDEX 16
FR
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170226166 A1 |
August 10, 2017 |
|
|
Family ID: |
37685889 |
Appl. No.: |
15/437890 |
Filed: |
February 21, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13739321 |
Jan 11, 2013 |
|
|
|
15437890 |
|
|
|
|
12084572 |
May 15, 2009 |
|
|
|
PCT/IB2006/003147 |
Nov 8, 2006 |
|
|
|
13739321 |
|
|
|
|
60734267 |
Nov 8, 2005 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 23/00 20180101;
A61K 38/00 20130101; C07K 14/43504 20130101; A61P 25/06 20180101;
A61P 23/02 20180101; A61K 38/1767 20130101; A61P 25/04 20180101;
A61P 43/00 20180101; A61P 29/00 20180101 |
International
Class: |
C07K 14/435 20060101
C07K014/435 |
Claims
1-28. (canceled)
29. An isolated and purified nucleic acid sequence comprising i) a
nucleotide sequence encoding a mu-conotoxin peptide consisting of
the amino acid sequence:
Xaa1-Xaa2-Cys-Cys-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Cys-Xaa8-Xaa9-Xaa10-Xaa11-Cys--
Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Cys-Cys-Xaa17 [SEQ ID NO:1], wherein
Xaa1 is any N-modified amino acid, Xaa2 is glycine, Xaa3 is absent
or is any acidic amino acid or any of its amide form, Xaa4 is
absent or is glycine, Xaa5 is absent or is proline or
hydroxy-proline, Xaa6 is absent or is any basic amino acid, Xaa7 is
absent or is glycine, Xaa8 is absent or is any non-aromatic
hydroxyl amino acid, Xaa9 is absent or is any non-aromatic hydroxyl
amino acid, Xaa10 is absent or is any basic amino acid, Xaa11 is
absent or is any aromatic amino acid, Xaa12 is absent or is any
basic amino acid, Xaa13 is absent or is any acidic amino acid or
any of its amide form, Xaa14 is absent or is any basic amino acid,
or any sulfur-containing amino acid, Xaa15 is absent or is any
hydrophobic or apolar amino acid, or any non-aromatic hydroxyl
amino acid, Xaa16 is absent or is any basic amino acid, Xaa17 is
absent or is any apolar amino acid, or an amide group, ii) a
nucleic acid sequence complementary to i), iii) a degenerated
nucleic acid sequence of i) or ii), iv) a nucleic acid sequence
capable of hybridizing under stringent conditions to or iii), v) a
nucleic acid sequence encoding a truncation or an analog of the
mu-conotoxin peptide, vi) and/or a fragment of i), ii), iii), or v)
encoding a biologically active fragment of the mu-conotoxin
peptide.
30. A vector comprising the isolated and purified nucleic acid
sequence of claim 29.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application is a divisional of U.S. patent
application Ser. No. 13/739,321, filed 11 Jan. 2013, which is a
continuation of U.S. patent application No. 12/084,572, filed on 15
May 2009, which is a National Stage Entry of International Patent
Application No. PCT/IB2006/003147, filed on 8 Nov. 2006, which
claims the benefit of U.S. Provisional Application No. 60/734,267
filed 8 Nov. 2005. The entire disclosures of each of the
above-recited applications are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to novel mu-conotoxin
peptides, biologically active fragments thereof, salts thereof,
combinations thereof and for variants thereof. The invention also
relates to their use in pharmaceutical composition for the
treatment or prevention of pain, and their use in the preparation
of an anesthetic.
BACKGROUND OF THE INVENTION
[0003] Venoms of the marine cone snail of the genus Conus are a
rich and extremely diverse source of bioactive components. With
more than 800 species of Conus available worldwide, cone snail
venoms appear as one of the richest source of naturally occurring
peptides exhibiting a wide array of biological activity. The
conopeptides target numerous and various molecular entities
including voltage-sensitive ion channels, ligand-gated ion channels
and G-protein-coupled receptors, with high affinity and specificity
(McIntosh et al., 1999; Olivera et al., 1985; Olivera et al.,
1990). Among all existing conopeptides, only a minority has been
extensively characterized from isolation, primary structure
elucidation to precise molecular target identification. However,
increasing attention has been brought to this research area as
conopeptides provide new and important tools for dissecting the
function of previously uncharacterised channels. This also allows
opportunities for entirely new biomedical application with the use
of new drugs acting on original physiological targets. This can be
exemplified by the discovery and use of omega-conotoxins for
differentiating particular calcium subtypes and the further use of
one of them as a drug (Prialt.RTM.) in pain management (Kerr and
Yoshikami, 1984; Olivera et al., 1984; Olivera et al., 1987).
[0004] The publication of the first representatives of the
mu-conopeptide family occurred in 1983 with the characterization of
the geographutoxins exhibiting a myotoxic activity (Sato et al.,
1983). This was followed by the isolation and identification of
several other mu-conopeptides since then. To date, a total of 9
mu-conopeptides have been so far characterized from 6 different
cone snail species, including mainly piscivorous species and one
molluscivorous species.
[0005] All these mu-conopeptides display a common primary structure
demonstrated by the conserved position of the cysteine residues in
the sequence. The disulfide bonding is between Cys1-Cys4, Cys2-Cys5
and Cys3-Cys6. This fold leads to a constrained tertiary structure
that has been studied for several representatives of the
mu-conopeptide family (Hill et al., 1996; Keizer et al., 2003;
Nielsen et al., 2002; Ott et al., 1991; Wakamatsu et al., 1992). It
has been demonstrated in numerous studies that the mu-conopeptides
target more or less specifically various voltage-sensitive sodium
channels (Becker et al., 1989; Bulaj et al., 2005; Cruz et al.,
1985; Cruz et al., 1989; Fainzilber et al., 1995; French et al.,
1996; Safo et al., 2000; Sato et al., 1991; West et al., 2002).
Whatever the subtype of sodium channels targeted, the
pharmacological effect always consists in a blockade of the channel
conductance leading to an inhibition of the voltage-sensitive
channel functionality.
[0006] Voltage-sensitive sodium channels (VSSCs) are transmembrane
proteins fundamental for cell communication as they generate action
potentials and enable its propagation in most vertebrate and
invertebrate excitable cells. Presently 9 genes have been
identified that code for mammalian VSSCs (Yu and Catterall, 2003).
VSSCs are classified according to their sensitivity to tetrodotoxin
(TTX), a toxin isolated in particular from the puffer fish. VSSCs
blocked by TTX are known as TTX-sensitive, while the others are
TTX-resistant channels. Each subtype of VSSC has a specialised
function depending on its cellular and tissue localization.
[0007] VSSCs have a major role in the transmission of the action
potential in muscles as well as in nerves, thus providing a key
target in anaesthesia. Drugs such as lidocaine or procaine act
through the inhibition of VSSCs present in sensory fibres (Scholz,
2002). However, inhibition does not occur equally in all fibres due
to the presence of numerous VSSCs subtypes differently affected by
the drugs. Among them, TTX-resistant VSSC subtypes have a
predominant role in the transmission of pain and are currently not
specifically targeted by any known drug. Furthermore, the short
duration of time of lidocaine and procaine as well as the
well-documented side-reactions or allergy in response to their
application make them difficult to use as anaesthesics in specific
cases. In this context, compounds allowing specific inhibition of
TTX-resistant VSSCs would appear as a major achievement for pain
control. As an example, the subtype Nav1.8 contributes to the
initiation and maintenance of hyperalgesia. In early stages of
neuropathic pain, the expression of Nav1.8 is reduced in the
primary afferent neurones which are injured, while expression
levels of Nav1.8 are maintained in adjacent neurones (Decosterd et
al., 2002; Gold et al., 2003). However, two days following sciatic
nerve injury there is a significant upregulation of Nav1.8
expression as well as a proportional increase in the TTX-resistant
compound action potential, at a conduction velocity consistent with
C fibres (Gold et al., 2003). This strongly supports an important
role for Nav1.8 in neuropathic pain.
[0008] The VSSCs thus represent useful targets which inhibition or
modulation allow anaesthesia, analgesia and pain control (Baker and
Wood, 2001; Julius and Basbaum, 2001; Lee, 1976).
[0009] A large number of peptides as isolated mu-conotoxins are
known from Patent Application WO 02/07678 (University of Utah
Research Foundation and Cognetix, Inc.). However, this document
provides an ambiguous and at times misleading description of the
peptides so that it is difficult to rely on its disclosures. For
the large part, most of the peptides described therein appear to
have been only identified by molecular biology techniques, by the
isolation and cloning of DNA coding for mu-conotoxin peptides,
translating and determining the toxin sequence. Reliance only on
such techniques can cause errors, since in nature the active amino
acid residues may result from posttranslational modification of the
encoded peptide, some which can not be directly discovered from the
nucleotide sequence.
[0010] Recently, Patent Application WO 2004/0099238 (The University
of Queensland) also disclosed novel mu-conotoxin peptides and
derivatives thereof with their use as neuronally active sodium
channel inhibitors (antagonists), in assays and probes and also in
the treatment of conditions involving pain, cancer, epilepsy and
cardiovascular diseases. This application also disclosed the use of
these novel muconotoxin peptides in radio-ligand binding assays
(RLB). It will be appreciated by a skilled person in the art that
these results do not imply any biological activity of the
mu-conotoxins but only a binding effect since it is known from the
literature that some compounds (including a conotoxin) bind to
their channel/receptor site without any biological activity
(Fainzilber et al., 1994; Shichor et al., 1996). Moreover, potency
indicated in these binding experiments would not be relevant to
inhibitory potency in vitro or in vivo, thus leaving the reader
ignorant of any potential biological inhibitory potency.
Furthermore, the only inhibitory activity (IC50) on the expressed
VSSCs channels mentioned are superior to 1 to 3 .mu.M thus
suggesting an even higher concentration for use in ex vivo or in
vivo preparation.
[0011] Thus novel compounds with potent and long-lasting biological
activity for application as anesthetics which have a good safety
profile, only low or no side effects and the possibility to
retreat, whenever necessary are still needed.
[0012] This object has been achieved by providing novel
mu-conotoxin peptides, a biologically active fragment thereof, a
salt thereof, a combination thereof and/or variants thereof. The
peptides of the invention, which present a long duration of
effects, can be useful in the preparation of an anesthetic and in
the treatment of a pain.
SUMMARY OF THE INVENTION
[0013] The invention provides a mu-conotoxin peptide essentially
comprising the amino acid sequence:
Xaa1-Xaa2-Cys-Cys-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Cys-Xaa8-Xaa9-Xaa10-Xaa11-Cys--
Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Cys-Cys-Xaa17 [SEQ ID No 1], a
biologically active fragment thereof, a combination thereof and/or
variants thereof.
[0014] Furthermore, the invention provides an isolated and purified
nucleic acid sequence comprising a nucleotide sequence encoding the
amino sequence of the peptide of the invention.
[0015] The invention further provides a pharmaceutical composition
comprising as an active substance a pharmaceutically effective
amount of at least one peptide according to the invention and the
use of said pharmaceutical composition, for the preparation of a
medicament for the treatment or prevention of a disease associated
with voltage-sensitive sodium channels.
[0016] The invention also provides the use of the pharmaceutical
composition of the invention in the preparation of an anesthetic
and its use in a method for providing musculoskeletal relaxation in
a patient undergoing a surgical procedure, requiring
anesthesia.
[0017] Another aspect of the present invention relates to a method
for the treatment or prevention of a pain.
DESCRIPTION OF THE FIGURES
[0018] FIG. 1 represents an electrospray ionization mass spectrum
of the native mu-conopeptide CnIIIA. Mass measurement was carried
out on a QTOF I mass spectrometer in positive ion mode and TOF-MS
configuration. The mass indicated is the measured monoisotopic
molecular mass. A potassium adduct of CnIIIA can be noticed.
[0019] FIG. 2 depicts the control of the identity of the synthetic
and native mu-conopeptide CnIIIA. (a) Co-elution experiments by
reverse-phase HPLC of synthetic, native and 50:50 mixture of both
peptides. (B) MS/MS of the reduced synthetic CnIIIA (up) along with
the reduced native CnIIIA (down), showing identical fragmentation
behaviour.
[0020] FIG. 3 shows the effect of mu-conopeptide CnIIIA on the
mouse hemidiaphragm contraction. (a) Effect of CnIIIA on the
muscular contraction provoked by the direct stimulation of the
mouse hemidiaphragm. Traces of contraction recorded in the absence
and in the presence of 100 to 600 nM of CnIIIA. (B) Dose-response
curve of the effect of CnIIIA on the contraction. For each
concentration of CnIIIA, the maximal amplitude of the contraction
is expressed on the basis of the control value. The theoretical
curve was established from the equation indicated, the Hill number
(n.sub.H) being 1.78 and the CnIIIA concentration necessary for 50%
inhibition of the contraction (K.sub.D) being 150 nM. Mean
value.+-.SEM of n experiments.
[0021] FIG. 4 shows the effects of mu-conopeptide CnIIIA on the
action potential and the synaptic responses recorded at the frog
Cutaneous pectoris muscle preparation. (A) Effects of CnIIIA on the
muscular action potential recorded at the frog Cutaneous pectoris
muscle: action potential traces and EPP are recorded in response to
motor nerve stimulation, before and at different time points after
application of 1 .mu.M of CnIIIA to the bathing solution. A
progressive block of the muscular action potential can be noticed.
(B) Effects of CnIIIA on the synaptic responses recorded at the
frog Cutaneous pectoris muscle: average traces of MEPPs recorded in
the absence and presence of 1 and 2 .mu.M of CnIIIA.
[0022] FIG. 5 represents the effect of mu-conotoxin CnIIIA on the
global action potential (GAP) of sciatic nerves isolated from mice.
(A) GAP records in response to 0.05 ms stimulations at intensities
that vary between 0.1 and 15 V in control conditions (no toxin
added) and when nerves are treated with various concentrations of
conotoxin CnIIIA (0.1 to 50 .mu.M). (B) Amplitude of GAP in
response to different intensities of 0.05 ms stimulations and to
different concentrations of the CnIIIA toxin (left panel). (C) This
table summarize the different parameters of the GAP recorded after
0.05 ms stimulations at various intensities (0.1 to 15 V).
