Note: Descriptions are shown in the official language in which they were submitted.
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CYCLISED ALPHA-CONOTOXIN PEPTIDES
Background of the Invention
This invention relates to a-conotoxin peptides, and in particular to cyclised
a-conotoxin
peptides useful in the therapeutic treatment of humans. The invention
especially relates
to oral and enteral preparations comprising these peptides, the use of these
peptides in
the manufacture of pharmaceutical preparations, and the use of these
pharmaceutical
preparations in the prophylaxis or treatment of conditions or diseases in
humans.
The marine snails of the genus Conus (cone snails) use a sophisticated
biochemical
strategy to capture their prey. As predators of either fish, worms or other
molluscs, the
cone snails inject their prey with venom containing a cocktail of small
bioactive peptides.
These toxin molecules, which are referred to as conotoxins, interfere with
neurotransmission by targeting a variety of ion-channels or receptors. They
typically
contain 12-30 amino acids arranged in linear sequence. The venom from any
single Conus
species may contain more than 100 different peptides. The conotoxins are
divided into
classes on the basis of their physiological targets. For example, the a-
conotoxin and yr-
conotoxins target nicotinic ACh receptors, causing ganglionic and
neuromuscular
blockade, while the w-conotoxin class of peptides target voltage-sensitive
Ca2+-channels,
inhibiting neurotransmitter release.
Most conotoxin peptides contain either four (4) or six (6) cysteine residues
which are
bonded in pairs to form either two (2) or three (3) disulfide bonds
respectively, although
there are some examples having two cysteine residues bonded to form a single
disulfide
bond (i.e., conopressins), as well as some having greater than three disulfide
bonds. The
peptides of some of the "activity" classes described above share a structural
motif,
possessing the same number of cysteine residues and the same disulfide bond
connectivity.
For this reason a new "superfamily" classification system has been developed.
For
example, the w-conotoxins and members of the X, 8 and -conotoxin classes have
six
cysteine residues which are bonded in pairs to form three disulfide bonds
between cysteine
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residues I and IV, II and V, and III and VI, where the six Roman numerals
represent the six
cysteine residues numbering from the N-terminus. Conotoxin peptides having
this
structural motif belong to the O-superfamily of conotoxins. Similarly, p-
conotoxins and
most a-conotoxins have four cysteine residues bonded in pairs to form two
disulfide bonds
between cysteine residues I and III, and II and IV. These conotoxin peptides
belong to the
"A-superfamily" of conotoxins. The present invention relates to a-conotoxins
in the A-
superfamily, i.e. a-conotoxin peptides having two disulfide bonds formed
between
cysteine residues I and III, and II and IV. As indicated above, conotoxin
peptides bind to a
range of different ion channel receptors in mammals and accordingly they have
several
potential therapeutic applications, including pain relief in humans. However,
in general
peptides have several difficulties associated with their use as drugs,
including generally
poor bioavailability, susceptibility to cleavage by proteases, and unwanted
side effects.
One w-conotoxin, MVIIA (also known as SNX-111, Ziconitide and Prialt),
recently
received approval by the United States Food and Drug Administration for the
treatment of
intractable pain associated with cancer, AIDS and neuropathies. The route of
administration is currently restricted to intrathecal infusion into the spinal
cord because of
some of the abovementioned difficulties, and because the receptors targetted
by this drug
are located within the CNS.
Another co-conotoxin which has commenced clinical trial is CVID (AM336 -
Zenyth
Pharmaceuticals) which is reported to have improved selectivity for N-type
calcium
channels over P/Q-type channels relative to Ziconitide. However, conotoxins of
the w
class have still been associated with undesirable side effects in some
patients. Two other
conotoxin peptides (CGX-1 160 and CGX-1007 isolated from Conus geographus) are
also
undergoing clinical trials, however administration of these peptides is also
restricted to the
intrathecal route. Another peptide being investigated for pain is a conotoxin
of the x-class
(Xen 2174 - Xenome Ltd). Again, administration of this peptide is limited to
the
intrathecal route.
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The a-conotoxins are a sub-family of conotoxins that typically range in size
from 12 to 16
amino acids, and usually have an amidated C-terminus. The a-conotoxins
interact with
both muscle and neuronal nicotinic acetylcholine receptors (nAChRs) which have
been
implicated in a range of disorders including Alzheimer's disease,
schizophrenia, depression
and small cell lung carcinoma, as well as playing a role in analgesia and
addiction. a-
conotoxin peptides and their potential uses are widely described in the
literature (see Lloyd
and Williams, 2000, J Pharmacol Exp Ther 292 (2) 461).
The number of residues between the cysteine residues is used to distinguish
different
classes of a-conotoxins. Based on the number of residues between the second
and third
cysteine residues (loop 1) and the third and fourth residues (loop 2) they are
divided into
a3/5, a4/3, a4/4, a4/6 and 4/7 structural subfamilies. Two examples of a-
conotoxin
peptides having 4/7 loop arrangement are MII and Vcl.l (also known as ACV1 -
Metabolic Pharmaceuticals Limited). One of the smallest a-conotoxin peptides
is IM1
which has a 4/3 loop arrangement.
Several a-conotoxin peptides have been studied to ascertain their selectivity
for the various
subtypes of nicotinic acetylcholine receptors, and in particular their
selectivity for
peripheral nAChR subtypes over central subtypes. nAChRs are expressed at low
levels
throughout the CNS and PNS, but various subtypes have different distributions.
The
mammalian nAChRs are composed of combinations of subunits. Seven of these
subunit
types are the major components involved in ligand binding (a2, a3, a4, a6, a7,
a9 and a10)
while 4 subunit types (aS, 02, 03 and 04) are considered to be structural,
imparting
functional and pharmacological properties to the receptors. The different
subtypes
combine in a variety of ways (generally as heterologous pentamers) to form
receptors
having particular pharmacological and electrophysiological properties. The a3
subunit is
considered to be a peripheral subunit (due to its presence in the PNS) while
the a7 subunit
is considered to be a subunit prevalent in the CNS.
The a-conotoxin Vcl.l was first discovered using a PCR screen of cDNAs from
the
venom ducts of Conus victoriae. The cysteine spacing within the sequence of
Vcl.l
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indicates that it is a member of the 4/7 subclass of a-conotoxins, which
includes the
extensively studied conotoxins MII, EpI and PnIB. The three dimensional
structure of
Vcl.1 comprises a small a-helix spanning residues P6 to D11 and is braced by
the I-II, III-
IV disulfide connectivity seen in other a-conotoxins (unpublished
observations). In
addition to an amidated C-terminus, which is common to most a-conotoxins, it
is also
possible to postranslationally modify residues Pro6 and G1u14 in linear Vcl.l
to
hydroxyproline and y-carboxyglutamate respectively. This post translatioiially
modified
analogue of Vcl.l is implicated in nerve regeneration but not pain (WO
02/079236).
Linear Vcl.l is not naturally post-translationally modified.
Linear Vcl.l, an antagonist of neuronal nAChRs in bovine chromaffin cells, has
been
shown to alleviate neuropathic pain in three rat models of human neuropathic
pain and to
accelerate the functional recovery of injured neurons (Satkunanathan et al.,
2005, Brain
Research 1059 (2) 149-158). As an analgesic, Vcl.1 has been reported be more
active
than Ziconotide (Sandall et al., 2003, Biochemistry 42, 6904-6911). More
recently, Vcl.l
was shown to antagonize the nicotine-induced increase in axonal excitability
of
unmyelinated C-fiber axons in isolated segments of peripheral human nerves
(Lang et al.,
2005, Neuroreport 16, 479-483). As mentioned above neuronal nAChRs are
pentameric
ligand-gated ion channels composed of combinations of a (a2 to alO) and R((32
to (34)
subunits that are found throughout both the central and peripheral nervous
systems.
Electrophysiological and immunohistochemical data indicate the functional
expression of
nAChRs composed of a3, a5 and (34 but not a4, (32 or a7 subunits in axons of
unmyelinated C fibers (Lang et al., 2005, Neuroreport 16, 479-483; and Lang et
al., 2003,
Neurophysiol 90, 3295-3303). Blockade of nAChRs on unmyelinated peripheral
nerve
fibers may have an analgesic effect on unmyelinated sympathetic and/or sensory
axons.
Interestingly, synthetic post translationally modified Vcl.l (ptmVcl.1) was
reported to not
inhibit the neuronal-type nicotinic response in chromaffin cells and was
inactive in two rat
neuropathic pain assays. Linear Vcl.1 or ACVl has commenced phase 2 human
clinical
trials.
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Nicotinic agonists have been previously reported to possess analgesic
activity. Examples
of such nicotinic agonists are epibatadine and ABT-594. It is postulated that
these agents
act by desensitising the nicotinic receptor, resulting in a reduction of ion
flux through the
receptor. Under these conditions the agonists are effectively acting as
antagonists of the
nAChRs and for this reason antagonists of nAChRs have been sought as potential
analgesic compounds. Conotoxin peptide Vcl.1 is said to be such an antagonist.
Unlike the previous conotoxin peptides which have been investigated for the
treatment of
pain and other conditions, Vcl.l is said to have the advantage that it can be
administered
subcutaneously or intramuscularly, rather than intrathecally. This is said to
provide a
significant advantage for Vcl.1 over previous conotoxin peptides, including
Ziconitide.
However, conotoxin peptide Vcl.l is said to lack oral bioavailability.
According to a
document published on Metabolic Pharmaceuticals' website dated January 2006
"ACV1 -
A novel therapeutic for neuropathic pain, Technical Summary of Preclinical
Data" ACV 1
is not orally available and current development is as a subcutaneous
injectable treatment.
Vcl.l is also reported to be effective in an animal model of inflammatory pain
and to
accelerate the recovery of injured nerves and tissues
Accordingly, there is still a need for effective method of treating patients
with a-conotoxin
peptides via oral or enteral routes, particularly in relation to the
production of analgesia,
the treatment or prevention of neuropathic pain and in the acceleration of
recovery from
nerve injury.
