U.S. patent application number 13/115711 was filed with the patent office on 2012-11-01 for conotoxin peptides useful as inhibitors of neuronal amine transporters.
This patent application is currently assigned to XENOME LIMITED. Invention is credited to Paul Francis Alewood, Richard James Lewis, Iain Andrew Sharpe.
Application Number | 20120277166 13/115711 |
Document ID | / |
Family ID | 3810492 |
Filed Date | 2012-11-01 |
United States Patent
Application |
20120277166 |
Kind Code |
A1 |
Lewis; Richard James ; et
al. |
November 1, 2012 |
CONOTOXIN PEPTIDES USEFUL AS INHIBITORS OF NEURONAL AMINE
TRANSPORTERS
Abstract
The invention relates to an isolated, synthetic or recombinant
.chi.-conotoxin peptide having the ability to inhibit a neuronal
amine transporter, nucleic acid molecules encoding all or part of
such peptides, antibodies to such peptides and uses and methods of
treatment involving them.
Inventors: |
Lewis; Richard James;
(Woolloongabba, AU) ; Alewood; Paul Francis;
(Moggill, AU) ; Sharpe; Iain Andrew; (Taringa,
AU) |
Assignee: |
XENOME LIMITED
Indooroopilly
AU
|
Family ID: |
3810492 |
Appl. No.: |
13/115711 |
Filed: |
May 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11934723 |
Nov 2, 2007 |
7994128 |
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13115711 |
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10918096 |
Aug 13, 2004 |
7326682 |
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11934723 |
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09787986 |
Jun 27, 2001 |
6794361 |
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PCT/AU99/00844 |
Oct 1, 1999 |
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10918096 |
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Current U.S.
Class: |
514/21.5 ;
530/327 |
Current CPC
Class: |
A61P 25/24 20180101;
A61P 9/06 20180101; A61P 25/22 20180101; A61P 25/34 20180101; A61P
29/00 20180101; C07K 14/43504 20130101; A61P 9/00 20180101; A61K
38/00 20130101; A61P 25/00 20180101; A61P 43/00 20180101; C07K 7/08
20130101; A61P 13/00 20180101; A61P 13/02 20180101; A61P 29/02
20180101; A61P 9/04 20180101 |
Class at
Publication: |
514/21.5 ;
530/327 |
International
Class: |
C07K 7/08 20060101
C07K007/08; A61P 29/00 20060101 A61P029/00; A61P 9/00 20060101
A61P009/00; A61P 25/00 20060101 A61P025/00; A61K 38/10 20060101
A61K038/10; A61P 13/00 20060101 A61P013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 2, 1998 |
AU |
PP 6274 |
Claims
1-6. (canceled)
7. An isolated, synthetic or recombinant .chi.-conotoxin peptide
having the activity inhibiting a neuronal amine transporter,
wherein said .chi.-conotoxin peptide comprises MrIA as set forth in
SEQ ID NO: 1 (NGVCCGYKLCHOC) or a modified sequence of SEQ ID NO: 1
which has undergone a conservative substitution of an amino acid
residue, wherein ".largecircle." represents 4-hydroxyproline, and
wherein the first and fourth cysteine residues of SEQ ID NO: 1 are
connected to form a disulfide bond and the second and third
cysteine residues of SEQ ID NO: 1 are connected to form a disulfide
bond.
8. The .chi.-conotoxin peptide according to claim 7 having the
ability to inhibit a neuronal noradrenaline transporter.
9. The .chi.-conotoxin peptide according to claim 8 which is a
selective inhibitor of neuronal noradrenaline transporter.
10. The .chi.-conotoxin peptide according to claim 8 having
negligible or no anticholinergic effect.
11. The .chi.-conotoxin peptide according to claim 8 having
negligible or no activity as a sodium channel blocker.
12. The .chi.-conotoxin peptide according to claim 8 having
negligible or no activity as an inhibitor of dopamine
transporter.
13. (canceled)
14. A composition comprising a .chi.-conotoxin peptide and a
pharmaceutically acceptable carrier or diluent, wherein said
.chi.-conotoxin peptide is a peptide according to claim 7.
15. The composition according to claim 14 which is a pharmaceutical
composition.
16-17. (canceled)
Description
[0001] The present invention relates to novel peptides and
derivatives thereof useful as inhibitors of neuronal amine
transporters of neurotransmitters such as noradrenaline, serotonin,
dopamine, glutamic acid and glycine. The invention also relates to
pharmaceutical compositions comprising these peptides, nucleic acid
probes useful in finding active analogues of these peptides, assays
for finding compounds having neuronal noradrenaline transporter
inhibitory activity and the use of these peptides in the
prophylaxis or treatment of conditions such as but not limited to
incontinence, cardiovascular conditions and mood disorders.
[0002] 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 mollusks, the cone snails
inject their prey with venom containing a cocktail of small
bioactive peptides. These toxin molecules, which are referred to as
conotoxins, interfere with neurotransmission by targeting a variety
of receptors and ion-channels. The venom from any, single Conus
species may contain more than 100 different peptides. The
conotoxins are divided into classes on the basis of their
physiological targets. To date, ten classes have been described.
The .omega.-conotoxin class of peptides target and block
voltage-sensitive Ca.sup.2+-channels inhibiting neurotransmitter
release. The .alpha.-conotoxins and .psi.-conotoxins target and
block nicotinic ACh receptors, causing ganglionic and neuromuscular
blockade. Peptides of the .mu.-conotoxin class act to block
voltage-sensitive Na.sup.+-channels inhibiting muscle and nerve
action potentials. The .delta.-conotoxins target and delay the
inactivation of voltage-sensitive Na.sup.+-channels, enhancing
neuronal excitability. The .kappa.-conotoxin class of peptides
target and block voltage-sensitive K.sup.+-channels, and these also
cause enhanced neuronal excitability. The conopressins are
vasopressin receptor antagonists and the conantokins are NMDA
receptor antagonists. More recently, the prototype of a new
.gamma.-conotoxin class, which targets a voltage-sensitive
nonspecific cation channel, and of a new .sigma.-conotoxin class,
which antagonizes the 5HT.sub.3 receptor, have been described.
[0003] It has now been found that a new class of conotoxin exists,
hereinafter referred to as the .chi.-conotoxin class, which are
characterised by having the ability to inhibit neuronal amine
transporters.