.sup.(1)Ratio between the maximum amplitude recorded after a 15 V
stimulation with or without mu-conotoxin. .sup.(2)Intensity of
stimulation corresponding to 50% of the maximal amplitude following
a 15 V-stimulation. Mean value.+-.SEM of n sciatic nerves.
[0023] FIG. 6 represents the effect of mu-conotoxin CnIIIA on the
GAP of mice sciatic nerves, recorded in the presence of various
concentrations (0.1 to 50 .mu.M) of conotoxin. The maximal
amplitudes of GAP, recorded at various concentrations (0.1 to 100
.mu.M) of toxin, were expressed according to the control value. The
theoretical curve was calculated according to the following
equation: A.sub.CnIIIA/A.sub.C=1/[1+([mu-conotoxin CnIIIA]/K.sub.D)
n.sub.H]. The Hill number (n.sub.H) was 1.02 and the toxin
concentration required to block 50% of the GAP (K.sub.D) was 1.53
.mu.M. Mean value.+-.SEM of n sciatic nerves.
[0024] FIG. 7 depicts the study of the reversibility of the
mu-conotoxin CnIIIA effect on the GAP of sciatic nerves isolated
from mice. The GAP were recorded following a 0.05 ms stimulation at
various intensities (0.1 to 15 V) in control conditions versus
nerves treated with 2, 10 or 50 .mu.M of CnIIIA toxin or washed
during 16 h or 24 h in fresh mammalian Ringer's solution. Mean
values.+-.SEM of n sciatic nerves.
[0025] FIG. 8 shows the effect of mu-conotoxin CnIIIA on the global
action potential (GAP) of olfactory nerves isolated from the
European pike (Esox lucius). (A) GAP records in response to 8 ms
stimulations at intensities that vary between 1 and 15 V in control
conditions (no toxin added) and when nerves are treated with
various concentrations of conotoxin CnIIIA (0.02 to 1 .mu.M). (B)
Amplitude of GAP in response changes according to different
intensities of 8 ms stimulations and to different concentrations of
the CnIIIA toxin with different durations of contact (see the left
panel). (C) This table summarize the different parameters of the
GAP recorded after a 8 ms stimulation at various intensities (1 to
15 V). .sup.(1)Ratio between the maximum amplitude recorded after a
15 V stimulation with or without mu-conotoxin. .sup.(2)Intensity of
stimulation corresponding to 50% of the maximal amplitude following
a 15 V-stimulation. Mean value.+-.SEM of n olfactory nerves.
[0026] FIG. 9 shows the effect of mu-conotoxin CnIIIA on the GAP of
olfactory nerves isolated from the European pike (Esox lucius)
recorded in the presence of various concentrations (0.01 to 10
.mu.M) of conotoxin, and expressed relatively to control values.
The maximal amplitudes of GAP, recorded at various concentrations
(0.1 to 10 .mu.M) of toxin, were expressed according to the control
value. The theoretical curve was calculated according to the
following equation: A.sub.CnIIIA/A.sub.C=1/[1+([mu-conotoxin
CnIIIA]/K.sub.D) n.sub.H]. The Hill number (n.sub.H) was 1.09 and
the toxin concentration required to block 50% of the GAP (K.sub.D)
was 0.15 .mu.M. Mean value.+-.SEM of n olfactory nerves.
[0027] FIG. 10 shows the surface anaesthetic effect of
.mu.-conotoxin CnIIIA and its comparison to that of lidocaine. The
intensity of the anaesthetic effect is expressed as the total
number of stimuli that fail to induce the oculo-palpebral reflex
with each concentration tested. Data represent the mean
values.+-.S.E.M. of 6 different determinations.
[0028] FIG. 11 shows the Digit Abduction Score (DAS) obtained in
vivo on mice.
[0029] FIG. 12 shows the grip strength assessment obtained in vivo
on mice.
[0030] FIG. 13 shows the relative mean contraction inhibition of
the muscle measured for each peptide (100 nM) by comparison to
CnIIIA (100 nM) after 40 min. incubation. CnIIIA has been
normalized to 100% for easy comparison. All peptides SmIIIA, PIIIA
and T3.1 display a lower activity than CnIIIA.
[0031] FIG. 14 A) shows Nav1.4 current traces in function of time
in the presence of 500 nM of CnIIIA. B) Nav1.4 current/Nav1.4 max.
current is plotted over time with a CnIIIA concentration of 500 nM.
The currents have been normalised to 1. The holding potential was
-90 mV and test potential -10 mV.
[0032] FIG. 15 A) shows the sodium currents recorded in HEK cells
as a control. B) in the presence of 50 nM CnIIIA and C) the sodium
current inhibition by CnIIIA is shown in function of time. Note
that the washing step after the steady-state level is not efficient
in suppressing the blocking effect.
DETAILED DESCRIPTION OF THE INVENTION
[0033] As used herein, "a" or "an" means "at least one" or "one or
more."
[0034] The terms "peptide", "protein", "polypeptide",
"polypeptidic" and "peptidic", as used herein, are used
interchangeably to designate a series of amino acid residues
connected to the other by peptide bonds between the alpha-amino and
carboxy groups of adjacent residues.
[0035] As used herein, the term "comprise" is generally used in the
sense of include, that is to say permitting the presence of one or
more features or components.
[0036] Conopeptides is an alternative term interchangeable with
conotoxins and conotoxin peptides. The conotoxins are some of the
most potent and diverse neurotoxins known, having an incredibly
wide range of actions. Interestingly, a strong division exists not
only between the mollusk eating and the fish eating species but
also between species within a group or even individuals of the same
species. The toxins from the fish hunting cone snails are also more
bioactive upon the human system than the mollusc hunting cone
snails, with deaths having occurred.
[0037] Three main classes of paralytic toxins have been the focus
of intense investigation where all three interfere with neuronal
communication but with different targets: alpha-conotoxins
(.alpha.-conotoxins), binding to and inhibiting the nicotinic
acetylcholine receptor; mu-conotoxins (.mu.-conotoxins), directly
abolishing muscle action potential by binding to the postsynaptic
sodium channels; and omega-conotoxins (.omega.-conotoxins),
decimating the release of acetylcholine through the prevention of
voltage activated entry of calcium into the nerve terminal.
[0038] Mu-conopeptides are isolated from the venoms of marine cone
snails of the genus Conus. The primary structure of mu-conopeptides
is organized with 15-30 amino acid folded by three disulfide
bridges. These peptides target a variety of voltage-sensitive
sodium channels that may be present either in muscles or in the
nervous system. A number of the members of the mu-conopeptide class
have been identified and their sequences published. GIIIA, GIIIB
and GIIIC from C. geographus venom are potent blockers of skeletal
muscle, but not neuronal VSSCs (Cruz et al., 1985). PIIIA from C.
purpurescens was found to inhibit muscle and to a lesser extent
neuronal TTX-Sensitive VSSCs (Shon et al., 1998).
[0039] Unfortunately, these conotoxins are not particularly potent
at neuronal VSSCs and are either selective for skeletal muscle
VSSCs (GIIIA, GIIIB and GIIIC) or are not able to discriminate
between skeletal muscle and neuronal VSSC subtypes (PIIIA).
Furthermore, it has been demonstrated that these peptides lack
three-dimensional (3D) structural stability and are prone to
conformational exchange in solution (Nielsen et al., 2002). In the
course of identifying and characterizing new conopeptides,
Applicants have shown that a family of mu-conopeptides revealed
extremely potent properties as a blocking agent of the mammalian
neuromuscular junction as well as a powerful inhibitor of the
action potential in motor neurons as well as in sensory neurons.
This new mu-conotoxin peptide essentially comprises the amino acid
sequence:
Xaa1-Xaa2-Cys-Cys-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Cys-Xaa8-Xaa9-Xaa10-Xaa11-Cys--
Xaa12-Xaa13-Xaa14-Xaa15-Xaa16-Cys-Cys-Xaa17 [SEQ ID No 1], a
biologically active fragment thereof, a salt thereof, a combination
thereof and/or variants thereof, and wherein Cys represents a
cystein. Usually, Xaa1 is any N-modified acidic amino acid.
Preferably, this modification is selected from the group comprising
acetylation, formylation, myristoylation or pyrrolidone.
Formylation applies to methionine (N-formylmethionine). Acetylation
applies to many residues including methionine (N-acetylmethionine),
threonine (N-acetylthreonine,), serine (N-acetylserine), aspartic
acid (N-acetylaspartate), glutamic acid (N-acetylglutamate),
glycine, valine and alanine. Myristoylation applies to
N-myristoylglycine
[0040] Most preferably the acidic amino acid modified is
pyroglutamate (pGlu or Z).
[0041] Xaa2 is preferably a glycine (Gly).
[0042] Xaa3 is any acidic amino acid or any of its amide form.
Preferably, Xaa3 is an asparagine (Asn).
[0043] Xaa4 is usually a glycine (Gly).
[0044] Xaa5 is usually a proline or an hydroxyl-proline.
[0045] Xaa6 is any basic amino acid. Preferably Xaa6 is lysine
(Lys).
[0046] Xaa7 is usually a glycine (Gly).
[0047] Xaa8 is any non-aromatic hydroxylamino acid. Preferably,
Xaa8 is a serine (Ser).
[0048] Xaa9 is any non-aromatic hydroxylamino acid. Preferably,
Xaa8 is a serine (Ser).
[0049] Xaa10 is any basic amino acid. Preferably, Xaa10 is a lysine
(Lys).
[0050] Xaa11 is any aromatic amino acid. Preferably, Xaa11 is a
tryptophan (Trp).
[0051] Xaa12 is any basic amino acid. Preferably, Xaa12 is an
arginine (Arg).
[0052] Xaa13 is any acidic amino acid or any of its amide form.
Preferably, Xaa13 is an aspartic acid (Asp).
[0053] Xaa14 is any basic amino acid or any sulfur-containing amino
acid. Preferably, Xaa14 is a methionin (Met) or a histidine
(His).
[0054] Xaa15 is any hydrophobic or apolar amino acid, or any
non-aromatic hydroxyl amino acid. Preferably Xaa15 is an alanine
(Ala).
[0055] Xaa16 is any basic amino acid. Preferably, Xaa16 is an
arginine (Arg).
[0056] Xaa17 is apolar amino acid, or an amide group. Xaa17 may
also be absent.
[0057] Optionally, in the mu-conotoxin 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), Cys/(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 native Cys bridges with lactam
bridges. Thioether analogs may be readily synthesized using
halo-Ala residues commercially available from RSP Amino Acid
Analogues.
[0058] The present invention also relates to a mu-conotoxin wherein
at least one amino acid consisting of amino acids Xaa3, Xaa4, Xaa5,
Xaa6 and Xaa7, or any combination thereof, is absent (group 1).
[0059] Also envisioned is a mu-conotoxin wherein at least at least
one amino acid consisting of amino acids Xaa8, Xaa9, Xaa10 and
Xaa11, or any combination thereof, is absent (group 2).
[0060] Further completed is a mu-conotoxin peptide of the
invention, wherein at least one amino acid consisting of amino
acids Xaa12, Xaa13, Xaa14, Xaa15 and Xaa16, or any combination
thereof, is absent (group 3).
[0061] Alternatively, the three above amino acids from group 1, 2
or 3, or combinations thereof, may be absent in the same
mu-conotoxin peptide of the invention.
[0062] Exemplary hydrophobic amino acids with aliphatic R-groups
include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu)
and isoleucine (Ile).
[0063] Exemplary amino acids with non-aromatic hydroxyl include
serine (Ser) and threonine (Thr).
[0064] Exemplary sulfur-containing amino acids include cysteine
(Cys) and methionine (Met).
[0065] Exemplary acidic amino acids and their amide forms include
aspartic acid (Asp), asparagine (Asn), glutamic acid (Glu),
glutamine (Gln) and pyroglutamic acid (pGlu).
[0066] Exemplary basic amino acids include arginine (Arg), lysine
(Lys) and histidine (His).
[0067] Exemplary aromatic amino acids include phenylalanine (Phe),
tyrosine (Tyr) and tryptophane (Trp).
[0068] Exemplary of imino acids include, for example, Proline (Pro)
and Hydroxyproline (Hyp or Hpro or O).
[0069] The present invention also considers a "biologically active
fragment" of the mu-conotoxin peptide, which refers to a sequence
containing less amino acids in length than the sequence of the
peptide. This sequence can be used as long as it exhibits
essentially the same properties or biological activity as the
native sequence from which it derives. Preferably this sequence
contains less than 99%, preferably less than 90%, in particular
less than 60% and more particularly less than 30% of amino acids in
length than the respective sequence of the peptide of the
invention.
[0070] Also envisioned is a salt of the mu-conotoxin peptide of the
invention, such as acid addition salts or metal complexes, e.g.,
with zinc, iron or the like (which are considered as salts for
purposes of this application). Illustrative of such acid addition
salts are hydrochloride, hydrobromide, sulphate, phosphate,
maleate, acetate, citrate, benzoate, succinate, malate, ascorbate,
tartrate and the like.
[0071] Further encompassed in the present invention is a "prodrug"
which is an entity representing an inactive form of an active
mu-conotoxin peptide of the invention. In other words, the
invention concerns a stable and soluble peptidic folding precursor
(composition) which has the potential of producing a desired
physiological effect on cells, but is initially inert (i.e. does
not produce said effect), and only after undergoing some
modifications becomes physiologically active and produces said
physiological effect on cells i.e. becomes pharmaceutically active
after biotransformation.
[0072] Biotransformation of the mu-conotoxin peptide may be carried
out under physiological conditions (in vitro and in vivo) and is a
result of a reaction with an enzyme, or a body fluid such as
gastric acid, blood etc., thus undergoing an enzymatic oxidation,
reduction, hydrolysis etc. or a chemical hydrolysis to convert into
the active compound by acyl migration reaction.
