Patent Application: US-5421208-A

Abstract:
this invention relates to novel α - conotoxin - like peptides comprising the following amino acid sequence : xaa 1 - cys - cys - ser - xaa 2 - xaa 3 - xaa 4 - cys - xaa 5 - xaa 6 - xaa 7 - xaa 8 - xaa 9 - xaa 10 - xaa 11 - cys - nh 2 in which xaa 1 is gly or asp ; xaa 3 is pro , hyp or glu ; each of xaa 2 to xaa 8 and xaa 11 is independently any amino acid ; xaa 9 is pro , hyp or glu ; xaa 10 is asp , glu or γ - carboxyglu ; xaa 11 is optionally absent ; and the c - terminus is optionally amidated , with the proviso that the peptide is not α - conotoxin ep1 or α - conotoxin im1 . the peptides are useful in the treatment or prevention of pain , in recovery from nerve injury , and in the treatment of painful neurological conditions such as stroke .

Description:
for the purposes of this specification , the term “ peptide and peptide analogue ” includes compounds made up of units which have an amino and carboxy terminus separated in a 1 , 2 , 1 , 3 , 1 , 4 or larger substitution pattern . this includes the 20 naturally - occurring or “ common ” □- amino acids , in either the l or d configuration , the biosynthetically - available or “ uncommon ” amino acids not usually found in proteins , such as hyp , 5 - hydroxylysine , citrulline and ornithine ; synthetically - derived α - amino acids , such as α - methylalanine , norleucine , norvaline , c α - and n - alkylated amino acids , homocysteine , and homoserine ; and many others as known in the art . this term also includes compounds that have an amine and carboxyl functional group separated in a 1 , 3 or larger substitution pattern , such as β - alanine , γ - amino butyric acid , freidinger lactam ( freidinger et al . 1982 ), the bicyclic dipeptide ( btd ) ( freidinger et al . 1982 ; nagai and sato , 1985 ), amino - methyl benzoic acid ( smythe and von itzstein , 1994 ), and others well known in the art . statine - like isosteres , hydroxyethylene isosteres , reduced amide bond isosteres , thioamide isosteres , urea isosteres , carbamate isosteres , thioether isosteres , vinyl isosteres and other amide bond isosteres known to the art are also useful for the purposes of the invention . a “ common ” amino acid is an l - amino acid selected from the group consisting of glycine , leucine , isoleucine , valine , alanine , phenylalanine , tyrosine , tryptophan , aspartate , asparagine , glutamate , glutamine , cysteine , methionine , arginine , lysine , proline , serine , threonine and histidine . these are referred to herein by their conventional three - letter or one - letter abbreviations . an “ uncommon ” amino acid includes , but is not restricted to , one selected from the group consisting of d - amino acids , homo - amino acids , n - alkyl amino acids , dehydroamino acids , aromatic amino acids ( other than phe , tyr and trp , ortho -, meta - or para - aminobenzoic acid , ornithine , citrulline , norleucine , γ - glutamic acid , aminobutyric acid ( abu ), and α , α - disubstituted amino acids . as stated above , the present invention includes peptides in which one or more of the amino acids has undergone side - chain modifications . examples of side - chain modifications contemplated by the invention include modifications of amino groups such as reductive alkylation by reaction with an aldehyde followed by reduction with nabh 4 , amidination with methylacetimidate ; acylation with acetic anhydride ; carbamoylation of amino groups with cyanate ; modification of amino groups with 2 , 4 , 6 - trinitrobenzene sulfonic acid ( tnbs ); and acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride . the carboxyl group may be modified by carbodiimide activation via o - acylisourea formation followed by subsequent derivatization , for example to a corresponding amide . sulfhydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide ; performic acid oxidation to cysteic acid ; formation of a mixed disulfide with other thiol compounds ; reaction with maleimide , maleic anhydride or other substituted maleimide ; formation of mercurial derivatives using 4 - chloromercuribenzoate , 4 - chloromercuriphenylsulfonic acid , phenylmercury chloride , 2 - chloromercuri - 4 - nitrophenol and other mercurials ; carbamoylation with cyanate at alkaline ph . any modification of a cysteine residue must not affect the ability of the peptide to form the necessary disulfide bonds . however , it is possible to replace the sulfhydryl group of cysteine with the selenium equivalent , a selenohydryl group , so that the peptide forms a diselenium bond in place of one or more of the disulfide bonds . tryptophan residues may be modified , for