Summary of the Invention
The present invention is based on the surprising finding that cyclisation of
an a-conotoxin
peptide to produce a compound with an amide cyclised backbone gives rise to a
cyclised
peptide with oral bioavailability or efficacy. This is the first time that any
conotoxin
peptide has been presented in a form which allows oral or enteral delivery.
Although
previous reports in relation to conotoxin peptides have suggested that with
appropriate
protection conotoxin peptides could be delivered orally, there has been no
report or
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disclosure in the literature of any such orally or enterally administrable
composition.
Accordingly in a first aspect the present invention provides an oral or
enteral
pharmaceutical preparation comprising at least one synthetically cyclised a-
conotoxin
peptide having an amide cyclised backbone such that the peptide has no free N-
or C-
terminus, said peptide having the ability to inhibit a nicotinic acetylcholine
receptor and
comprising four cysteine residues bonded in pairs to form two disulfide bonds,
wherein the
N-terminus of the corresponding linear/non-cyclised conotoxin peptide is
linked to the C-
terminus by a peptide linker, in a vehicle which is pharmaceutically suitable
for oral or
enteral administration.
Preferably the peptide linker is such that between six and eight natural or
unnatural amino
acids span the distance between cysteine residues I and W.
The invention further provides a method for the treatment or prevention of
pain comprising
the step of orally or enterally administering a pharmaceutical preparation as
described
above.
In a further embodiment, the invention provides a method for accelerating the
recovery of
nerve damage comprising the step of orally or enterally administering a
pharmaceutical
preparation as described above.
The invention further provides a method for the treatment or prevention of
Aizheimer's
disease, schizophrenia, depression, small cell lung carcinoma, cardiovascular
disorders,
gastric motility disorders, urinary incontinence, nicotine addiction, mood
disorders (such
as bipolar disorder, unipolar depression, dysthymia, Tourette Syndrome and
seasonal effect
disorder) or inflammation comprising the step of orally or enterally
administering a
pharmaceutical preparation as described above.
In a further embodiment, the invention provides the use of a cyclised a-
conotoxin peptide
in the manufacture of a medicament for oral or enteral administration for the
treatment or
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prevention of pain or in the acceleration of recovery from nerve damage.
The invention also provides the use of a cyclised a-conotoxin peptide in the
manufacture
of a medicament for oral or enteral administration for the treatment or
prevention of
Alzheimer's disease, schizophrenia, depression, small cell lung carcinoma,
cardiovascular
disorders, gastric motility disorders, urinary incontinence, nicotine
addiction, mood
disorders (such as bipolar disorder, unipolar depression, Tourette syndrome,
dysthymia and
seasonal effect disorder) or inflammation.
Description of Preferred Embodiments
The a-conotoxin class is a very large class of conotoxin peptides with many
hundreds of
examples being described in the literature. Most of the a-conotoxin peptides
are members
of the A-superfamily, having four cysteine residues bonded in pairs forming
two disulfide
bonds between cysteine residues I and III and between cysteine residues II and
IV. Some
examples of a-conotoxin peptides which have been described in the literature
are set out in
Table 1 below.
Table 1
Vcl.1 GCCSDPRCNYDHPEIC
Vcl.lptm GCCSDORCNYDHPyIC
Vgl.1 DCCSNPPCAHNNPD-C
Anl.1 GCCSHPACYANNQDYC
PreVcl.1 GCCSDPRCNYDHPEICG
SII.4 GGCCSYPPCNVSYPEIC
Dil.1 GCCSNPPCAHNNPD-CR
Om1.1 GCCSYPPCFATNPD-C
Vr1.1 DCCSNPPCSQNNPD-CM
Vrl.2 DCCSNPPCAHNNPD-CR
Bt1.4 GCCSHPACSVNHPELC
Dal.1 GCCSHPACNVDHPEIC
TIB GCCSHPACSGNNPEFCRQ
Pnl.1 GCCSHPPCAMNNPDYC
Cr1.2 GCCSNPVCHVEHPELCRRRR
Tx1.2 GCCSRPPCIANNPDLC
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Bt1.8 GGCCSHPACSVTHPELC
Lv1.4 EDCCSDPRCSVGHQDLC
Lv1.7 EDCCSDPRCSVGHQDMC
Mr1.4 GCCSHPACSVNNQDIC
01.7 GCCSHPPCAQNNQDYC
Om1.5 GCCSDPSCNVNNPDYC
Rg1.4 GCCSHPVCKVRYPDLC
Pn1.2 GCCSHPPCFLNNPDYC
Da1.2 GCCSRPACIANNPDLC
EPI GCCSDPRCNMNNPDYC
Pn1A GCCSLPPCAANNPDYC
Pn1B GCCSLPPCALSNPDYC
AulA GCCSYPPCFATNSDYC
Au1B GCCSYPPCFATNPD-C
Au1C GCCSYPPCFATNSGYC
MII GCCSNPVCHLEHSNLC
PIA RDPCCSNPVCTVHNPQIC
GIC GCCSHPACFASNPDYC
GID IRDyCCSNPACRVNNOHVC
AnIA CCSHPACAANNQDYC
AnIB GGCCSHPACAANNQDYC
AnIC GGCCSHPACFASNPDYC
PeIA GCCSHPACSVNHPELC
BuIA GCCST-PPCAVLY---C
In the above table y represents y-carboxyglutamate and 0 represents 4-hydroxy
proline.
The remaining symbols are those commonly used to designate naturally occurring
amino
acids. Most of the above peptides have an amidated C-terminus.
The cyclisation of conotoxin peptides to improve stability was first described
by Craik et
al. in International Patent Application No. PCT/AU99/00769 filed on 14
September, 1999
(WO 00/15654). Accordingly the cyclised a-conotoxin peptides formulated
according to
the present invention may be prepared using the methodology described in that
patent
application, the entire contents of which is incorporated herein by reference.
As used herein, unless the context requires otherwise, the term "linear" when
used in
connection with a conotoxin peptide means that the peptide is in a non-
cyclised state, i.e.
the N-terminus and C-terminus have not been linked (directly or with a linker)
to form an
amide cyclised backbone. Although the presence of one or more disulfide bonds
in an a-
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conotoxin peptide will introduce a degree of circularity to the peptide, a
peptide with such
a disulfide bond is still to be regarded as "linear" if there is no
cyclisation of the backbone
of the peptide through linking of the N- and C-termini.
The terms "oral/enteral bioavailability", "orally/enterally administerable"
and "suitable for
"oral/enteral administration" as used herein refer to the ability of the
cyclised peptide to be
administered via the oral or enteral routes to provide a pharmaceutically
relevant effect,
such as the treatment or prevention of pain, Alzheimer's disease etc. or in
the acceleration
of recovery from nerve damage. Although the effect is believed to occur
through the
interaction of the peptide with its known target, the term is not intended to
impose such a
restriction on the scope of this invention. Other or alternative targets may
be involved, and
peptide metabolites may also be involved in providing the pharmaceutically
relevant
effect.
The linear a-conotoxin peptide may be any a-conotoxin peptide which is capable
of being
cyclised. It may have the sequence of a naturally occurring a-conotoxin
peptide, or it may
be a derivative thereof. Preferably the a-conotoxin peptide is one which, in
its non-
cyclised form, has an activity associated with the therapeutic treatment of
mammals, such
as humans. Since the cyclisation of the peptide has the potential to alter the
activity of the
peptide, or introduce new activities, it is possible that some cyclised
conotoxin peptides
may have modified improved therapeutic properties relative to "linear"
conotoxins. In
some cases the cyclised conotoxin peptide will have a disulfide connectivity
different to
the linear a-conotoxin peptide, for example Cys I to IV II to III (ribbon)
connectivity and
Cis I to II, III to IV (beads) connectivity. The peptide may also be presented
as a
combination of isomers.
According to one embodiment of the invention the linear a-conotoxin peptide
which is
subject to cyclisation is a 4/7 or 4/6 peptide comprising the sequence set out
below:
Xaal CCS Xaa2 P Xaa3 C Xaa4 Xaa5 Xaa6 Xaa7 Xaa8 Xaay Xaa10 C SEQ ID NO: 1
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in which Xaal is glycine or aspartate, Xaa2 to Xaa7 represent any naturally
occurring or
unnatural amino acid, Xaa8 represents proline, hydroxyproline or glutamine,
Xaa9
represents aspartate, glutamate or y-carboxyglutamate and Xaalo represents any
naturally
occurring or unnatural amino acid or may be absent.
More preferably Xaal to Xaalo are selected as follows:
Xaal is glycine or aspartate,
Xaa2 is selected from aspartate, asparagine, histidine, tyrosine, arginine or
lysine,
even more preferably from aspartate, asparagine and histidine, and most
preferably aspartate,
Xaa3 is selected from arginine, proline, alanine, valine or serine, more
preferably
arginine, proline or alanine and most preferably arginine,
Xaa4 is selected from asparagine, alanine, tyrosine, histidine, phenylalanine,
serine, isoleucine or lysine, more preferably asparagine, alanine or tyrosine
and most preferably asparagine,
Xaa5 is selected from tyrosine, histidine, alanine, valine, glutamine,
glycine,
leucine, serine, thionine, asparagine, aspartate, glutamate, lysine or
arginine,
more preferably a hydrophilic amino acid residue, and most preferably
tyrosine,
Xaa6 is selected from aspartate, asparagine, serine, tyrosine, glutamate,
glycine,
arginine or histidine, more preferably aspartate or asparagine and most
preferably aspartate,
Xaa7 is selected from histidine, asparagine or tyrosine, more preferably
histidine,
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Xaa8 is selected from proline, hydroxyproline, glutamine or serine, more
preferably proline,
Xaa9 is selected from glutamate, y-carboxyglutamate, aspartate, glycine or
asparagine, more preferably glutamate, y-carboxyglutamate and most
preferably, glutamate,
Xaalo is selected from isoleucine, tyrosine leucine or des-Xaalo, more
preferably
isoleucine.
In particularly preferred embodiments
Xaal is glycine or aspartate,
Xaa2 is aspartate,
Xaa3 is arginine,
Xaa4 is asparagine,
Xaa5 is tyrosine,
Xaa6 is aspartate,
Xaa7 is histidine,
Xaa8 is proline,
Xaa9 is glutamate,
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Xaaio is isoleucine.