[0073] The present invention also includes a variant of the
mu-conotoxin peptide of the invention. The term "variant" refers to
a peptide having an amino acid sequence that differ to some extent
from a native sequence peptide, that is an amino acid sequence that
vary from the native sequence by conservative amino acid
substitutions, whereby one or more amino acids are substituted by
another with same characteristics and conformational roles. The
amino acid sequence variants possess substitutions, deletions,
side-chain modifications and/or insertions at certain positions
within the amino acid sequence of the native amino acid sequence.
Conservative amino acid substitutions are herein defined as
exchanges within one of the following five groups: [0074] I. Small
aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro,
Gly [0075] II. Polar, positively charged residues: His, Arg, Lys
[0076] III. Polar, negatively charged residues: and their amides:
Asp, Asn, Glu, Gln [0077] IV. Large, aromatic residues: Phe, Tyr,
Trp [0078] V. Large, aliphatic, nonpolar residues: Met, Leu, Ile,
Val, Cys.
[0079] It is to be understood that some non-conventional amino
acids may also be suitable replacements for the naturally occurring
amino acids. For example Lys residues may be substituted by
ornithine, homoarginine, nor-Lys, N-methyl-Lys, N,N-dimethyl-Lys
and N,N,N-trimethyl-Lys. Lys residues can also be replaced with
synthetic basic amino acids including, but not limited to,
N-1-(2-pyrazolinyl)-Arg, 2-(4-piperinyl)-Gly, 2-(4-piperinyl)-Ala,
2-[3-(2S) pyrrolininyl]-Gly and 2-[3-(2S)pyrrolininyl]-Ala. Tyr
residues may be substituted with 4-methoxy tyrosine (MeY),
meta-Tyr, ortho-Tyr, nor-Tyr, 1251-Tyr, mono-halo-Tyr, di-halo-Tyr,
O-sulpho-Tyr, O-phospho-Tyr, and nitro-Tyr.
[0080] Tyr residues may also be substituted with the 3-hydroxyl or
2-hydroxyl isomers (meta-Tyr or ortho-Tyr, respectively) and
corresponding O-sulpho- and O-phospho derivatives. Tyr residues can
also be replaced with synthetic hydroxyl containing amino acids
including, but not limited to 4-hydroxymethyl-Phe,
4-hydroxyphenyl-Gly, 2,6-dimethyl-Tyr and 5-amino-Tyr. Aliphatic
amino acids may be substituted by synthetic derivatives bearing
non-natural aliphatic branched or linear side chains CnH2n+2 where
n is a number from 1 up to and including 8. Examples of suitable
conservative substitutions by non-conventional amino acids are
given in WO2004/0099238 (see Table 1).
TABLE-US-00001 TABLE 1 Non-conventional amino acid Code
Non-conventional amino acid Code L-.alpha.-aminobutyric acid Abu
L-.alpha.-methylhistidine Mhis .alpha.-amino-.alpha.-methylbutyrate
Mgabu L-.alpha.-methylisoleucine Mile aminocyclopropane- Cpro
L-.alpha.-methylleucine Mleu carboxylate L-.alpha.-methylmethionine
Mmet aminoisobutyric acid Aib L-.alpha.-methylnorvaline Mnva
aminonorbornyl- Norb L-.alpha.-methylphenylalanine Mphe carboxylate
L-.alpha.-methylserine Mser cyclohexylalanine Chexa
L-.alpha.-methyltryptophan Mtrp cyclopentylalanine Cpen
L-.alpha.-methylvaline Mval D-alanine DAla N-(N-(2,2-diphenylethyl)
Nnbhm D-arginine DArg carbamylmethylglycine D-asparagine DAsn
1-carboxy-1-(2,2-diphenyl- Nmbc D-aspartic acid DAsp
ethylamino)cyclopropane D-cysteine DCys L-N-methylalanine Nmala
D-glutamine DGln L-N-methylarginine Nmarg D-glutamic acid DGlu
L-N-methylaspartic acid Nmasp D-histidine DHis L-N-methylcysteine
Nmcys D-isoleucine DIle L-N-methylglutamine Nmgln D-leucine DLeu
L-N-methylglutamic acid Nmglu D-lysine DLys L-N-methylhistidine
Nmhis D-methionine DMet L-N-methylisolleucine Nmile D-ornithine
DOrn L-N-methylleucine Nmleu D-phenylalanine DPhe L-N-methyllysine
Nmlys D-proline DPro L-N-methylmethionine Nmmet D-serine DSer
L-N-methylnorleucine Nmnle D-threonine DThr L-N-methylnorvaline
Nmnva D-tryptophan DTrp L-N-methylornithine Nmorn D-tyrosine DTyr
L-N-methylphenylalanine Nmphe D-valine DVal L-N-methylproline Nmpro
D-.alpha.-methylalanine DMala L-N-methylserine Nmser
D-.alpha.-methylarginine DMarg L-N-methylthreonine Nmthr
D-.alpha.-methylasparagine DMasn L-N-methyltryptophan Nmtrp
D-.alpha.-methylaspartate DMasp L-N-methyltyrosine Nmtyr
D-.alpha.-methylcysteine DMcys L-N-methylvaline Nmval
D-.alpha.-methylglutamine DMgln L-N-methylethylglycine Nmetg
D-.alpha.-methylhistidine DMhis L-N-methyl-t-butylglycine Nmtbug
D-.alpha.-methylisoleucine DMile L-norleucine Nle
D-.alpha.-methylleucine DMleu L-norvaline Nva
D-.alpha.-methyllysine DMlys .alpha.-methyl-aminoisobutyrate Maib
D-.alpha.-methylmethionine DMmet
.alpha.-methyl-.gamma.-aminobutyrate Mgabu
D-.alpha.-methylornithine DMorn .alpha.-methylcyclohexylalanine
Mchexa D-.alpha.-methylphenylalanine DMphe
.alpha.-methylcyclopentylalanine Mcpen D-.alpha.-methylproline
DMpro .alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylserine DMser .alpha.-methylpenicillamine Mpen
D-.alpha.-methylthreonine DMthr N-(4-aminobutyl)glycine Nglu
D-.alpha.-methyltryptophan DMtrp N-(2-aminoethyl)glycine Naeg
D-.alpha.-methyltyrosine DMty N-(3-aminopropyl)glycine Norn
D-.alpha.-methylvaline DMval N-amino-.alpha.-methylbutyrate Nmaabu
D-N-methylalanine DNmala .alpha.-napthylalanine Anap
D-N-methylarginine DNmarg N-benzylglycine Nphe D-N-methylasparagine
DNmasn N-(2-carbamylethyl)glycine Ngln D-N-methylaspartate DNmasp
N-(carbamylmethyl)glycine Nasn D-N-methylcysteine DNmcys
N-(2-carboxyethyl)glycine Nglu D-N-methylglutanmine DNmgln
N-(carboxymethyl)glycine Nasp (-carboxyglutamate Gla
N-cyclobutylglycine Ncbut 4-hydroxyproline Hyp N-cyclodecylglycine
Ncdec 5-hydroxylysine Hlys N-cylcododecylglycine Ncdod
2-aminobenzoyl(anthraniloyl) Abz N-cyclooctylglycine Ncoct
N-cyclopropylglycine Ncpro Cyclohexylalanine Cha
N-cycloundecylglycine Ncund Phenylglycine Phg
N-(2,2-diphenylethyl)glycine Nbhm 4-phenyl-phenylalanine Bib
N-(3,3-diphenylpropyl)glycine Nbhe L-pyroglutamic acid pGlu
N-(1-hydroxyethyl)glycine Nthr L-Citrulline Cit
N-(hydroxyethyl)glycine Nser L-1,2,3,4-tetrahydroiso- Tic
N-(imidazolylethyl))glycine Nhis quinoline-3-carboxylic acid
N-(3-indolylyethyl)glycine Nhtrp L-Pipecolic acid (homo proline)
Pip N-methyl-.gamma.-aminobutyrate Nmgabu D-N-methylmethionine
Dnmmet L-homoleucine Hle N-methylcyclopentylalanine Nmcpen L-Lysine
(dimethyl) DMK D-N-methylphenylalanine Dnmphe L-Napthylalanine Nal
D-N-methylproline Dnmpro L-dimethyldopa or DMD D-N-methylthreonine
Dnmthr L-dimethoxyphenylalanine N-(1-methylethyl)glycine Nval
L-thiazolidine-4-carboxylic acid THZ N-methyla-napthylalanine
Nmanap N-methylpenicillamine Nmpen L-homotyrosine hTyr
N-(.rho.-hydroxyphenyl)glycine Nhtyr L-3-pyridylalanine PYA
N-(thiomethyl)glycine Ncys L-2-furylalanine FLA penicillamine Pen
L-histidine(benzyloxymethyl) HBO L-.alpha.-methylalanine Mala
L-histidine(3-methyl) HME L-.alpha.-methylasparagine Masn
D-N-methylglutamate Dnmglu L-.alpha.-methyl-t-butylglycine Mtbug
D-N-methylhistidine Dnmhis L-methylethylglycine Metg
D-N-methylisoleucine Dnmile L-.alpha.-methylglutamate Mglu
D-N-methylleucine Dnmleu L-.alpha.-methylhomophenylalanine Mhphe
D-N-methyllysine Dnmlys N-(2-methylthioethyl)glycine Nmet
N-methylcyclohexylalanine Nmchexa L-.alpha.-methyllysine Mlys
D-N-methylornithine Dnmorn L-.alpha.-methylnorleucine Mnle
N-methylglycine Nala L-.alpha.-methylornithine Morn
N-methylaminoisobutyrate Nmaib L-.alpha.-methylproline Mpro
N-(1-methylpropyl)glycine Nile L-.alpha.-methylthreonine Mthr
N-(2-methylpropyl)glycine Nleu L-.alpha.-methyltyrosine Mtyr
D-N-methyltryptophan Dnmtrp L-N-methylhomophenylalani Nmhphe
D-N-methyltyrosine Dnmtyr N-(N-(3,3- Nnbhe D-N-methylvaline Dnmval
diphenylpropyl)carbamylmethylglycine L-t-butylglycine Tbug
O-methyl-L-serine Omser L-ethylglycine Etg O-methyl-L-homoserine
Omhser L-homophenylalanine Hphe O-methyl-L-tyrosine MeY
L-.alpha.-methylarginine Marg .gamma.-aminobutyric acid Gabu
L-.alpha.-methylaspartate Masp L-.alpha.-methylaspartate Masp
O-methyl-L-homotyrosine Omhtyr L-.alpha.-methylcysteine Mcys
L-.E-backward.-homolysine BHK L-.alpha.-methylglutamine Mgln
L-ornithine Orn N-cycloheptylglycine Nchep N-cyclohexylglycine
Nchex N-(3-guanidinopropyl)glycine Narg D-N-methylserine DNmser
[0081] Insertions encompass the addition of one or more naturally
occurring or non conventional amino acid residues, although
preferably not cysteine residues.
[0082] Deletion encompasses the deletion of one or more amino acid
residues, although preferably not cysteine residues.
[0083] As stated above the present invention includes peptides in
which one or more of the amino acids other than Cys has undergone
side chain modifications. Examples of side chain modifications
contemplated by the present invention include modifications of
amino groups such as by reductive alkylation by reaction with an
aldehyde followed by reduction with NaBH4; amidination with
methylacetimidate; acylation with acetic anhydride; carbamoylation
of amino groups with cyanate; trinitrobenzylation of amino groups
with 2,4,6 trinitrobenzenesulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride;
and pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH4; and N-acetylation.
[0084] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0085] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivatisation, for example, to a corresponding amide.
[0086] Acidic amino acids may be substituted with tetrazolyl
derivatives of glycine and alanine, as described in WO02/060923
(COGNETIX INC; Univ. Utah Res Found.).
[0087] The tyrosine residue may be altered, for example by
methoxylation at the 4-position. Tyrosine may also be altered by
nitration with tetranitromethane to form a 3-nitrolyrosine
derivative.
[0088] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0089] Proline residue may be modified by, for example,
hydroxylation in the 4-position.
[0090] Other variants contemplated by the present invention include
a range of glycosylation variants. Altered glycosylation patterns
may result from expression of recombinant molecules in different
host cells. Ser, Thr and Hyp residues may be modified to contain an
O-glycan, while Asn and Gln residues can be modified to form a
N-glycan. In accordance with the present invention, the term
"glycan" refers to an N-, S- or O-linked mono-, di-, tri-, poly- or
oligosaccharide that can be attached to any hydroxy, amino or thiol
group of natural of modified amino acids by synthetic or enzymatic
methodologies known in the art. The monosaccharides making up the
glycan can include D-allose, D-altrose, D-glucose, D-mannose,
D-gulose, D-idose, D-galactose, D-talose , D-galactosamine,
D-glucosamine , D-N-acetyl-glucosamine (GIcNAc),
D-N-acetyl-galactosamine (GalNac), D-fucose or D-arabinose. These
saccharides may be structurally modified i.e., with one or more
O-sulphate, O-phosphate, O-acetyl or acidic groups such as sialic
acid, including combinations thereof. The glycan may also include
similar polyhydroxyl groups, such as D-penicillamine 2,5 and
halogenated derivatives thereof or polypropylene glycol
derivatives. The glycosidic linkage is beta and 1-4 or 1-3,
preferably 1-3.
[0091] The linkage between the glycan and the amino acid may be
alpha or beta, preferably alpha and is 1-.
[0092] Furthermore, since an inherent problem with native peptides
(in L-form) is the degradation by natural proteases, the peptide of
the invention may be prepared in order to include D-forms and/or
"retro-inverso isomers" of the peptide. Preferably, retro-inverso
isomers of short parts, variants or combinations of the peptide of
the invention are prepared.