example , by oxidation with n - bromosuccinimide or alkylation of the indole ring with 2 - hydroxy - 5 - nitrobenzyl bromide or sulfenyl halides . tyrosine residues may be in a sulfated or phosphorylated form . a non - exhaustive list of amino acids having modified side chains and other modified or non - naturally occurring amino acids is set out in table 1 . these types of modifications may be useful to stabilize the peptide following administration to a subject . other modifications may be made to the peptide in order to stabilize it or to enhance its other properties , for example membrane penetration or solubility . such modifications include modifying the side chain of one or more amino acids to attach other types of group , for example one or more lipophilic groups . such attachment may be made through a linking group designed to space the other group or groups away from the peptide so as not to interfere with the activity of the peptide . all such modified forms of the peptide are within the scope of the invention , provided that the modified peptide retains the ability to inhibit the activity of a nachr , to prevent pain , and / or accelerate the rate of recovery from nerve injury . those skilled in the art will readily be able to determine how to modify the peptides of the invention , and to identify those modified peptides which have the necessary activity . for oral administration the active ingredients may require protection before administration . for example , the peptide may be formulated with an assimilable edible carrier , or the peptide may be enclosed in hard or soft shell gelatin capsule , or compressed into tablets , or 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 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 . in addition , the active compound ( s ) may be incorporated into sustained - release preparations and formulations . the pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions , and sterile powders for the extemporaneous preparation of sterile injectable solutions . 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 . prolonged absorption of the injectable compositions can be achieved 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 , intraperitoneal , subcutaneous , intracerebral , intrathecal , epidural injection or infusion . 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 . parenteral dosage forms are preferred , including those suitable for intravenous , intrathecal , intracerebral , intramuscular , intraperitoneal , subcutaneous or epidural delivery . 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 . a unit dosage form can , for example , contain the principal active compound in amounts ranging from 0 . 25 μg to about 2000 mg . expressed in proportions , the active compound is generally present from about 0 . 25 μg / ml 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 . peptide vc1 . 1 was identified through the application of the following molecular biology techniques to conus victoriae . a cdna library was created from mrna isolated from venom duct tissue . oligonucleotide primers were used to amplify α - conotoxin precursors using the polymerase chain reaction . the pcr product was cloned into a suitable plasmid vector and individual clones sequenced . the amino acid sequence encoded by the cdna was then elucidated . this is described in further detail in example 1 . peptide vc1 . 1 inhibits the neuronal nachr . this was determined using a bovine chromaffin cell neuronal nachr assay system . bovine chromaffin cells are known to express neuronal nachrs ( campos - caro et al . 1997 ). when stimulated with nicotine the catechol - amines noradrenaline and adrenaline are released . chromaffin cells were isolated from the bovine adrenal medulla ( livett et al ., 1987a ) and stimulated with nicotine in the presence of the peptide vc1 . 1 . catecholamines released by chromaffin cells were separated using reverse phase high performance liquid chromatography and quantitated using electrochemical detection ( livett et al ., 1987b ). it was found that the peptide vc1 . 1 inhibited catecholamine release evoked by nicotine . this is described in example 2 . peptide vc1 . 1 also has analgesic activity . the chronic constriction injury model ( cci ) of bennett and xie ( 1987 ) was used to test the ability of vc1 . 1 to inhibit mechanically induced hyperalgesia in rats . rats were anesthetized and the right sciatic nerve exposed and tied with four chromic gut ligatures . this procedure produces hyperalgesia in the rat &# 39 ; s paw . rats were then injected intramuscularly in the mid thigh region with either saline or saline containing vc1 . 