[0004] Compounds which inhibit neurotransmitter reuptake have been
found to be useful in the treatment of lower urinary tract
disorders, such as urinary incontinence, detrusor instability and
interstitial cystitis. One such compound is "imipramine" which, in
addition to inhibiting noradrenaline reuptake, has been shown to
affect calcium channel blockade, and to exhibit anticholinergic,
local anaesthetic activity and a number of other effects. Other
compounds capable of inhibiting noradrenaline reuptake are
described in U.S. Pat. No. 5,441,985. These compounds are said to
have a reduced anticholinergic effect relative to imipramine.
[0005] In the case of the peptides of the present invention this
inhibition of neurotransmitter reuptake is achieved by selectively
inhibiting the neuronal neurotransmitter transporter, such as the
noradrenaline transporter, which functions to rapidly clear
released noradrenaline from the synapse back into neurons.
[0006] The peptides of the present invention are the first peptides
to have activity in inhibiting an amine transporter. All other
conotoxin peptides characterised to date target ion channels or
receptors on cell surfaces.
[0007] According to one aspect of the present invention there is
provided an isolated, synthetic or recombinant .chi.-conotoxin
peptide having the ability to inhibit a neuronal amine
transporter.
[0008] Preferably, the neuronal amine transporter is the neuronal
noradrenaline transporter.
[0009] The .chi.-conotoxin peptide may be a naturally occurring
peptide isolated from a cone snail, or a derivative thereof.
[0010] Preferably the .chi.-conotoxin peptide is .chi.-MrIA or
.chi.-MrIB, or a derivative thereof. .chi.-MrIA and .chi.-MrIB may
be isolated from the venom of the mollusk hunting cone snail, Conus
marmoreus.
[0011] They are both peptides of 13 amino acid residues in length,
and contain 2-disulphide bonds; the peptides show most homology to
members in the .alpha.-conotoxin class, which act as nicotinic ACh
receptor antagonists.
[0012] The amino acid sequences of .chi.-MrIA and .chi.-MrIB are as
follows:
TABLE-US-00001 SEQ ID NO. 1 .chi.-MrIA NGVCCGYKLCHOC SEQ ID NO. 2
.chi.-MrIB VGVCCGYKLCHOC
[0013] The C-terminus may be a free acid or amidated.
[0014] In the sequences above the "O" refers to 4-hydroxy proline
(Hyp). This amino acid residue results from post translational
modification of the encoded peptide and is not directly encoded by
the nucleotide sequence.
[0015] Preferably, the .chi.-conotoxin peptide is a selective
inhibitor of the neuronal noradrenaline transporter. The terms
"selective" and "selectively" as used herein mean that the activity
of the peptide as an inhibitor of neuronal noradrenaline
transporter is considerably greater than any activity at the
.alpha..sub.1-adrenoceptors.
[0016] U.S. Pat. No. 5,441,985 indicates that inhibitors of
noradrenaline reuptake which have a negligible anticholinergic
effect are particularly useful in the treatment of lower urinary
tract disorders. It has been found that .chi.-MrIA also has no
detectable anticholinergic effect.
[0017] Accordingly in a preferred embodiment of the invention there
is provided an isolated, synthetic or recombinant .chi.-conotoxin
peptide having the ability to selectively inhibit neuronal
noradrenaline transporter, and having negligible or no
anticholinergic effect.
[0018] .chi.-MrIA has also been found to have no activity as a
sodium channel blocker or as an inhibitor of dopamine transporter.
The absence in .chi.-MrIA of these additional pharmacological
activities commonly associated with other noradrenaline transporter
inhibitors and in preferred peptides according to the invention,
makes these peptides useful pharmacological tools.
[0019] The .chi.-conotoxin peptides according to the invention may
be naturally occurring peptides, such as .chi.-MrIA and .chi.-MrIA
or may be derivatives of naturally occurring peptides.
[0020] The term "derivative" as used herein in connection with
naturally occurring .chi.-conotoxin peptides, such as .chi.-MrIA
and .chi.-MrIB, refers to a peptide which differs from the
naturally occurring peptides by one or more amino acid deletions,
additions, substitutions, or side-chain modifications. Such
derivatives which do not have the ability to inhibit neuronal
noradrenaline transporter do not fall within the scope of the
present invention.
[0021] 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
SerThProHypGlyAla, ValIleLeu, HisLysArg, AsnGlnAspGlu or PheTrpTyr.
It is to be understood that some non-conventional amino acids may
also be suitable replacements for the naturally occurring amino
acids. For example ornithine, homoarginine and dimethyllysine are
related to His, Arg and Lys.
[0022] Substitutions encompassed by the present invention may also
be "non-conservative", in which an amino acid residue which is
present in a peptide is substituted with an amino acid having
different properties, such as naturally-occurring amino acid from a
different group (e.g. 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.
[0023] Amino acid substitutions are typically of single residues,
but may be of multiple residues, either clustered or dispersed.
[0024] Preferably, amino acid substitutions are conservative.
[0025] 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.
[0026] 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 NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with
2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride;
and pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4.
[0027] 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.
[0028] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivitization, for example, to a corresponding amide.
[0029] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH. Any modification of cysteine residues
must not affect the ability of the peptide to form the necessary
disulphide bonds. It is also possible to replace the sulfhydryl
groups of cysteine with selenium equivalents such that the peptide
forms a diselenium bond in place of one or more of the disulphide
bonds.
[0030] 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 sulphonyl halides. Tyrosine
residues on the other hand, may be altered by nitration with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0031] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with dimethylpyrocarbonate.
[0032] Praline residue may be modified by, for example,
hydroxylation in the 4-position.
[0033] A list of some amino acids having modified side chains and
other unnatural amino acids is shown in Table 1.
TABLE-US-00002 TABLE 1 Non-conventional amino acid Code
Non-conventional amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate
L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa
L-N-methylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcyclopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cyclododeclglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethy)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethy)glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nva
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mtrp L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe
carbamylmethyl)glycine carbamylmethyl)glycine
1-carboxy-1-(2,2-diphenyl- Nmbc O-methyl-L-serine Omser
ethylamino)cyclopropane O-methyl-L-homoserine Omhser
[0034] These types of modifications may be important to stabilise
the peptide if administered to an individual or used as a
diagnostic reagent.
[0035] 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.
[0036] The .chi.-conotoxin of the present invention are typically
amidated at the C-terminal, however compounds with a free carboxyl
terminus or other modifications at the C-terminal are considered to
be within the scope of the present invention. Preferably the
peptides are amidated or have a free carboxyl at the
C-terminal.
[0037] Preferably the derivatives of naturally occurring
.chi.-conotoxin peptides will retain the Cys residues and
characteristic disulphide bonding pattern. Derivatives may include
additional Cys residues provided they are protected during
formation of the disulphide bonds.