[0093] Protecting the peptide from natural proteolysis should
therefore increase the effectiveness of the specific heterobivalent
or heteromultivalent compound. A higher biological activity is
predicted for the retro-inverso containing peptide when compared to
the non-retro-inverso containing analog owing to protection from
degradation by native proteinases. Furthermore they have been shown
to exhibit an increased stability and lower immunogenicity [Sela M.
and Zisman E., (1997) Different roles of D-amino acids in immune
phenomena-FASEB J. 11, 449].
[0094] Retro-inverso peptides are prepared for peptides of known
sequence as described for example in Sela and Zisman, (1997).
[0095] By "retro-inverso isomer" is meant an isomer of a linear
peptide in which the direction of the sequence is reversed and the
chirality of each amino acid residue is inverted; thus, there can
be no end-group complementarity.
[0096] The invention also includes analogs in which one or more
peptide bonds have been replaced with an alternative type of
covalent bond (a "peptide mimetic") which is not susceptible to
cleavage by peptidases. Where proteolytic degradation of the
peptides following injection into the subject is a problem,
replacement of a particularly sensitive peptide bond with a
noncleavable peptide mimetic will make the resulting peptide more
stable and thus more useful as an active substance. Such mimetics,
and methods of incorporating them into peptides, are well known in
the art.
[0097] Also useful are amino-terminal blocking groups such as
t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl,
suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl,
fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl,
methoxysuberyl, and 2,4,-dinitrophenyl.
[0098] The combination of the mu-conotoxin of the invention, or of
particular biologically active fragments thereof, are envisioned
and can be made to improve the potency, selectivity or stability of
existing peptides of the invention.
[0099] Preferably, the mu-conotoxin peptide is selected from the
group comprising
TABLE-US-00002 [SEQ ID No 2]
pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-
Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys and [SEQ ID No 3]
pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-
Lys-Trp-Cys-Arg-Asp-Met-Ala-Arg-Cys-Cys.
[0100] Usually, the C-terminus of these peptide are amidated.
[0101] It should also be understood that the terms mu-conotoxin
peptide or mu-conotoxins are not limited to naturally occurring
toxic peptides obtained from the genus Conus but rather simply
indicates an initial source from which the peptides have been or
can be derived. The mu-conotoxin peptide of the invention, as well
as a fragment, combination and a variant thereof can be prepared by
a variety of methods and techniques known in the art such as for
example chemical synthesis or recombinant techniques as described
in Maniatis et al. 1982, Molecular Cloning, A laboratory Manual,
Cold Spring Harbor Laboratory and Amblard et al. 2005.
[0102] When recombinant techniques are employed to prepare
mu-conotoxin peptides in accordance with the present invention,
nucleic acid molecules or biologically active fragments thereof
encoding the polypeptides are preferably used.
[0103] Therefore the present invention also relates to an isolated
and purified nucleic acid sequence comprising a nucleotide sequence
encoding the amino acid sequence as described above.
[0104] "An isolated and purified nucleic acid sequence" refers to
the state in which the nucleic acid molecule encoding the
mu-conotoxin peptide of the invention, or nucleic acid encoding
such mu-conotoxin peptide will be, in accordance with the present
invention. Nucleic acid will be free or substantially free of
material with which it is naturally associated such as other
polypeptides or nucleic acids with which it is found in its natural
environment, or the environment in which it is prepared (e.g. cell
culture) when such preparation is by recombinant nucleic acid
technology practised in vitro or in vivo.
[0105] The term "nucleic acid" is intended to refer either to DNA
or to RNA.
[0106] In case the nucleic acid is DNA, then DNA which can be used
herein is any polydeoxynucleotide sequence, including, e.g.
double-stranded DNA, single-stranded DNA, double-stranded DNA
wherein one or both strands are composed of two or more fragments,
double-stranded DNA wherein one or both strands have an
uninterrupted phosphodiester backbone, DNA containing one or more
single-stranded portion(s) and one or more double-stranded
portion(s), double-stranded DNA wherein the DNA strands are fully
complementary, double-stranded DNA wherein the DNA strands are only
partially complementary, circular DNA, covalently-closed DNA,
linear DNA, covalently cross-linked DNA, cDNA,
chemically-synthesized DNA, semi-synthetic DNA, biosynthetic DNA,
naturally-isolated DNA, enzyme-digested DNA, sheared DNA, labeled
DNA, such as radiolabeled DNA and fluorochrome-labeled DNA, DNA
containing one or more non-naturally occurring species of nucleic
acid.
[0107] DNA sequences that encode the mu-conotoxin peptide, or a
biologically active fragment thereof, can be synthesized by
standard chemical techniques, for example, the phosphotriester
method or via automated synthesis methods and PCR methods.
[0108] The purified and isolated DNA sequence encoding the
mu-conotoxin peptide according to the invention may also be
produced by enzymatic techniques. Thus, restriction enzymes, which
cleave nucleic acid molecules at predefined recognition sequences
can be used to isolate nucleic acid sequences from larger nucleic
acid molecules containing the nucleic acid sequence, such as DNA
(or RNA) that codes for the mu-conotoxin peptide or for a fragment
thereof.
[0109] Encompassed by the present invention is also a nucleic acid
in the form of a polyribonucleotide (RNA), including, e.g.,
single-stranded RNA, double-stranded RNA, double-stranded RNA
wherein one or both strands are composed of two or more fragments,
double-stranded RNA wherein one or both strands have an
uninterrupted phosphodiester backbone, RNA containing one or more
single-stranded portion(s) and one or more double-stranded
portion(s), double-stranded RNA wherein the RNA strands are fully
complementary, double-stranded RNA wherein the RNA strands are only
partially complementary, covalently crosslinked RNA,
enzyme-digested RNA, sheared RNA, mRNA, chemically-synthesized RNA,
semi-synthetic RNA, biosynthetic RNA, naturally-isolated RNA,
labeled RNA, such as radiolabeled RNA and fluorochrome-labeled RNA,
RNA containing one or more non-naturally-occurring species of
nucleic acid.
[0110] The isolated and purified nucleic acid sequence, DNA or RNA,
also comprises an isolated and purified nucleic acid sequence
having substantial sequence identity or homology to a nucleic acid
sequence encoding the mu-conotoxin peptide of the invention.
Preferably, the nucleic acid will have substantial sequence
identity for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, or
85% nucleic acid identity; more preferably 90% nucleic acid
identity; and most preferably at least 95%, 96%, 97%, 98%, or 99%
sequence identity.
[0111] Identity as known in the art and used herein, is a
relationship between two or more amino acid sequences or two or
more nucleic acid sequences, as determined by comparing the
sequences. It also refers to the degree of sequence relatedness
between amino acid or nucleic acid sequences, as the case may be,
as determined by the match between strings of such sequences.
Identity and similarity are well known terms to skilled artisans
and they can be calculated by conventional methods (for example see
Computational Molecular Biology, Lesk, A. M. ed., Oxford University
Press, New York, 1988; Biocomputing: Informatics and Genome
Projects, Smith, D. W. ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part I, Griffin, A. M. and
Griffin, H. G. eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G. Academic Press, 1987;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds. M.
Stockton Press, New York, 1991, Carillo, H. and Lipman, D., SIAM J.
Applied Math. 48:1073, 1988). Methods which are designed to give
the largest match between the sequences are generally preferred.
Methods to determine identity and similarity are codified in
publicly available computer programs including the GCG program
package (Devereux J. et al., Nucleic Acids Research 12(1): 387,
1984); BLASTP, BLASTN, and FASTA (Atschul, S. F. et al. J. Molec.
Biol. 215: 403-410, 1990). The BLAST X program is publicly
available from NCBI and other sources (BLAST Manual, Altschul, S.
et al. NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al. J.
Mol. Biol. 215: 403-410, 1990).
[0112] Also encompassed by the present invention is a nucleic acid
sequence complementary to the isolated and purified nucleic acid
sequence encoding mu-conotoxin peptide of the invention.
[0113] Also within the scope of the invention is a degenerated
nucleic acid sequence having a sequence which differs from a
nucleic acid sequence encoding the mu-conotoxin peptide of the
invention, or a complementary sequence thereof, due to degeneracy
in the genetic code. Such nucleic acid encodes functionally
equivalent mu-conotoxin peptide but differs in sequence from the
sequence due to degeneracy in the genetic code. This may result in
silent mutations which do not affect the amino acid sequence. Any
and all such nucleic acid variations are within the scope of the
invention.
[0114] In addition, also considered is a nucleic acid sequence
capable of hybridizing under stringent conditions, preferably high
stringency conditions, to a nucleic acid sequence encoding the
mu-conotoxin peptide of the invention, a nucleic acid sequence
complementary thereof or a degenerated nucleic acid sequence
thereof. Appropriate stringency conditions which promote DNA
hybridization are known to those skilled in the art, or can be
found in Current Protocols in Molecular Biology, John Wiley &
Sons, N.Y. (1989), 6.3.1-6.3.6. For example, 6.0.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.0.times. SSC at 50.degree. C. may be employed. The
stringency may be selected based on the conditions used in the wash
step. By way of example, the salt concentration in the wash step
can be selected from a high stringency of about 0.2.times. SSC at
50.degree. C. In addition, the temperature in the wash step can be
at high stringency conditions, at about 65.degree. C.
[0115] The present invention also includes an isolated and purified
nucleic acid encoding a mu-conotoxin peptide of the invention
comprising a nucleic acid sequence encoding a truncation or an
analog of a mu-conotoxin peptide. The term "truncation" refers to a
sequence encoding a peptide containing less amino acid than the
native but exhibiting the same properties.
[0116] The invention also encompasses allelic variants of the
disclosed isolated and purified nucleic sequence; that is,
naturally-occurring alternative forms of the isolated and purified
nucleic acid that also encode peptides that are identical,
homologous or related to that encoded by the isolated and purified
nucleic sequences. Alternatively, non-naturally occurring variants
may be produced by mutagenesis techniques or by direct
synthesis.
[0117] A biologically active fragment of the disclosed isolated and
purified nucleic sequence is also considered and refers to a
sequence containing less nucleotides in length than the nucleic
acid sequence encoding the mu-conotoxin peptide, a nucleic acid
sequence complementary thereof or a degenerated nucleic acid
sequence thereof. This sequence can be used as long as it exhibits
the same properties as the native sequence from which it derives.
Preferably this sequence contains less than 90%, preferably less
than 60%, in particular less than 30% amino acids in length than
the respective isolated and purified nucleic sequence of the
mu-conotoxin peptide.
[0118] Yet another concern of the present invention is to provide
an expression vector comprising the isolated and purified nucleic
acid sequence encoding the mu-conotoxin peptide. The choice of an
expression vector depends directly, as it is well known in the art,
on the functional properties desired, e.g., mu-conotoxin peptide
expression and the host cell to be transformed or transfected.
[0119] Surprisingly, Applicants have shown that mu-conotoxin
peptides as described herein demonstrated useful and potent
biological activity for application as anaesthetics. These peptides
clearly show a better activity than the currently used local
anaesthetics such as procaine or lidocaine and a much longer
duration time of activity. These mu-conotoxin can thus be applied
to specific cases where long and efficient anaesthesia is required.
They can also be used as alternatives in case of undesired
side-reactions or allergy in response to classical anaesthetics
such as procaine or lidocaine (Finucane B. T., 2005).
[0120] These mu-conotoxins also demonstrate better potency in
biological activity by comparison to the data available in the
scientific literature, in the patents cited above and the appended
examples.
[0121] Accordingly, the present invention is also directed to a
pharmaceutical composition comprising as an active substance a
pharmaceutically effective amount of at least one mu-conotoxin
peptide as described, optionally in combination with
pharmaceutically acceptable carriers, diluents and/or
adjuvants.
[0122] "A pharmaceutically effective amount" refers to a chemical
material or compound which, when administered to a human or animal
organism induces a detectable pharmacologic and/or physiologic
effect.
[0123] The respective pharmaceutically effect amount can depend on
the specific patient to be treated, on the disease to be treated
and on the method of administration. Further, the pharmaceutically
effective amount depends on the specific peptide used, especially
if the peptide additionally contains a drug as described or not.
The treatment usually comprises a multiple administration of the
pharmaceutical composition, usually in intervals of several hours,
days or weeks. The pharmaceutically effective amount of a dosage
unit of the polypeptide usually is in the range of 0.001 ng to 100
.mu.g per kg of body weight of the patient to be treated.
Preferably in the range of 0.1 ng to 10 .mu.g per kg of body
weight.
[0124] Preferably, in addition to at least one mu-conotoxin peptide
as described herein, the pharmaceutical composition may contain one
or more pharmaceutically acceptable carriers, diluents and
adjuvants.
[0125] Acceptable carriers, diluents and adjuvants which
facilitates processing of the active compounds into preparation
which can be used pharmaceutically are non-toxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl
orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.RTM., PLURONICS.RTM. or polyethylene glycol (PEG).
[0126] The form of administration of the pharmaceutical composition
may be systemic or topical. For example, administration of such a
composition may be various parenteral routes such as subcutaneous,
intravenous, intradermal, intramuscular, intraperitoneal,
intranasal, transdermal, buccal routes or via an implanted device,
and may also be delivered by peristaltic means.
[0127] The present invention also contemplates an implant device
comprising the mu-conotoxin or the pharmaceutical composition of
the invention.
[0128] The pharmaceutical composition, as well as the anesthetic,
comprising a mu-conotoxin peptide, as described herein, as an
active agent may also be incorporated or impregnated into a
bioabsorbable matrix, with the matrix being administered in the
form of a suspension of matrix, a gel or a solid support. In
addition the matrix may be comprised of a biopolymer.
[0129] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi permeable
matrices of solid hydrophobic polymers containing the mu-conotoxin
peptide, which matrices are in the form of shaped articles, e.g.
films, microspheres, implants or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and [gamma]ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0130] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished for example by filtration
through sterile filtration membranes.
[0131] It is understood that the suitable dosage of a mu-conotoxin
peptide of the present invention will be dependent upon the age,
sex, health, and weight of the recipient, kind of concurrent
treatment, if any and the nature of the effect desired.