1 . a basile analgesy - meter ( ugo basile , comerio , italy ), which exerts a force that increases at a constant rate , was used to apply pressure to the rat &# 39 ; s paw . the force applied before the rat withdrew its paw was measured . it was found that following intramuscular administration of vc1 . 1 , nerve induced mechanical hyperalgesia was significantly attenuated both in the short ( 1 - 24 hrs ) and long term ( day 1 - 21 ). the ability of vc1 . 1 to reduce mechanically induced hyperalgesia was compared with ω - mviia , a conotoxin that has been reported to be 1000 times more potent than morphine ( bowersox et al . 1996 ). vc1 . 1 was found to be approximately 3 times more effective than ω - mviia at relieving mechanically induced hyperalgesia . this is described in example 3 . peptide vc1 . 1 is also effective in inhibiting sensory nerve function as determined by measuring the vascular response to nerve stimulation . microvascular blood flow was measured in the skin using laser doppler flowmetry from the base of blisters raised on the hind footpad of anaesthetized rats using a suction pressure of − 40 kpa . sensory nerve modulation of vascular function was assessed using antidromic electrical nerve stimulation performed on a sciatic nerve preparation and by perfusing sensory neuropeptides over the blister base . this is described in example 4 . peptide vc1 . 1 and its post - translationally modified form ( seq id no . 3 ) were also effective in accelerating the rate of recovery from nerve injury . this is discussed in example 5 . the invention will now be described in detail by way of reference only to the following non - limiting examples and drawings . specimens of the cone snail conus victoriae were collected from broome , western australia . the snails were part of the legal by - catch of a commercial fisherman , and approximately 20 snails were used . the venom ducts and bulbs of the cone snails were removed by dissection , before being snap - frozen in liquid nitrogen . poly - a mrna was extracted using a dynabeads mrna direct kit in accordance with the manufacturer &# 39 ; s specifications . double - stranded cdna was prepared from the isolated mrna utilising a marathon ™ cdna amplification kit . first strand dna was constructed utilising the marathon cdna synthesis primer and moloney murine leukaemia virus reverse transcriptase . second strand dna synthesis was achieved using e . coli dna polymerase , e . coli dna ligase and e . coli rnase h . blunt ended double stranded cdna was prepared using t4 dna polymerase . dna encoding the vc1 . 1 was amplified using oligonucleotide primers designed to anneal to the 5 ′ pre region 5 ′- atgggcatgcggatgatgtt - 3 ′ ( seq id no : 5 ) and the 3 ′ untranslated region 5 ′- cggaaagtgaagcaggtcag - 3 ′ ( seq id no : 6 ). the pcr contained 5 u of taq polymerase ( hoffmann - la roche ); 5 μl of 10 × reaction buffer ( 100 mm tris - hcl , 15 mm mgcl 2 , 500 mm kcl , ph 8 . 3 ); 400 μg of primers ; 2 μl of 10 mm dntps ; and an appropriate amount of cdna in a total volume of 50 μl of milliq water . the conditions for the pcr reaction were : 94 ° c . for 2 min ; followed by 30 cycles of 94 ° c . for 30 sec , an annealing step of 55 ° c . for 30 sec , 72 ° c . for 45 sec ; concluding with a final step of 72 ° c . for 5 mins . 50 ng pcr product was ligated into the pcr2 . 1 vector ( original ta cloning ® kit ) at a molar ratio of 2 : 1 . the reaction mixture was incubated overnight at 14 ° c . in a final volume of 10 μl of 6 mm tris - hcl , ph7 . 5 , 6 mm mgcl 2 , 5 mm nacl , 0 . 1 mg / ml bsa , 7 mm β - mercaptoethanol , 0 . 1 mm atp , 2 mm dithiothreitol , 1 mm spermidine with 4 u of t4 dna ligase . the pcr2 . 1 plasmid was transformed into competent invαf ′ e . coli cells in accordance with the manufacturer &# 39 ; s specifications ( original ta cloning ® kit ). cells were then spread onto lb plates containing ampicillin and x - gal ( 5 - bromo - 4 - chloro - 3 - indolyl - β - d - galactoside ). recombinant colonies were selected , incubated overnight at 37 ° c . in lb broth , and screened using a ultraclean ™ mini plasmid prep kit in accordance with the manufacturer &# 39 ; s protocols . clones containing inserts of interest were identified by agarose gel electrophoresis and sequenced by the dideoxy chain termination method using an abi prism dye terminator cycle sequence ready reaction kit . a geneamp pcr system 2400 ( perkin elmer ) was used for thermal cycling . each sequencing reaction underwent 25 cycles of : 96 ° c . for 10 sec , 50 ° c . for 5 sec , 60 ° c . for 4 min . sequences were analysed on a perkin elmer 377 sequencer . the nucleotide sequence of the clone encoding vc1 . 