In a preferred embodiment the linear alpha conotoxin peptide subject to
cyclisation is an
a4/7 conotoxin peptide. The peptide may have selectivity for peripheral
subtypes at
nAChR over central subtypes. For example the peptide may have selectivity for
0
subtype over 0 subtypes.
In a further preferred embodiment the linear alpha conotoxin peptide subjected
to
cyclisation is Vc1.1.
The cyclised conotoxin peptides according to the present invention will
generally consist
of an a-conotoxin peptide in which the N- and C-termini are linked via a
linking moiety,
although in some cases it may be possible to directly connect the N- and C-
termini of a
naturally occurring a-conotoxin peptide or derivative thereof without the need
for an
additional linking moiety. The linking moiety, if present, may be a peptide
linker such that
cyclisation produces an amide-cyclised peptide backbone. These peptides will
have no
free N- or C-termini.
Considerable variation in the peptide sequence of the linking moiety is
possible. Since this
linking region does not bind to or occlude the primary active site of the a-
conotoxin it can
be modified to alter physiochemical properties, and potentially reduce side
effects of the
conotoxins.
In linking the N- and C-termini of the conotoxin it may in some cases be
necessary or
desirable to remove one or more of the N- or C-termini residues. Such
modification of the
linear conotoxin sequence is within the scope of the present invention.
The linking moiety will necessarily be of sufficient length to span the
distance between the
N- and C-termini of the conotoxin peptide. In the case of peptide linkers the
length will
generally be in the order of 2 to 10 amino acids. In some cases longer or
shorter peptide
linkers may be required. In one embodiment the linking moiety is composed of
glycine
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and/or alanine residues in addition to any amino acid residues already present
in the linear
a-conotoxin.
For a-conotoxin peptides of the A-superfamily it has been found that the
distance between
cysteine residues I and IV is substantially conserved. For such conotoxin
peptides,
allowing for bond angles and distance, the linker length is preferably chosen
such that the
number of amino acids between cysteine residue I and IV is between six and
eight.
Accordingly, for the a-conotoxins Vcl.1 and MII the additional amino acid
residues
required for the linker would be between five and seven, allowing for the
single amino acid
already present at the N-terminus.
Accordingly in a further aspect the present invention provides synthetically
cyclised a-
conotoxin peptide having an amide cyclised backbone such that the peptide has
no free N-
or C- terminus, said peptide having the ability to inhibit a nicotinic
acetylcholine receptor
and comprising four cysteine residues bonded in pairs to form two disulfide
bonds,
wherein the N-terminus of the corresponding linear/non-cyclised conotoxin
peptide is
linked to the C-terminus by a peptide linker of such that between six and
eight amino acid
residues span the distance between cysteine residues I and IV.
Preferably the number of amino acids in the linker is selected such that there
are seven
amino acids between the first and the fourth cysteine residues. Depending on
the sequence
of the linear peptide, some or all of these residues may be derived from the
linear
sequence. Accordingly if the conotoxin peptide has one amino acid at the N-
terminus
adjacent the first cysteine residue, the number of additional amino acids
required for the
linker would be six.
Of course it would also be possible to substitute one or more of the N-
terminal residues
with another residue which would form part of the linker.
It is possible, according to the present invention, to modify or potentiate
the activity of a
conotoxin peptide by selection of a particular size and/or type of peptide
linker. Small
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changes in the conformation of the conotoxin caused by the introduction of a
linking group
can alter the binding affinities of the peptides for their particular binding
sites.
Conversely, where the activity is to be as close to the activity of the parent
conotoxin
peptide as possible, a linker will be selected which minimises any change in
conformation.
The linker may also provide a "handle" on the peptide which does not interfere
substantially with the primary biological effect. The linker can provide a
place for
functionalising the molecule to improve biophysical properties.
There are several ways in which linear conotoxins may be cyclised. These
include the
following:
1. Cyclisation of the reduced peptide followed by oxidation to form the
required
disulfide bonds.
In this approach an extended linear peptide is first synthesised "on resin"
using solid phase
peptide synthesis methods. This extended linear peptide comprises the native
sequence
starting at a cysteine residue at, or closest to, the N-terminus and a C-
terminal extension
which comprises the new linking moiety. The solid phase synthesis actually
starts in the
reverse order- ie at the C-terminus of the extended linear peptide. Following
cleavage
from the resin, the extended conotoxin is cyclised to a thioester intermediate
which
subsequently rearranges to an amide-cyclised peptide. This reduced peptide is
then
oxidised to form the disulfide bonds. A schematic diagram of the reaction
involved in the
cyclisation is shown in Figure 1. The linear peptide is cleaved from the resin
with the
linker to the resin (R) still attached. R corresponds to the linker between
the peptide and
the resin and is different from the linking moiety used in the cyclisation.
The first reaction
involves the formatiqn of a thioester between the thiol of the N-terminal
cysteine and the
carboxy terminus. This then undergoes an S, N acyl migration to form the
cyclic peptide
with a native peptide bond.
2. Oxidation of the reduced linear peptide, followed by cyclisation.
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In this approach an extended peptide is assembled using solid phase peptide
synthesis. The
extended linear peptide comprises the native conotoxin sequence with extra
residues added
at the N- and/or C-termini. The (new) N and C termini should preferably be
glycine
residues. The peptide is folded, and in the case of the conotoxin-like
peptides, the termini
of the folded molecule are generally close together in space. This facilitates
the cyclisation
of the peptide in solution using standard chemistry. Complications may occur
when large
numbers of lysine, glutamic acid or aspartic acid residues are present in the
sequence and
method 1 is then preferable.
3. Ligation of a linker onto an existing conotoxin, followed by cyclisation.
In this method the starting material is a mature conotoxin. A peptide linker
is synthesised
and ligated with the conotoxin using published procedures for the ligation of
peptides. The
extended peptide is then cyclised and oxidised.
In the process described above the steps can be performed in any order,
provided the
product is a cyclised a-conotoxin peptide having the required disulfide bonds.
For
example, in process 1 the cleavage and cyclisation steps may be performed
simultaneously
or in either order. Similarly in process 2 the cyclisation and folding steps
could be
performed simultaneously, or in either order.
It is also possible to form the disulfide bonds selectively using protecting
groups on the
cysteine residues. Selective protection of the cysteine residues in this way
allows the
production of a particular disulfide bond pattern. Examples of groups capable
of
protecting cysteine residues include acetamidomethyl (Acm), 4-methylbenzyl
(MeBzl) and
4-methoxybenzyl (Mob).
Also, in view of the cyclic nature of the final products, synthetic procedures
may involve
cyclic permutation of the a-conotoxin peptides or of the extended
peptide/linker sequences.
For example, the designs of the extended linear peptide for a-conotoxins could
commence
by adding a linker to the C-terminal residue of the a-conotoxin, cyclically
permuting the
CA 02566832 2006-11-16
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N-terminal residue(s) to the C-terminal, to provide an N-terminal cysteine,
and cyclising as
described.
The term "derivative" as used herein in connection with naturally occurring
conotoxin
peptides, such as MII, refers to a peptide which differs from the naturally
occurring
peptides by one or more amino acid deletions, additions, substitutions, or
side-chain
modifications.
Substitutions encompass amino acid alterations in which an amino acid is
replaced with a
different naturally-occurring or a non-conventional amino acid residue. Such
substitutions
may be classified as "conservative", in which case an amino acid residue
contained in a
polypeptide is replaced with another naturally-occurring amino acid of similar
character
either in relation to polarity, side chain functionality, or size, for example
SerHThrHProHHypHGly+-+Ala, Va1HI1eHLeu, HisHLysHArg,
AsnHGInHAspHGlu or PheHTrpHTyr. It is to be understood that some non-
conventional amino acids may also be suitable replacements for the naturally
occurring
amino acids. For example ornithine, homoarginine and dimethyllysine are
related to His,
Arg and Lys.
Substitutions encompassed by the present invention may also be "non-
conservative", in
which an amino acid residue which is present in a polypeptide is substituted
with an amino
acid having different properties, such as a naturally-occurring amino acid
from a different
group (eg. substituting a charged or hydrophobic amino acid with alanine), or
alternatively,
in which a naturally-occurring amino acid is substituted with a non-
conventional amino
acid.
Amino acid substitutions are typically of single residues, but may be of
multiple residues,
either clustered or dispersed.
Preferably, amino acid substitutions are conservative.
CA 02566832 2006-11-16
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Additions encompass the addition of one or more naturally occurring or non-
conventional
amino acid residues. Deletion encompasses the deletion of one or more amino
acid
residues.
As stated above the present invention includes peptides in which one or more
of the amino
acids has undergone sidechain modifications. Examples of side chain
modifications
contemplated by the present invention include 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-
trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic
anhydride
and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-
5-phosphate
followed by reduction with NaBH4.
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.
The carboxyl group may be modified by carbodiimide activation via 0-
acylisourea
formation followed by subsequent derivitisation, for example, to a
corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with
iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid;
formation of
mixed disulfides with other thiol compounds; reaction with maleimide, maleic
anhydride
or other substituted maleimide; formation of mercurial derivatives using 4-
chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury
chloride, 2-
chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate
at alkaline
pH. Any modification of cysteine residues must not affect the ability of the
peptide to
form the necessary disulfide bonds. It is also possible to replace the
sulphydryl groups of
cysteine with selenium equivalents such that the peptide forms a diselenium
bond in place
of one or more of the disulfide bonds.
CA 02566832 2006-11-16
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Tryptophan residues may be modified by, for example, oxidation with N-
bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl
bromide
or sulphenyl halides. Tyrosine residues on the other hand, may be altered by
nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished
by
alkylation with iodoacetic acid derivatives or N-carbethoxylation with
diethylpyrocarbonate.
Proline residues may be modified by, for example, hydroxylation in the 4-
position.
A list of some amino acids having modified side chains and other unnatural
amino acids is
shown in Table 2.