[0132] The appropriate dosage form will depend on the disease, the
peptide, and the mode of administration; possibilities include
tablets, capsules, lozenges, dental pastes, suppositories,
inhalants, solutions, ointments, creams and parenteral depots.
[0133] In the case of inhalants, then it is preferably in the form
of a spray. A nasal formulation of the mu-conotoxin of the
invention is made in order to provide, for example, efficient
epithelial sodium channel inhibition. The amount injected via nasal
spray is dependant of the subject characteristics, such as age and
weight. Determination of an effective dose range is routine for
those of skill in the art.
[0134] As an example of a specific formulation, the amount of
mu-conotoxin in a daily nasal spray formulation with a volume
between about 30 to about 300 .mu.L, can deliver a daily dose of
mu-conotoxin of between about 1 .mu.g to about 10 .mu.g. It will be
appreciated that the daily spray volume can be administered in one,
two, or more separate deliveries to achieve the desired total daily
spray volume. It will further be appreciated that the spray volume
and the amount of mu-conotoxin in the nasal formulation are each
individually adjustable to achieve the desired daily dosage.
[0135] Since amino acid modifications of the amino acids of the
mu-conotoxin peptide are also encompassed in the present invention,
this may be useful for cross-linking the mu-conotoxin peptide of
the invention to a water-insoluble matrix or the other
macromolecular carriers, or to improve the solubility, adsorption,
and permeability across the blood brain barrier. Such modifications
are well known in the art and may alternatively eliminate or
attenuate any possible undesirable side effect of the peptide and
the like.
[0136] While a preferred pharmaceutical composition of the present
invention comprises a mu-conotoxin peptide as an active agent, an
alternative pharmaceutical composition may contain an isolated and
purified nucleic acid sequence encoding the mu-conotoxin peptide,
as described herein, as an active agent. This pharmaceutical
composition may include either the sole isolated and purified DNA
sequence, an expression vector comprising said isolated and
purified DNA sequence or a host cell previously transfected or
transformed with an expression vector described herein. In this
latter example, host cell will preferably be isolated from the
patient to be treated in order to avoid any antigenicity problem.
These gene and cell therapy approaches are especially well suited
for patients requiring repeated administration of the
pharmaceutical composition, since the said purified and isolated
DNA sequence, expression vector or host cell previously transfected
or transformed with an expression vector can be incorporated into
the patient's cell which will then produce the protein
endogenously.
[0137] Usually, the pharmaceutical composition as described herein
is used for the treatment or prevention of a pain. The pain to be
treated or prevented will be selected, for example, from the group
comprising migraine, acute pain, persistent pain, chronic pain,
neuropathic pain or nociceptive pain.
[0138] Alternatively, the pharmaceutical composition as described
herein is used for treating cystic fibrosis or
oto-rhino-laryngological diseases.
[0139] Since the mu-conotoxin of the invention is a sodium channel
inhibitor, it can be applied to the airway epithelium and nasal
membrane for blocking the enhancement of sodium intake by the
epithelial sodium channel. This has the effect of lowering the
mucous viscosity and promote a better clearance of the external
biological fluid, such as lung fluids and nasal fluids. In this
respect, the mu-conotoxin inhibits at low concentrations the sodium
channels present in membranes associated with cystic fibrosis
disease and with inflammatory states where mucous production is
above normal levels. Epithelial sodium channels modulate clearance
of mucous lung or nasal fluids. Application of different
concentrations of pharmaceutical composition comprising the
mu-conotoxin of the invention in the micromalor and sub-micromolar
range would induce better clearance of the accumulation of
biological fluids in mucus. Mu-conotoxin thus has a therapeutic
potential in treating oto-rhino-laryngological inflammatory states
presenting abnormal fluid secretions in mucus. Mu-conotoxin
application is also dedicated to the potential treatment of
abnormal lung secretions arising in cystic fibrosis.
[0140] Also encompassed by the present invention is the use of the
pharmaceutical composition of the invention, in the preparation of
a medicament for the treatment or prevention of a disorder
associated with voltage-sensitive sodium channels.
[0141] The mu-conotoxin peptide of the invention will generally be
used in an amount to achieve the intended purpose. For use to treat
or prevent a pain, the peptide or the pharmaceutical compositions
thereof, is administered or applied in a therapeutically effective
amount. A "therapeutically effective amount" is an amount effective
to ameliorate or prevent the symptoms. Determination of a
therapeutically effective amount is well within the capabilities of
those skilled in the art, especially in light of the detailed
disclosure provided herein.
[0142] For systemic administration, a therapeutically effective
amount or dose can be estimated initially from in vitro assays. For
example, a dose can be formulated in animal models to achieve a
circulating concentration range that includes the IC50 as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans.
[0143] Initial doses can also be estimated from in vivo data, e.g.
animal models, using techniques that are well known in the art. One
ordinarily skill in the art could readily optimise administration
to humans based on animal data and will, of course, depend on the
subject being treated, on the subject's weight, the severity of the
disorder, the manner of administration and the judgement of the
prescribing physician.
[0144] Further encompassed by the present invention is the use of
the pharmaceutical composition of the invention, in the preparation
of an anesthetic.
[0145] The present disclosure also provides a method for providing
musculoskeletal relaxation in a patient undergoing a surgical
procedure requiring anesthesia which comprises administering to a
patient in need thereof a pharmaceutically effective amount of at
least one mu-conotoxin peptide of the invention or a
pharmaceutically acceptable salt thereof.
[0146] "Administered" or "administering", as it applies in the
present invention means "giving" or "contacting" and refers to
contact of a pharmaceutical, therapeutic, or anesthetic composition
to the subject, preferably a human.
[0147] Usually, in the method described above, the at least one
mu-conotoxin peptide is administered as a local anesthetic.
Preferably, the at least one mu-conotoxin peptide is used in, for
example, ophthalmology, in the treatment of dystonia, in
otolaryngology, in the treatment of anal fissures, in dermatology,
in traumatology, in cosmetic surgery, in the treatment of
fibromyalgia and chronic myofascial pain as well as in the
treatment of all pains.
[0148] Preferably, the at least one mu-conotoxin peptide is
administered as an ocular anesthetic.
[0149] Also encompassed in the present invention is a method for
local anesthesia, said method comprising administering a
pharmaceutically effective amount of at least one mu-conotoxin
peptide of the invention or a pharmaceutically acceptable salt
thereof. Preferably, said pharmaceutically effective amount of at
least one mu-conotoxin peptide of the invention or the
pharmaceutical composition provides a long and duration of effect
as disclosed in the Examples.
Preferably, the long duration of effect is about 30 min to 48 hours
depending on the subject to be treated and/or the concentration of
mu-conotoxin of the invention used. However, in any case said
duration is longer than any duration described until now for
classical anesthetics such as lidocaine of procaine. Preferably,
the duration is 30 min to 12 hours.
[0150] Further encompassed by the present invention is an
anesthetic comprising the pharmaceutical composition or the
mu-conotoxin peptide of described in the present disclosure.
[0151] Preferably said anesthetic is suitable for subcutaneous,
intravenous, intradermal, intramuscular, intraperitoneal,
intranasal, transdermal, buccal routes or an implanted device.
[0152] Usually, the anesthetic is in the form of tablets, capsules,
lozenges, dental pastes, suppositories, inhalants, solutions,
ointments, creams and parenteral depots. Preferably, the inhalant
is a spray.
EXAMPLES
Example 1
Material and Methods
Materials
[0153] Specimens of Conus consors were collected in chesterfield
Island (New Calcdonia) and immediately frozen at -80.degree. C. The
venom was obtained from freshly dissected venom duct apparatus, and
extracted with 0.08% trifluoroacetic acid (TFA) in water. Extracts
obtained from several venom ducts were centrifuged to remove
insoluble particles. Supernatants from all extractions were
combined, lyophilised, weighed, and stored at -80.degree. C. until
required for use.
Chromatography
[0154] Fractionation of the crude lyophilised venom was performed
using a Thermo Separation Product (TSP) high pressure liquid
chromatography system equipped with a TSP-150 UV detector. Elution
buffers used for reverse-phase chromatography were the following:
buffer A, H.sub.2O/0.1% TFA; buffer B, H.sub.2O/CH.sub.3CN 40/60
0.1% TFA. Semi-preparative runs on the crude venom were performed
with a C18 Vydac 218TP510 column using the following gradient. The
program was 0-8% B/5 min., 8-80% B/70 min., 80-100% B/10 min.,
followed by 100% B/10 min. (flow rate, 2 ml/min). Further
purification steps using an analytical C18 Vydac 2181P54 column was
carried out with gradient such as 0-10% B/5 min., 10-20% B/10 min.,
20-40% B/40 min. Fractions were detected at 220 nm.
Amino Acid Composition and Edman Sequencing
[0155] Peptide samples were hydrolyzed by addition of 200 ml of 6 M
HCl at the bottom of the vial which was evacuated, sealed and
heated at 120.degree. C. for 16 h. The hydrolysates were analysed
on an automatic analyser (Applied Biosystems, model 130A) equipped
with an on-line derivatiser (model 420A) for the conversion of the
free amino acids into their phenylthiocarbamoyl derivatives.
Sequencing trials were performed by Edman's degradation on an
automatic Applied Biosystems 477A microsequencer. Before
sequencing, the homogeneous peptide was reduced by dithiothreitol
in 6 M guanidine hydrochloride, 0.5 M Tris/HCl, 2 mM
ethylenediamine tetraacetic acid (EDTA) (pH 7.5) for 1 h and then
treated with 4-vinylpyridine (1.5 .mu.M) at room temperature for 3
h. The peptide derivative was purified by reverse-phase HPLC using
a C18 Vydac column (4.6 mm.times.25 cm, 5 .mu.m particle size).
Mass Spectrometry
[0156] Molecular mass measurements were performed on a QTOF I
instrument (Micromass/Waters, USA) equipped with an electrospray
ion source. Sample analysis was carried out in positive mode using
a carrier infusion solvent of H2O/CH3CN/HCOOH (49.9/49.9/0.2).
Single MS experiments with the TOF-MS configuration was used for
simple molecular mass determination. Tandem mass spectrometry was
carried out for structural investigations. In this configuration,
parent mass was first selected using the quadrupole, and the
collision induced dissociation was performed by manually adjusting
the collision energy. In this case, the native sample was
previously reduced using 100 mM dithiothreitol (DTT) in an ammonium
bicarbonate buffer (pH 7.8) at 56.degree. C. for 3 h. The reduced
peptide was then desalted using a ZipTip (Millipore, USA) according
to the manufacturer protocol. The multiply-charged spectra obtained
were transformed into singly-charged data with the aid of the
software MassLynx (Micromass/Waters, USA) using the MaxEnt3 option.
Manual and semi-automatic data treatment was then operated for
sequence characterisation.
Peptide Synthesis
[0157] Solid-phase synthesis was performed on a custom-modified
433A peptide synthesizer from Applied Biosystems, using in situ
neutralization/2-(1H-benzotriazol-1-yl)-1,1,1,3,3-tetramethyluronium
hexa fluoro-phosphate (HBTU) activation protocols for stepwise Boc
chemistry chain elongation. After chain assembly was completed, the
peptide was deprotected and cleaved from the resin by treatment
with anhydrous HF for 1 hr at 0.degree. C. with 5% p-cresol as a
scavenger. After cleavage, the peptide was precipitated with
ice-cold diethylether, dissolved in aqueous acetonitrile and
lyophilized. The peptide was purified by RP-HPLC with a Vydac C18
column by using a linear gradient of buffer B (acetonitile/10%
H.sub.2O/0.1% trifluoroacetic acid) in buffer A (H.sub.2O/0.1%
trifluoroacetic acid) and UV detection at 214 nm. Samples were
analyzed by electrospray mass spectrometry with a Platform II
instrument (Micromass, Manchester, England).
[0158] For the oxidative folding of the peptide, the material
(about 0.5 to 1 mg/mL) was dissolved in 0.5M GuHCl, 100 mM Tris, pH
7.8 containing 0.5 mM reduced and 0.1 mM oxidized glutathione.
After gentle stirring overnight at room temperature, the protein
solution was purified by RP-HPLC as described above. The overall
yield of the folding step was of approximately 35%.
Frog and Mouse Neuromuscular Preparations
[0159] The cutaneous pectoris muscle and associated nerve were
removed from double pithed male frogs (Rana esculenta) weighing
20-25 g. and pinned to the base of a 2 ml tissue bath superfused
with a standard solution containing (in mM): NaCl, 115.0; KCl, 2.0;
CaCl.sub.2, 1.8 and HEPES buffer, 5.0 (pH 7.25). In some
experiments, excitation-contraction was uncoupled by treating the
cutaneous pectoris neuromuscular preparations with 2 M formamide.
Left and right hemidiaphragm muscles with their associated phrenic
nerves were isolated from Swiss-Webster mice (20-25 g) that were
killed by dislocation of the cervical vertebrae followed by
immediate exsanguination. The two hemidiaphragms were separated and
each was mounted in a Rhodorsil (Rhone-Poulenc, St. Fons,
France)-lined organ bath (2 ml volume) superfused with a
physiological solution (mammalian Krebs-Ringer's solution) of the
following composition (in mM): NaCl, 154.0; KCl, 5.0; CaCl.sub.2,
2; MgCl.sub.2, 1.0; HEPES buffer, 5.0; glucose, 11.0. The solution,
gassed with pure O.sub.2, had a pH of 7.4.
Mouse Sciatic Nerve Preparation
[0160] Both sciatic nerves (left and right) were dissected from
mice killed by dislocation of the cervical vertebrae. The nerves
were rinsed with oxygenated mammalian Krebs-Ringer's solution at
room temperature for 30 min prior use.
Pike Olfactory Nerve Preparation
[0161] Left and right olfactory nerves were removed from
decapitated pikes (Esox lucius). Each nerve was rinsed with an
oxygenated pike Ringer's solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM
CaCl.sub.2, 1 mM Na.sub.2HPO.sub.4 buffer, 5 mM HEPES, 1 mM
MgCl.sub.2, adjusted at pH 7.3 with NaOH), at room temperature for
a 30-min period prior use.