1 ( seq id no : 3 ) and deduced amino acid sequence for the vc1 . 1 precursor protein ( up to the stop codon ) ( seq id no : 6 ) are shown in fig1 . the nucleotide sequence giving rise to the deduced mature peptide is underlined , and the stop codon tga immediately follows the underlined sequence . this sequence has the following characteristics : ( a ) the coding sequence is the first 201 base pairs up to and including a stop codon of seq id no : 3 . ( b ) the coding sequence translates to 66 amino acid residues ( seq id no : 4 ): m g m r m m f t v f l l v v l a t t v v s s t s g r r e f r g r n a a a k a s d l v s l t d k k r g c c s d p r c n y d h p e i c g ( c ) the synthesized 16 amino acid residue peptide vc1 . 1 ( seq id no : 2 ) derives from the 17 amino acid residues preceding the stop codon , with the c - terminal glycine residue usually being removed and replaced by amidation of the resulting c - terminal cysteine , consistent with the c - terminus of known α - conotoxins which are active as neuronal nachr antagonists . ( d ) the synthesized peptide has four cysteine residues in a cc -( x ) 4 - c -( x ) 7 - c framework . adrenal chromaffin cells were isolated from adult bovine adrenal glands as described by livett et al ., ( 1987a ). isolated cells were plated out on collagen coated 24 - well plates at a density of 2 . 8 × 10 5 cells / cm 2 . three - to four - day old cultured chromaffin cells were allowed to equilibrate to room temperature for 5 mins . the incubation media was removed by two consecutive washes in locke &# 39 ; s buffer ( 154 mm nacl , 2 . 6 mm kcl , 2 . 15 mm k 2 hpo 4 , 0 . 85 mm kh 2 po 4 , 10 mm d - glucose , 1 . 18 mm mgso 4 . 7h 2 o , 2 . 2 mm cacl 2 . 2h 2 o , 0 . 5 % bovine serum albumin , ph 7 . 4 ) for five mins . cells were then incubated with various concentrations ( 0 . 01 μm , 0 . 1 μm , 1 μm , 10 μm ) of the peptide vc1 . 1 for five mins , before stimulation with either 4 μm nicotine or 56 mm kcl for a further five mins . the incubation mixture was separated from the cells and acidified with 2m perchloric acid ( pca ) to give a final concentration of 0 . 4m pca . the catecholamines remaining in the chromaffin cells were released by lysing the cells with 0 . 01m pca , and then acidified by addition of an equal volume of 0 . 8m pca . precipitated proteins were removed by centrifugation at 10 , 000 rpm for 10 mins . to measure the basal release of catecholamines , a control containing neither nicotine , kcl or vc1 . 1 was used . to determine the maximal release of catecholamines , a second control was stimulated with 4 μm nicotine or 56 mm kcl in the absence of vc1 . 1 . catecholamines present in each sample were separated by reverse phase high performance liquid chromatography ( rp - hplc ) utilizing a c18 column ( bio - rad ; 150 mm × 4 . 6 mm , 5 μm particle size ) and isocratic elution with 10 % methanol in the mobile phase ( 70 mm kh 2 po 4 , 0 . 1 mm naedta , 0 . 2 % heptane sulfonic acid ). catecholamines eluting from the column were identified by their retention time , and quantified by electrochemical detection ( 650 mv bas model lc - 3a ). known adrenaline and noradrenaline standards were used to calculate the amount of catecholamines in each sample , and these were expressed as a percentage of the total cell content . the results are shown in fig2 . consistent with its membership in an a - lineage superfamily , this novel α - conotoxin was a potent inhibitor of the nicotinic response in an in vitro cell - based functional assay for the neuronal - type nicotinic receptor , but did not inhibit the response due to 56 mm k + , indicating that voltage - activated ion channels such as the n - type voltage - gated ca ++ ion channels which are the target for the ω - conotoxins such as ziconotide are unlikely to be involved . thus it was found that vc1 . 1 inhibited catecholamine release evoked by nicotine . at a concentration of 10 μm vc1 . 1 inhibited noradrenaline release by 86 % and adrenaline release by 97 %. the ic 50 of vc1 . 1 was calculated to be 1 - 3 μm . vc1 . 1 did not have an inhibitory action when catecholamine release was evoked with kcl , as shown in fig3 . this demonstrated that vc1 . 1 does not target voltage gated ion channels , and confirmed that it targets the nicotinic acetylcholine receptor . vc1 . 1 displaces 3 h - epibatidine ( a nicotinic receptor agonist ) from its binding site on the nachr bovine adrenal glands obtained from the local abattoir were dissected and the medulla removed and placed into ice - cold 10 % w / v 0 . 32m sucrose buffer , supplemented with 1 mm edta , 0 . 