[0038] In modification to form derivatives of naturally occurring
.chi.-conotoxin peptides it is useful to compare the amino acid
sequences of active naturally occurring peptides to determine
which, if any, of the residues are conserved between active
species. Substitution of these conserved residues, while not
prohibited, is less favoured than substitutions of non-conserved
residues.
[0039] Derivatives where Ala replaces one or more residues can be
used to identify the pharmacophore. Preferably only one or two
amino acids is replaced with Ala at a time. Additional new peptides
can be made where charged, polar or hydrophobic residues,
respectively, are replaced to assist defining more precisely the
type of interactions involved in the binding of this
pharmacological class of peptide to its receptor. Non-conservative
replacements, where charge is reversed, or polar residues replace
hydrophobic residues, can further identify residues involved in
binding. All of these peptides have potential to show improved
potency, or greater selectivity. Non-native amino acid changes
could also be included to improve potency, selectivity and/or
stability.
[0040] Exposed residues are most likely to be involved in receptor
binding and can be systematically replaced. Particular emphasis is
placed on changing residues involved in binding and residues just
on the periphery of the pharmacophore, using longer side chain
forms or non-conserved changes to pick up additional binding
interactions for improved potency and/or selectivity. Reducing or
enlarging loop sizes and the tail of MrIA or MrIB further modifies
activity.
[0041] It is noted that MrIA and MrIB are composed of a tail
(residues 1-3), and two loops (residues 6-9 and 11-12), however the
.chi.-conotoxin peptides, and derivatives of the present invention
are not restricted to those having this particular arrangement of
amino acids and disulphide bonds. Other arrangements are also
possible, and provided the resultant peptide has the requisite
activity, a peptide will fall within the scope of the present
invention. Preferably the peptides will have at least two cysteine
residues and at least one disulphide bond, or more preferably four
cysteine residues and two disulphide bonds.
[0042] The connectivity of the disulfide bonds in these peptides
may be A-B/C-D, A-C/B-D or A-D/B-C, the latter being preferred for
MrIA and MrIB. A, B, C and D refer to the first, second, third and
fourth Cys residues involved in disulphide bond formation,
respectively.
[0043] These peptides can also be labelled and used to establish
binding assays to identify new molecules that act at the same site.
For example, labelled ligand of MrIA or MrIB could have tritium
included or may have radio-active iodine or similar attached
through a Tyr or other appropriate residue. A Tyr scan through each
peptide will establish a suitable location for incorporation of the
Tyr. The inhibition of binding of such labelled peptides to tissue
homogenates or expressed transporters by compounds or mixtures
would permit identification of new peptides active at this site,
including peptides present in serum and nerve and muscle tissue of
mammals, including human tissues. The assay will also allow
identification of non-peptide molecules that also act at the same
site as MrIA and MrIB, and that may have utility as orally active
forms of these peptides. Labelled peptides will additionally permit
autoradiographic studies to identify the location of the peptide
binding across various tissues.
[0044] Portions of these sequences can be used to search ESTR data
bases to identify in mammals peptides or proteins that contain
related sequence information that could be used to identify
endogenous ligands that act in a similar manner in mammals.
[0045] The .chi.-conotoxins of the present invention may be
prepared using standard peptide synthetic methods followed by
oxidative disulfide bond formation. For example, the linear
peptides may be synthesised by solid phase methodology using BOC
chemistry, as described by Schnoltzer et al (1992). Following
deprotection and cleavage from the solid support the reduced
peptides are purified using preparative chromatography. The
purified reduced peptides are oxidised in buffered systems, for
example as described in example 2. The oxidised peptides were
purified using preparative chromatography.
[0046] References describing the synthesis of conotoxins include
Sato et al, Lew et al and WO 91/07980.
[0047] The .chi.-conotoxins may also be prepared using recombinant
DNA technology. A nucleotide sequence encoding the desired peptide
sequence may be inserted into a suitable vector and protein
expressed in an appropriate expression system. In some instances,
further chemical modification of the expressed peptide may be
appropriate, for example C-terminal amidation. Under some
circumstances it may be desirable to undertake oxidative bond
formation of the expressed peptide as a chemical step following
peptide expression. This may be preceded by a reductive step to
provide the unfolded peptide. Those skilled in the art may readily
determine appropriate conditions for the reduction and oxidation of
the peptide.
[0048] The invention father provides an isolated nucleic acid
molecule comprising a sequence of nucleotides encoding or
complementary to sequence encoding a .chi.-conotoxin peptide as
described above.
[0049] In a further aspect of the present invention there is
provided a nucleic acid probe comprising a sequence of nucleotides
encoding or complementary to a sequence encoding all or part of a
.chi.-conotoxin peptide.
[0050] In a particularly preferred embodiment the nucleic acid
probe comprises a sequence of nucleotides encoding or complementary
to a sequence encoding the sequence shown in SEQ ID NO: 1 or SEQ ID
NO: 2.
[0051] As used herein a reference to a "probe" includes reference
to a primer used in amplification or a probe for use in direct
hybridization.
[0052] Still another aspect of the present invention is directed to
antibodies to the .chi.-conotoxin peptides according to the
invention. Such antibodies may be monoclonal or polyclonal and may
be selected from naturally occurring antibodies to the peptides or
may be specifically raised to the peptides using standard
techniques. In the case of the latter, the peptides may first need
to be associated with a carrier molecule. The antibodies of the
present invention are particularly useful as therapeutic or
diagnostic agents.
[0053] In this regard, specific antibodies can be used to screen
for the peptides according to the invention. Techniques for such
assays are well known in the art and include, for example, sandwich
assays and ELISA. Knowledge of peptide levels may be important for
monitoring certain therapeutic protocols.
[0054] It may also be possible to prepare antiidiotypic antibodies
using techniques known to the art. These antiidiotypic antibodies
and their use as therapeutic agents represent a further aspect of
the present invention.
[0055] The nucleic acid molecules of the present invention may be
DNA or RNA. When the nucleic acid molecule is in DNA form, it may
be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of
the present invention are generally mRNA.
[0056] Although the nucleic acid molecules of the present invention
are generally in isolated form, they may be integrated into or
ligated to or otherwise fused or associated with other genetic
molecules such as vector molecules and in particular expression
vector molecules. Vectors and expression vectors are generally
capable of replication and, if applicable, expression in one or
both of a prokaryotic cell or a eukaryotic cell. Preferably,
prokaryotic cells include E. coli, Bacillus sp and Pseudomonas sp.
Preferred eukaryotic cells include yeast, fungal, mammalian and
insect cells.
[0057] Accordingly, another aspect of the present invention
contemplates a genetic construct comprising a vector portion and a
gene capable of encoding a peptide according to the invention.