Mechanical Recordings on Mouse Neuromuscular Preparations
[0162] In this type of experiments, one end of the mouse
hemidiaphragm muscle is pined to the tissue bath and the other end
(tendon) is attached to an isometric transducer (FT03, Grass
Instruments). Contractions are evoked by stimulation of the motor
nerve, via a suction electrode, with current pulses of 0.15 ms
duration at a 0.1 Hz frequency. The resting tension of each
preparation is adjusted so that maximal contractile response is
obtained in normal conditions, with direct stimulation of the
muscle or indirect stimulation via the motor nerve. Tension signals
recorded from the transducer are amplified, collected and digitized
with the aid of a computer equipped with a digital interface
(DT-2821, Data Translation). Experiments are carried out at room
temperature.
Electrophysiology Recordings on Frog and Mouse Neuromuscular
Preparations
[0163] The motor nerve of isolated neuromuscular preparations was
stimulated via a suction microelectrode, adapted to the diameter of
the nerve, with pulses of 0.05-0.1 msec duration and supramaximal
voltage (typically 3-8 V). These pulses were generated by a S-44
stimulator (Grass Instruments, West Warwick, U.S.A.) linked to a
stimulus isolation unit. Membrane potentials and synaptic
potentials were recorded from endplate regions at room temperature
(22-24.degree. C.) with intracellular microelectrodes filled with 3
M KCl (8-120 resistance) using conventional techniques and an
Axoclamp-2A system (Axon Instruments, Foster city, Calif., U.S.A.).
Recordings were made continuously from the same endplate before and
throughout application of the conotoxin tested. Electrical signals
after amplification were displayed on a digital oscilloscope and
simultaneously recorded on video tape with the aid of a modified
digital audio processor (Sony PCM 701 ES) and a video cassette
recorder (Sony SLC9F). Data were collected and digitized with the
aid of a computer equipped with an analogue and digital I/O
interface board (DT2821, Data Translation Marlboro, U.S.A.) at a
sampling rate of 25 kHz. Computerized data acquisition and analysis
was performed with a program kindly provided by Dr. John Dempster
(University of Strathclyde, Scotland). Endplate potentials (EPPs)
and miniature endplate potentials (MEPPs) were analyzed
individually for amplitude and time course. For each condition
studied, 3-6 individual experiments were performed and the results
were averaged to give the presented mean.+-.standard error of the
mean (S.E.M.). Statistical testing was performed by using student's
test with P<0.05 being taken to indicate significance.
Electrophysiology Recordings on Mouse and Pike Nerves
[0164] The sciatic nerve from mice or the pike olfactory nerve was
mounted onto two pairs of platinum wires (internal diameter 0.5 mm)
connected to a Plexiglas chamber. For stimulation, the first pair
of electrodes was connected to a stimulator (S-88, Grass
Instruments) that was delivering rectangular pulses of current at
various amplitude and time. For the recording of the global action
potential (GAP), the second pair of electrodes was connected to a
home made differential amplifier at high gain. An additional
platinum wire was connecting both pairs of electrodes to the
ground. In response to the electric stimulation, the nervous
activity was collected, digitised and recorded on a computer
equipped with an analogue and digital converter with the aid of the
software program Axon Pclamp version 6.0 (Axon instruments). All
experiments were performed at room temperature. During recording,
the nerve was maintained in a humid chamber without any close
contact to any solution to avoid any short circuit. Between each
recording, the nerve was placed at 4.degree. C., in a small
container filled with either Ringer's or test solution.
In Vivo Experiments on Rabbit Eyes
[0165] The local anaesthetic activity of CnIIIA on superficial
nerve terminal endings was determined on the rabbit cornea in vivo.
For this, adult male Chilean rabbits with coloured eyes weighing
1.5-2 kg were used. The test solution was instilled into the
conjunctival sac of one of the eyes and left there for 2 min.
Stimuli were applied to the cornea by pressure from a nylon hair
stimulator at a frequency of about 2 Hz until the oculo-palpebral
reflex was evoked. Each period of stimulation consisted of 100
stimuli, or less if the oculo-palpebral reflex was evoked. An
interval of at least 5 min separated two stimulation periods. The
intensity of the anaesthetic action was expressed as the total
number of stimuli that could be applied to the cornea from the
administration of a test or anaesthetic solution until the
reappearance of the oculo-palpebral reflex. This method allowed
also determining the duration of the effect. Lidocaine HCl
(Sigma-Aldrich) was used in saline solution and its pH value was
adjusted to 6.9.+-.0.01 with 1 N NaOH.
Example 2
Results
Isolation, Purification and Characterization of a Novel
Mu-Conopeptide
[0166] The dried venom was dissolved in 0.08% TFA in water and
loaded in batches of 10 mg on a semipreparative C.sub.18 Vydac
column. A fraction eluting, at approximately 20 min in the
chromatogram, was further purified at an analytical scale. This
fraction revealed a potent preliminary activity on frog
neuromuscular junction. Application of this fraction into this ex
vivo preparation induced a block of the muscle contraction provoked
by stimulating the motor nerve. This fraction was eventually
purified to homogeneity as demonstrated by the UV chromatogram and
the ESI-MS mass spectrum (FIG. 1). The fraction was then subjected
to Edman degradation several times, but did not give any result.
Amino acid analysis of the fraction led to the identification of
several different amino acids (results not shown), thus indicating
that the fraction was of peptidic nature but with a probable
blocked N-terminus. To further characterize the compound, the
fraction was reduced using DTT and the sample was desalted prior
analysis. Tandem mass spectrometry was then performed on the
desalted sample. Selection of the 4.times. charged species at m/z
596 and manual adjustment of the collision energy allowed proper
and homogeneous fragmentation of the peptide. Manual interpretation
of the data led to the assignment of a peptide sequence bearing 22
amino acids. The sequence shared homology with previously published
mu-conopeptides and was thus named CnIIIA (table 4).
Chemical Synthesis of CnIIIA
[0167] The peptide was assembled as described above (see Peptide
synthesis). The synthetic peptide was purified to homogeneity by
reverse-phase HPLC using a gradient of ACN in acidified water.
Peptide purity and integrity were controlled by ESI-MS. Several
conditions were explored for the oxidative refolding of the linear
peptide. The peptide was dissolved in a Tris buffer (100 mM) at pH
7.8 with guanidinium chloride (0.5M), and left either under air
oxidation at 4.degree. C. or in mixtures containing various ratio
of reduced/oxidized gluthation. The final refolding experiment was
carried out using Tris 100 mM, guanidinium chloride 0.5M and
reduced/oxidized gluthation 0.5 mM/0.1 mM. This mixture was left
under stirring overnight at room temperature. It was then acidified
using acetic acid, and concentrated using a C.sub.18 SepPak
cartridge following manufacturer protocols. The final folded
peptide was purified by reverse-phase chromatography at a
semi-preparative scale. It appeared homogeneous and led to an
approximate yield of 35% starting from the linear entity. The
purity of the synthetic compound was assessed by HPLC and ESI-MS
analysis. The authenticity of the synthetic peptide with the
natural form was confirmed by HPLC co-elution and MS/MS analysis
(FIG. 2). Synthetic CnIIIA was thus used for the different
biological assays.
Effect on the Mouse Hemidiaphragm Contraction
[0168] The activity of CnIIIA was assessed on the muscle
contraction induced by direct mouse hemidiaphragm stimulation (FIG.
3). In each condition (absence or presence of various CnIIIA
concentrations), the contraction was recorded in response to
stimulations of 250 .mu.s and of variable intensity. This allowed
to determine the supramaximal stimulation intensity, i.e. the
intensity necessary to obtain maximal contraction amplitude. For
each CnIIIA concentration, muscle contraction recordings were
carried out 2 h. after peptide application to the preparation in
order to saturate toxin receptor sites. As shown in FIG. 3A, the
amplitude of the contraction decreases in the presence of 100 and
300 nM CnIIIA up to a complete inhibition with 600 nM CnIIIA. By
comparison, a concentration of at least 2 .mu.M mu-conotoxin GIIIA
or GIIIB is necessary for complete block of the same preparation in
identical conditions. The CnIIIA thus appears at least 4 times more
potent than existing mu-conotoxins tested in this ex vivo model.
The dose-response curve of the effect of CnIIIA reveals that the
CnIIIA concentration producing half maximal inhibition of the mouse
hemidiaphragm contraction is 150 nM (FIG. 3B). Similar results were
obtained when muscle contraction was induced by the stimulation of
the motor nerve (results not shown).
Effect on Synaptic Responses at the Mouse Neuromuscular
Junction
[0169] In order to assess the CnIIIA selectivity of action between
muscle and nerve tissues, intracellular recordings of synaptic
responses were performed at the mouse hemidiaphragm neuromuscular
junction, after application of 600 nM CnIIIA. Firstly, results
showed that the membrane resting potential of fibres was unchanged
compared to controls. This indicates that the inhibition of muscle
contraction does not result from a depolarising effect of CnIIIA.
Secondly, miniature endplate potentials (MEPPs) could be detected
in the presence of CnIIIA, thus demonstrating that the sensitivity
of the nicotinic acetylcholine receptors was not altered at doses
producing complete blockade of the muscle contraction. Finally, in
the presence of 600 nM of CnIIIA, the nerve stimulation was able to
give rise to phasic synaptic responses. Hence, endplate potentials
(EPPs), similar to controls, could be recorded. This indicates that
the nerve conduction was not altered at this conotoxin
concentration. Moreover, extracellular current recordings allowed
the detection of a presynaptic current, which reflects the presence
of the presynaptic nerve action potential in motor nerve endings. A
postsynaptic current was also observed, due to the opening of
cationic synaptic channels in the muscle membrane.
Effects on the Muscle Action Potential and Synaptic Responses at
Frog Neuromuscular Preparations
[0170] The effects of CnIIIA were studied on the frog cutaneous
pectoris nerve-muscle preparation (FIG. 4). In this preparation,
uncoupling the excitation-contraction is easily achieved by
formamide treatment, thus allowing intracellular recordings of
muscle action potentials induced by nerve stimulation without
movement. Results showed that in the presence of 2 .mu.M CnIIIA,
the membrane resting potential of fibres is similar to controls.
Application of 1 .mu.M CnIIIA to the preparation, for 5-20 min.
caused a decrease in amplitude and an increase in duration of
action potentials evoked by motor nerve stimulation. After 25 min,
the muscle action potential was completely abolished whereas EPPs
could still be recorded. Similar effects, although occurring
faster, were observed in the presence of higher concentrations of
CnIIIA (2 .mu.M). It should be noted that the latency time between
nerve stimulation and muscle response was not significantly
affected by the conotoxin. CnIIIA applications (1 and 2 .mu.M) also
did not alter the amplitude and the frequency of MEPPs compared to
controls. These results strongly suggest that the inhibition of the
skeletal muscle contraction, produced by the conotoxin CnIIIA,
results from a preferential blockade of the muscle action
potential, which is thus more sensitive to the conotoxin than the
nerve action potential.
Effect on the Global Action Potential (GAP) of the Mouse Sciatic
Motor Nerve
[0171] Applicants optimized the duration and intensity of the
stimulation to get a GAP that represents the activity of all the
fibres constituting the nerve. For a given duration of stimulation
(0.10, 0.05 or 0.01 ms), the GAP amplitude increased with
increasing intensity of the stimulation applied (0.1 to 15 V), as a
consequence of an enhancement in the number of fibres recruited. In
response to a stimulation intensity equal or superior than 7 V, the
GAP amplitude reached a maximum value which remained constant
whatever the stimulation duration (0.05 or 0.10 ms). This means
that all the fibres of the nerve responded to the stimulation. In
contrast, a 0.01 ms-stimulation was not sufficient to recruit all
the fibres, as the maximum amplitude of the GAP was only 92% of
that recorded after a stimulation of 0.05 or 0.10 ms. To study the
effect of CnIIIA on the GAP, the duration of the stimulation was
thus set to 0.05 ms and the intensity applied increased from 0.1 to
15 V. However, in order to attest that our experimental conditions
were optimal for any concentration of CnIIIA studied, stimulations
of 0.10 ms at various intensities were also applied. At
concentrations ranging from 0.1 to 50 .mu.M, CnIIIA was found to
decrease the GAP amplitude which reached an almost zero value when
the nerve was treated during 30 to 60 min with 50 .mu.M CnIIIA
(FIGS. 5A, B). In addition, the stimulation intensity necessary to
reach 50% of the GAP maximum amplitude increased with increasing
conotoxin concentration (FIG. 5C). Finally, CnIIIA did not
significantly modify the propagation velocity of GAP.
[0172] Altogether, these results show conclusively that the
mu-conotoxin CnIIIA acts on mice sciatic nerves by decreasing the
response of individual fibres without altering significantly their
membrane excitability.
[0173] The dose-response curve of the effect of CnIIIA on the mouse
sciatic nerve revealed that a concentration of 1.53 .mu.M of
conotoxin reduced by 50% the maximum GAP amplitude of the sciatic
nerves (FIG. 6). These data show that the motor nerve response is
ten times less sensitive to CnIIIA than the muscle contraction
response. These results also indicate that the mu-conotoxin is
about 1000 times more potent than classical anaesthetics such as
lidocaine on the mouse sciatic nerve. Millimolar concentrations of
lidocaine are indeed necessary to obtain similar inhibitory effect
on mouse sciatic nerve.
[0174] The reversibility of the effect of CnIIIA was evaluated by
recording the GAP of sciatic nerves firstly in the presence of
various concentrations of conotoxin (2, 10 and 50 .mu.M), and
secondly at various times (from 2 to 24 h) after the immersion of
nerves in a mammalian Ringer's solution devoid of CnIIIA. Even
after a 24 h washing, only a slight increase in the GAP amplitude
was observed (FIG. 7). To test whether the absence of reversibility
could be due to a spontaneous decrease of the GAP amplitude as a
function of time ("run-down" phenomenon), the GAP of sciatic nerves
was recorded at various times (from 2 to 66 h) after bathing the
nerves only in a mammalian Krebs-Ringer's solution. Under these
conditions, no significant modification of the GAP amplitude was
observed.