1 mm pmsf and 0 . 01 % sodium azide . the medulla was homogenized using a polytron homogeniser , and the homogenate centrifuged at 100 g for 10 minutes at 4 ° c . the resultant supernatant was decanted ( s1 ) and the pellet resuspended in ice - cold sucrose buffer ( 5 ml / g of original medulla weight ). this resuspended pellet was centrifuged at 100 g for 10 minutes at 4 ° c . and the supernatants s1 and s2 were combined and centrifuged at 12000 g for 30 minutes at 4 ° c . the resultant pellet was resuspended in 50 mm phosphate buffer ( 40 mm k 2 hpo 4 , 10 mm kh 2 po 4 ) containing 0 . 01 % sodium azide , 0 . 1 mm pmsf and 1 mm bovine serum albumin and centrifuged at 12000 g for 30 minutes at 4 ° c . this step was repeated and the washed pellet resuspended in 50 mm phosphate buffer and stored at − 70 ° c . a bradford assay was performed to determine protein concentration , using bovine serum albumin as standard . this study used 3h - epibatidine ( 3h - epi ) and other ligands in binding studies for pharmacological characterization of the nachrs expressed on bovine adrenal medullary membranes . epibatidine has a high affinity for neuronal nachrs , particularly those containing α3 , but a low affinity for α7 nachrs ( gerzanich et al ., 1995 ). the stored adrenal medulla membranes were thawed and incubated ( 300 - 500 μg protein / tube ) at 37 ° c ., for 2 hours in the presence of 1 nm 3 h - epibatidine and the test ligand ( concentration range 1 nm - 1 mm ). 3 h - epibatidine was the radioligand , of choice as it has broad nachr subtype affinity . after the incubation period each assay tube was filtered and washed with phosphate buffered saline ( 150 mm nacl , 8 mm k 2 hpo 4 , 2 mm kh 2 po 4 ) in a brandell filtration system . glass fibre filters used in the filtration step had previously been soaked overnight in phosphate - buffered saline containing 5 % polyethylene imine ( pei ). the bound radioactivity remaining on the membranes was quantitated by liquid scintillation . non - specific binding was that remaining on the membranes after displacement with 2 mm nicotine . total binding was obtained by substituting the displacing test ligand with milliq water . specific binding was determined by subtracting the non - specific from the total binding . saturation binding experiments ( n = 3 ) determined a kd value using non - linear regression analysis ( prism , graphpad , san diego , calif .). from the displacement experiments hill coefficients and ki values were determined using non - linear regression analysis . ki values were determined using the cheng - prusoff rule whereby : ki = ic50 /( 1 +[ 3 h - epibatidine ])/ kd of 3 h - epibatidine . our results show that 3 h - epibatidine binding to bovine adrenal medullary membranes fits a single affinity model with a hill coefficient of 1 . 08 . vc1 . 1 and vc1 . 1ptm were examined for their ability to displace 3 h - epibatidine . the plasma membranes are known to contain neuronal α - nachrs . as shown in fig5 , α - conotoxin vc01 displaced 3 h - epibatidine binding , whereas vc1 . 1 ptm did not . conotoxin vc1 . 1 displaced the binding of the non - selective nicotinic receptor ligand 3 h - epibatidine in a concentration - dependent manner , displaying both a high affinity and a lower affinity two - component binding profile . vc1 . 1ptm displaced the first component , but did not significantly displace the second component of the binding , suggesting that two distinct binding sites may be involved in the binding of vc1 . 1 to these membranes . this differential action of vc1 . 1 and vc1 . 1 ptm is also apparent in fig7 , where vc1 . 1 inhibits a painful stimulus but vc1 . 1ptm is without effect . in other studies we have found that cytosine , 1 , 1 - dimethyl - 4 - phenylpiperazinium iodide ( dmpp ), carbachol , and nicotine , but not the α7 selective ligands , α - conotoxin 1 ml ( purchased commercially from sigma or auspep ) and α - bungarotoxin , displaced 3h - epibatidine binding . taken together , these studies indicate that vc01 probably binds to receptors of the α3α4 or α3α5α4 subtypes , whereas the receptor population not labelled by 3 h - epi most probably contains the α7 subtype , perhaps in combination with other subunits . a unilateral peripheral neuropathy was produced by the chronic constriction injury ( cci ) method of bennett and xie ( 1987 ). rats ( n = 4 - 8 per group ) were anaesthetized with sodium pentobarbital ( 60 mg / kg ). the right sciatic nerve was exposed at the level of the middle of the thigh by blunt dissection through the biceps femoris muscle . proximal to the sciatic trifurcation , about 7 mm of nerve was freed of adhering tissue and 4 ligatures ( 4 . 