[0058] Preferably, the gene portion of the genetic construct is
operably linked to a promoter on the vector such that said promoter
is capable of directing expression of the gene portion in an
appropriate cell.
[0059] The present invention extends to such genetic constructs and
to prokaryotic or eukaryotic cells comprising same.
[0060] Chimeras of .chi.-conotoxins such as MrIA, with other
conotoxins or additionally with other peptides or proteins, can be
made to engineer the activity into other molecules, in some
instances to produce a new molecule with extra functionality. This
would preferably be done using the segment or segments of the
sequence of these peptides that contain the pharmacophore. Where
the pharmacophore is discontinuous, the segments making up the
pharmacophore should be positioned in the new construct to allow
binding to the receptor. Chimeras with other conotoxins may include
additional Cys residues and additional disulphide bonds.
[0061] It is common for conotoxin peptides within an activity class
to have a similar pattern of disulphide bonding, with peptide loops
between the respective cysteine residues. For .chi.-MrIA and
.chi.-MrIB disulphide bonds link the first and fourth, and the
second and third cysteine residues. This pattern is different from
the binding pattern observed for .alpha.-conotoxin peptides.
Despite this difference chimeric derivatives may be prepared by
substituting a loop of a .chi.-conotoxin peptide with the loop
comprising a sequence from another peptide, including
.alpha.-conotoxin.
[0062] The invention also includes dimers, trimers, etc. of
.chi.-conotoxin peptides as well as .chi.-conotoxin peptides bound
to other peptides.
[0063] Preferably the .chi.-conotoxin peptides according to the
invention have 10 to 30 amino acids, more preferably 11 to 20.
[0064] The complete gene sequence for the naturally occurring
.chi.-conotoxin peptides may be obtained using a combined 5' RACE
and 3' RACE strategy coupled with cloning and DNA sequencing.
[0065] The .chi.-conotoxin peptides according to the present
invention are active in inhibiting neuronal noradrenaline
transporter. Accordingly the invention provides the use of a
.chi.-conotoxin peptide as an inhibitor of neuronal noradrenaline
transporter, and in the treatment or prophylaxis of diseases or
conditions in relation to which the inhibition of neuronal
noradrenaline transporter is associated with effective treatment.
Such activity in pharmacological agents is associated with activity
in the prophylaxis or treatment of diseases or conditions of the
urinary or cardiovascular systems, or mood disorders, or in the
treatment or control of pain or inflammation.
[0066] Accordingly the present invention provides a method for the
treatment or prophylaxis of urinary or cardiovascular conditions or
diseases or mood disorders or for the treatment or control of pain
or inflammation including the step of administering to a mammal an
effective amount of an isolated, synthetic or recombinant
.chi.-conotoxin peptide having the ability to inhibit neuronal
isolated, noradrenaline transporter.
[0067] Examples of diseases or conditions of the urinary system
include urinary and fecal incontinence. Examples of cardiovascular
diseases or conditions include arrhythmias of various origins and
coronary heart failure. Examples of mood disorders include
depression, anxiety and cravings, such as smoking. Examples of pain
include chronic pain, neuropathic pain and inflammatory pain.
[0068] Preferably the mammal is in need of such treatment although
the peptide may be administered in a prophylactic sense.
[0069] The invention also provides a composition comprising an
isolated, synthetic or recombinant .chi.-conotoxin peptide having
the ability to inhibit neuronal noradrenaline transporter, and a
pharmaceutically acceptable carrier or diluent.
[0070] Preferably the composition is in the form of a
pharmaceutical composition.
[0071] There is also provided the use of an isolated, synthetic or
recombinant .chi.-conotoxin peptide having the ability to inhibit
neuronal noradrenaline transporter in the manufacture of a
medicament for the treatment or prophylaxis of urinary or
cardiovascular conditions or diseases, or mood disorders, of for
the treatment or control of pain or inflammation.
[0072] It is also noted that noradrenaline transporter is expressed
not only by nerve cells, but also by other tissues including the
placenta, pulmonary endothelial cells and the uterus. The peptides
according to the present invention may also be effective in
inhibiting these noradrenaline transporters, and may be useful in
treating conditions in which these transporters are implicated.
[0073] As will be readily appreciated by those skilled in the art,
the route of administration and the nature of the pharmaceutically
acceptable carrier will depend on the nature of the condition and
the mammal to be treated. It is believed that the choice of a
particular carrier or delivery system, and route of administration
could be readily determined by a person skilled in the art. In the
preparation of any formulation containing the peptide actives care
should be taken to ensure that the activity of the peptide is not
destroyed in the process and that the peptide is able to reach its
site of action without being destroyed. In some circumstances it
may be necessary to protect the peptide by means known in the art,
such as, for example, micro encapsulation. Similarly the route of
administration chosen should be such that the peptide reaches its
site of action.
[0074] The pharmaceutical forms suitable for injectable use include
sterile injectable solutions or dispersions, and sterile powders
for the extemporaneous preparation of sterile injectable solutions.
They should be stable under the conditions of manufacture and
storage and may be preserved against oxidation and the
contaminating action of microorganisms such as bacteria or
fungi.
[0075] Those skilled in the art may readily determine appropriate
formulations for the peptides or modified peptides of the present
invention using conventional approaches. Identification of
preferred pH ranges and suitable excipients, for example
antioxidants, is routine in the art (see for example Cleland et al,
1993). Buffer systems are routinely used to provide pH values of a
desired range and include carboxylic acid buffers for example
acetate, citrate, lactate and succinate. A variety of antioxidants
are available for such formulations including phenolic compounds
such as BHT or vitamin E, reducing agents such as methionine or
sulphite, and metal chelators such as EDTA.
[0076] The solvent or dispersion medium for the injectable solution
or dispersion may contain any of the conventional solvent or
carrier systems for peptide actives, and may contain, for example,
water, ethanol, polyol (for example, glycerol, propylene glycol and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about where necessary by the
inclusion of various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal
and the like. In many cases, it will be preferable to include
agents to adjust osmolality, for example, sugars or sodium
chloride. Preferably, the formulation for injection will be
isotonic with blood. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin. Pharmaceutical forms suitable for injectable use may be
delivered by any appropriate route including intravenous,
intramuscular, intracerebral, intrathecal, epidural injection or
infusion.
[0077] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients such as these
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the various
sterilized active ingredient into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, preferred methods of
preparation are vacuum drying or freeze-drying a of a previously
sterile-filtered solution of the active ingredient plus any
additional desired ingredients.