[0175] Altogether, these data strongly suggest that mu-conotoxin
CnIIIA is firmly associated to a receptor site on mouse sciatic
nerves, i.e. the dissociation of the complex conotoxin/receptor
occurs rather slowly.
Effect on the Global Action Potential (GAP) of the Olfactory Nerves
of Pike
[0176] The sensory olfactory nerve of the European pike (Esox
lucius) contains approximately five millions of relatively
homogenous (95%) unmyelinated axons with an average diameter of
0.20 .mu.m. This nerve has a high density of axonal membrane
packing, and is therefore an exceptional model for biophysical,
electrophysiological and pharmacological investigations. The
optimal conditions (intensity and duration of stimulations) for
recording the GAP of all the fibres constituting this sensory nerve
were previously reported (Benoit et al., 2000). All the fibres of
the olfactory nerve were recruited for an intensity and duration of
stimulation of 8-9 V and 7-8 ms, respectively. Under such
conditions, the maximum amplitude of the response was 2.77.+-.0.15
mV (n=37), and propagated at a velocity of 12.+-.0.5 cm/s (n=37)
which is 60 times slower than in the sciatic nerve composed of
myelinated axons. Therefore, the effect of CnIIIA was studied on
the GAP of the pike olfactory nerve using stimulations of 8 ms
duration and of 1-15 V intensities. A decrease of the GAP amplitude
was observed when the olfactory nerves were treated with increasing
concentrations of conotoxin, and no GAP could be recorded with 10
.mu.M of conotoxin applied for 30-60 min (FIGS. 8A, B). In
addition, the intensity of stimulation corresponding to 50% of
maximum GAP amplitude (i.e. recorded at 15 V) increased with
increasing concentrations of conotoxin. Finally, CnIIIA did not
significantly modify the propagation velocity of the GAP (FIG.
8C).
[0177] Altogether, these results show conclusively that CnIIIA acts
on the pike olfactory nerve by decreasing the response of
individual fibres without altering significantly their membrane
excitability.
[0178] The dose-response curve of the effect of CnIIIA on the pike
olfactory nerve revealed that a concentration of 0.15 .mu.M of
conotoxin reduced by 50% the maximum GAP amplitude of olfactory
nerves (FIG. 9). These data show that the response of unmyelinated
axons constituting the olfactory sensory nerve is as sensitive to
CnIIIA as the mouse muscle (see FIG. 3B) and is ten times more
sensitive to the mu-conotoxin than the response of myelinated axons
constituting the sciatic motor nerve (see FIG. 6). The
reversibility of the effect of CnIIIA was evaluated by recording
the GAP of pike olfactory nerves firstly in the presence of various
concentrations of conotoxin (1, 2 and 10 .mu.M), and secondly at
various times (from 12 to 24 h) after the immersion of nerves in a
pike Ringer's solution devoid of CnIIIA. Even after a 24 h washing,
no increase in the GAP amplitude could be detected. To test whether
the absence of reversibility was due to a spontaneous decrease of
the GAP amplitude in function of time ("run-down" phenomenon), the
GAP of olfactory nerves was recorded after bathing the nerves only
in a pike Ringer's solution, under various experimental conditions:
(i) at room temperature in the experimental chamber for a period of
30-60 min (n=10), (ii) at room temperature in the pike Ringer's
solution for less than 3 h (n=14), and (iii) at 4.degree. C. in the
pike Ringer's solution for 48 h (n=66) and for 72 h (n=46).
Whatever the experimental conditions were, no significant
modification of the GAP amplitude was observed.
[0179] Altogether, these data strongly suggest that mu-conotoxin
CnIIIA is firmly associated to a receptor site on the pike
olfactory nerve, i.e. the dissociation of the complex
conotoxin/receptor occurs rather slowly.
Surface Anaesthetic Effect of CnIIIA in Rabbit Eyes, and Comparison
to that of Lidocaine
[0180] Results obtained on the rabbit cornea indicate that the
duration of anaesthetic action of lidocaine at concentrations of
2.5, 5.0 and 10 g/l was 5.3, 14.2 and 22.3 min, respectively.
CnIIIA was not only more active than lidocaine on equimolar basis,
but also its duration of action lasted longer, as shown in Table 2
below and in FIG. 10. The intensity of the anaesthetic action of
CnIIIA, expressed as the sum of the number of stimuli applied to
the corneal surface until the reappearance of the blinking reflex,
was also more important than for lidocaine. Interestingly, the
corneal reflex recovered without detectable damage of the mucous
surface after CnIIIA.
TABLE-US-00003 TABLE 2 Drug Dose (g/L) Mean Stimuli (#) S.E.M. n
CnIIIA 0.025 300 .+-.16 6 Lidocaine 2.5 15 .+-.21 6 5 245 .+-.10 6
10 420 .+-.16 6
In Vitro Experiments on Sodium Current Recorded from HEK Cells by
Patch-Clamp
[0181] Patch-clamp current recordings were performed in HEK 293
cells stably expressing the rat skeletal muscle Na channel .alpha.
subunit (.mu.l, Nav1.4) (Yamagishi et al., 1997). These cells
display robust Na currents (>2 nA), are sensitive to saxitoxin
(STX) and derivatives (Velez et al., 2001), and have a small size
(diameter <20 .mu.m), allowing an appropriate control of the
holding potential.
[0182] Whole-cell patch-clamp recordings (Hamill et al., 1981) were
performed at room temperature (20-22.degree. C.) on HEK 293 cells
stably expressing Nav1.4 channels. Patch pipettes made from
borosilicate glass and pulled on a P-97 puller (Sutter Instrument
Company, Novato, Calif.) had a 1.5-3.0 M.OMEGA. tip resistances
when filled with internal physiological solution. Membrane currents
were recorded using an Axopatch 200-B patch-clamp amplifier (Axon
Instruments, Union City, Calif.). Peak sodium currents were
elicited by 10-ms depolarizing pulses from a holding voltage of
-100 to -10 mV. A P/4 protocol was used to subtract linear
capacitative and leak currents. Membrane currents were filtered
with an integrated 8-pole low-pass Bessel filter at 10 kHz. The
filtered signals were digitized by a 12 bit A/D converter (Digidata
1200B, Axon Instruments) and stored using pCLAMP software (Axon
Instruments). Recordings were analyzed using Origin 7 software
(OriginLab Corp., Northampton, Mass.).
[0183] The cells were continuously perfused at 1 ml min.sup.-1 with
a control external solution containing (in mM): 70 NaCl, 70
tetraethylammonium chlorhidrate, 5 KCl, 3 CaCl.sub.2, 1 MgCl.sub.2,
10 mM glucose, 10 HEPES (pH 7.4). The patch pipette contained (in
mM) 140 CsF, 5 NaCl, 1 MgCl.sub.2, 10 EGTA, 10 HEPES buffer (pH
7.2). Na currents were recorded under control conditions and after
perfusion with different concentrations of .mu.-conotoxin CnIIIA
(.mu.-CnIIIA) or with saxitoxin diacetate (STX) (Sigma-Aldrich
Chemical Corp).
[0184] As shown in a typical experiment (FIG. 15), .mu.-CnIIIA (50
nM) applied by bath superfusion blocked sodium currents (FIGS. 15A
and 15B) elicited by a family of depolarizing pulses from -100 to
-10 mV. .mu.-CnIIIA blocked sodium current in a
concentration-dependent manner as determined by sigmoidal nonlinear
regression curve fitting for concentration-response data. The
effective concentration that reduced 50% peak sodium current
(EC.sub.50) was 14.0 nM. As shown in FIG. 1C, washout began after
peak sodium currents had reached a steady-state level in the
presence of .mu.-CnIIIA did not reversed upon washing with a
peptide-free medium. In contrast, STX action on sodium currents was
completely reversed within 2-3 min perfusion with a STX-free
solution. In typical experiments, .mu.-CnIIIA was persistent while
STX effect was reversible upon washing out from the medium.
In Vivo Experiments on Mice--Digit Abduction Score (DAS) Assay
[0185] These experiments were performed on adult (between 2 and 3
months old) male or female Swiss-Webster mice (20-40 g). Each
lightly anesthetized mouse received a single intramuscular
injection of 50 or 100 .mu.L physiological solution containing
.mu.-CnIIIA or procaine into the antero-lateral region of the left
hind limb. After the injection, functional recoveries were
monitored by using the DAS assay (Aoki, 2001). Briefly, mice were
suspended by the tail to elicit a characteristic startle response
in which the animal extends its hind limbs and abducts its hind
digits. Following .mu.-CnIIIA or procaine injection, the degree of
digit abduction of the left and right hind limbs was determined as
a function of time, and scored on a five-point scale (0=normal to
4=maximal reduction in digit abduction and leg extension) by an
observer who was masked to treatment.
In Vivo Experiments on Mice--Grip Strength Assessment
[0186] Each lightly anesthetized mouse received a single
intramuscular injection of 50 .mu.L physiological solution
containing .mu.-CnIIIA or procaine into the antero-lateral region
of each front limb. Muscle strength was measured, before and at
various times after the injection, using a grip strength meter for
mice (600 g range; Technical and Scientific Equipment GmbH, Bad
Homburg, Germany), connected to a laptop computer. The test was
carried out essentially as originally described for rats (Tilson
& Cabe, 1978). Briefly, mice were held on the base of the tail
and allowed to firmly grab the pulling bar of the device with both
forepaws. The mouse was then pulled gently backwards until it
released its grip. The peak force of each trial was considered the
grip strength. Each mouse performed three trials, which were about
30 s apart. The averaged value of the trials was expressed
relatively to the corresponding control, and used for statistical
analysis (mean.+-.SEM of 2-3 mice).
[0187] The results (FIG. 12) show that a decrease of 50% of the
relative strength occurred about 5 and 10 min after intramuscular
injection of 108-111 pmoles .mu.-CnIIIA and 22-26 pmoles procaine
per g of mouse, respectively. In addition of occurring about 2
times faster, the effect was also more pronounced in the presence
of the peptide than in the presence of the local anaesthetic.
Therefore, intramuscular injection of .mu.-CnIIIA is at least about
5000 fold more effective than procaine to produce, in vivo, a
decrease of the muscle strength of mice.
Sodium Channel Expression
[0188] For expression in X. laevis oocytes, the Nav1.4/pUI-2
vectors were linearized with NotI and transcribed with the T7
mMESSAGE mMACHINE kit (Ambion).
Electrophysiological Studies on Cloned Channels
[0189] Oocytes were injected with 50 nl of cRNA at a concentration
of 1 ng/nl using a microinjector from Drummond Scientific
(Broomall, Pa.). The solution used for incubating the oocytes
contained 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl.sub.2, 2 mM MgCl.sub.2,
and 5 mM HEPES, pH 7.4, supplemented with 50 mg/l gentamycin
sulfate and 180 mg/l theophylline. Two-electrode voltage-clamp
(TEVC) recordings were performed at room temperature (18-22.degree.
C.) using a GeneClamp 500 amplifier (Molecular Devices) controlled
by a pClamp data acquisition system (Molecular Devices). Whole-cell
currents from oocytes were recorded 1 day after injection. Voltage
and current electrodes were filled with 3 M KCl. Resistances of
both electrodes were kept as low as possible. Bath solution
composition was 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl.sub.2, 2 mM
MgCl.sub.2, and 5 mM HEPES, pH 7.4. Currents were filtered at 1 kHz
with a four-pole, low-pass Bessel filter and sampled at 5 kHz. Leak
subtraction was performed using a -P/4 protocol. Currents were
evoked in oocytes expressing the cloned Nav1.4 voltage gated sodium
channel by depolarizations to the test potential of -10 mV for 100
ms from an holding potential of -90 mV. The pulse frequency was 0.2
Hz and the sampling frequency of 20 kHz. All experiments were
carried out at room temperature.
[0190] The results shown in FIG. 14 demonstrate the ability of
CnIIIA (500 nM) to block expressed Nav1.4 channels. From these
experiments, it can be concluded that the peptide CnIIIA is able to
block the voltage-gated sodium channel Nav1.4 isoform with an IC50
estimated in the range 1-50 nM. The peptide-channel interaction is
compatible to a bimolecular reaction in which the association rate
is concentration dependant. Finally, the reversibility of the
effect has been noted as very low.
Example 3
[0191] CnIIIA Comparison with Other Mu-Conotoxins
In Vitro Experiments on the Contraction of EDL Muscle of Mice
[0192] EDL muscles were isolated from mice killed by dislocation of
the cervical vertebrae followed by immediate exsanguination.
Isolated muscles were mounted in silicone-lined Plexiglass baths (4
ml volume) containing standard Krebs-Ringer physiological solution
of the following composition (in mM): 154 NaCl; 5 KCl; 2
CaCl.sub.2; 1 MgCl.sub.2; 5 HEPES buffer (pH 7.4); 11 glucose. The
solution was gassed with pure O.sub.2.
[0193] For twitch tension measurements, one of the tendons of EDL
muscle was tied with silk thread, via an adjustable stainless steel
hook, to an FT03 isometric transducer (Grass Instruments,
Astro-Med. Inc., West Warwick, R.I., USA), and the other tendon was
pinned to the silicone-lined bath with stainless steel pins.
Twitches were evoked by stimulating directly the muscle fibers by
current pulses of 0.2 ms duration and supramaximal intensity,
supplied by a S-44 stimulator (Grass Instruments, Astro-Med. Inc.,
West Warwick, R.I., USA) to an electrode array placed along the
muscle, at frequencies of 0.1 Hz. For each preparation
investigated, the resting tension was adjusted to obtain maximal
contractile responses and was monitored during the whole duration
of the experiment. Tension signals from the isometric transducer
were amplified, collected, and digitized with the aid of a computer
equipped with a DT2821 analogue to digital interface board (Data
Translation, Marlboro, Mass., USA). Computerized data acquisition
and analysis were performed with a program kindly provided by Dr.