Table 2
Non-conventional Code Non-conventional Code
amino acid amino acid
L-a-aminobutyric acid Abu L-a-methylhistidine Mhis
a-amino-a-methylbutyrate Mgabu L-a-methylisoleucine Mile
aminocyclopropane- Cpro L-a-methylleucine Mleu
carboxylate L-a-methylmethionine Mmet
aminoisobutyric acid Aib L-a-methylnorvaline Mnva
aminonorbomyl- Norb L-a-methylphenylalanine Mphe
carboxylate L-a-methylserine Mser
cyclohexylalanine Chexa L-a-methyltryptophan Mtrp
cyclopentylalanine Cpen L-a-methylvaline Mval
D-alanine DAIa N-(N-(2,2-diphenylethyl) Nnbhm
D-arginine DArg carbamylmethylglycine
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D-asparagine nAsn 1-carboxy-1-(2,2-diphenyl- Nmbc
D-aspartic acid DAsp ethylamino)cyclopropane
D-cysteine nCys L-N-methylalanine Nmala
D-glutamine nGln L-N-methylarginine Nmarg
D-glutamic acid nGlu L-N-methylaspartic acid Nmasp
D-histidine DHis L-N-methylcysteine Nmcys
D-isoleucine nIle L-N-methylglutamine Nmgln
D-leucine nLeu L-N-methylglutamic acid Nmglu
D-lysine nLys L-N-methylhistidine Nmhis
D-methionine DMet L-N-methylisolleucine Nmile
D-ornithine nOrn L-N-methylleucine Nmleu
D-phenylalanine DPhe L-N-methyllysine Nmlys
D-proline nPro L-N-methylmethionine Nmmet
D-serine DSer L-N-methylnorleucine Nmnle
D-threonine DThr L-N-methylnorvaline Nmnva
D-tryptophan DTrp L-N-methylornithine Nmom
D-tyrosine DTyr L-N-methylphenylalanine Nmphe
D-valine nVal L-N-methylproline Nmpro
D-a-methylalanine DMala L-N-methylserine Nmser
D-a-methylarginine DMarg L-N-methylthreonine Nmthr
D-a-methylasparagine DMasn L-N-methyltryptophan Nmtrp
D-a-methylaspartate DMasp L-N-methyltyrosine Nmtyr
D-a-methylcysteine DMcys L-N-methylvaline Nmval
D-a-methylglutamine nMgln L-N-methylethylglycine Nmetg
D-a-methylhistidine DMhis L-N-methyl-t-butylglycine Nmtbug
D-a-methylisoleucine DMile L-norleucine Nle
D-a-methylleucine DMleu L-norvaline Nva
D-a-methyllysine DMlys a-methyl-aminoisobutyrate Maib
D-a-methylmethionine DMmet a-methyl-y-aminobutyrate Mgabu
D-a-methylornithine nMom a-methylcyclohexylalanine Mchexa
D-a-methylphenylalanine DMphe a-methylcyclopentylalanine Mcpen
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D-a-methylproline DMpro a-methyl-a-napthylalanine Manap
D-a-methylserine DMser a-methylpenicillamine Mpen
D-a-methylthreonine DMthr N-(4-aminobutyl)glycine Nglu
D-a-methyltryptophan nMtrp N-(2-aminoethyl)glycine Naeg
D-a-methyltyrosine DMty N-(3-aminopropyl)glycine Norn
D-a-methylvaline DMval N-amino-a-methylbutyrate Nmaabu
D-N-methylalanine DNmala a-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 nNmcys N-(2-carboxyethyl)glycine Nglu
D-N-methylglutamine DNmgln N-(carboxymethyl)glycine Nasp
y-carboxyglutamate Gla N-cyclobutylglycine Ncbut
4-hydroxyproline Hyp N-cyclodecylglycine Ncdec
5-hydroxylysine Hlys N-cylcododecylglycine Ncdod
2-aminobenzoyl Abz N-cyclooctylglycine Ncoct
(anthraniloyl) N-cyclopropylglycine Ncpro
Cyclohexylalanine Cha N-cycloundecylglycine Ncund
Phenylglycine Phg N-(2,2-diphenylethyl)glycine Nbhm
4-phenyl-phenylalanine Bib N-(3,3-diphenylpropyl)glycine Nbhe
L-Citrulline Cit N-(hydroxyethyl)glycine Nser
L- 1,2,3,4-tetrahydroiso- Tic N-(imidazolylethyl))glycine Nhis
quinoline-3-carboxylic acid N-(3-indolylyethyl)glycine Nhtrp
L-Pipecolic acid (homo Pip N-methyl-y-aminobutyrate Nmgabu
proline) D-N-methylmethionine Dnmmet
L-homoleucine Hle N-methylcyclopentylalanine Nmcpen
L-Lysine (dimethyl) DMK D-N-methylphenylalanine Dnmphe
L-Naphthylalanine Nal D-N-methylproline Dnmpro
L-dimethyldopa or DMD D-N-methylthreonine Dnmthr
L-dimethoxyphenylalanine N-(1-methylethyl)glycine Nval
L-thiazolidine-4-carboxylic THZ N-methyla-napthylalanine Nmanap
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acid N-methylpenicillamine Nmpen
L-homotyrosine hTyr N-(p-hydroxyphenyl)glycine Nhtyr
L-3-pyridylalanine PYA N-(thiomethyl)glycine Ncys
L-2-furylalanine FLA penicillamine Pen
L-histidine(benzyloxymethyl) HBO L-a-methylalanine Mala
L-histidine(3-methyl) HME L-a-methylasparagine Masn
D-N-methylglutamate DNmglu L-a-methyl-t-butylglycine Mtbug
D-N-methylhistidine nNmhis L-methylethylglycine Metg
D-N-methylisoleucine nnmile L-a-methylglutamate Mglu
D-N-methylleucine DNmleu L-a-methylhomophenylalanine Mhphe
D-N-methyllysine DNmlys N-(2-methylthioethyl)glycine Nmet
N-methylcyclohexylalanine Nmchexa L-a-methyllysine Mlys
D-N-methylomithine nNmorn L-a-methylnorleucine Mnle
N-methylglycine Nala L-a-methylonriithine Mom
N-methylaminoisobutyrate Nmaib L-a-methylproline Mpro
N-(1-methylpropyl)glycine Nile L-a-methylthreonine Mthr
N-(2-methylpropyl)glycine Nleu L-a-methyltyrosine Mtyr
D-N-methyltryptophan DNmtrp L-N-methylhomophenylalani Nmhphe
D-N-methyltyrosine DNmtyr N-(N-(3,3-diphenylpropyl) Nnbhe
D-N-methylvaline DNmval carbamylmethylglycine
L-t-butylglycine Thug O-methyl-L-serine Omser
L-ethylglycine Etg O-methyl-L-homoserine Omhser
L-homophenylalanine Hphe O-methyl-L-tyrosine MeY
L-a-methylarginine Marg y-aminobutyric acid Gabu
L-a-methylaspartate Masp O-methyl-L-homotyrosine Omhtyr
L-a-methylcysteine Mcys L-3-homolysine BHK
L-a-methylglutamine Mgln L-ornithine Orn
N-cycloheptylglycine Nchep N-cyclohexylglycine Nchex
N-(3-guanidinopropyl)glycine Narg D-N-methylserine DNmser
CA 02566832 2006-11-16
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These types of modifications may be important to stabilise the peptide if
administered to an
individual or for use as a diagnostic reagent.
Other derivatives contemplated by the present invention include a range of
glycosylation
variants from a completely unglycosylated molecule to a modified glycosylated
molecule.
Altered glycosylation patterns may result from expression of recombinant
molecules in
different host cells.
Preferably the cyclised a-conotoxin peptides will retain the Cys residues and
characteristic
disulfide bonding pattern. Derivatives may include additional Cys residues
provided they
are protected during formation of the disulfide bonds.
Preferably the conotoxin peptides according to the invention have 12 to 40
amino acids,
more preferably 15 to 30.
The cyclised conotoxin peptides according to the present invention are useful
as
therapeutic agents.
The a-conotoxins bind to nicotinic acetylcholine receptors (nAChRs) which have
been
implicated in the treatment of pain and in the acceleration of recovery of
nerve damage.
Such receptors have also been implicated in the pathophysiology of several
neuropsychiatric disorders including schizophrenia, Alzheimer's disease,
Parkinson's
disease and Tourette's syndrome and thus the a-conotoxins have potential
therapeutic
indications for these diseases. It is possible that the cyclised a-conotoxin
peptide
according to the invention target other receptors or ion channels.
The cyclic conotoxin peptides are believed to be particularly useful for the
therapeutic
treatment or prophylaxis of neuropathic pain. Pain is considered neuropathic
if it arises
from within the nervous system itself. The involvement of the nervous system
can be at
various levels: nerves, nerve roots and central pain pathways in the spinal
cord and the
brain. Neuropathic pain may result from aberrant activity of damaged nerves
and is the
CA 02566832 2006-11-16
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most difficult form of pain to treat. Neuropathic pain is a form of chronic
pain, which is
persistently generated and serves no beneficial function for the affected
individual. Patients
suffering neuropathic pain typically present with allodynia (pain from a
normally non-
painful stimulus) and hyperalgesia (an increased response to a painful
stimulus).
There are several, well established animal pain models which demonstrate
characteristic
pain symptoms of neuropathy, allodynia and hyperalgesia. The chronic
constriction injury
(CCI) rat model of neuropathic pain involves applying loose ligatures to the
sciatic nerve,
resulting in inflammation which causes nerve constriction and neuropathy. The
partial
nerve ligation (PNL) model involves creating a tight ligation around part of
the sciatic
nerve, leaving the rest of the nerve uninjured and resulting in allodynic and
hyperalgesic
pain responses. The streptozotocin (STZ) model involves inducing diabetes on
pancreatic
(3 cells, resulting in neuropathic pain symptoms such as mechanical
hyperalgesia and tactile
allodynia.