0 chromic gut ) were tied loosely around it with about 1 mm spacing . the skin incision was closed using surgical clips . groups of control rats received sham procedures , in which the right sciatic nerves were isolated but not ligated . a synthetic form of ω - conotoxin mviia ( snx - 111 ; bowersox et al , 1996 ) was purchased from auspep pty ltd . animals were allowed to recover for one week before being injected intramuscularly at the mid thigh region with saline , or with either vc1 . 1 or the synthetic ω - conotoxin mviia dissolved in 0 . 2 ml saline . mechanical pain thresholds were determined with a slightly modified version of the randall - selitto method ( randall and selitto , 1957 ), using the basile analgesy - meter ( ugo basile , comerio , italy ). this instrument exerts a force on the rat &# 39 ; s paw , which increases at a constant rate ( a certain number of grams per second ). the animal was restrained gently between cupped hands , and calibrated pressure increased until the rat withdrew its hind paw . mechanical pain thresholds were measured before drug injection , 1 , 3 and 24 hours post - injection , and then daily over 7 days . after 7 days of drug treatment , intramuscular injections ceased , and the pain threshold was measured twice a week . vc1 . 1 attenuated mechanically - induced hyperalgesia by 89 %, 64 %, and 116 % after 1 hour , 3 hours and 24 hours , respectively , compared to saline - treated controls . these results are summarized in fig6 a and b . daily intramuscular injection of vc1 . 1 also had significant effects on nerve injury - induced mechanical hyperalgesia at day 11 , day 12 , day 13 and day 14 , corresponding to 140 %, 186 %, 156 % and 172 % respectively , as shown in fig7 a . vc1 . 1 was approximately three times more effective than synthetic ω - conotoxin mviia at relieving mechanically - induced hyperalgesia over this period of time as shown in fig9 b . in addition , vc1 . 1 attenuated mechanically - induced hyperalgesia for several weeks following cessation of daily intramuscular injections , as shown in fig7 b . this study compared the effects of three synthetic conotoxins , ω - conotoxin mviia , ω - conotoxin gvia and α - conotoxin vc1 . 1 , on the vascular response mediated via activation of unmyelinated primary afferent sensory nerves ( antidromic stimulation ) using low frequency electrical stimulation ( lfes ) at 20v , 2 ms for 1 min at 5 hz . the conotoxin peptides were perfused over the base of a blister raised on the footpad of an anaesthetized rat using a suction pressure of − 40 kpa . the footpad is innervated by peripheral terminals of the sciatic nerve , and these are accessible to the perfused compounds . changes in the microvascular blood flow were measured in the skin using laser doppler flowmetry from the base of the blister ( merhi , dusting and khalil , 1998 ). the three conopeptides were perfused for 30 min prior to electrical stimulation of the sciatic nerve , throughout the 1 min electrical stimulation period and for 20 min following the stimulation . the results illustrated in fig8 a show that vc1 . 1 ( 1 μm ) produced a significant inhibition of the vascular response to low frequency electrical stimulation of the sensory nerves . changes in the microvascular blood flow are presented as area under the response curve measured in cm2 for 20 min post - stimulation . all three conotoxins tested produced a significant inhibition of the vascular response in response to lfes when compared to the control . there was no significant difference between any of the three conotoxin treatment groups . the degree of inhibition was comparable to that produced by ω - conotoxin gvia and by ω - conotoxin mviia . as the vascular response measured in response to electrical stimulation of sensory nerves not only reflects the ability of these nerves to release neurotransmitters to cause vasodilatation , but also the ability of the smooth muscle cells to dilate , we used sodium nitroprusside ( snp ) as an internal control indicator of changes in smooth muscle reactivity . snp is a direct smooth muscle cell dilator , which acts independently of sensory nerves . snp was perfused for 10 min . over the blister base in the presence or absence of the conotoxin to see if the action of the conotoxin tested was dependent or independent of an action on smooth muscle reactivity . the snp ( 100 μm , dissolved in ringer &# 39 ; s solution ) was perfused over the blister base for 10 min followed by a 10 min washout with ringer &# 39 ; s until blood flow was returned to baseline . the results in the absence of vc1 . 