John Dempster (Department of Physiology & Pharmacology,
University of Strathclyde, Glasgow, Scotland). All experiments were
performed at 22.degree. C. After preparations were equilibrated for
15 min with the oxygenated physiological solution containing 10
.mu.M tubocurarine (to block nicotinic receptors), .mu.-conotoxins
(CnIIIA, TIIIA, T3.1, PIIIA, SmIIIA, or SIIIA) were added to the
medium.
[0194] To compare the relative effects of the different
.mu.-conotoxins investigated on EDL muscles, a similar
concentration (i.e. 100 nM) of .mu.-conotoxins was tested on muscle
contraction. When possible, concentration-response curves were
generated in individual muscles (the contraction measured in the
presence of various concentrations of a given .mu.-conotoxin being
expressed as percent of the control twitch response). Each
.mu.-conotoxin concentration was applied by perfusion and allowed
to equilibrate for 45-60 min. Sigmoidal nonlinear regression curve
fitting for concentration-response data allowed an estimation of
the effective concentration that reduces the twitch tension by 50%
(EC.sub.50).
Results (FIG. 13)
[0195] The relative mean contraction inhibition of the muscle is
given for each peptide (100 nM) by comparison to CnIIIA (100 nM)
after 40 min incubation. CnIIIA has been normalized to 100% for
easy comparison. It can be noted that all peptides SmIIIA, PIIIA
and T3.1 display a lower activity than CnIIIA (table below).
TABLE-US-00004 TABLE 3 .mu.-conotoxin Relative Mean % SEM % n
CnIIIA 100.00 1.34 19 SmIIIA 83.00 0.85 11 PIIIA 50.00 3.70 11 T3.1
3.30 1.12 12
[0196] Sigmoidal nonlinear regression curve fitting for
concentration-response data allowed an estimation of the effective
concentration that reduces the twitch tension by 50% (EC.sub.50)
and thus deduce the K.sub.D (in nM) for each peptide. The results
are presented in table below:
TABLE-US-00005 TABLE 4 .mu.-conotoxin K.sub.D (nM) CnIIIA 125
SmIIIA 130 PIIIA 500 T3.1 800
REFERENCE LIST
[0197] Amblard M. et al, 2005, Methods Mol Biol. 298, 3-24 [0198]
Baker, M. D. and Wood, J. N., 2001. Involvement of Na+ channels in
pain pathways. Trends Pharmacol. Sci. 1 (22), 27-31. [0199] Becker,
S., Atherton, E., Gordon, R. D., 1989. Synthesis and
characterization of mu-conotoxin III. Eur. J. Biochem. 1 (185),
79-84. [0200] Benoit, E., Charpentier G, Mateu L, Luzzati V, Kado
R, 2000. The pike olfactory nerve: a source of unmyelinated sensory
axons. Cybium, Rev. Eur. Ichtyol. 3 (24), 241-248. [0201] Bulaj,
G., West, P. J., Garrett, J. E., Marsh, M., Zhang, M. M., Norton,
R. S., Smith, B. J., Yoshikami, D., Olivera, B. M., 2005. Novel
conotoxins from Conus striatus and Conus kinoshitai selectively
block TTX-resistant sodium channels. Biochemistry 19 (44),
7259-7265. [0202] Cruz, L. J., Gray, W. R., Olivera, B. M., Zeikus,
R. D., Kerr, L., Yoshikami, D., Moczydlowski, E., 1985. Conus
geographus toxins that discriminate between neuronal and muscle
sodium channels. J. Biol. Chem. 16 (260), 9280-9288. [0203] Cruz,
L. J., Kupryszewski, G., LeCheminant, G. W., Gray, W. R., Olivera,
B. M., Rivier, J., 1989. mu-conotoxin GIIIA, a peptide ligand for
muscle sodium channels: chemical synthesis, radiolabeling, and
receptor characterization. Biochemistry 8 (28), 3437-3442. [0204]
Decosterd, I., Ji, R. R., Abdi, S., Tate, S., Woolf, C. J., 2002.
The pattern of expression of the voltage-gated sodium channels
Na(v)1.8 and Na(v)1.9 does not change in uninjured primary sensory
neurons in experimental neuropathic pain models. Pain 3 (96),
269-277. [0205] Fainzilber, M., Kofman, O., Zlotkin, E., Gordon,
D., 1994. A new neurotoxin receptor site on sodium channels is
identified by a conotoxin that affects sodium channel inactivation
in molluscs and acts as an antagonist in rat brain. J. Biol. Chem.
4 (269), 2574-2580. [0206] Fainzilber, M., Nakamura, T., Gaathon,
A., Lodder, J. C., Kits, K. S., Burlingame, A. L., Zlotkin, E.,
1995. A new cysteine framework in sodium channel blocking
conotoxins. Biochemistry 27 (34), 8649-8656. [0207] Finucane B. T.,
2005. Allergies to local anesthetics--the real truth. 50:869-874.
Canadian Journal of Anesthesia (50), 869-874. [0208] French, R. J.,
Prusak-Sochaczewski, E., Zamponi, G. W., Becker, S., Kularatna, A.
S., Horn, R., 1996. Interactions between a pore-blocking peptide
and the voltage sensor of the sodium channel: an electrostatic
approach to channel geometry. Neuron 2 (16), 407-413. [0209] Gold,
M. S., Weinreich, D., Kim, C. S., Wang, R., Treanor, J., Porreca,
F., Lai, J., 2003. Redistribution of Na(V)1.8 in uninjured axons
enables neuropathic pain. J. Neurosci. 1 (23), 158-166. [0210]
Hill, J. M., Alewood, P. F., Craik, D. J., 1996. Three-dimensional
solution structure of mu-conotoxin GIIIB, a specific blocker of
skeletal muscle sodium channels. Biochemistry 27 (35), 8824-8835.
[0211] Julius, D. and Basbaum, A. I., 2001. Molecular mechanisms of
nociception. Nature 6852 (413), 203-210. [0212] Keizer, D. W.,
West, P. J., Lee, E. F., Yoshikami, D., Olivera, B. M., Bulaj, G.,
Norton, R. S., 2003. Structural basis for tetrodotoxin-resistant
sodium channel binding by mu-conotoxin SmIIIA. J. Biol. Chem. 47
(278), 46805-46813. [0213] Kerr, L. M. and Yoshikami, D., 1984. A
venom peptide with a novel presynaptic blocking action. Nature 5956
(308), 282-284. [0214] Lee, A. G., 1976. Model for action of local
anaesthetics. Nature 5569 (262), 545-548. [0215] McIntosh, J. M.,
Olivera, B. M., Cruz, L. J., 1999. Conus peptides as probes for ion
channels. Methods Enzymol. (294), 605-624. [0216] Moczydlowski, E.,
Olivera, B. M., Gray, W. R., Strichartz, G. R., 1986.
Discrimination of muscle and neuronal Na-channel subtypes by
binding competition between [3H]saxitoxin and mu-conotoxins. Proc.
Natl. Acad. Sci. U.S.A 14 (83), 5321-5325. [0217] Nielsen, K. J.,
Watson, M., Adams, D. J., Hammarstrom, A. K., Gage, P. W., Hill, J.
M., Craik, D. J., Thomas, L., Adams, D., Alewood, P. F., Lewis, R.
J., 2002. Solution structure of mu-conotoxin PIIIA, a preferential
inhibitor of persistent tetrodotoxin-sensitive sodium channels. J.
Biol. Chem. 30 (277), 27247-27255. [0218] Olivera, B. M., Cruz, L.
J., de, S., V, LeCheminant, G. W., Griffin, D., Zeikus, R.,
McIntosh, J. M., Galyean, R., Varga, J., Gray, W. R., 1987.
Neuronal calcium channel antagonists. Discrimination between
calcium channel subtypes using omega-conotoxin from Conus magus
venom. Biochemistry 8 (26), 2086-2090. [0219] Olivera, B. M., Gray,
W. R., Zeikus, R., McIntosh, J. M., Varga, J., Rivier, J., de, S.,
V, Cruz, L. J., 1985. Peptide neurotoxins from fish-hunting cone
snails. Science 4732 (230), 1338-1343. [0220] Olivera, B. M.,
McIntosh, J. M., Cruz, L. J., Luque, F. A., Gray, W. R., 1984.
Purification and sequence of a presynaptic peptide toxin from Conus
geographus venom. Biochemistry 22 (23), 5087-5090. [0221] Olivera,
B. M., Rivier, J., Clark, C., Ramilo, C. A., Corpuz, G. P.,
Abogadie, F. C., Mena, E. E., Woodward, S. R., Hillyard, D. R.,
Cruz, L. J., 1990. Diversity of Conus neuropeptides. Science 4966
(249), 257-263. [0222] Ott, K. H., Becker, S., Gordon, R. D.,
Ruterjans, H., 1991. Solution structure of mu-conotoxin GIIIA
analysed by 2D-NMR and distance geometry calculations. FEBS Lett. 2
(278), 160-166. [0223] Safo, P., Rosenbaum, T., Shcherbatko, A.,
Choi, D. Y., Han, E., Toledo-Aral, J. J., Olivera, B. M., Brehm,
P., Mandel, G., 2000. Distinction among neuronal subtypes of
voltage-activated sodium channels by mu-conotoxin PIIIA. J.
Neurosci. 1 (20), 76-80. [0224] Sato, K., Ishida, Y., Wakamatsu,
K., Kato, R., Honda, H., Ohizumi, Y., Nakamura, H., Ohya, M.,
Lancelin, J. M., Kohda, D., 1991. Active site of mu-conotoxin
GIIIA, a peptide blocker of muscle sodium channels. J. Biol. Chem.
26 (266), 16989-16991. [0225] Sato, S., Nakamura, H., Ohizumi, Y.,
Kobayashi, J., Hirata, Y., 1983. The amino acid sequences of
homologous hydroxyproline-containing myotoxins from the marine
snail Conus geographus venom. FEBS Lett. 2 (155), 277-280. [0226]
Scholz, A., 2002. Mechanisms of (local) anaesthetics on
voltage-gated sodium and other ion channels. Br. J. Anaesth. 1
(89), 52-61. [0227] Shichor, I., Fainzilber, M., Pelhate, M.,
Malecot, C. O., Zlotkin, E., Gordon, D., 1996. Interactions of
delta-conotoxins with alkaloid neurotoxins reveal differences
between the silent and effective binding sites on voltage-sensitive
sodium channels. J. Neurochem. 6 (67), 2451-2460. [0228] Shon, K.
J., Olivera, B. M., Watkins, M., Jacobsen, R. B., Gray, W. R.,
Floresca, C. Z., Cruz, L. J., Hillyard, D. R., Brink, A., Terlau,
H., Yoshikami, D., 1998. mu-Conotoxin PIIIA, a new peptide for
discriminating among tetrodotoxin-sensitive Na channel subtypes. J.
Neurosci. 12 (18), 4473-4481. [0229] Wakamatsu, K., Kohda, D.,
Hatanaka, H., Lancelin, J. M., Ishida, Y., Oya, M., Nakamura, H.,
Inagaki, F., Sato, K., 1992. Structure-activity relationships of
mu-conotoxin GIIIA: structure determination of active and inactive
sodium channel blocker peptides by NMR and simulated annealing
calculations. Biochemistry 50 (31), 12577-12584. [0230] West, P.
J., Bulaj, G., Garrett, J. E., Olivera, B. M., Yoshikami, D., 2002.
Mu-conotoxin SmIIIA, a potent inhibitor of tetrodotoxin-resistant
sodium channels in amphibian sympathetic and sensory neurons.
Biochemistry 51 (41), 15388-15393. [0231] Yu, F. H. and Catterall,
W. A., 2003. Overview of the voltage-gated sodium channel family.
Genome Biol. 3 (4), 207
Sequence CWU 1
1
2123PRTArtificial SequenceSynthesized sequence or isolated from a
natural source (e.g., marine cone snail of the genus
Conus).MISC_FEATURE(1)..(1)Xaa is any N-modified amino
acidMISC_FEATURE(2)..(2)Xaa is glycineMISC_FEATURE(5)..(5)Xaa is
any acidic amino acid or an amide form
thereofMISC_FEATURE(6)..(6)Xaa is glycineMISC_FEATURE(7)..(7)Xaa is
proline or hydroxy-prolineMISC_FEATURE(8)..(8)Xaa is any basic
amino acidMISC_FEATURE(9)..(9)Xaa is
glycineMISC_FEATURE(11)..(11)Xaa is any non-aromatic hydroxyl amino
acidMISC_FEATURE(12)..(12)Xaa is any non-aromatic hydroxyl amino
acidMISC_FEATURE(13)..(13)Xaa is any basic amino
acidMISC_FEATURE(14)..(14)Xaa is any aromatic amino
acidMISC_FEATURE(16)..(16)Xaa is any basic amino
acidMISC_FEATURE(17)..(17)Xaa is any acidic amino acid or an amide
form thereofMISC_FEATURE(18)..(18)Xaa is any basic amino acid, or
any sulfur-containing amino acidMISC_FEATURE(19)..(19)Xaa is any
hydrophobic or apolar amino acid, or any non-aromatic hydroxyl
amino acidMISC_FEATURE(20)..(20)Xaa is any basic amino
acidMISC_FEATURE(22)..(22)Where Xaa at position 23 is absent, this
residue may be an amide form/amidated.MISC_FEATURE(23)..(23)Xaa is
absent or is any apolar or slightly polar amino acid. 1Xaa Xaa Cys
Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa
Xaa Xaa Xaa Cys Cys Xaa 20 222PRTArtificial SequenceSynthesized
sequence or isolated from a natural source (e.g., marine cone snail
of the genus Conus).MISC_FEATURE(1)..(1)Xaa stands for pyroglutamic
acid 2Xaa Gly Cys Cys Asn Gly Pro Lys Gly Cys Ser Ser Lys Trp Cys
Arg 1 5 10 15 Asp His Ala Arg Cys Cys 20
* * * * *