Assays useful for assessing compounds with the above mentioned activities may
be in vitro
or in vivo and are known to those skilled in the art. For example, assays
useful for
assessing activity at nAChRs include those described in WO 00/15654 (Hogg et
al., 1999,
J Biol Chem. 274(51):36559-64; and Hogg et al., 2003, Reviews of Physiology,
Biochemistry and Pharmacology 1: 1-46).
Preferably the mammal is in need of such treatment although the peptide may be
administered in a prophylactic sense.
As will be readily appreciated by those skilled in the art, the route of
administration (oral
and enteral) and the nature of the pharmaceutically acceptable vehicle will
depend on the
nature of the condition and the mammal to be treated. It is believed that'the
choice of a
particular carrier or delivery system, and route of administration, could be
readily
determined by a person skilled in the art.
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In the oral and enteral formulations of the present invention the active
peptide may be
formulated with an inert diluent or with an assimilable edible carrier, or it
may be enclosed
in hard or soft shell gelatin capsule, or it may be compressed into tablets,
or it may be
incorporated directly with the food of the diet. For oral therapeutic
administration, the
active compound may be incorporated with excipients and used in the form of
ingestible
tablets, buccal or sublingual tablets, troches, capsules, elixirs,
suspensions, syrups, wafers,
and the like. It will be appreciated that some of these oral formulation
types, such as
buccal and sublingual tablets, have the potential to avoid liver metabolism.
However the
cyclised peptides of the present invention may also be delivered to the
stomach where liver
metabolism is likely to be involved. Such compositions and preparations
preferably
contain at least 1% by weight of active compound. The percentage of the
compositions
and preparations may, of course, be varied and may conveniently be between
about 5 to
about 80% of the weight of the unit. The amount of active compound in such
therapeutically useful compositions is such that a suitable dosage will be
obtained.
The tablets, troches, pills, capsules and the like may also contain the
components as listed
hereafter: A binder such as gum, acacia, corn starch or gelatin; excipients
such as
dicalcium phosphate; a disintegrating agent such as corn starch, potato
starch, alginic acid
and the like; a lubricant such as magnesium stearate; and a sweetening agent
such a
sucrose, lactose or saccharin may be added or a flavouring agent such as
peppermint, oil of
wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it
may contain,
in addition to materials of the above type, a liquid carrier. Various other
materials may be
present as coatings or to otherwise modify the physical form of the dosage
unit. For
instance, tablets, pills, or capsules may be coated with shellac, sugar or
both. A syrup or
elixir may contain the active compound, sucrose as a sweetening agent, methyl
and
propylparabens as preservatives, a dye and flavouring such as cherry or orange
flavour. Of
course, any material used in preparing any dosage unit form should be
pharmaceutically
pure and substantially non-toxic in the amounts employed. In addition, the
active
compound(s) may be incorporated into sustained-release preparations and
formulations.
Liquid formulations may also be administered enterally via a stomach or
oesophageal tube.
CA 02566832 2006-11-16
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Enteral formulations may be prepared in the form of suppositories by mixing
with
appropriate bases, such as emulsifying bases or water-soluble bases. It is
also possible, but
not necessary, for the cyclised peptides of the present invention to be
administered
topically, intranasally, intravaginally, intraocularly and the like.
Pharmaceutically acceptable vehicles include any and all solvents, dispersion
media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents and
the like. The use of such media and agents for pharmaceutical active
substances is well
known in the art. Except insofar as any conventional media or agent is
incompatible with
the active ingredient, use thereof in the therapeutic compositions is
contemplated.
Supplementary active ingredients can also be incorporated into the
compositions.
It is especially advantageous to formulate the oral or enteral compositions in
dosage unit
form for ease of administration and uniformity of dosage. Dosage unit form as
used herein
refers to physically discrete units suited as unitary dosages for the
mammalian subjects to
be treated; each unit containing a predetermined quantity of active material
calculated to
produce the desired therapeutic effect in association with the required
phannaceutically
acceptable vehicle. The specification for the novel dosage unit forms of the
invention are
dictated by and directly dependent on (a) the unique characteristics of the
active material
and the particular therapeutic effect to be achieved, and (b) the limitations
inherent in the
art of compounding active materials for the treatment of disease in living
subjects having a
diseased condition in which bodily health is impaired as herein disclosed in
detail.
As mentioned above the principal active ingredient is compounded for
convenient and
effective administration in effective amounts with a suitable phannaceutically
acceptable
vehicle in dosage unit form. A unit dosage form can, for example, contain the
principal
active compound in amounts ranging from 0.25 g to about 2000 mg. Expressed in
proportions, the active compound is generally present in from about 0.25 g to
about 2000
mg/ml of carrier. In the case of compositions containing supplementary active
ingredients,
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the dosages are determined by reference to the usual dose and manner of
administration of
the said ingredients.
In some aspects of the invention, administration forms and routes other than
oral or enteral
are contemplated, for example topical application such as creams, lotions,
transdermal
patches, sprays and gels, or compositions suitable for inhalation or
intranasal delivery, for
example solutions or dry powders.
Parenteral dosage forms may also be employed, including those suitable for
intravenous,
subcutaneous, intrathecal, intracerebral or epidural delivery.
The composition may also be formulated for delivery via slow release implants,
including
implantable pumps, such as osmotic pumps.
Detailed Description of the Invention
The invention will now be described with reference to the accompanying
examples and
figures which describe the production of some cyclised conotoxin peptides and
their
biological activity. However, it is to be understood that the particularity of
the following
description is not to supersede the generality of the preceding description of
the invention.
Referring to the figures:
Figure 1 is a scheme for peptide cyclisation via a C-terminal thioester. The
free sulfur of
an N-terminal cysteine interacts with the C-terminal thioester to form an
intermediate
which undergoes an S,N, acyl migration to form a cyclised peptide with a
native peptide
bond.
Figure 2 is a representation of the three-dimensional structures of the
conotoxins MII,
cMII-6 and cMII-7, and includes an overlay of these structures. The disulfide
bonds are
represented by ball and sticks. The structures were determined by NMR
spectroscopy.
CA 02566832 2006-11-16
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Figure 3 is a graph depicting the concentration-response curves for inhibition
of nicotine (5
M)-evoked catecholamine release from isolated bovine chromaffin cells by
increasing
concentrations of MII, cMII-5, cMII-6 and cMII-7. The results indicate that
the activity of
the cyclic peptides is comparable to linear MII.
Figure 4 is a graph depicting the relative stability of MII, cMII-6 and cMII-7
against attack
by proteolytic enzymes as assessed by incubating the peptide with
endoproteinase, Endo-
GIuC. The amount of intact peptide remaining was determined by RP-HPLC.
Figure 5 is a graph depicting the relative stability of MII, cMII-6 and cMII-7
against
enzymes in 50% human plasma. The amount of intact peptide remaining was
determined
by RP-HPLC.
Figure 6 is a representation of the three dimensional structure of both Vcl.1
and cyclic
Vcl.1-6. The structures were determined using NMR spectroscopy. The structures
indicate the native conformation of Vcl.l-6 is retained in cyclised Vcl.1-6.
Figure 7 is a graph depicting the relative biological activity of cyclised
Vcl.1-6 and Vc1.1,
as assessed by measuring catchecholamine release from bovine adrenal
chromaffin cells.
Chromaffm cells were incubated with indicated amount of peptides for 20 min
and then
stimulated with 10 M nicotine for 20 min in the continuing presence of
inhibitors.
Aliquots were removed and assayed fluorimetrically for catecholamine secretion
(n=6
experiments) as previously described (Meunier et al., 2002).
Figure 8 is a graph depicting the concentration-response curves for inhibition
of nicotine (5
M)-evoked catecholamine release from isolated bovine chromaffin cells by
increasing
concentrations of Vcl.1, cVcl.1-5A, cVcl .1 -5B, cVcl.1-6A and cVcl.l-6B.
CA 02566832 2006-11-16
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Figure 9 is a graph depicting increased mean paw withdrawal thresholds (PWTs)
in the
ipsilateral hindpaw of CCI-rats from <6g to - lOg for single bolus s.c. doses
of Vcl.l and
cVcl.1 (100 g/kg).
Figure 10 is a graph depicting increased mean paw withdrawal thresholds (PWTs)
in the
ipsilateral hindpaw of CCI-rats from <6g to - lOg for single bolus oral doses
of Vc1.I and
cVcl.1 (100 g/kg).
Figure 11 is a graph depicting mean paw withdrawal thresholds (PWTs) in the
contralateral
hindpaw of CCI-rats for single bolus s.c. or oral doses of Vcl.l and cVcl.l
(100 g/kg). in
CCI-rats.
Figure 12 is a graph depicting mean PWTs for single oral bolus doses of Vcl.1
(0.03-
3mg/kg), which produced dose-dependent relief of tactile allodynia in the
ipsilateral
hindpaw of CCI-rats.
Figure 13 is a graph depicting the extent and duration of anti-allodynia (APWT
AUC
values) plotted against dose to produce a dose-response curve; the approximate
ED50 dose
was estimated to be 1 mg/kg.
Example 1: Cyclic MII analogues
(a) Design and Synthesis
The size of the linker required to span the N and C termini of the native MII
peptide was
determined using well-known molecular modelling methodology. The three-
dimensional
model of MII was downloaded from the Protein Data Bank (PDB). The molecular
modelling program, Accelrys (Insight II Modeling Environment, Release 2000,
San Diego,
Accelrys Inc. 2001), was used to determine an appropriate linker length.
Briefly, this
involved building in an amino acid linker between the N- and C-termini, then
locking the
conformation of the parent conotoxin structure and perfonning a simple energy
minimisation. The conformation of the parent conotoxin was then unlocked and
the entire
peptide structure reminimized. Models with a linker size that is too small
result in
CA 02566832 2006-11-16
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structures with higher energies than those with a linker length that does not
perturb the
native structure. It was determined that a linker containing a minimum of five
residues was
required to span the N- and C-termini. Therefore peptides containing linkers
of five
(GGAAG), six (GGAAGG) and seven (GGAGAAG) residues in addition to the N-
terminal glycine were synthesised. This corresponds to a total intercysteine
length
spanning six to eight residues. Distance measurements revealed that each
residue in the
linker would need to span approximately 2.0 to 2.4 angstroms (note that this
is less than an
extended conformation which would span approximately 3.5 angstroms and
therefore
represents a relaxed conformation). Glycines and alanines were chosen for the
linker
- amino acids as they are relatively inert amino acids with small side-chains
that are less
likely to interact with other residues in the native peptide, or interfere
with folding
compared to larger or highly functionalised amino acid residues.