1 ( 24 . 5 ± 1 . 4 ) were not significantly different to the results in the presence of vc1 . 1 ( 23 . 0 ± 1 . 6 ) ( n = 22 ). the results , shown in fig8 b , show that perfusion of vc1 . 1 did not alter the response to snp , indicating that the inhibitory effect of vc1 . 1 on the vascular response to sensory nerve stimulation is mediated via an action which is independent of changes in smooth muscle reactivity . the most likely explanation is that vc1 . 1 inhibits the release of sensory neurotransmitters from the stimulated sensory nerves . we propose , based on the above results , that the most likely mechanism of action of vc1 . 1 is one which involves blocking the neuronal type nicotinic acetylcholine receptors on sensory nerves . this is not to exclude other mechanisms ( for example possible blocking of n - type ca ++ channels ), and indeed appears likely since a functional association of specific presynaptic nachrs and voltage - gated calcium channels has been demonstrated ( kulak et al . 2001 ). previous studies in our laboratory ( khalil et al ., 1999 ) showed that recovery from chronic constriction nerve injury can be determined by a reduction in hyperalgesia and a return to normal of the peripheral vascular response in an area innervated by the injured nerve . the vascular response can be examined by the perfusion of a vasodilator ( substance p , which acts via a preterminal mechanism ) over a base of a blister raised over an area innervated by the injured nerve . fig1 b and 12 b show the vascular response to perfusion of substance p at 1 μm after week 8 . the vc1 . 1 treated group showed 83 % and the vc1 . 1ptm treated group showed 85 % recovery compared to control . the vascular response in both saline ( 47 %) and mviia ( 54 %) treated groups remained significantly different from the control , suggesting incomplete functional recovery . the sciatic nerve includes sensory , sympathetic and motor nerves ; however this particular vascular test specifically reflects the function of sensory components of the nerve as substance p induces a vasodilator effect via a sensory - dependent mechanism . therefore , a return of this vascular response to normal reflects functional recovery of sensory nerves demonstrated by their ability to mount an inflammatory vascular response . the results show that both vc1 . 1 and vc1 . 1ptm were effective in returning the vascular response to normal after 8 weeks . peptide vg1 . 1 was isolated from the venom ducts of conus virgo ( collected at lizard island , north queensland ) by the same molecular approach as described for the isolation of vc1 . 1 from conus victoriae ( see example 1 above ). clones were selected , and the nucleotide sequence of the clone encoding vg1 . 1 was amplified using rtpcr / race technology . the amino acid sequence for vg1 . 1 was deduced and synthesized commercially ( auspep , melbourne ) by solid - phase peptide synthesis , and the disulfide bonds allowed to form by air oxidation . the deduced peptide vg1 . 1 has the structure : the synthesized peptide was tested as described for vc1 . 1 for its ability to : ( 1 ) inhibit the neuronal - type nicotinic acetylcholine receptor response in bovine adrenal medullary chromaffin cells in culture ; ( 2 ) compete functionally for the nicotinic response of chromaffin cells ( release of adrenaline and noradrenaline ); and ( 3 ) inhibit the vascular response to electrical stimulation of the sciatic nerve in a rat and to prevent pain in the cci model of neuropathic pain in the rat . the results are shown in fig1 and 13 . peptide an1 . 1 was isolated as described above for vg1 . 1 , except that the source of the peptide was the venom ducts of conus anemone collected from edithburgh , st vincent &# 39 ; s gulf , south australia ). the deduced structure of peptide an1 . 1 is : gly - cys - cys - ser - his - pro - ala - cys - tyr - ala - asn - asn - gln - asp - tyr - cys - nh 2 ( seq id no : 9 ), in which the c - terminus cysteine is amidated . the deduced sequence was chemically synthesized commercially ( auspep , melbourne ) and air oxidized . the synthetic peptide was tested in vitro and in vivo as described for vc1 . 1 . the results are shown in fig1 and 14 . the sequences of vc1 . 1 , vc1 . 1 ptm , an1 . 1 and vg1 . 1 can be aligned as follows , to illustrate the high degree of homology : in a preliminary experiment to determine whether the novel α - conotoxins had any adverse systemic side - effects , the effect of vc1 . 1 ( 1 μm in 200 μl im ) on resting systolic blood pressure in the rat was measured . fig1 shows that vc1 . 1 has no effect upon systemic blood pressure , i . e ., vc1 . 