[0078] When the active ingredients are suitably protected they may
be orally administered, for example, with an inert diluent or with
an assimilable edible carrier, or it may be enclosed in hard or
soft shell gelatin capsule, or it may be compressed into tablets,
or it may be incorporated directly with the food of the diet. For
oral therapeutic administration, the active compound may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and preparations
preferably contain at least 1% by weight of active compound. The
percentage of the compositions and preparations may, of course, be
varied and may conveniently be between about 5 to about 80% of the
weight of the unit. The amount of active compound in such
therapeutically useful compositions in such that a suitable dosage
will be obtained.
[0079] 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.
[0080] The present invention also extends to any other forms
suitable for administration, for example topical application such
as creams, lotions and gels, or compositions suitable for
inhalation or intranasal delivery, for example solutions or dry
powders.
[0081] Parenteral dosage forms are preferred, including those
suitable for intravenous, intrathecal, intracerebral or epidural
delivery.
[0082] Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents and the
like. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, use thereof in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0083] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the novel dosage unit
forms of the invention are dictated by and directly dependent on
(a) the unique characteristics of the active material and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
material for the treatment of disease in living subjects having a
diseased condition in which bodily health is impaired as herein
disclosed in detail.
[0084] The principal active ingredient is compounded for convenient
and effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form. A unit
dosage form can, for example, contain the principal active compound
in amounts ranging from 0.25 .mu.g to about 2000 mg. Expressed in
proportions, the active compound is generally present in from about
025 .mu.g to about 200 mg/ml of carrier. In the case of
compositions containing supplementary active ingredients, the
dosages are determined by reference to the usual dose and manner of
administration of the said ingredients.
[0085] The invention will now be described with reference to the
accompanying drawings and examples, however it is to be understood
that the particularity of the following description is not to
supersede the generality of the preceding description of the
invention.
[0086] Referring to the figures:
[0087] FIG. 1 is a graphical representation showing the typical
effect of .chi.-MrIA on the time course of the contraction of the
bisected rat prostatic vas deferens to field stimulation with a
single supramaximal pulse (55 V, 1 ms). .chi.-MrIA (30 nM-3 .mu.M)
was added to the organ bath cumulatively in half log unit
steps.
[0088] FIG. 2 is a graphical representation of the effect of
.chi.-MrIA, -MrIA on contractile responses of bisected portions of
the rat epididymal vas deferens to exogenous
.alpha..sub.1-adrenoceptor agonists. (a) log concentration-response
curves to noradrenaline in the absence (.largecircle.) and the
presence of 1 .mu.M (.DELTA.) or 3 .mu.M (.quadrature.) in
.chi.-MrIA, -MrIA. (b) log concentration-response curves to
methoxamine in the absence (.largecircle.) and the presence
(.quadrature.) of 3 .mu.M .chi.-MrIA. Each data point in (a) and
(b) represents the mean.+-.SEM of observations from 4-5 individual
tissue preparations. Some error bars are obscured by the
symbols.
[0089] FIG. 3 is a graphical representation showing inhibition by
.chi.-MrIA on the desipramine sensitive accumulation of
[.sup.3H]-noradrenaline by Chinese Hamster Ovary (CHO) cells
transfected with the cDNA clone for the human neuronal
noradrenaline transporter. Accumulation of [.sup.3H]-noradrenaline
is expression as a percentage of the cellular uptake in the absence
of .chi.-MrIA. Data points represent the mean.+-.SEM or
observations from 4 separate experiments.
[0090] FIG. 4 is a diagrammatic representation showing derivation
of coneshell venom peptide sequences. 5'RACE PCR using the primers
AP1+CHI-1B produce the 5' UTR and leader peptide sequence which is
then used to generate the PCR primers specific for .chi.-conotoxin.
The 3' UTR using the primers CHI-1A+ANCHOR completed the derivation
of the remaining mature peptide sequence and 3' UTR sequence.
EXAMPLES
Drugs
[0091] The drugs used in this and the following examples include:
desipramine hydrochloride (Sigma); indomethacin (Sigma);
methoxamine hydrochloride (Sigma); (-)-noradrenaline bitartrate
(Sigma); [.sup.3H]-1-noradrenaline (specific activity 2200 Ci/mM;
New England Nuclear, Boston, Mass., U.S.A.); tetrodotoxin (Sigma);
yohimbine hydrochloride (Sigma).
Statistical Analysis
[0092] Data for the examples below are expressed as the mean and
standard error of 4-6 experiments. Sigmoidal curve-fitting for the
calculation of EC.sub.50 values was performed by non-linear
regression using the software package Igor Pro (WaveMetrics).
Differences between means were assessed by Student's t test
(two-tailed) using the software package Prism (GraphPad). Values of
P<0.05 were taken to indicate statistically significant
differences.
Example 1
Rat Vas Deferens Preparation
[0093] Male Wistar rats (250-350 g) were killed by a blow to the
head and the vasa deferentia were removed. Each tissue was divided
into bisected epididymal and prostatic portions. The tissue
segments were mounted in 5 mL organ baths under a tension of 0.5 g.
The bathing solution had the following composition (mM): NaCl, 119;
KCl, 4.7; MgSO.sub.4, 1.17; KH.sub.2PO.sub.4, 1.18; NaHCO.sub.3,
25.0; glucose, 5.5; CaCl.sub.2, 2.5; EDTA, 0.026; was maintained at
37.degree. C. and bubbled with 5% v/v. CO.sub.2 in O.sub.2. The
preparations were equilibrated for at least 45 min prior to the
commencement of experimentation. Contractions were registered by
means of an isometric force transducer (Narco Bio-System F-60) and
were recorded on a Power Macintosh computer using Chart version
3.5.4/s software and a MacLab/8s data acquisition system
(ADInstruments) at a sampling frequency of 200 Hz.
[0094] The bisected prostatic segments were used to examine the
effect of .chi.-MrIA on the electrically evoked contraction of the
smooth muscle mediated by sympathetic neurotransmission. The tissue
preparations were straddled with platinum stimulating electrodes.
Electrical field stimulation (EFS) was made at 3 min intervals
using a single 55 V pulse of 1 ms duration generated by a Grass S44
Stimulator. The contractions could be blocked by tetrodotoxin (0.1
.mu.M), indicating they were neurally mediated. Increasing
concentrations of the peptide were added to the organ bath
cumulatively in half log unit increments. Each dose was applied
once the effect of the previous dose on the response to electrical
stimulation had attained a steady level.