(b) Synthesis of cyclic MII analogues
The peptides were synthesised using BOC/HBTU chemistry with in situ
neutralisation
utilising a thioester linker (Schnolzer et al., (1992) Int. J. Pept. Protein
Res. 40, 180-193;
Dawson et al., (1994) Science 266, 776-9; Tam et al., (1999) J. Am. Chem. Soc.
121,
4316-4324; Yan, L.Z. and Dawson, P.E. (2001) J Am Chem Soc 123, 526-33).
This linker was synthesised by treating Gly PAM resin with 4 equivalents of S-
trityl-(3-
mercaptopropionic acid, 4 equivalents of HBTU and 5 equivalents of DIEA in DMF
(2 x
minutes). The trityl group is then removed by treating the resin with
TFA/triisopropylsilane/HZO (96:2:2) for 2 x 20 minutes. The C-terminal residue
of the
peptide chain was then added to the linker using standard coupling conditions.
Peptides
25 with a C-terminal thioester can also be made using FMOC chemistry by
following standard
literature procedures [Ingenito, R., Bianchi, E., Fattori, D. and Pessi, A.
(1999) J Am
Chem Soc 121, 11369-74; Shin, Y., Winans, K.A., Backes, B.J., Kent, S.B.H.,
Ellman,
J.A. and Bertozzi, C.R. (1999) J Am Chem Soc 121, 11684-89; Clippingdale,
A.B.,
Barrow, C.J. and Wade, J.D. (2000) J Pept Sci 6,225-34; Camarero, J.A.,
Hackel, B.J., De
30 Yoreo, J.J. and Mitchell, A.R. (2004) J Org Chem 69, 4145-51]. Once the
peptide chain
was assembled it was cleaved from the resin using HF with cresol and
thiocresol as
CA 02566832 2006-11-16
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scavengers. The crude peptide mixture was then purified by RP-HPLC utilising a
gradient
of 0-80% B over 80 minutes (A = 0.05% TFA in water, B = 90% acetonitrile,
9.95% water,
0.05% TFA) using a C18 column. Fractions were collected, analysed by MS and
those
containing the desired product were pooled and lyophilised.
Oxidation and cyclisation of the purified linear reduced peptide was achieved
by
dissolving the peptide in either 0.1 M NH4HCO3 (pH 8.1) or 50/50 0.1 M
NH4HCO3/aPrOH
at a concentration of approximately 0.3 mg/mL. The reaction mixture was
stirred overnight
at room temperature and the resulting mixture purified by RP-HPLC using the
conditions
described above. The peptides have the sequence given below.
The six residue linker cyclic MIl formed two distinct isomers in an
approximate ratio of
45:55. In contrast, the crude oxidation HPLC profile of the five residue
linker cyclic MII
appeared as a poorly dispersed set of peaks. Finally, the seven residue linker
cyclic MII
formed one predominant (>90%) isomer which, from the 'H NMR data, had a well
ordered
structure. The small number of peaks in the crude oxidation mixture for the
MII analogues
with a linker size of six residues (in addition to the N-terminal glycine)
provides a
preliminary indication that this is the optimal minimal size for MII. For the
crude mixtures,
individual peaks were collected and analysed by MS. Those containing peptides
of a mass
corresponding to the cyclised and oxidised peptide were subjected to
analytical RP-HPLC.
Fractions were then combined based on the HPLC analysis and lyophilised.
The 1D 1H NMR spectrum of the cyclic peptides obtained in sufficient amounts
were
measured. One isomer of the five residue linker peptide appeared to have a
well-ordered
structure based on the 'H NMR spectrum. In the case of the six residue linker
peptide the
later eluting, more abundant isomer appeared well ordered based on the 'H NMR
spectrum. The seven residue cyclic MII peptide was also well ordered based on
the 'H
NMR spectrum.
The sequence of the cyclic peptide analogues of MII are shown below:
CA 02566832 2006-11-16
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(a) cMII-5
Gly Cys Cys Ser Asn Pro Val Cys His Leu Glu His Ser Asn Leu Cys Gly Gly Ala
Ala Gly
SEQ ID No. 2
(b)cMII-6
Gly Cys Cys Ser Asn Pro Val Cys His Leu Glu His Ser Asn Leu Cys Gly Gly Ala
Ala Gly
Gly SEQ ID No. 3
(c) cMII-7 -
Gly Cys Cys Ser Asn Pro Val Cys His Leu Glu His Ser Asn Leu Cys Gly Gly Ala
Gly Ala
~
Ala Gly
SEQ ID No. 4
(c) Structural characterisation of the cyclic MII analogues.
Two-dimensional NMR spectral data were then obtained for the three well
ordered
analogues (now referred to as cMII-5, cMII-6 and cMII-7). A complete NMR
assignment
was made and the chemical shift of the Ha and HN protons was compared to those
of
native MII. A comparison of chemical shift data gives an indication of how
similar the
structures are. The results revealed that the chemical shifts of cMII-5 were
poorly
matched to those of MII, indicating a change in structure. In contrast, the
chemical shifts
of cMII-6 and cMII-7 were highly correlated to those of the native conotoxin.
This
indicates that cMII-6 and cMII-7 adopt a similar structure to native MII,
whereas cMII-5
clearly deviates from the native structure.
From this it was evident, based on the oxidation profile and the NMR data,
that a linker of
five added residues was too short and interfered with the correct folding of
the conotoxin
while a linker with six or seven added residues was of sufficient size to
allow the cyclic
analogue to form a native-like structure. The full three dimensional (3D)
structures of both
cMII-6 and cMII-7 was determined to confirm the close correlation of structure
between
the native MII and their cyclic counterparts.
CA 02566832 2006-11-16
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The 3D structure of cMII-6 was determined using NMR spectroscopy. Spectra were
obtained on a Bruker DMX 750 spectrometer at 280 and 287 K. The homonuclear
spectra
recorded included double quantum filtered DQF-COSY [Rance, M., Sorensen, O.W.,
Bodenhausen, G., Wagner, G., Ernst, R.R. and Wiithrich, K. (1983) Biochem.
Biophys.
Res. Commun. 117, 479-485.], TOCSY using a MLEV17 spin lock sequence [Bax, A.
and
Davis, D.G. (1985) J. Magn. Reson. 65, 355-360.] with an isotropic mixing time
of 80 ms;
ECOSY [ Griesinger, C., S,arensen, O.W. and Ernst, R.R. (1987) J. Magn. Reson.
75, 474-
492], and NOESY [Jeener, J., Meier, B.H., Bachmann, P. and Ernst, R.R. (1979)
J. Chem.
Phys. 71, 4546-4553; Kumar, A., Ernst, R.R. and Wuthrich, K. (1980) Biochem.
Biophys.
Res. Commun. 95, 1-6] with mixing times of 150 and 350 ms. In DQF-COSY and
ECOSY
experiments, the water resonance was suppressed by low power irradiation
during the
relaxation delay. For the TOCSY and NOESY experiments, water suppression was
achieved using a modified WATERGATE sequence [Piotto, M., Saudek, V. and
Sklenar,
V. (1992) J. Biomol. NMR 2, 661-665]. Two-dimensional spectra were generally
collected
over 4096 data points in the f2 dimension and 512 increments in the fl
dimension over a
spectral width corresponding to 12 ppm. For identification of slowly
exchanging amides, a
series of one-dimensional and TOCSY spectra were run immediately after
dissolving the
sample in D20. All spectra were processed on a Silicon Graphics workstation
using
XWINNMR (Bruker). The fl dimension was zero-filled to 2048 real data points
with the
fl and f2 dimensions being multiplied by a sine-squared function shifted by 90
prior to
Fourier transformation. Processed spectra were analyzed and assigned using the
program
XEASY [ Eccles, C., Guntert, P., Billeter, M. and Wuthrich, K. (1991) J.
Biomol. NMR 1,
111-30]. Spectra were assigned using the sequential assignment protocol
[Wuthrich, K.
(1986) Wiley-Interscience, New York]. The process was facilitated, in part,
using the
automatic assignment program NOAH, which is part of the DYANA package
[Guntert, P.,
Mumenthaler, C. and Wuthrich, K. (1997) J. Mol. Biol. 273, 283-98]. Cross-
peaks in
NOESY spectra recorded in 90% H20, 10% D20 with mixing times of 350 and 150 ms
were integrated and calibrated in XEASY, and distance constraints were derived
using
DYANA. Backbone dihedral angle restraints were derived from 3JHNx coupling
constants
measured from line shape analysis of antiphase cross-peak splitting in the DQF-
COSY
CA 02566832 2006-11-16
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spectrum. Angles were restrained to -120 30 for 3J.a, >8.5 Hz and to -600
f30 for
3JHNHa <5 Hz. Stereospecific assignments of (3-methylene protons and xl
dihedral angles
were derived from 3Jap coupling constants, measured from ECOSY spectra, in
combination
with NOE peak intensities. Preliminary structures were calculated using a
torsion angle
simulated annealing protocol within DYANA. Final structures were calculated
using
simulated annealing and energy minimization protocols within CNS version 1.0
[Brunger,
A.T., Adams, P.D. and Rice, L.M. (1997) Structure 5, 325-336].The starting
structures
were generated using random phi, psi dihedral angles and energy-minimized to
produce
structures with the correct local geometry. This protocol involves a high
temperature phase
comprising 4000 steps of 0.015 ps of torsion angle dynamics, a cooling phase
with 4000
steps of 0.015 ps of torsion angle dynamics during which the temperature was
lowered to 0
K, and finally an energy minimization phase comprising 5000 steps of Powell
minimization. Structures were refined using further molecular dynamics and
energy
minimization in a water shell, as described by Linge and Nilges [Linge, J.P.
and Nilges, M.