1 does not have a systemic circulatory effect . the association of nicotinic receptor function with pain is relatively new . earlier work had established that strong nicotinic agonists were analgesics , but the case is not so obvious or well established for nicotinic antagonists . mice lacking a particular nicotinic receptor subunit in the brain , have reduced pain responses ( marubio et al ., 1999 ). there is now good evidence that nachrs are also expressed on the somata of cultured sensory dorsal root ganglion ( drg ) neurons ( genzen et al ., 2001 ), and it is possible that nachrs are also expressed on the terminals of drg afferents . the spinal cord contains a subpopulation of inhibitory cholinergic interneurons which make presynaptic contact on to the terminals of primary afferents in the dorsal horn ( reviewed by genzen et al ., 2001 ). acetylcholine in the spinal cord might therefore activate nachrs expressed on the central terminals of drg afferents . the sources of acetylcholine which could activate nachrs on peripheral nerve terminals have not been identified , although there is evidence that many non - neuronal cells either contain or can manufacture acetylcholine ( wessler et al ., 1999 ). in addition , changeux and colleagues have recently shown that up to 40 % of neuronal nachrs are constitutively active , and do not require ach for activation ( changeux et al . 2001 ). nicotinic antagonists are effective in dampening down the constitutive level of activity . this population of constitutively active receptors may be differentially expressed in higher numbers in a neuroma following peripheral nerve damage . in addition , nicotinic receptor desensitization , which is responsible for the analgesic effects of strong nicotinic agonists such as epibatidine and abt - 594 , occurs at much lower agonist concentrations than those needed for nicotinic receptor activation ( see genzen et al ., 2001 ), and may play an important role in modulating sensory activity . others have reported irrative and autonomic responses or hyperalgesia when such agonists are administered at subanalgesic doses ( masner , 1972 ; khan et al ., 1994 ). while peripheral applications of nicotinic agonists can cause the excitatory effects described above , behavioural experiments have demonstrated that nicotinic agonists can also have analgesic properties . interestingly , epibatidine bears a resemblance to nicotine . taking note of the similar structure of epibatidine , but aware that it was too toxic for human use , daly and his colleagues in association with abbott laboratories began to create nicotine - like chemicals hoping that one may be effective as a painkiller ( for review , see plotkin , 2000 ). of the hundreds of molecules devised and tested , one stood out : abt - 594 . not only did it lack the toxicity of epibatidine , but proved effective against several types of pain , including pain caused by nerve damage against which even opiates are relatively ineffective . unlike opiates , abt - 594 appears to be non - addictive , enhances alertness rather than causing sedation and has relatively little effect upon the respiratory system . it lacks the major side effects of morphine , such as constipation and addiction , but has yet to receive regulatory approval . abt - 594 , like epibatidine , is an nicotinic agonist with analgesic potency greater than morphine , and can produce analgesia when administered systemically , intradermally or centrally in the nucleus raphe magnus , a site involved in descending modulatory system of analgesia . whereas the excitatory effects of nicotinic agonists are primarily due to nachr activation with subsequent neuronal depolarization leading to na + or ca 2 + channel activation , we propose that the inhibitory effects of strong nicotinic agonists like epibatidine and derivatives such as abt - 594 , may be due to receptor desensitization and na + or ca 2 + channel closure . this has not been suggested previously , and indicated that nicotinic antagonists , such as the α - conotoxins are useful as analgesics for clinical use against pain . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . all references cited herein , some of which are listed below , are incorporated herein by reference in their entirety . albuquerque e x , alkondon m , pereira e f , castro n g , schrattenholz a , barbosa c t , bonfante - cabarcas r , aracava y , eisenberg h m , maelicke a ( 1997 ) properties of neuronal nicotinic acetylcholine receptors : pharmacological characterization and modulation of synaptic function . j . pharmacol . exp . ther . 280 1117 - 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