Effect of .chi.-MrIA on Sympathetic Neurotransmission
[0095] The bisected portions of the prostatic rat vas deferens
responded to field stimulation with a biphasic contraction. In this
preparation, the two components of the biphasic contraction were
well separated temporally. The first part of the response was the
dominant one, and reached a maximum level approximately 200 ms
after delivery of the electrical pulse. The second phase of the
contraction peaked approximately 500 ms after stimulation. Our
attempts to identify the pharmacological activity of .chi.-MrIA
began with an investigation of its effects in the field stimulated
rat vas deferens. The effect of .chi.-MrIA on the response of the
preparation to field stimulation is shown in FIG. 1. The conotoxin
(30 nM-3 .mu.M) acted to increase the second phase of the
contraction. This effect was found to be concentration dependent.
By subtracting the control response from traces obtained in the
presence of .chi.-MrIA, the specific enhancement by the peptide of
only the second component of the contraction becomes apparent. A
concentration-response curve for .chi.-MrIA acting to specifically
potentiate the second component can also be constructed.
[0096] The action of .chi.-MrIA on the electrically evoked response
was highly specific, enhancing only the second component of the
biphasic response. This late phase of the contraction is recognised
to be mediated by noradrenaline, and is selectively inhibited by
prazosin and other .alpha..sub.1-adrenoceptor antagonists. The
first phase of the contraction, which is due to the activation of
postjunctional P.sub.2x-purinoceptors by released ATP, was not
similarly enhanced. The magnitude of the noradrenergic component of
the contraction is modulated by the amount of noradrenaline
released by sympathetic nerve firing, the density of
post-junctional .alpha..sub.1-adrenoceptors and their coupling to
effector systems, and the rate at which noradrenaline is cleared
from the synapse.
[0097] Antagonism at presynaptic .alpha..sub.2-adrenoceptors is
well recognised to enhance the electrically evoked release of
noradrenaline from sympathetic nerves by blocking the activation of
a negative feedback system by released noradrenaline. However,
.alpha..sub.2-adrenoceptor antagonism can not be the mechanism of
action of .chi.-MrIA. Unlike .chi.-MrIA, .alpha..sub.2-adrenoceptor
antagonists such as yohimbine and idazoxan act to enhance equally
the purinergic and noradrenergic components of the contraction of
the rat vas deferens. Furthermore, the response to a single pulse,
as opposed to a train of stimuli, is not subject to regulation by
this negative feedback mechanism since there would be no agonist
present at these receptor sites at the time of stimulation.
Accordingly yohimbine (1 .mu.M) has no effect on the evoked
responses in this assay.
Example 2
Preparation to Examine Effect of .chi.-MrIA on Responses to
.alpha..sub.1-Adrenoceptor Agonists
[0098] The rat vas deferens was used as described above, except
that the bisected epididymal segments were not electrically
stimulated. These preparations were instead used to establish
concentration response curves to noradrenaline and methoxamine in
the absence and presence of .chi.-MrIA. In this tissue,
noradrenaline and methoxamine cause contraction of the smooth
muscle via activation of postjunctional
.alpha..sub.2-adrenoceptors. .chi.-MrIA at a concentration of
either 1 .mu.M or 3 .mu.M was applied to the organ bath and allowed
to equilibrate with the tissue for 20 min prior to cumulative
additions of noradrenaline or methoxamine. A single concentration
response curve was determined per preparation, with contralateral
tissue segments to which .chi.-MrIA was not applied serving as
controls.
Effect of .chi.-MrIA on Responses to .alpha..sub.1-Adrenoceptor
Agonists
[0099] It was possible to determine whether the action of
.chi.-MrIA occurs upstream or downstream of neurotransmitter
release by examining the effect of the peptide on the response to
exogenously applied noradrenaline. Since .chi.-MrIA enhanced the
potency of bath-applied noradrenaline, we can conclude that the
conotoxin acts by potentiating the response to noradrenaline,
rather than by promoting its release from neuronal stores. This
potentiation could occur as a consequence of increased
.alpha..sub.1-adrenoceptor responsiveness or impaired termination
of the action of noradrenaline. The .alpha..sub.1-adrenoceptor
agonist methoxamine was used to ascertain which of these was the
mechanism of action of .chi.-MrIA. This .alpha..sub.1-adrenoceptor
agonist differs from noradrenaline in that it is not a substrate
for the neuronal noradrenaline transporter. This transporter
functions to eliminate noradrenaline and other catecholamines from
the synapse by uptake into the nerve terminals, and represents the
major mechanism for terminating the action of noradrenaline at the
adrenoceptors of sympathetically innervated tissues. Because
methoxamine is not subject to removal by this mechanism, inhibition
of the transporter does not enhance the potency of its actions.
[0100] The effect of .chi.-MrIA on the responses of the bisected
segments of the rat epididymal vas deferens to two
.alpha..sub.1-adrenoceptor agonists was investigated. Log
concentration-response curves to noradrenaline in the absence and
presence of .chi.-MrIA are shown in FIG. 2a. At a concentration of
1 .mu.M, .chi.-MrIA acted to increase the sensitivity of the tissue
to noradrenaline, shifting the concentration response curve to the
left. The degree of potentiation observed was larger in experiments
with 3 .mu.M .chi.-MrIA. Neither concentration of the conotoxin
altered the maximum response of the tissue to noradrenaline.
.chi.-MrIA at a concentration of 3 .mu.M had no effect on the
concentration response curve to methoxamine (FIG. 2b). The
observation that .chi.-MrIA does not potentiate the action of
methoxamine, in contrast to its effect on responsiveness to
noradrenaline, is consistent with .chi.-MrIA being an inhibitor of
the neuronal noradrenaline transporter.
[0101] The lack of effect of .chi.-MrIA on the concentration
response curve to methoxamine also demonstrates that .chi.-MrIA has
no effect at .alpha..sub.1-adrenoceptors, which would be evident as
a parallel shift of the curve to the right. This distinguishes
.chi.-MrIA from some other inhibitors of noradrenaline transport,
particularly those used as antidepressants. The therapeutic target
of many of the antidepressant drugs is the neuronal noradrenaline
transporter. However, many of these compounds, especially the
tricyclic antidepressants, and to a lesser extent some newer drugs
which are structurally unrelated to the conventional tricyclics,
are recognised to, act at other sites such as
.alpha..sub.1-adrenoceptors and muscarinic ACh receptors.