(1999) J. Biomol. NMR 13, 51-9]. The refinement in explicit water involves the
following
steps. First heating to 500 K via steps of 100 K, each comprising 50 steps of
0.005 ps of
Cartesian dynamics. Second, 2500 steps of 0.005 ps of Cartesian dynamics at
500 K before
a cooling phase where the temperature was lowered in steps of 100 K, each
comprising
2500 steps of 0.005 ps of Cartesian dynamics. Finally, the structures were
minimized with
2000 steps of Powell minimization. Structures were analyzed using Promotif and
Procheck
[Hutchinson, E.G. and Thomton, J.M. (1996) Protein Sci. 5, 212-220; Laskowski,
R.A.,
Rullmannn, J.A., MacArthur, M.W., Kaptein, R. and Thornton, J.M. (1996) J.
Biomol.
NMR 8,477-86].
A comparison of cMI1-6 with MII show native conformation of MII is retained in
toto in
cMII-6. The striking degree of similarity between the two peptides is
illustrated in Figure
4.
(d) Selective synthesis of cMII-6
CA 02566832 2006-11-16
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Although non-selective oxidation is the preferred method used in the synthesis
of cyclic
conotoxins, they can also be made using directed disulfide bond formation as
demonstrated
for cMII-6. The peptide was synthesised as described above but the sidechains
of
cysteines 3 and 16 were protected with Acm groups. The peptide was cleaved
from the
resin, purified and oxidised/cyclised using the conditions described above.
The second
disulfide bond was formed selectively between cysteines at positions 3 and 16
by
deprotection/oxidation using 12. The peptide (20mg) was dissolved in 50%
aqueous acetic
acid at a concentration of 1 mg/mL and the flask flushed with nitrogen. To the
flask was
added 2mL of 1M HCI followed by sufficient 0.1M 12 in 50% aqueous acetic acid
to make
the solution a pale yellow (approximately 1 mL). The reaction was then stirred
under
nitrogen at room temperature for 1.5 hours. The reaction was quenched by the
addition of
1M ascorbic acid until the reaction mixture became colourless. The mixture was
then
diluted with buffer A and purified by RP-HPLC to yield the cyclic, fully
oxidised peptide.
This peptide co-eluted with cMII-6 and hence confinned the disulfide
connectivity of
cMIl-6 as corresponding to that of the native peptide ie., Cys2-Cys8 and Cys3-
Cysl6 (1-3,
2-4).
(e) Biological activity of cMII-6
Chromaffin cells were prepared from bovine adrenal glands and maintained in 24-
well
plates (Nunc) as described in standard literature procedures [Lawrence, G.W.,
Weller, U.
and Dolly, J.O. (1994) Eur J Biochem 222, 325-33; Meunier, F.A., Mattei, C.,
Chameau,
P., Lawrence, G., Colasante, C., Kreger, A.S., Dolly, J.O. and Molgo, J.
(2000) J Cell Sci
113 ( Pt 7), 1119-25; Meunier, F.A., Feng, Z.P., Molgo, J., Zamponi, G.W. and
Schiavo,
G. (2002) Embo J 21, 6733-43.]. Intact cells were washed briefly once with
buffer A
(mM): NaCl, 145; KC1, 5, Na2HPO4, 1.2; glucose, 10; HEPES-NaOH, 20 (pH 7.4)
and
incubated with native and cyclised conotoxins for 20 min in the presence of 2
mM CaC12
and stimulated by nicotine (5 M) for 20 min. Aliquots of the supematant were
taken at
the end of each experiment and cells were lyzed with 1% (v/v) Triton X-100
(Sigma).
Both sets of samples were assayed fluorimetrically for catecholamines, and the
amount
released expressed as a percentage of control as described in the literature
(see above).
CA 02566832 2006-11-16
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The results of the biological assay are shown in Figure 3 and indicate that
the activity of
the cyclic peptides are comparable to that of linear MII.
(o Stability of cMII-6
The stability of cMII-6 and cMIl-7 against attack by proteolytic enzymes was
assessed by
incubating the peptide with endoproteinase Glu-C (Endo Glu-C). MII, cMII-6 and
cMII-7
have an identical potential processing site, on the opposite face from the
termini (in MII)
and the linker (in cMII-6 and cMII-7). The peptides were dissolved in 0.1M
NH4HCO3
(pH 8.0) buffer at a concentration of 20 g/mL. Endo G1uC was then added at a
peptide:enzyme ratio (wt/wt) of 50:1 and the solution incubated at 37 C.
Aliquots (3 L)
were taken out and quenched with 5% formic acid (57 L) every hour from 0-10.
Samples
were then analysed by LC/MS and the amount of intact peptide remaining at each
time
point determined. Each trial was performed in triplicate with the appropriate
positive (a
linear non-disulfide peptide with a EndoGluC cleavage point) and negative
(peptide in
buffer with no enzyme) controls.
The results of the stability assay are illustrated in Figure 4. The cyclised
peptides remain
entirely intact over the full ten hour period whereas native MII has a half
life of
approximately 10 hours. The enhanced stability demonstrated by cMII-6 and cMII-
7 is
surprising given that the putative processing sites on MII and the cyclised
peptides are
identical (ie., on the C-terminal side of Glutamic Acid) and distant from the
termini (in
MII) and the peptide linker in cMII-6 and cMII-7. Additionally, it may be
expected that
any enhanced stability would be against only against exoproteases that are
active against
the N-terminus (MII is amidated at its C-terminus), yet Glu-C is an
endoprotease. Thus,
the cyclic conotoxins appear to have enhanced stability beyond that which may
be
expected from protection of the termini alone.
(g) Stability of cMII-6 and cMII-7 against enzymes in human plasma
To test the resistance of cMII-6 and cMII-7 against proteolytic attack, and
compare this
with MII, these conotoxins were incubated in human blood plasma. MIl, cMII-6
and cMII-
7(10 M) were incubated in 50% human plasma for a period of 24 hours. Aliquots
were
CA 02566832 2006-11-16
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taken at several time points, quenched with 15% aqueous trichloroacetic acid
and
centrifuged. The supernatant was then analysed by RP-HPLC.
Each trial was performed in triplicate. Figure 5 shows the results of this
experiment. The
stability of cMII-6 and cMII-7 is dramatically greater than MII. Native MII
had a half-life
of approximately 16 hours whereas the amount of cMII-6 and cMII-7 remaining
after 24
hours was close to 90%.
Example 2: Synthesis and characterisation of cyclic Vcl.l
(a) Design of cyclic Vc1.1
The a-conotoxin Vcl.l is a member of the 4/7 class of a-conotoxins which
includes MII,
and has potential for the treatment of pain (Sandall DW, Satkunanathan N,
Keays DA,
Polidano MA, Liping X, Pham V, Down JG, Khalil Z, Livett BG, Gayler KR,
Biochemistry 2003 42(22):6904-11). There was no structural data available for
Vcl.l in
the literature and hence the procedure described above for the design of the
cyclic MII
analogues could not be followed. However, because other conotoxins with this
same
framework have been studied structurally, the optimal linker length for Vcl.1
was
estimated by simple distance measurements on other members of the same family,
including MII and PnIA. There are four structures of a4/7 conotoxins in the
Protein Data
Bank excluding duplicate structures and the conotoxin GID which has an
extended flexible
"tail" on the N-terminus. By averaging the distance between the N- and C-
termini of these
structures of these four compounds the value obtained is quite consistent
(12.2 0.8 A).
Therefore it can be estimated that suitable linker lengths for cyclising Vcl.l
would be
similar to those used for cyclising MII. eg. approximately six to seven
residues. Again,
these six residues are additional to an existing Glycine residue at the N-
terminus.
(b) Synthesis and structural characterisation of cyclic Ycl.l and Yc1.1
The synthesis of cyclic Vcl.l was carried out using the synthetic procedures
described for
cyclic MII (example 1). The cyclisation/oxidation buffer used for cyclic Vcl.l
was 0.1M
NH4HCO3 (pH 8.1). The cyclisation/oxidation yielded one predominant isomer
(now
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referred to as cVcl.1-6) that was purified and analysed by 1H NMR
spectroscopy. The
sequence of Vc1.1 (which is C-terminally amidated) and cVcl.l-6 are shown
below.
Gly Cys Cys Ser Asp Pro Arg Cys Asn Tyr Asp His Pro Glu Ile Cys
SEQ ID NO. 5
Gly Cys Cys Ser Asp Pro Arg Cys Asn Tyr Asp His Pro Glu Ile Cys Gly Gly Ala
Ala Gly Gly
I
SEQ ID NO. 6
Linear Vc1.1 was also synthesised using BOC/HBTU chemistry with in situ
neutralization
on MBHA-amide resin. The peptide was folded in 0.1 M NH4HCO3 at room
temperature
overnight and yielded a single isomer. The disulfide connectivity of synthetic
Vcl.l and
cVcl.1-6 were both confirmed to be Cys2-Cys8, Cys3-Cysl6 using standard
reduction/alkylation methodologies [G6ransson, U. and Craik, D.J. (2003) J.
Biol. Chem.
278, 48188-96] with MS/MS sequencing. The three dimensional NMR structure was
then
determined for comparison to cVc1.1-6. The three dimensional structure of both
Vc1.1 and
cVcl.1-6 was determined using NMR spectroscopy as described above for cyclic
MII. The
native conformation of Vc 1.1-6 is retained in cVc 1.1-6 as shown in Figure 6.
(c) Disulfide Mapping of Vcl.1
Vc1.1 was partially reduced by incubating with TCEP in citrate buffer at low
pH. The
reaction mixture was purified by RP-HPLC and the one-disulfide species
alkylated with N-
ethylmaleimide. The alkylated peptide was then fully reduced and analysed by
MS/MS.
The M/MS data clearly showed that C2 and C8 had been alkylated with N-
ethylmaleimide.
Fragmentation patterns from both ends of the peptide chain were observed and
fully
supported the proposed alkylation pattern. Therefore it was concluded that the
disulfide
connectivity of Vcl.1 was C2 to C8 and C3 to C16. This is consistent with the
I-III, II-IV
disulfide bonding pattern seen in other a-conotoxins.