Example 3
Guinea-Pig Ileum
[0102] Male guinea-pigs (285-425 g) starved overnight were killed
by a blow to the head and exsanguinated. Segments of the ileum of
approximately 1.5 cm length were removed and the luminal contents
cleared by gentle washing using a syringe filled with bathing
solution. The preparations were placed in 5 mL organ baths
containing bathing solution of the following composition (mM):
NaCl, 136.9; KCl, 2.68; CaCl.sub.2, 1.84; MgCl.sub.2, 1.03;
glucose, 5.55; NaHCO.sub.3, 11.9; and KH.sub.2PO.sub.4, 0.45;
warmed to 37.degree. C. and bubbled with 5% v/v CO.sub.2 in
O.sub.2. Indomethacin (10 .mu.M) was included in the bathing
solution to produce a stable baseline. The tissues were placed
under a tension of 1.0 g and allowed to equilibrate for 40 min
prior to the commencement of experimentation. Doses of nicotine (4
.mu.M) were then applied at 15 min intervals until the responses
were observed to be consistent. .chi.-MrIA (3 .mu.M) was then
added, 25 min after which another dose of nicotine was applied. The
responses to nicotine were measured isometrically and digitised at
a sampling rate of 10 Hz.
Effect of .chi.-MrIA on Responses to Nicotine in the Guinea-Pig
Ileum
[0103] .chi.-MrIA (3 .mu.M) had no significant effect on the
responses of ileal segments to nicotine. In the absence of the
conotoxin, the mean response was 3.83.+-.0.76 g, compared to
4.07.+-.0.80 g when .chi.-MrIA was present (p>0.1; paired
t-test; n=4). The .alpha.-conotoxins block nicotinic ACh receptors
of either the neuronal or muscle subtype. It was demonstrated that
.chi.-MrIA does not target neuronal nicotinic ACh receptors using
isolated segments of guinea-pig ileum. Because of the dependence of
the contractile response on muscarinic receptor activation, it
would be expected that the response of the guinea-pig ileum to
nicotine would be attenuated in the presence of .chi.-MrIA if the
conotoxin also acted as a muscarinic ACh receptor antagonist. In
this preparation, the nicotine-induced release of ACh and various
other neurotransmitters which activate postjunctional receptors was
unaffected by .chi.-MrIA. Thus, and in contrast to many other
transport inhibitors, .chi.-MrIA lacks anti-muscarinic
activity.
Example 4
Mouse Phrenic Nerve-Hemidiaphragm Preparation
[0104] Male Quackenbush mice (20-30 g) were killed by cervical
dislocation. Left and right hemidiaphragms were removed with the
phrenic nerves attached. The base of each hemidiaphragm was
positioned between two parallel platinum stimulating electrodes and
the phrenic nerve was placed through two small platinum loops for
field stimulation. The preparation was incubated at 37.degree. C.
in a 5 mL organ bath bubbled with 5% v/v CO.sub.2 in O.sub.2. The
composition of the bathing solution was (mM): NaCl, 135.0; KCl,
5.0; CaCl.sub.2, 2.0; MgCl.sub.2, 1.0; glucose, 11.0; NaHCO.sub.3,
15.0; and KH.sub.2 PO.sub.4, 1.0. The tissues were placed under 1.0
g resting tension. After allowing at least 30 min for
equilibration, alternating direct and indirect stimulation was made
at 10 s intervals using a 30 V pulse of 2 ms duration to the
muscle, and a 3 V pulse of 0.2 ms duration to the nerve,
respectively. The effect of a single dose of .chi.-MrIA at a
concentration of 3 .mu.M on the directly and indirectly elicited
contractions was examined. Responses were digitised and recorded as
described for the vas deferens preparations.
Effect of .chi.-MrIA on Responses to Electrical Stimulation of the
Mouse Phrenic Nerve-Hemidiaphragm
[0105] Contractions elicited by field stimulation of the phrenic
nerve, or by direct muscle stimulation, were unaffected by 3 .mu.M
.chi.-MrIA (n=4). .chi.-MrIA does not block the muscle nicotinic
ACh receptors in the mouse phrenic-nerve hemidiaphragm preparation.
The lack of activity of MrIA at skeletal muscle or motor nerve
distinguishes it from the majority of conotoxin peptides
characterised to date which are paralytic toxins and hence have a
clear role in prey capture.
Example 5
Cellular Uptake of [.sup.3H]-Noradrenaline
[0106] Chinese hamster ovary (CHO) cells were grown in 24 well
plates (Falcon) in 10% v/v fetal calf serum. On reaching 60-70%
confluence, the cells were transiently transfected (Lipofectamine,
Gibco) with an expression vector (pcDNA3, Invitrogen) incorporating
the full length cDNA for the human neuronal noradrenaline
transporter (Pacholczyk et al., (1991) Nature, 350, 350-4). A cDNA
clone of the neuronal noradrenaline transporter was used (Valium
Institute, Portland, Oreg., USA). Cellular uptake studies were
performed 36 hours after transfection. The CHO cells were initially
washed with transport buffer containing (mM): NaCl, 157; KCl, 2.7;
NaH.sub.2PO.sub.4, 11.8; MgCl.sub.2, 1.0 and CaCl.sub.2, 0.1; and
of pH 7.4. The cells were then incubated with transport buffer
containing 50 nM [.sup.3H]-noradrenaline (supplemented with
unlabelled noradrenaline as required) and 100 .mu.M ascorbic acid.
.chi.-MrIA (0.1 nM-1 .mu.M) or desipramine (10 .mu.M) were also
included as appropriate. After 20 minutes at room temperature, the
cells were rapidly washed with ice-cold phosphate buffered saline
and then lysed in 0.1% v/v Triton-X. The cell lysates were taken
for liquid scintillation counting to determine their level of
radioactivity. Additionally, an aliquot of the cell lysate was used
to measure protein concentration (BioRad DC protein assay). The
specific uptake of [.sup.3H]-noradrenaline by the noradrenaline
transporter was defined as the component sensitive to desipramine
(10 .mu.M).
Effect of .chi.-MrIA on Cellular Accumulation of
[.sup.3H]-Noradrenaline
[0107] The accumulation of noradrenaline into CHO cells expressing
the human neuronal noradrenaline transporter was reduced to less
than 0.5% of the control amount by desipramine (10 .mu.M),
demonstrating that the accumulation was due almost entirely to
specific uptake via the cloned transporter. The noradrenaline
transporter was confirmed as the target of the conotoxin in
cellular uptake studies. .chi.-MrIA (0.1 nM-1 .mu.M) inhibited the
accumulation of radiolabelled noradrenaline in a
concentration-dependent manner (FIG. 3), with a log IC.sub.50 value
of -8.17.+-.0.0275 (n=4). The concentration of .chi.-MrIA required
to inhibit the accumulation by 50% was found to be approximately 7
nM. This concentration is approximately one order of magnitude
lower than that needed for desipramine to produce the same
effect.