CA 02566832 2006-11-16
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(d) Biological activity of cVcl.1-6
The biological activity of cVcl.1-6 was analysed by measuring catchecholamine
release
from bovine adrenal chromaffin cells as described for the cyclic MII example.
The results
of the assay are shown in Figure 7 which demonstrates that the activity of
cVcl.l-6 is
identical, within experimental error, to that of linear Vcl.l.
(e) Stability of cVc1.1-6
The ability of cVcl.l-6 to resist attack by proteolytic enzymes can be
assessed using the
experiments outlined for cyclic MII. Resistance to breakdown in manunalian
gastric juices
and plasma can also be assessed using the protocols given in Example 1.
(/) Biological activity of cVc.1
The biological activities of Vcl.l, cVcl.1-5A, cVcl.1-5B, cVcl.l-6A and cVcl.1-
6B
were assayed using chromafilm cells as described above in relation to cMII-6
(see example
1(e)). The "A" isomers are the isomers having the native 1-3, 2-4 disulfide
bond
connectivity while the "B" isomers have the ribbon 1-4, 2-3 connectivity. The
results are
shown in Figure 8.
(g) Selectivity of a-CTX Vc1.1 inhibition of recombinant nAChR subtypes
Vcl.1 inhibition of ACh-induced currents was examined in Xenopus oocytes
expressing
various nAChRs subunit combinations. The ACh-evoked response was assessed
every 10
min and the toxin was bath applied 4 mins in prior to co-application of the
agonist plus
toxin. Vcl.1 (10 M) failed to inhibit ACh-evoked currents mediated by either
the central
nAChR subtypes, a402 and a404, or the skeletal muscle nAChR subtype, apyS (n =
7-
12). Similarly, 10 M Vc1.1 inhibited only 14 f 2% of the ACh-evoked current
mediated
by the homopentameric neuronal nAChR, a7 (n = 11). However, 10 M Vc1.1
inhibited
the peripheral nAChR subtypes a3P2 and a3P4 to a similar extent, 58 7% (n =
8) and 56
7% (n = 12) of control, respectively. A similar potency was observed upon
addition of
the a5 subunit to the nAChR combination, a3a5(32 (n = 7) but Vcl.l exhibited >
5-fold
lower potency to inhibit a3a5(34 (n = 15). The ACh-induced current amplitude
was only
CA 02566832 2006-11-16
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inhibited by -50% by 30 M Vcl.1 at the a6 containing nAChR subtype, a3a6P2 (n
=
13). Bath application of Vcl.1 at concentrations of <_100 nM did not
antagonise ACh-
evoked currents nor elicit a detectable response alone (ie. >50 nA) for a3-
containing
nAChRs.
(h) Oral and subcutaneous efficacy of cyclised Yc1.1-6A
A study was undertaken to compare the anti-allodynic (pain-relieving) effect
of Vc1.1 and
cyclised Vcl.l when administered by subcutaneous (s.c.) and oral bolus doses
using a
well-defined model of neuropathic pain (CCI-rat).
Tactile allodynia, the defining symptom of neuropathic pain, was induced by
tying four
loose ligatures around the sciatic nerve of the left leg of the rat to induce
a chronic
constriction injury (CCI) resulting in hypersensitivity to light touch in the
hindpaw on the
same side ( i.e. the ipsilateral hindpaw). Tactile allodynia was assessed
using calibrated
von Frey filaments involving the application of graded non-noxious pressure to
the
ipsilateral hindpaw of CCI-rats. In non-injured rats and in the non-injured
(contralateral)
hindpaw of CCI-rats, the von Frey paw withdrawal threshold (PWT) is -12g
whereas by
14 days post-CCI surgery, the ipsilateral PWT is < 6g. The treatment goal is
to alleviate
tactile allodynia such that the PWT for the ipsilateral hindpaw is increased
from 6 to 12 g.
Vc1.1 and cVc1.1 administered as a single bolus dose (100 g/kg) by the s.c or
the oral
route produced significant relief of tactile allodynia as demonstrated by the
increase in the
mean peak ipsilateral PWT from < 6 g to - 10 g. The cyclizing of Vcl.1
appeared to result
in a longer duration of action compared with the parent linear peptide
independent of the
route of administration (Figures 9 and 10). Neither Vc1.1 nor cVcl.l when
administered
orally or s.c. increased PWTs in the contralateral hindpaw, consistent with
expectations
(Figure 11).
The results demonstrate that cyclised Vcl.l was significantly more active than
linear
Vcl.1 in this in vivo model of neuropathic pain. It also has a longer half
life than linear
Vcl.1, is more stable, and is orally bioavailable in an animal.
CA 02566832 2006-11-16
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(i) Anti-allodynic efficacy and potency of cyclic Vcl.1-6A
A study was undertaken to document the anti-allodynic efficacy of single oral
bolus doses
of Vcl.1-6A in CCI-rats.
Each CCI-rat (n=6) received up to five single oral bolus doses of Vcl.l-6A
with a
minimum 2-day washout period between doses, in order to identify the effective
dose
range. Using this approach, effective anti-allodynic doses were between
0.3mg/kg and
3mg/kg for Vcl.1-6A. Larger doses were not tested due to insufficient peptide
availability. Control animals received oral bolus doses of vehicle (n=3). The
Vc1.1-6A or
vehicle were administered in a volume of 500 L using 1 ml Hamilton glass
syringes and
PWTs were determined utilizing the procedure outlined in 2(h) above.
CCI-rats received a single oral bolus dose of Vcl.l-6A at 1 mg/kg (n=3).
Control animals
received single oral bolus doses of vehicle (n=3). The Vcl.1-6A or vehicle
were
administered in a volume of 500 L using a Hamilton glass syringe and PWTs
were
determined utilizing the procedure outlined in 2(h).
After completion of the experimental protocol, rats were euthanised with 100%
CO2
followed by cervical dislocation. Rat carcasses were frozen until removal by
The
University of Queensland biological waste removal service.
Mean ( SEM) PWT versus time curves were plotted for each dose of Vcl.1-6A and
vehicle in CCI-rats. PWT values were also normalized by subtracting the
respective pre-
dosing baseline values and the area under the normalized response versus time
curves
(AUC) were estimated using trapezoidal integration. The AUC values were
plotted against
dose and the ED50 value was estimated
CA 02566832 2006-11-16
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As expected, tactile allodynia manifested as a significant (P<0.05) decrease
in the mean
( SEM) PWT in the ipsilateral hindpaw (4.8 0.2g) at 2ldays post CCI-surgery
relative to
the respective mean ( SEM) PWT for the contralateral (non-injured) hindpaw
(10.6
0.2g). The treatment target was full reversal of tactile allodynia i.e. von
Frey PWTs of - 11
g in the ipsilateral hindpaw.
Following administration of single oral bolus doses of Vcl.1-6A (0.3 to 3
mg/kg) to CCI-
rats according to a'washout' protocol, there was a rapid onset of dose-
dependent relief of
tactile allodynia in the ipsilateral hindpaw. For all doses tested, peak ainti-
allodynia in the
ipsilateral hindpaw occurred at -0.5h post-dosing and the corresponding
durations of
action were in the range 1- 1.25 h. At the highest dose of Vcl.1-6A tested
(3mg/kg),
mean ( SEM) PWTs increased from 5.2 ( 0.4g) pre-dose to the peak effect (9.5
1 g) at
0.5 h post-dosing. The mean ( SEM) extent and duration of anti-allodynia (OPWT
AUC
values) in the ipsilateral hindpaw of CCI-rats increased in a dose-dependent
manner. The
-ED50 dose of Vc 1.1-6A was estimated at 1 mg/kg and this dose was selected
for
administration to drug-naive CCI-rats. Oral administration of Vcl.1-6A in
doses up to 3
mg/kg in CCI-rats produced insignificant antinociception in the contralateral
hindpaw.
Following administration of single oral bolus doses of Vcl.1-6A at lmglkg in
drug-naive
CCI-rats, there was a rapid onset of relief of tactile allodynia in the
ipsilateral hindpaw.
Consistent with the findings from the 'washout' protocol, peak anti-allodynia
in the
ipsilateral hindpaw occurred at -0.5 h post-dosing and the corresponding mean
duration of
action was 1.5 h. Specifically, the mean ( SEM) PWT values in the ipsilateral
hindpaw of
CCI-rats increased from 4(f Og) pre-dose to 8.7 ( 0.7) g at the time of peak
effect (0.5 h).
Oral administration of Vcl.1-6A at 1 mg/kg in drug-naive CCI-rats produced
insignificant
antinociception in the contralateral hindpaw.
In the study, single oral bolus doses of Vcl.l -6A at the doses tested (0.3-3
mg/kg) did not
produce discernible adverse behavioural effects in CCI-rats.
CA 02566832 2006-11-16
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Administration of single oral bolus doses of Vc1.1-6A at 0.3-3 mg/kg in CCI-
rats produced
dose-dependent anti-allodynic responses in the ipsilateral (injured) hindpaws
without
producing antinociception in the corresponding contralateral hindpaw, The
observation
that Vcl.1-6A does not produce antinociception in the contralateral (non-
injured)
hindpaws is consistent with the notion that Vcl.1-6A produces its pain-
relieving effects via
modulation of the pro-nociceptive (pro-pain) pathway rather than by amplifying
endogenous pain inhibitory mechanisms.
The reference in this specification to any prior publication (or information
derived from it),
or to any matter which is known, is not, and should not be taken as an
acknowledgment or
admission or any form of suggestion that that prior publication (or
information derived
from it) or known matter forms part of the conunon general knowledge in the
field of
endeavour to which this specification relates.
Those skilled in the art will appreciate that the invention described herein
is susceptible
to variations and modifications other than those specifically described. It is
to be
understood that the invention includes all such variations and modifications
which fall
within the spirit and scope. The invention also includes all of the steps,
features,
compositions and compounds referred to or indicated in this specification,
individually or
collectively, and any and all combinations of any two or more of said steps or
features.