[0108] Cocaine and .chi.-MrIA are both naturally occurring
compounds, however, they are quite dissimilar. Cocaine is an
alkaloid extracted from the leaves of the coca plant, whereas
.chi.-MrIA is a peptide directly encoded by an animal gene. In
addition to its effect at the uptake transporter, cocaine is known
to possess potent local anaesthetic properties. This is due to
blockade of both sodium and potassium channels. No evidence was
found for local anaesthetic activity of .chi.-MrIA in any of the
assays. It was found that .chi.-MrIA had neither contractile nor
relaxant effects on the tone of the vas deferens by itself. Similar
studies revealed that .chi.-conotoxin does not inhibit the dopamine
transporter.
Example 6
[0109] Tritiated mazindol binding to the noradrenaline transporter
was measured in cells expressing the transporter protein (see
Example 5). The influence of .chi.-MrIA from 10.sup.-6 to 10.sup.9
on tritiated mazindol binding was measured. .chi.-MrIA had no
effect on tritiated mazindol binding, indicating that it acts
non-competitively, at a site distinct from traditional
noradrenaline transport inhibitors, such as desipramine, mazindol
and Cocaine.
Example 7
Derivation of Gene Sequence for the .chi.-Conotoxin Peptides
[0110] The complete gene sequence for the .chi.-MrIA was isolated
using a combined 5' RACE (Random Amplification of cDNA Ends) and 3'
RACE strategy coupled with cloning and DNA sequencing.
5' RACE
[0111] The oligonucleotide primer CH1-1B were designed from the
mature peptide sequence. The relationship of the oligonucleotides
to the peptide is as follows, together with the oligonucleotide
sequence:
TABLE-US-00003 SEQ ID NO. 3 .chi.-MrIA - NGVCCGYKLCHPC SEQ ID NO. 4
CHI-1b 5'- CANGGRTGRCANARYTTRTA -3' SEQ ID NO. 5 AP1 5'-
CCATCCTAATACGACTCACTATAGGGC -3' (where N/ A/C/G/T, R / A/G, Y /
C/T,)
[0112] Polymerase Chain Reaction (PCR) was carried out using the
oligonucleotide CH1-1B in combination with the AP1 oligonucleotide
on cDNA templates derived from the mRNA isolated from coneshell
venom ducts. The PCR products, which represent the 5' region of the
MrIA gene were isolated, purified, cloned into bacterial vectors
and sequenced. Gene sequence for MrIA was obtained from C.
marmoreus (FIG. 4).
3' RACE
[0113] The DNA sequence for the 5'-regions of the gene was used to
design oligonucleotides that were capable of detecting the MrIA
sequence, and sequences from other closely related peptides. The
positioning of the oligonucleotides relative to the gene sequence
is shown in FIG. 4. The oligonucleotide CH1-1A was used in PCR in
conjunction with the ANCHOR oligonucleotide to produce DNA
fragments corresponding to the leader peptide, mature peptide and
3' untranslated (3'UTR) regions of the gene. PCR of venom duct cDNA
templates from C. marmoreus produced DNA fragments corresponding to
the MrIA peptide.
[0114] The DNA sequences for the oligonucleotides are:
TABLE-US-00004 SEQ ID NO. 6 CHI-1A 5'- ACAGGCAGAATGCGCTGTCTCCC -3'
SEQ ID NO. 7 ANCHOR 5'- AACTGGAAGAATTCGCGGCCGCAGGAAT -3'
Complete Sequence for .chi.-MrIA
[0115] Gene sequence for .chi.-MrIA produced using 5' RACE and 3'
RACE represent overlapping fragments of the gene. These fragments
were combined, to produce a consensus sequence for the gene. The
consensus sequence is the full cDNA for the gene, and includes 5'
UTR, the leader peptide, the mature peptide and the 3' UTR. The
.chi.-MrIA leader and mature peptide oligonucleotide sequence is
shown in SEQ ID NO. 8, while the leader and mature peptide amino
acid sequence is shown in SEQ ID NO. 9.
TABLE-US-00005 SEQ ID NO. 8
ATGCGCTGTCTCCCAGTCTTGATCATTCTTCTGCTGCTGACTGCATCTGC
ACCTGGCGTTGTTGTCCTACCGAAGACCGAAGATGATGTGCCCATGTCAT
CTGTCTACTGTAATGGAAAGAGTATCCTACGAGGAATTCTGAGGAACGGT
GTGTGCTGTGGCTATAAGTTGTGCCATCCATGTTAA SEQ ID NO. 9
MRCLPVLIILLLLTASAPGVVVLPKTEDDVPMSSVYCNGKSILRGILRNG VCCGYKLCHPC
[0116] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integers or steps.
[0117] 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.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
Sequence CWU 1
1
9113PRTConus marmoreusPEPTIDE(12)Xaa at position 12 is 4-hydroxy
proline 1Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
10213PRTConus marmoreusPEPTIDE(12)Xaa at position 12 is 4-hydroxy
proline 2Val Gly Val Cys Cys Gly Tyr Lys Leu Cys His Xaa Cys1 5
10313PRTConus marmoreus 3Asn Gly Val Cys Cys Gly Tyr Lys Leu Cys
His Pro Cys1 5 10420DNAArtificial SequenceDescription of Artificial
Sequence Oligonucleotide probe 4canggrtgrc anaryttrta
20527DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide probe 5ccatcctaat acgactcact atagggc
27623DNAArtificial SequenceDescription of Artificial Sequence
Oligonucleotide probe 6acaggcagaa tgcgctgtct ccc 23728DNAArtificial
SequenceDescription of Artificial Sequence Oligonucleotide probe
7aactggaaga attcgcggcc gcaggaat 288186DNAConus marmoreus
8atgcgctgtc tcccagtctt gatcattctt ctgctgctga ctgcatctgc acctggcgtt
60gttgtcctac cgaagaccga agatgatgtg cccatgtcat ctgtctactg taatggaaag
120agtatcctac gaggaattct gaggaacggt gtgtgctgtg gctataagtt
gtgccatcca 180tgttaa 186961PRTConus marmoreus 9Met Arg Cys Leu Pro
Val Leu Ile Ile Leu Leu Leu Leu Thr Ala Ser1 5 10 15Ala Pro Gly Val
Val Val Leu Pro Lys Thr Glu Asp Asp Val Pro Met 20 25 30Ser Ser Val
Tyr Cys Asn Gly Lys Ser Ile Leu Arg Gly Ile Leu Arg 35 40 45Asn Gly
Val Cys Cys Gly Tyr Lys Leu Cys His Pro Cys 50 55 60
* * * * *